Identification and characterization of a monocyte-derived neutrophil- …

ORAL PAPERSThrombin-Activatable Fibrinolysis Inhibitor

O2A-1Identification of a protein factor that regulates humanthrombin-activatable fibrinolysis inhibitor mRNAstabilityKuo ACY1, Novakovic D2, Koschinsky ML2 and Boffa MB2

1Queen’s University, Kingston; 2University of Windsor, Windsor,

Canada

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a basic carboxy-

peptidase zymogen that constitutes a molecular link between coagu-

lation and fibrinolysis, and between coagulation and inflammation.

The 3¢-untranslated region (UTR) of the human TAFI mRNA plays

a key role in regulating TAFI mRNA abundance. Three different

polyadenylation sites are used in TAFI mRNA processing with the

longest transcript being of the lowest stability and abundance and the

shortest transcript being of the highest stability and abundance.

Down-regulation of TAFI mRNA abundance by the inflammatory

cytokines IL-1b and IL-6 occurs through modulation of TAFI

mRNA stability. In the current study, we aimed to pinpoint cis-act-

ing elements in the TAFI 3¢-UTR and to identify protein factors

binding to these sites. We constructed a series of plasmids encoding

mRNAs containing rabbit b-globin sequences (as a reporter) fused to

sequences of the TAFI 3¢-UTR (encompassing 5¢ and internal dele-

tions). These plasmids were stably transfected into HepG2 (human

hepatoma) cells and the stability of the fusion transcripts measured.

We identified one element conferring stability and three elements con-

ferring instability. We then performed a series of gel mobility shift

analyses using RNA probes encompassing the three instability ele-

ments and HepG2 cell cytoplasmic extracts. Supershift assays identi-

fied the protein bound to the site between the second and third

polyadenylation sites as tristetraprolin (TTP). Mutation of the TTP

site abolished TTP binding in gel mobility shift assays and also stabi-

lized b-globin/TAFI fusion transcripts. TTP is a member of the

CCCH tandem zinc finger proteins and has been shown to regulate

the mRNA stability of a number of inflammatory proteins; TTP is

regulated by MAP kinase pathways, thus providing a plausible mech-

anism for the regulation of TAFI mRNA by inflammatory cytokines.

O2A-2Effect of TAFIa inhibition on tissue factor-inducedthromboembolism in the mouseRupin A, Vallez MO, Mennecier P, Richard I, Gloanec P andVerbeuren TJServier, Suresnes, France

Activated TAFI (TAFIa) or carboxypeptidase U cleaves C-terminal

lysine residues at the surface of partially degraded fibrin clots inhibit-

ing fibrinolysis before its propagation phase. The aim of this study

was to evaluate the role of TAFIa in an acute pulmonary thrombo-

embolism model induced by the systemic intravenous injection of

recombinant tissue factor (100 lL, Innovin�; Dade Behring) in the

tail vein of the mouse. Recombinant human tissue plasminogen acti-

vator (tPA, Alteplase, Boehringer Ingelheim) injected intravenously

(0.1 mL) 30 s before tissue factor increases survival of the mice in a

dose-dependent manner from 0.5 mg/kg (P < 0.05, Fisher test).

Interestingly, in the presence of an ineffective dose of tPA

(0.25 mg/kg), oral pretreatment of mice 1 h before tissue factor injec-

tion with a potent and selective TAFIa inhibitor (UK-396082, Ki

TAFIa = 12 nM and Ki Carboxypeptidase N > 100 lM) also

increases survival in a dose-dependent manner from 10 mg/kg

(P < 0.05, Fisher test). These results demonstrate that TAFIa plays

a role in acute pulmonary embolism induced by tissue factor in the

mouse. Moreover, our data confirm that prevention of TAFIa activ-

ity by an oral TAFIa inhibitor represents a novel therapeutic strategy

to prevent pulmonary thrombotic complications induced by activa-

tion of coagulation and fibrinolysis as found in patients with pulmo-

nary embolism.

O2A-3Evaluation of the profibrinolytic properties of ananti-TAFI monoclonal antibody in a mouse pulmonaryembolism modelVercauteren EB, Peeters M, Emmerechts J, Declerck PJ andGils AKatholieke Universiteit Leuven, Leuven, Belgium

Introduction Thrombin Activatable Fibrinolysis Inhibitor (TAFI)

plays an important role in the regulation of coagulation and fibrino-

lysis. Activated TAFI attenuates fibrinolysis by removing carboxyter-

minal lysines from partially degraded fibrin resulting in limited

plasmin formation.

Objective: To evaluate the profibrinolytic properties of MA-

TCK26D6.

Methods and Results: One-side ELISA and Surface Plasmon Reso-

nance analysis revealed that MA-TCK26D6, a monoclonal antibody

raised against human TAFI, cross-reacts with mouse TAFI. Chromo-

genic assays showed that MA-TCK26D6, using an 8-fold molar ratio

over TAFI, inhibits the plasmin-mediated activation of human and

mouse TAFI by 96 � 2% and 75 � 4%, respectively, and the throm-

bin/thrombomodulin-mediated activation by 26 � 17% and < 2%,

respectively. In in vitro clot lysis assays, MA-TCK26D6 (65 lg/mL)

reduces clot lysis time by 94 � 5% and 69 � 22% in human and

mouse plasma, respectively. The reduction of clot lysis time obtained

with the antibody is expressed relative to the reduction of clot lysis

time by potato tuber carboxypeptidase inhibitor (PTCI; 25 lg/mL), a

well-characterized TAFIa inhibitor. In a pulmonary embolism model,

mice were injected intravenously with a suboptimal concentration of

t-PA (0.1 mg/kg) before thromboplastin (2.5 mg/kg) injection to

induce thrombus formation in the lungs. Injection of MA-TCK26D6

(19 mg/kg) prior to t-PA and thromboplastin injections decreased

fibrin deposition in both lungs significantly, resulting in a significant

increase in the percentage of mice showing normal physical activity

(Table 1).

Conclusion: This is the first report on a monoclonal antibody that is

able to impair the activation of both human and mouse TAFI and

that can efficiently increase the percentage of mice with normal physi-

cal activity in a mouse pulmonary embolism model.

Table 1 for O2A-3

no MA MA-TCK26D6

Fibrin deposition left lungs 123 � 33 lg/mL 24 � 7 lg/mL*

Fibrin deposition right lungs 80 � 24lg/mL 33 � 9 lg/mL*

Mice with normal physical activity 26% 54%**

*P < 0.05; **P < 0.01, compared to control mice without MA

injection

ª 2010 The Authors. Journal compilation ª 2010 International Society on Thrombosis and Haemostasis 8 (Suppl. 1) (2010) 1–44

ORAL PAPERS____________________________________________________________________________________

O2A-4Novel in vivo and in vitro profibrinolytic effect ofMMP-10 mediated by TAFIOrbe J1, Rodriguez JA1, Orset C2, Barrenetxe J1, Angles-Cano E2,Colucci M3, Vivien D2 and Paramo JA1

1CIMA University of Navarra, Pamplona, Spain; 2INSERM U9192

Serine Proteases and Pathophysiology of the Neurovascular Unit,

Caen, France; 3Section of General and Experimental Pathology,

University of Bary, Bari, Italy

Introduction: Extracellular proteolysis, mediated by the plasminogen/

plasmin and the metalloproteinase systems, plays a key role in many

pathophysiological processes. The role of metalloproteinases (MMPs)

on fibrinolysis has been scarcely studied. We examined the profibrin-

olytic effect of thrombin-activated MMP-10 (stromelysin-2) in vivo

and in vitro.

Methods: t-PA-mediated fibrinolysis was measured in human plasma

and blood by turbidimetry and thromboelastography, respectively.

Degradation of TAFI and other fibrinolytic proteins by MMP-10

was assessed on tricine gels. TAFI activity was determined with a

chromogenic substrate (Pentapharma). Tail-bleeding time and throm-

boembolic ischemic stroke induced by local injection of thrombin

(1 U/mL) in the middle cerebral artery were investigated in wild-type

(WT) and MMP-10 knockout (MMP-10-/-) mice.

Results: MMP-10 (1:10 ratio) degraded and inactivated purified

human TAFI but not other fibrinolytic proteins (plasminogen, t-PA

and u-PA). MMP-10 (100–400 nM) dose-dependently shortened the

lysis time of normal plasma clots (P < 0.05) but not of TAFI-defi-

cient clots. On the contrary, a monoclonal antibody anti-MMP-10

prolonged the lysis time of plasma clots, even in the absence of added

MMP-10, suggesting a role of the endogenous strmelysis-2. MMP-10

(200 nM) caused a significant and TAFI-dependent (PTCI-sensitive)

shortening of lysis time in whole blood (30.5 � 19.7%, P = 0.01).

Compared to WT animals, MMP-10-/- mice displayed higher levels

of circulating TAFI (P < 0.01) and delayed reperfusion after throm-

bin-induced thromboembolic stroke (40%, P < 0.05). Surprisingly,

MMP-10-/- mice showed a significantly shorter tail bleeding time that

was reverted by MMP-10 injection (2 nM).

Conclusions: We show for the first time that MMP-10 plays a previ-

ously unrecognized profibrinolytic effect favouring clot lysis in vivo

and in vitro. Our results indicate that inhibition of TAFI activity by

MMP-10 may contribute to enhanced clot lysis and support a profi-

brinolytic effect of this protease.

O2A-5Intact thrombomodulin-mediated regulation offibrinolysis during and after liver transplantation,despite a profoundly defective thrombomodulin-mediated regulation of coagulationLisman T1, Ruitenbeek K1, Adelmeijer J1, Hendriks HGD1,Meijers JCM2 and Porte RJ1

1University Medical Center Groningen, Groningen;2AMC, Amsterdam, The Netherlands

Liver disease is associated with substantial hemostatic changes, which

aggravate during liver transplantation. We have recently measured

thrombin generation in absence and presence of thrombomodulin in

plasma samples taken during and after liver transplantation and

observed a striking and as yet unexplained inability of thrombomod-

ulin to downregulate thrombin generation in patient plasma. Since

thrombomodulin is not only vital for the activation of protein C, but

also for activation of thrombin activatable fibrinolysis inhibitor

(TAFI), we investigated thrombomodulin-mediated regulation of

fibrinolysis during liver transplantation using a plasma-based clot

lysis assay. Ten adult patients undergoing liver transplantation and

eleven healthy volunteers from our laboratory were included in this

study. Blood samples from the patients were taken perioperatively

and at days 1, 5 and 10 after surgery. Clot lysis assays were per-

formed in the presence or absence of thrombomodulin and in the

presence or absence of the TAFI inhibitor CPI. During liver trans-

plantation, clot lysis time in the absence of thrombomodulin steadily

decreased and was shortest after reperfusion. At the end of surgery,

clot lysis time transiently increased and subsequently returned

towards normal in the postoperative period. In the presence of CPI,

clot lysis time decreased in controls and in patients at the start of

surgery, but was not different from clot lysis time in the absence of

CPI after reperfusion indicating that no TAFI activation takes place

in samples taken at this time point. Upon addition of thrombomodu-

lin, clot lysis time in controls and in patients at the start of surgery

doubled, and clot lysis time after reperfusion increased almost 5-fold,

and these increases were fully attributable to TAFI activation.

In conclusion, we have demonstrated intact thrombomodulin-medi-

ated regulation of fibrinolysis during liver transplantation, even

though thrombomodulin-mediated regulation of coagulation is pro-

foundly disturbed in these patients.

O2A-6Soluble thrombomodulin improves the hemostaticbalance in whole blood from canines with hemophiliaAFoley JH1, Petersen KU2, Lillicrap D1 and Nesheim ME1

1Queen’s University, Kingston, Canada; 2PAION Deutschland

GmbH, Aachen, Germany

Background: Hemophilia A is a debilitating disease that affects

approximately 1 in 5000 males. Severe hemophiliacs are prone to

spontaneous soft tissue, joint and intracranial bleeding which

increases the morbidity and mortality of the disease. Another major

complication of hemophilia is the development of inhibitors to fVIII

which makes the primary form of treatment ineffective. Soluble

thrombomodulin (Solulin), an indirect stabilizer of blood clots, may

stabilize the clot to an extent where less fVIII (or fIX, fVIIa, aPCC,

etc.) may be needed to control bleeding in hemophilia.

Methods: Clot-lysis experiments were conducted in normal and hem-

ophilic (�fVIII neutralizing inhibitors) dog plasma and whole blood

and clotting and fibrinolysis were monitored by turbidity and throm-

belastography, respectively.

Results: Canine TAFI exists in plasma at a concentration of 100 nM

and the half-life of canine TAFIa is 23 min. In hemophilic plasma,

the clot-lysis time and the TAFIa potential increased linearly with

the Solulin concentration. At 200 nM Solulin the TAFIa dependent

prolongation of lysis reached a plateau. In hemophilic whole blood,

the area under the clot-lysis curve (AUCL) can be used as a measure

of clot firmness and 100 nM Solulin increased this parameter by

greater than 5-fold. The clot lysis time in whole blood was increased

from 20 min in the absence of Solulin to > 3 h in the presence of

100 or 250 nM Solulin. Finally, in hemophilic whole blood with

fVIII-neutralizing inhibitors (> 150 BU), the AUCL was normalized

with 390 nM Solulin and the clot-lysis time was increased from 18 to

46 min.

Conclusions: Solulin increases both the clot lysis time and clot firm-

ness in hemophilic plasma and whole blood. This improvement in the

hemostatic balance suggests that Solulin may be used to suppress

fibrinolysis and stabilize clots in hemophilia.


2 ORAL PAPERS____________________________________________________________________________________

Cellular Aspects

O2B-1Tissue-type plasminogen activator (t-PA) andtenecteplase but not urokinase or desmoteplasemodulate permeability across a human model of theblood-brain-barrierNiego B1, Medcalf RL1 and Petersen KU2

1Monash University, Melbourne, Australia; 2PAION Deutschland

GmBH, Aachen, Germany

t-PA is well-established for its role in the treatment of ischaemic

stroke in the CNS and can modulate blood brain barrier (BBB)

permeability in vitro and in vivo using rodent systems. This feature

of t-PA may be deleterious in patients with ischaemic stroke by

increasing the risk for haemorrhagic transformation during throm-

bolytic therapy. The mechanism underlying this effect of t-PA is

not fully understood, nor is it known if this phenomenon is shared

by other plasminogen activators. We have developed a human BBB

model whereby transformed human astrocytes are co-cultured for

72 h on the underside of a 3-micron porous membrane in contact

with primary human brain microvascular endothelial cells, seeded in

the inner (luminal) chamber. BBB permeability was estimated by

quantitating the transfer of FITC-labeled albumin across the mem-

brane. Addition of t-PA (0.1–1 lM) to the luminal side of the insert

caused a concentration-dependent increase in albumin transfer

within 8 h. Catalytic activity was required, as an inactive t-PA vari-

ant (ct-PA) produced no change in permeability. The t-PA effect

was blocked by a2-antiplasmin or aprotinin, indicating a require-

ment for plasmin generation. t-PA-induced increase in permeability

was also associated with changes in astrocyte morphology. The

t-PA variant, Tenecteplase (1 lM) also produced a significant

increase in permeability, although to a lesser extent than t-PA.

Reteplase, a variant consisting only of the second kringle and prote-

ase domains, was significantly less active. Urokinase, as well as des-

moteplase (both at 1 lM), were without effect and had no influence

on astrocyte morphology. Hence, the effect of t-PA on a human

model of the BBB requires plasmin generation, and is unique to

t-PA, its closest variant Tenecteplase and to a lesser extent retep-

lase. The lack of effect of desmoteplase is of particular interest as

this fibrin-selective thrombolytic is under phase-3 clinical develop-

ment for patients with ischaemic stroke

O2B-2Tissue-type plasminogen activator (t-PA) inducesintracranial bleeding through stromelysin-1 (MMP-3)induction in endothelial cells via low-densitylipoprotein receptor familySuzuki Y1, Nagai N2, Yamakawa K1, Kawakami J1, Lijnen HR3

and Umemura K1

1Hamamatsu University School of Medcine, Hamamatsu; 2Kinki

University School of Medicine, Osakasayama, Japan; 3KU

Leuven, Leuven, Belgium

Background: Tissue-type plasminogen activator (t-PA) is approved

for treatment of ischemic stroke patients, but it increases the risk of

intracranial bleeding (ICB). Here, we investigated the role of matrix

metalloproteinase -3 (MMP-3) in ICB increased by tPA.

Method: ICB was studied by a thrombotic middle cerebral artery

occlusion (MCAO) model in mice with genetic deficiencies of strom-

elysin-1 (MMP-3-/-) with or without intravenous t-PA (10 mg/kg)

administration 4 h after MCAO. Receptor-associated protein (RAP,

1 or 2 mg/kg), an antagonist of the low-density lipoprotein receptor

family (LDLRs) was also intravenously administered 5 min before

t-PA administration. In vitro, MMP-3 induction by t-PA treatment

(10 lg/mL) with or without RAP (200 nM) under 6 h oxygen-

glucose deprivation (OGD) followed by 18 h normoxia (Nor) was

assessed in bEnd.3, a mouse brain derived endothelial cell line.

Expression of LRP, a member of LDLRs, was measured both

in vitro and in vivo.

Results: In MMP-3+/+ WT mice given solvent, ICB was

4.3 � 2.9 mm3 (mean � SD, n = 7), which was significantly

increased with t-PA treatment to 9.7 � 4.7 mm3 (n = 9, P < 0.05),

whereas ICB in MMP-3-/- mice was not altered by t-PA treatment

(5.7 � 2.7 mm3, n = 9). Pretreatment with RAP prevented the

increased ICB induced by t-PA (2.0 � 2.0 and 1.9 � 1.1 mm2,

respectively, n = 8 each). In bEnd.3 cells, MMP-3 induction was

also significantly enhanced after OGD (1.7 � 0.14-fold increase),

which was further increased by t-PA treatment (2.4 � 0.16-fold,

P < 0.05 vs. OGD, n = 5). Furthermore, we observed upregulation

of LRP by ischemic stress, and suppression of MMP-3 induction

associated with t-PA by pretreatment with RAP, both in vitro and

in vivo.

Conclusion: These findings indicate that t-PA deteriorates ICB via

MMP-3 induction in endothelial cells and that antagonists of LDLRs

may have the potential to suppress ICB caused by t-PA treatment in

patients with stroke.

O2B-5Hypoxia and upregulation of hypoxia inducible factor1 alpha stimulate angiogenesis within resolvingvenous thrombiEvans CE, Humphries J, Mattock K, Waltham M, Wadoodi A,Saha P and Smith AKCL, London, UK

Objective: Angiogenesis is an important process in thrombus resolu-

tion, but the primary stimulus for neovascularisation is unknown.

Our aims were to determine whether: (i) hypoxia and hypoxia-induc-

ible factor (HIF) 1a are induced in resolving thrombus; (ii) this stim-

ulates production of factors that regulate angiogenesis; and (iii)

upregulating HIF1a enhances resolution.

Methods: The levels of hypoxia, HIF1a, and vascular endothelial

growth factor (VEGF) were measured in mouse thrombi at 1, 3, 7,

and 14 days after induction (n = 10/group). Oxygen tension was

measured with an oxygen sensor. HIF1a and VEGF were localised

by immunostaining and quantified by ELISA. An additional group

of thrombosed mice received daily intraperitoneal injections of either

L-mimosine, which prevents HIF1a degradation, or vehicle control

(n = 30/group). Expression of HIF1a, VEGF, and nine other HIF1-

mediated factors were measured by ELISA and proteome array at

days 1 and 7. Macrophage infiltration and thrombus resolution was

measured by image analysis at day 7.

Results: Oxygen tension in the thrombus was negatively correlated

with HIF1a (RS = -0.77, P < 0.0001); while HIF1a positively corre-

lated with VEGF (R = 0.85, P < 0.0005), during natural resolution.

HIF1a (P < 0.005) and VEGF (P < 0.005) was 2-fold greater in the

thrombus of mice treated with L-mimosine compared with controls,

and insulin-like growth factor binding protein 1 (IGFBP1, P < 0.01)

and stromal cell-derived factor 1 (SDF1, P < 0.04) were also

increased. Thrombus weight (P < 0.001) and volume (P < 0.05)

were reduced by a third in L-mimosine-treated mice compared with

controls; while vein recanalisation was 2-fold greater (P < 0.05),

which was associated with macrophage recruitment into the vein

wall.

Conclusions: Hypoxia and HIF1a induced in the naturally resolv-

ing thrombus are associated with angiogenic factor expression.


ORAL PAPERS 3____________________________________________________________________________________

Accumulation of HIF1a increases the expression of factors that

regulate neovascularisation and enhances thrombus resolution.

HIF1a may represent a novel target for treatments that promote

resolution and reduce the incidence of post-thrombotic syndrome.

O2B-6Deficiency of thrombospondin-2 does not affectmurine adipose tissue angiogenesis or developmentvan Hul M and Lijnen HRKU Leuven, Leuven, Belgium

Thrombospondins (TSP) are large, secreted, multimodular, calcium-

binding glycoproteins that are believed to have in vivo anti-angiogenic

properties. Interestingly, TSP-2 is upregulated in gonadal adipose tis-

sue of obese mice and TSP-2 deficiency is associated with elevated

levels of MMP-2, that was recently shown to be involved in adipose

tissue development in mice. In this study we have evaluated the

potential contribution of TSP-2 to adipose tissue related angiogenesis

and fat development. Therefore, TSP-2 deficient (TSP-2-/-) and wild-

type littermate (TSP-2+/+) mice were kept on normal chow (SFD)

or on high fat diet (HFD) for 15 weeks, followed by analysis of sub-

cutaneous (SC) and gonadal (GON) fat.

TSP-2 deficiency had no significant effect on total body weight or on

SC or GON adipose tissue mass of mice kept on either SFD or

HFD. The composition of SC and GON adipose tissues of TSP-2-/-

and TSP-2+/+ mice was comparable in terms of size and density of

adipocytes or blood vessels. The lack of an effect of TSP-2 deficiency

could not be explained by compensatory increases of TSP-1 expres-

sion in the TSP-2-/- mice. TSP-2 deficiency had no effect on adipose

tissue mRNA expression of gelatinase A (MMP-2), whereas gelati-

nase B (MMP-9) was downregulated in SC and GON adipose tissues

of TSP-2-/- mice on HFD. Zymography with extracts of SC and

GON adipose tissues of both genotypes did, however, not reveal sig-

nificant differences in MMP-9 or MMP-2 activity.

Taken together, these data indicate that TSP-2 is not an important

mediator of adipose tissue associated angiogenesis or fat mass accu-

mulation.

O2B-7Polymerization of serine protease inhibitors iscritically dependent on structural stability of deeplyburied residues inside surface cavitiesSingh P, Jairajpuri MA and Khan MdSJMI, New Delhi, India

Serine proteinase inhibitors (Serpins) are a unique superfamily of

protease inhibitor whose native state is metastable state which trans-

forms into stable state when they inhibit target proteases. Serpins like

neuroserpin, antithrombin, alpha-1antitrypsin, alpha-antichymotryp-

sin,C1-inhibitors and plasminogen activator inhibitor are involved in

important biological processes like blood coagulation, fibrinolysis,

cell migration, cell differentiation, embryo implantation, competent

activation and tumour suppression. Mutation in serpins can lead to

aberrant intermolecular linkages that can compromise the specific

function and also lead to polymer formation. Polymerization of ser-

pin is associated with disease like emphysema/cirrhosis, angiodema,

familial dementia, chronic obstructive bronchitis and thrombosis.

Critical understanding of the factors and mechanisms promoting ser-

pin misfolding and those regulating serpins conformational changes

are essential for elucidating the etiology of serpin based diseases. In

this study we have taken a dataset of all the known naturally occur-

ring point mutants in serpins that are prone to polymerization. First

we did accessible surface area (ASA) analysis to know the status of

burial at these positions and showed that most of the amino acids

are completely buried residues in native state. A cavity based analysis

showed that these residues are generally present in cavities which

change in size during mechanism of inhibition and polymerization.

We calculated the corresponding free energy change due to point

mutation in these natural variants, and showed that overwhelming

majority of amino acids involved in polymerization destabilizes the

protein. A comprehensive cavity analysis of various conformational

states of serpin showed of a very large cavity in the shutter region of

polymerized and cleaved state but not in native, latent and cavity fill-

ing variants indicating their potential as target for hindering polymer-

ization. We further show that docking and experimental studies that

indeed cavities can be filled to retard the native to polymer transi-

tion.


4 ORAL PAPERS____________________________________________________________________________________

Plenary Lectures (1)

O3-1The urokinase receptor: from structure-functionrelationships to in vivo imagingPloug MRigshospitalet, Copenhagen, Denmark

The urokinase-type plasminogen activator receptor (uPAR) is a gly-

colipid-anchored membrane glycoprotein that has been implicated in

a number of human pathologies, including dissemination of cancer.

uPAR belongs to the Ly-6/uPAR (LU) protein domain family and is

one of five multidomain members that are encoded by a small gene

cluster on chromosome 19q13 (1). The primary function of uPAR is

to focus uPA-mediated plasminogen activation to cell surfaces and

thus assist in extravascular fibrinolysis. Other proposed functions of

uPAR include stimulation of cell adhesion and migration by direct

interactions with the extracellular matrix. At the molecular level these

properties are accomplished by a high-affinity binding site for uPA

and a non-overlapping binding site for the somatomedin B domain in

vitronectin (Vn). A detailed structural insight into the molecular

interplay between uPAR, uPA and Vn has recently emerged from

crystallographic and biochemical studies on these complexes (2–5).

These studies clearly show that all three homologous LU-domains in

uPAR play important roles for the correct assembly of the composite

ligand-binding sites for uPA and Vn. Importantly, these studies also

define possible druggable target sites in uPAR for small molecules (5)

and provide guidelines for the development of small reporter probes

applicable for non-invasive imaging of uPAR expression in vivo by

positron emission tomography. In this presentation, I will review

recent advances in our knowledge of structure-function relationships

in the interaction between uPAR and its ligands (uPA and Vn) and

discuss how this information can guide translational research in pre-

clinical intervention studies of uPAR function.

1 Jacobsen & Ploug, Curr. Med. Chem. 2008; 15:2559–2573.

2 Llinas et al., EMBO J. 2005; 24:1655–1663.

3 Huai et al., Science 2006; 311:656–659.

4 Huai et al., Nat. Struct. Mol. Biol. 2008; 15:422–423.

5 Lin et al., J. Biol. Chem. 2010; 285:10982–10992.

O3-2Clinical relevance of fibrin structure for coagulationand fibrinolysisLord STUNC-Chapel Hill, Chapel Hill, NC, USA

The clinical relevance of fibrin structure has been demonstrated in

studies of populations with common diseases and individuals with

unusual diseases. The former, epidemiological studies link fibrin clot

structure to most, if not all, thrombotic diseases coronary artery dis-

ease, ischemic heart disease, stroke and thromboembolic disease. The

latter, represented by patients with Hemophilia where reduced throm-

bin generation manifests in an altered clot structure, link fibrin clot

structure to bleeding diseases. Studies of individuals with alterations

in fibrinogen structure and/or fibrinogen concentration link fibrin

structure to both bleeding and thrombotic diseases. Currently, how-

ever, the molecular pathophysiology that mediates these links

between fibrin structure and disease remains poorly defined. Bio-

chemical analyses have shown that many aspects of fibrin polymeriza-

tion influence clot structure. Polymerization is a kinetically controlled

process, influenced by the concentrations of thrombin and fibrinogen.

Understanding how the effective in vivo concentrations of thrombin

and fibrinogen are controlled and how these concentrations influence

polymerization provides a potential means to control clot structure.

Polymerization is mediated though specific interactions between fibrin

monomers and between early fibrin polymers. Identifying the nature

of these interactions may also provide a means to control clot struc-

ture. In addition, the link between structure and disease almost cer-

tainly reflects clot stability, but it is unknown whether susceptibility

to fibrinolysis or susceptibility to shear-induced fragmentation or

both is of greater consequence. Identifying the interactions that con-

trol clot endurance will provide opportunities to modify clot stability,

to enhance clot stability in bleeding diseases and control clot degra-

dation in thrombotic diseases.


ORAL PAPERS 5____________________________________________________________________________________

Quality of Fibrin: Sensitivity to Lysis

O4A-1Role of fibrin clot structure in susceptibility to lysisAriens RAS, Ajjan R, Standeven KF, Uitte de Willige S, Mutch NJand Philippou HUniversity of Leeds, Leeds, UK

A blood clot is composed of an intricate matrix of fibrin fibres,

which twist and branch out to form a three-dimensional network

that holds the clot together. The structure of the fibrin clot plays a

major role in the resistance of the clot to fibrinolysis by plasmin.

Several methods including turbidity and confocal microscopy have

been developed to investigate the relationship between fibrin struc-

ture and lysis. Studies on fibrinogen polymorphisms, fibrinogen gly-

cation, fibrin cross-linking by FXIIIa, and polyphosphates have

shed light on mechanisms involved in the modulation of fibrinolysis

by fibrin structure. Factors that influence fibrinolysis include: (i) the

density or porosity of the fibrin network determines the speed at

which fibrinolytic factors permeate the clot, (ii) the binding of tissue

plasminogen activator, plasminogen, and other factors to fibrin can

be modified by fibrin structure, and (iii) cross-linking of a2-antiplas-min to fibrin by FXIIIa contributes to resistance of the clot to lysis.

In turn, the process of fibrinolysis itself influences the structure and

quality of fibrin, thereby providing interplay between proteolysis

and fibrin structure. Further studies are required to develop a more

complete understanding of the relationship between fibrin structure

and fibrinolysis and its role in thrombosis. Modulation of the resis-

tance of fibrin to lysis could provide an important mechanism by

which to enhance (natural or therapeutic) thrombolysis and reduce

the risk for thrombosis.

O4A-2Effects of total fibrinogen and fibrinogen c’ onthrombin generation in plasmaCastoldi E1, Uitte de Willige S2, Dirven RJ3, Ariens RAS2,Rosing J1 and Bertina R3

1Maastricht University, Maastricht, The Netherlands; 2University

of Leeds, Leeds, UK; 3Leiden University, Leiden, The

Netherlands

Background: The fibrinogen c’ isoform is characterised by a nega-

tively charged C-terminal tail that binds with high affinity to throm-

bin exosite II, thereby inhibiting some of thrombin’s procoagulant

activities (e.g. factor VIII activation). Therefore, fibrinogen molecules

containing this isoform (cA/c’, 10–15% of total fibrinogen) have an

anticoagulant as well as a procoagulant function.

Aim: To investigate the effects of total fibrinogen and fibrinogen c’on thrombin generation in plasma.

Methods: Purified human fibrinogen was purchased from a commer-

cial source. cA/cA and cA/c’ fibrinogen were separated using anion

exchange chromatography and depleted of FXIII by ammonium sul-

phate precipitation. Thrombin generation was measured in: (i)

whole and defibrinated normal plasma; (ii) fibrinogen-deficient

plasma reconstituted with increasing amounts of cA/cA or cA/c’fibrinogen; and (iii) normal plasma before and after addition of c’-derived synthetic peptides (wild-type, mono-sulfated wild-type,

reverse and truncated). Thrombin generation was initiated with

either tissue factor (TF) or kaolin and monitored continuously using

a fluorogenic substrate for thrombin (Calibrated Automated Throm-

binography).

Results: Comparison of thrombin generation curves obtained in full

and defibrinated plasma indicated that fibrinogen prolongs the lag

time and increases the peak height of thrombin generation. The same

was observed when fibrinogen-deficient plasma was reconstituted with

total fibrinogen. Titrations of the individual fibrinogen isoforms in

fibrinogen-deficient plasma suggested that these effects were largely

attributable to cA/c’ fibrinogen. Accordingly, the wild-type c’ peptide(especially if sulfated) dose-dependently prolonged the lag time and

increased the peak height of thrombin generation, while the truncated

and reverse peptides were ineffective. All effects were more pro-

nounced when thrombin generation was initiated with kaolin or with

a low TF concentration, where the intrinsic pathway (factor VIII)

importantly contributes to thrombin formation. Differently, hardly

any effect of fibrinogen was observed at high TF concentrations. In

line with the effect of fibrinogen on the lag time of thrombin genera-

tion (which is a measure of the clotting time), the (sulfated) wild-type

c’ peptide also dose-dependently prolonged the aPTT but not the PT

of normal plasma.

Conclusions: Fibrinogen (particularly cA/c’) prolongs the lag time

and increases the peak height of thrombin generation initiated with

kaolin or with a low TF concentration. While the prolongation of

the lag time illustrates the anticoagulant action of fibrinogen, the

increase in peak height is likely due to the fact that fibrin-bound

thrombin is protected against inhibition by antithrombin.

O4A-3Clots formed from c’-fibrinogen are resistant to lysisbecause of impaired plasminogen activation by t-PAKim PYS, Leslie BA, Stafford AR, Vu T, Fredenburgh JC andWeitz JIThrombosis and Atherosclerosis Research Institute, McMaster

University, Hamilton, Canada

Background: About approximately 15% of circulating fibrinogen

contains a c-chain variant with an extended C-terminus and is desig-

nated c’-Fg. Although fibrin clots formed from c’-Fg have been

reported to be more resistant to lysis than those prepared from the

predominant cA-Fg, the mechanism responsible for this difference is

unknown.

Methods: Samples containing cA- or c’-Fg at various concentrations,

plasminogen, tissue-type plasminogen activator (t-PA) and a2-anti-plasmin were incubated with thrombin and CaCl2 at 37 �C and tur-

bidity was monitored at 400 nM. The time to half maximal decrease

in turbidity was designated as the lysis time. Similar experiments were

performed using (i) plasmin instead of plasminogen/t-PA, and (ii)

batroxobin, a snake venom extract that only releases fibrinopeptide-

A from fibrinogen, instead of thrombin, which releases both fibrino-

peptides-A and -B.

Results: t-PA-mediated lysis times increased with higher concentra-

tions of both cA- or c’-Fg, but the lysis times of clots formed from

c’-Fg were 1.4-fold longer than those from cA-Fg. This difference

was lost when plasmin was substituted for t-PA/plasminogen or

when batroxobin was substituted for thrombin. These findings sug-

gest that the slower lysis of clots formed from c’-Fg results from

reduced capacity to stimulate plasminogen activation by t-PA and

that this is attributable to impaired fibrinopeptide-B release from

c’-Fg.Conclusion: Our findings suggest that (i) fibrinopeptide-B release is

an important determinant of the capacity of fibrin to serve as a stim-

ulator of plasminogen activation by t-PA, thereby supporting the

concept that fibrinopeptide-B release exposes cryptic t-PA and/or

plasminogen binding sites on fibrin, and (ii) the relative resistance of

clots formed from c’-Fg to lysis reflects impaired fibrinopeptide-B

release. Therefore, these studies provide additional insight into the

stimulatory effects of fibrin on plasminogen activation by t-PA, and

suggest that c’-Fg levels may be an important determinant of the

resistance of thrombi to lysis.


6 ORAL PAPERS____________________________________________________________________________________

O4A-4Hampered dissolution of fibrin formed undermechanical stressKolev K1, Varju I1, Szabo L2, Machovich R1, Silva M3 andLongstaff C3

1Semmelweis University, Budapest, Hungary; 2Chemical

Research Center, Hungarian Academy of Sciences, Budapest,

Hungary; 3National Institute for Biological Standards and

Control, South Mimms, Potters Bar, UK

Recent data indicate that stretching forces cause a dramatic decrease

in clot volume accompanied by gross conformational changes of

fibrin structure. The present study attempts to characterize the lytic

susceptibility of fibrin exposed to mechanical stress. The relevance of

these structural variants was substantiated by scanning electron

microscopic (SEM) evaluation of human thrombi removed during

surgery. In 40% of the examined thrombi the fibrin fibers on the sur-

face of the clot were oriented in the direction of the shear forces

resembling fibrin architecture observed under clot stretching, whereas

the interior fibers formed a random 3D spatial meshwork. For our in

vitro dissolution studies these structural variations were modelled

with fibrin prepared in elastic silicon rubber tubes, which allow

adjustable mechanical stress. Following 2- and 3-fold longitudinal

stretching (2 · S, 3 · S) the volume of the fibrin clot decreased by

90% and in parallel the median fiber diameter and pore area in the

SEM images of the fibrin network decreased 2- to 3-fold. Application

of tissue plasminogen activator (tPA) to the surface of the clot, which

contains plasminogen, resulted in plasmin generation which was mea-

sured in the fluid phase. After 30 min activation 12.6 pmol/mm2 plas-

min was released from the non-stretched clot (NS), 5.5 pmol/mm2

from 2 · S and 2.3 pmol/mm2 from 3 · S clot. In the initial 15 min

of tPA-initiated fibrin lysis 160 ng/mm2 fibrin degradation products

were released from NS fibrin and 47 ng/mm2 from both 2 · S and

3 · S clots. Confocal microscopic images of fibrin surfaces showed

that a green fluorescent protein-fusion variant of tPA accumulated in

the interfacial layer of NS fibrin but not stretched fibrin. In conclu-

sion, mechanical stress confers proteolytic resistance to fibrin, which

is related to hampered tPA binding and penetration in the denser

fibrin network and consequently modified plasminogen activation at

the fluid-gel interface.

O4A-5Composition of coronary thrombi in acute myocardialinfarctionSilvain J1, Collet JP1, Nagaswami C2, Beygui F1, Edmondson K2,Bellemain-Appaix A1, Pena A1, Barthelemy O1, Montalescot G1

and Weisel JW2

1INSERM CMR937, Pitie-Salpetriere Hospital (AP-HP),

Universite Paris 6, Paris, France; 2University of Pennsylvania

School of Medicine, Philadelphia, PA, USA

Background: The dynamic process of intracoronary thrombus forma-

tion in ST-elevation Myocardial Infarction (STEMI) patients is

poorly understood. While it is known that time is of the essence in

the treatment of these patients, the reasons are poorly understood.

Methods and Results: Intracoronary thrombi (n = 45) were obtained

by thromboaspiration in 288 consecutive STEMI patients presenting

for primary percutaneous intervention (PCI) within 12 h of symptom

onset. Thrombi were analyzed using high definition pictures taken

with a scanning electron microscope. Plasma biomarkers (TnI,

CRPus, IL-6, PAI-1, sCD40 ligand and TNF-a) and plasma fibrin

clot viscoelastic properties were measured simultaneously on periph-

eral blood.

Thrombi were composed of fibrin (55.918%), platelets (16.818%),

erythrocytes (11.59%), cholesterol crystal (5.28.4%) and leukocytes

(1.32.0%). The median ischemic time from symptom onset to PCI

was 175 min (IQR 140–297). Ischemic time impacted thrombi compo-

sition, resulting in a positive correlation with intracoronary thrombus

fibrin content, r = 0.38, P = 0.01 and a negative correlation with

platelet content r = -0.34, P = 0.02. Thus, fibrin content increased

with ischemic time, ranging from 48.421% (< 3 h) up to 66.99%

(> 6 h) (P = 0.02), while platelet content decreased from 24.923%

(< 3 h) to 9.16% (> 6 h) (P = 0.07). The platelet activation marker

sCD40 ligand was positively correlated to platelet content in the

thrombus (r = 0.40, P = 0.02) and negatively correlated with fibrin

content (r = -0.36; P = 0.04). Multivariate analysis indicated that

ischemic time was the only predictor of thrombus composition with a

2-fold increase of fibrin-rich thrombus per ischemic hour [adjusted

OR2 (1.03–3.7) P = 0.01].

Conclusions: In acute STEMI, platelet and fibrin contents of the

occlusive thrombus vary over time, which may have a direct impact

on the efficacy of drugs or devices used for coronary reperfusion. For

example, these results show that the decreased effectiveness of throm-

bolytic drugs with time from onset of symptoms is likely due to an

increase in fibrin content of the thrombi over time.


ORAL PAPERS 7____________________________________________________________________________________

Fibrinolysis and Innate Immunity

O4B-1Fibrinolysis and innate immunityvan der Poll TAcademic Medical Center, University of Amsterdam,

Amsterdam, The Netherlands

Components of the fibrinolytic system have properties that go

beyond fibrinolysis. In this lecture the roles of several fibrinolytic

mediators in in vivo models of severe infection will be discussed.

Plasminogen activator inhibitor type I (PAI-1) has been implicated in

the pathogenesis of sepsis as elevated circulating PAI-1 levels are

highly predictive for an unfavourable outcome in sepsis patients.

Recently, studies using PAI-1 deficient mice and mice with transiently

enhanced expression of PAI-1 have pointed to a protective rather

than a detrimental role of this mediator in severe gram-negative

pneumonia and sepsis. PAI-1 deficiency impaired host defense during

Klebsiella pneumonia and sepsis as reflected by enhanced lethality and

increased bacterial growth and dissemination in mice with a targeted

deletion of the PAI-1 gene. Conversely, transgenic overexpression of

PAI-1 in the lung using a replication defective adenoviral vector

markedly improved host defense against Klebsiella pneumonia and

sepsis.

Tissue-type plasminogen activator (tPA) has been found to affect

antibacterial defense during abdominal sepsis caused by Escherichia

coli in mice: tPA deficient mice demonstrated an impaired defense

against E. coli peritonitis as indicated by higher bacterial loads and a

reduced survival. The protective function of tPA was independent of

its capacity to convert plasminogen into plasmin since plasminogen

gene deficient mice were indistinguishable from Wt mice in this

model. Similarly, pulmonary overexpression of human tPA markedly

improved host defense against Klebsiella pneumonia.

The urokinase-type plasminogen activator receptor (uPAR) mediates

leukocyte adhesion to the vascular wall or extracellular matrix com-

ponents. UPAR deficient mice demonstrated a strongly reduced neu-

trophil influx in models of bacterial pneumonia, which was

accompanied by an enhanced growth and dissemination of bacteria.

Conclusion: Mediators of the fibrinolytic system impact on innate

immunity by various mechanisms that at least in part are unrelated

to their function in lysis of fibrin clots.

O4B-2The occupancy of EPCR by protein C switches thePAR-1-dependent proinflammatory function ofthrombin to a protective responseRezaie RSt. Louis University School of Medicine, St. Louis, MO, USA

Thrombin is a multifunctional enzyme in plasma which clots fibrin-

ogen to stop bleeding during vascular injury. In addition to this

role, thrombin also regulates the anticoagulant and fibrinolytic path-

ways when it binds to its high affinity endothelial cell surface recep-

tor thrombomodulin (TM) to activate two plasma zymogens,

protein C and thrombin-activatable fibrinolysis inhibitor. Thrombin

also regulates inflammatory pathways when it activates the G-pro-

tein coupled receptor, protease-activated receptor 1 (PAR-1),

expressed on the surface of endothelial and other cell types. The

traditional view is that the activation of PAR-1 by thrombin on

vascular endothelial cells initiates a series of intracellular signaling

responses that culminate in the activation of the nuclear factor-jB(NF-jB) pathway, disruption of cellular permeability and expression

of proinflammatory molecules by endothelial cells. However, we

recently demonstrated that the PAR-1-dependent proinflammatory

signaling function of thrombin, observed in vitro in the endothelial

cell-culture systems, may not reflect a true physiological response

for thrombin based on the observation that the occupy of endothe-

lial protein C receptor (EPCR) by its natural ligand protein C

potently inhibited the PAR-1-dependent barrier enhancing and pro-

inflammatory functions of thrombin in the same cellular models.

Interestingly, we now demonstrate that the EPCR occupancy also

inhibits the PAR-1-dependent rapid release of P-selectin and

angiopoietin 2 (Ang2) from Weibel-Palade bodies, thereby down-

regulating the interaction of neutrophils with endothelium and up-

regulating the Ang1/Tie2 protective signaling pathway. Furthermore,

we demonstrate that the pretreatment of endothelial cells with the

catalytically inactive Ser-195 to Ser mutant of the zymogen protein

C leads to the PAR-1-dependent up-regulation of expression of

both Ang1 and Tie2 in endothelial, the same response which has

been attributed to activated protein C. Based on our results, we

hypothesize a PAR-1-dependent protective role for the low con-

centrations of thrombin in maintaining the integrity of the EPCR-

containing vasculature.

O4B-3The human fibrinolytic system is a target forproteases secreted by the pathogenic bacteriumPseudomonas aeruginosaMagdolen V, Seweryn P, Schmitt M and Beaufort NTechnical University Munich, Muenchen, Germany

A number of pathogenic bacteria interact with and engage the host

matrilytic and fibrinolytic plasminogen activation system. We hypoth-

esized that proteases secreted by Pseudomonas aeruginosa might con-

tribute to the activation of this major extracellular proteolytic

system, thereby participating in host tissue destruction and bacterial

dissemination.

We observed that the pseudomonal thermolysin-like metalloprotease

LasB converts the human zymogen of the urokinase-type plasmino-

gen activator (uPA), into its active form. Accordingly, while the sec-

retome from a LasB-expressing strain efficiently activates pro-uPA,

the secretome from an isogenic LasB-deficient strain is markedly less

potent in pro-uPA activation. Still, both secretomes induce some me-

talloprotease-independent activation of the human zymogen. This

later involves a trypsin-like protease, which we identified as the serine

protease ‘protease IV’(PIV).

Along with this, LasB converts plasminogen into mini-plasminogen

and angiostatin-related species, while, as previously reported, it pro-

cesses the uPA receptor, inactivates the plasminogen activator inhibi-

tor 1, and activates pro-matrix metalloproteinase 2. By contrast, PIV

does not target these factors at all.

To conclude, LasB and PIV, although belonging to different protease

families, both target and activate the host fibrinolytic system, a path-

way that is likely to contribute to bacterial virulence.

O4B-4Fibrinolysis impairs host defense during severemurine gram-negative sepsis (melioidosis)Kager M, Wiersinga WJ, Roelofs JJ, van t Veer C andvan der Poll TAcademic Medical Center, Amsterdam, The Netherlands

Background: Melioidosis, an endemic disease in Southeast Asia, is

caused by the gram-negative bacterium Burkholderia (B.) pseudomal-

lei. Melioidosis is associated with pneumonia and bacterial dissemina-

tion to distant sites, often leading to severe sepsis. Our previous work


8 ORAL PAPERS____________________________________________________________________________________

(Wiersinga et al. JTH 2008; 6: 32) revealed that infected patients

demonstrate evidence of coagulation activation with concurrent inhi-

bition of fibrinolysis. Mediators of fibrinolysis may not only influence

coagulation during infection but also leukocyte function.

Objective: To investigate the role of different proteins involved with

fibrinolysis during murine infection with B. pseudomallei, in particular

the role of tissue-type plasminogen activator, plasminogen activator

inhibitor type 1 (PAI-1) and a2-antiplasmin (A2AP).

Methods: Normal wild-type mice and mice deficient for either tPA,

PAI-1 or A2AP were infected intranasally with B. pseudomallei to

induce melioidosis. Mice were sacrificed after 24, 48 or 72 h and sur-

vival studies were performed. Lungs, liver, spleen and plasma were

harvested to measure bacterial loads, cellular influxes, pathology

scores, cytokine levels and coagulation parameters.

Results: tPA knockout (KO) mice, having less basal fibrinolysis, had

a strong survival advantage compared to wild-type mice. They also

had less bacterial outgrowth in liver and blood together with an anti-

inflammatory cytokine profile in the blood. On the contrary, both

PAI-1 KO mice and A2AP KO mice, that both have enhanced basal

fibrinolysis, showed significantly more bacterial outgrowth and a pro-

inflammatory cytokine profile in lungs and blood 48 and 72 h after

inoculation. Moreover, in PAI-1 KO mice a non-lethal bacterial load

appeared to be lethal during a survival study.

Conclusion: Fibrinolysis is an important component of the host

response during melioidosis. Our murine studies show that both tPA,

PAI-1 and A2AP play crucial roles, showing that less fibrinolysis

seems to be beneficial during this infection. Targeting fibrinolysis

could be a new therapy for this severe disease.

O4B-5Caveolin deficiency leads to increased activation ofcoagulation and decreased fibrinolysis in miceLupu F1, Lupu C1, Ivanciu L1 and Lijnen HR2

1Oklahoma Medical Research Foundation,Oklahoma City, USA;2Center for Molecular and Vascular Biology, Katholieke

Universiteit Leuven, Leuven, Belgium

Caveolin 1 (CAV1) is the main organizer of the specialized vesicular

microdomains of plasmalemma, named caveolae that are involved in

a variety of cellular processes. We have shown previously that caveo-

lae regulate the initiation of coagulation by controlling the endothe-

lial cell (EC)-bound TFPI. Here we used CAV1 deficient mice to

study the in vivo role of caveolin in the regulation of hemostasis in

normal and experimental inflammation. CAV1 knockout (CAV1-/-)

and wild type (WT) mice were challenged with LPS (1 mg/kg body

weight) or exposed to hypoxia. We observed that in contrast to WT

mice, non-challenged CAV1-/- animals have increased plasma fibrino-

gen consumption and fibrin deposition in the lung. Thrombin-anti-

thrombin and D-dimer levels confirmed that CAV1-/- mice have a

prothrombotic state that is further increased after LPS challenge.

In vitro analysis of immortalized lung microvascular EC from WT

and CAV1-/- mice incubated with/without LPS showed that EC of

CAV1-/- mice had less TFPI on the cell surface, and higher

TF-dependent coagulant activity than EC isolated from WT mice.

Zymography assay for plasmin generation on lung cryosections dem-

onstrated lower levels of t-PA dependent plasmin generation in

CAV-/- mice as compared to WT. Tissue and plasma analysis of

t-PA, u-PA and PAI1, measured at mRNA, protein and enzyme

activity levels suggest that the observed decrease in plasmin genera-

tion is due to decreased t-PA and increased PAI-1 production in

CAV1-/- mice. En-face confocal microscopy also showed less t-PA

staining. Quantitation of t-PA protein in lung homogenates con-

firmed that CAV1-/- mice have 2.5-fold less t-PA than WT mice. The

observed increased PAI-1 plasma levels correlate with higher PAI-1

mRNA expression in the liver of CAV1-/- mice.

In conclusion, our data demonstrate that CAV1 deficiency in mice

leads to increased activation of coagulation due to impaired TFPI

function and decreased t-PA dependent fibrinolysis.


ORAL PAPERS 9____________________________________________________________________________________

Massive Hemorrhage in the Intensive Care: Treatment, Diagnostic

Methods and Basic Science

O5A-1Introduction: bleeding and thromboelastographyten Cate HDepartment of Internal Medicine and Cardiovascular Research

Institute Maastricht, Maastricht University Medical Center, The

Netherlands

Bleeding is a major source of health related morbidity and costs in

patients undergoing surgery and/or being treated with antithrombotic

medication. In addition, in rare congenital diseases like the hemophi-

lias, bleeding is a potential life threatening event. In all these situa-

tions, specific derangements of the blood coagulation system occur.

However, the attending physician is not always aware of these hemo-

static defects, which explains why major bleeding complications can

occur without adequate preparative measures being taken.

One of the laboratory methods that have been advocated for guiding

major surgical procedures, a major source of postoperative bleeding,

is thromboelastography (TEG). TEG, of which several commercial

applications have been developed (e.g. TEG� or ROTEM�), is one

of the so called capacity assays that reflect a major part of the blood

coagulation system ex vivo. This means that TEG quantifies both the

initiation, amplification and propagation phases of fibrin clot forma-

tion, but also gives options for measuring fibrinolysis upon addition

of clot lysing compounds. All these phases of blood clotting can be

expressed as specific variables, providing a pattern of clot formation

and lysis that reflects certain clinical conditions. TEG was already

clinically applied in 1985 and several algorithms have been published

in which clinical management after surgery was guided by TEG mea-

surements. However, in spite of its longtime application controlled

clinical studies are scarce. Such published small scale studies of mod-

erate quality suggest that there is some benefit to obtain when using

algorithm based TEG in the surgical arena, showing a tendency

towards reduced need for transfusion of blood products as a main

outcome. There remains however a strong need for properly designed

controlled trials to unequivocally demonstrate the clinical benefit in

terms of reduced bleeding and costs, for TEG, also in comparison to

other capacity assays like platelet function tests or thrombin genera-

tion assays.

O5A-2The clinical problem and trials with fibrinogenRahe-Meyer NHannover Medical School, Hannover, Germany

Until 2000 it was internationally agreed that the critical fibrinogen

plasma level in intraoperative bleeding were 1 g/L or even lower.

Fibrinogen concentrate was only available in some countries, fibrino-

gen therapy with plasma or cryo was tertiary after therapy with

platelets and thrombin generating enzymes, and observational data

seemed to confirm this. Since 2000 fibrinogen concentrate have

become more available, more clinicians experienced successful bleed-

ing therapy targeting at higher fibrinogen plasma levels, and corre-

sponding data were published.

The planning of clinical trials with fibrinogen is complicated by a set

of problems:

1 Intraoperatively acquired coagulation disorders are multi dimen-

sional – how can you examine only one coagulation factor?

2 Intraoperative bleeding situations are dynamic and standard labo-

ratory test take to much time – how can you rapidly measure the

fibrinogen level to guide your therapy?

3 How can you intraoperatively quantify bleeding to trigger the ther-

apy and to control its success?

4 Which target levels should be used and how can dosing be calcu-

lated?

Two recently published studies will be discussed. A FIBTEM-guided

post-cardiopulmonary bypass administration of fibrinogen concen-

trate in aortic surgery 1,2 as a first-line therapy of microvascular

bleeding had been performed there. In their study design answers

were given to the questions above. They have resulted in:

1 Improved intraoperative management of coagulopathic bleeding

2 Reduced requirements for allogeneic blood product transfusion

3 Reduced 24-h drainage volume

4 Efficacious first-line therapy (even under the conditions of reduced

thrombin generation and platelet function)

5 No thrombotic events even with fast, high dose administration of

fibrinogen concentrate.

There is emerging evidence that fibrinogen concentrate may prove

effective in the management of perioperative bleeding in cardiac

surgery. The application to other kinds of surgery is yet to be estab-

lished.

1 Rahe-Meyer N et al., J. Thorac. Cardiovasc. Surg. 2009; 138:694–

702.

2 Rahe-Meyer N et al., Br. J. Anaesth. 2009; 102:785–92.

O5A-3The effect of blood plasma substitutes on fibrinstructureFenger-Eriksen C1 and Sørensen B2

1Aarhus University Hospital, Aarhus, Denmark; 2Haemostasis

Research Unit, St. Thomas Hospital, London, UK

Introduction: Artificial plasma expanders like hydroxyethyl starch

(HES) or dextrans is often required in massively bleeding patients to

maintain haemodynamic stability. Blood loss and fluid resuscitation

dispose to development of dilutional coagulopathy through dilution,

loss and consumption of coagulation factors and cells involved in the

haemostatic procress. In addition specific adverse effect from HES

fluid resuscitation on fibrinogen function and fibrin polymerisation

has been reported in more studies.

Impact of colloids and crystalloids on fibrin polymerisation

An in vitro comparative analysis of whole blood haemodilution with

isotonic saline versus artificial colloids verified a coagulopathy char-

acterized by suppressed clot firmness, but unchanged clot initiation

and parameters of clot propagation, as evaluated by thromboelas-

tometry.

Similar thromboelastometric characteristics have been reported from

a study including 20 bleeding patients receiving fluid resuscitation

with HES until a dilution level at 30%. The same study reported that

levels of fibrinogen decreased significantly below the levels expected

from dilution. In another human study including 66 patients under-

going orthopaedic surgery fibrin polymerisation, as measured by

thromboelastometry, was significantly impaired in the group receiving

colloids as compared with Ringers lactate (1).

A series of laboratory, animal, retrospective as wells one randomized

controlled trial all have shown that fibrinogen concentrate restored

clot strength, re-established the architecture of the fibrin meshwork.

Intervention with fibrinogen concentrate has been shown to reduce

blood loss in a pig model of massive bleeding and to decrease trans-

fusion requirements in bleeding patients (2).

Conclusion: Artificial colloids impair haemostasis more than crystal-

loids. Acquired fibrinogen deficiency caused by fibrin polymerisation

defect seems to be the leading determinant in colloid induced dilu-

tional coagulopathy.


10 ORAL PAPERS____________________________________________________________________________________

1 Mittermayr M, Streif W, Haas T, Fries D, Velik-Salchner C, Klin-

gler A, et al., Hemostatic changes after crystalloid or colloid fluid

administration during major orthopedic surgery: the role of fibrino-

gen administration. Anesth. Analg. 007 Oct;105(4):905–17.

2 Fenger-Eriksen C, Ingerslev J, Sorensen B. Fibrinogen concentrate

– a potential universal haemostatic agent. Expert Opin. Biol. Ther.

2009; 9(10).

O5A-4Thrombelastography-based monitoring for massiveblood loss in children during elective surgery forcraniosynostosis repair, TEG�versus ROTEM�

Machotta A1, Geerts J2, Grimminck B2, Stigter RL2, Poley MJ3,Al MJ3 and Appel IM2

1Department of Anaesthesiology; 2Department of Paediatirc

Oncology/Haematology; 3The institute for Medical Technology

Assessment (iMTA), Erasmus MC/ Sophia Children’s Hospital,

Erasmus University Medical Centre, Rotterdam, The Netherlands

The re-introduction of thromboelastography (TEG) has led to

decreased transfusions of blood products in adults. No systematic

studies on TEG and TEG-guided intervention during pediatric sur-

gery have been done. Using citrated blood, we compared TEG/Hae-

moscope and ROTEM/Pentapharm in a prospective study of 44

otherwise healthy children with craniosynostosis undergoing elective

surgery at the Sophia Children’s Hospital. Blood samples (5 mL from

an arterial line) for TEG and ROTEM measurement were obtained

after: induction of anesthesia (T1), Ringer’s LS 10 mL/kg (T2), Veno-

fundin to a maximum of 30 mL/kg (T3), transfusion of red blood

cells (T4), and FFP (T5). Between T2 and T3 all children demon-

strated a significant decline in Hb from mean 6.5 to 3.8 mmol/L

(P < 0.0005). The blood loss was mean 440 mL, requiring mean

220 mL transfusion of red blood cells. TEG and ROTEM parameters

pointed equally to dilutional coagulopathy, no signs of fibrinolysis.

Paired Students t-test demonstrated earlier significant changes in RO-

TEM, but most relevant changes from T2-T3 were equally highly sig-

nificant in both devices.

Table 1 for O5A-4

TEG� ROTEM�-INTEM ROTEM�-EXTEM

T1–T2R 6.3–6.0 min

K 1.9–1.7 min

a 65–66 �MA 62–61 mm

CT 159–154 s

CFT 62–69 s*

a 77–76 �MCF 63–61 mm*

CT 60–62 s

CFT 84–94 s*

a 73–71 �*MCF 61–59 mm*

MA-FF 19–19 FIBTEM-MCF 14–13*

T2–T3R 6.0–6.0

K 1.7–2.8*

a 66–58**

MA 61–48**

CT 154–170*

CFT 69–165**

a 76–62**

MCF 61–49**

CT 62–94**

CFT 94–170**

a 71 –60**

MCF 59–48**

MA-FF 19–6.2**FIBTEM-MCF 13–5.2**

T3–T4R 6.0–6.1

K 2.8–4.7*

a 58–42**

MA 48–42*

CT 170–193**

CFT 165–278*

a 62–54*

MCF 49–41**

CT 94–123*

CFT 170–302*

a 60–53*

MCF 48–41**

MA-FF 6.2–5.4* FIBTEM-MCF 5.2–4.5*

T4–T5R 6.1–5.9

K 4.7–3.1*

a 42–55**

MA 42–47*

CT 193–153*

CFT 278–195

a 54–59*

MCF 41–46*

CT 123–83*

CFT 302–212*

a 53–55*

MCF 41–46**

MA-FF 5.4–7.0* FIBTEM-MCF 4.5–5.6*

*P < 0.05 **P < 0.0001

O5A-5Fibrinogen and post-partum hemorrhage: fromprediction to treatmentDucloy-Bouthors AS, Pilla C, Bauters A, Wibaut B and Jude BCentre Hospitalier Regional et Universitaire, Lille, France

Post-partum hemorrhage (PPH) remains a major cause of maternal

morbidity and mortality. Charbit et al (1) have shown the decrease of

fibrinogen to be an early predictor of the severity of PPH. Early diag-

nosis of the hypofibrinogenemia is facilitated by thromboelastometry

ROTEM (Pentapharm Germany).(2). The correction of coagulation

disorders associated with the uterotonic treatment, could improve the

PPH evolution but it is not yet demonstrated (3). We report a clinical

observation illustrating the follow up of the coagulopathy’s treat-

ment.

Case report: Mrs H.M. was a 36 years. primiparus. A large fibroma

and anterior placenta praevia had been discovered during pregnancy.

Caesarean section had to be transplacental. Prophylactic uterine arte-

rial catheters were placed. The caesarean section lasted 90 min and

2500 mls bleeding was compensated by 2500 mls vascular loading

with colloids. Abdominal haemorrhage started again 3 h later. Embo-

lization was performed promptly and succeeded to stop the uterine

and hypogastric arterial flow but haemorrhage continued up to

4500 mls. Severe hypofibrinogenemia < 0.5 g/L (N: 2.9–7 g/L) and

nul FIBTEM amplitude (N: 20–35 mm) was treated gradually by

repeated injections of fibrinogen concentrate and antifibrinolytic

drug, then fresh frozen plasma, platelet concentrate and recombinant

activated factor seven (rFVIIa). Close biological and thromboelasto-

metric monitoring of each step of the procoagulant treatment was

realized. Although the ph, temperature, platelet count and calcium

had been maintained in normal range, rFVIIa failed to stop the

abdominal haemorrhage till the plasma fibrinogen level reached a

baseline range of 1.9 g/L and FIBTEM amplitude 11 mm.

Conclusion: In this PPH, a closed follow-up of the hypofibrinogen-

emia and its treatment efficacy has been useful for the clinician and

has contributed to complete the embolization and to avoid hysterec-

tomy.

1 Charbit D, Mandelbrot L, Samain E, Baron G, Haddaoui B et al.,

The decrease of fibrinogen is an early predictor of severity oh post-

partum haemorrhage. J.Thromb. Haemost. 2007; 5:266–73.

2 Huissoud C, Carrabin N, Audibert F, Levrat A, Massignon D,

Berland M, Rudigoz R-C. Bedside assessment of fibrinogen level in

postpartum haemorrhage by thrombelastometry. BJOG 2009;

116:1097–1102.

3 WHO guidelines for the management of post-partum haemorrhage

and retained placenta. WHO Library cataloguing 2009; WQ 330.


ORAL PAPERS 11____________________________________________________________________________________

Cellular Proteolysis

O5B-1Update on the clinical relevance of the plasminogenactivation system in cancerSchmitt MTechnical University of Munich, Munich, Germany

Today’s integration of cancer-associated biomarkers into disease

management of cancer patients differs from cancer policies several

years ago. Different than before, personalized, tailored patient care is

now in focus of cancer medicine, observing detailed information

about a patient’s gene and/or protein expression profile, to allow

identification of patients at risk. Substantial progress has come

through molecular biology techniques, providing gene and/or protein

signatures of molecular alterations in each patient’s tumor. Though,

to allow personalized treatment of cancer patients, we need signifi-

cant, validated biomarkers to determine the course of the disease and

to predict response to a given therapy.

Regarding this issue, the prognostic/predictive value of cancer bio-

markers uPA (urokinase-type plasminogen activator) and its inhibitor

PAI-1, which are key members of the plasminogen activation system,

determined by ELISA in tumor tissue extracts, to tailor individual-

ized cancer therapy, was convincingly shown for several cancer types

in numerous clinically relevant, validated retrospective and prospec-

tive studies. Most of the research on clinical utility of uPA, its recep-

tor uPAR (CD87), and its inhibitor PAI-1 has been centered on

breast cancer specimens, resulting in a multicenter breast cancer ther-

apy trial (Chemo-N0) and an international multicenter pooled analy-

sis surveying original follow-up data of patients with either high or

low uPA/PAI-1 antigen values. Consequently, for the first time ever

for any cancer biomarker, for breast cancer, uPA and PAI-1 were

awarded the highest level of evidence, LOE-1.

Another international clinical trial, NNBC3, aiming at treating 4150

high-risk breast cancer patients stratified by high uPA/PAI-1 has fin-

ished recruitment. A second, still ongoing large chemotherapy trial

(Plan B) is comparing the clinical effectiveness of uPA/PAI-1 versus

the 21-gene test Oncotype DX. An orally applicable small synthetic

molecule (Mesuprone) directed towards the proteolytic activity of

uPA is currently in phase II clinical trials in patients afflicted with

different types of advanced cancer.

O5B-2Extracts of echinococcus multilocularis cysts induceproliferation and protease expression of humanumbilical vein endothelial cells in vitroMahdy Ali K1, Kaun C1, Rychli K2, Hohensinner PJ3, Weiss T4,Auer H5 and Wojta J1

1Medical University of Vienna; 2University of Veterinary

Medicine, Vienna, Austria; 3Vesalius Research Center,

K.U. Leuven, Leuven, Belgium; 4Center for Clinical Heart

Research, Ulleval University Hospital, Oslo, Norway;5Department of Hygiene, Medical University of Vienna, Vienna,

Austria

Objective: Echinococcus multilocularis (E. multilocularis), one of the

most dangerous helminthic parasites, causes alveolar echinococcosis

affecting the liver and destroying the parenchyme. In alveolar echino-

coccosis tumor-like cysts grow beyond the organic borders and

invade attached organs. Vascularization is essential for tumor

growth, therefore induction of angiogenesis is a common feature of

many tumors. For angiogenesis to succeed proteolysis of extracellular

matrix is essential. The aim of our study was to investigate possible

angiogenic properties of E. multilocularis and to detect a possible

involvement of proteolytic proteins in this process.

Methods: Human umbilical vein endothelial cells (HUVEC) were

treated with the extract of homogenized E. multilocularis cysts grown

in mice at different concentrations for 72 h. Cell proliferation was

quantified using a proliferation assay (EZ4U�; Biomedica) Tube for-

mation of HUVEC grown on Matrigel� in the absence or presence

of echinococcal cyst extract was determined. Quantitative PCR analy-

sis was performed to detect specific mRNA for urokinase type of

plasminogen activator (uPA), uPA receptor (uPAR) and matrix me-

talloproteinase 1 (MMP-1) in stimulated HUVEC.

Results: HUVEC treated with the extract showed increased prolifera-

tion (up to 2.3-fold) and capillary tube formation in comparison to

untreated HUVEC. Quantitative PCR revealed a significant increase

in mRNA levels specific for uPA (up to 4-fold), uPAR (up to 4.6-

fold) and MMP-1 (up to 7.6-fold).

Conclusion: We could show for the first time that echinococcal cyst

extract induces proliferation and tube formation of HUVEC in vitro.

Furthermore we could show an increased expression of several prote-

olytic molecules in such treated HUVEC, namely uPA, uPAR and

MMP-1, which are known to modulate angiogenesis. We speculate

that the helminthic parasite E. multilocularis has the ability to induce

angiogenesis and that the uPA/uPAR system as well as the MMP

system may be critically involved in the tumor-like growth of this

parasite.

O5B-3Modulation of uPAR signaling to ERK/MAPK byendocytic receptors of the LDLR familyGeetha N1, Mihaly-Bison J1, Blasi F2 and Binder BR1

1Medical University of Vienna, Vienna, Austria; 2Molecular

Genetics Unit, DIBIT- San Raffaele Hospital, Milan, Italy

The interactions of uPA/uPAR/PAI-1 complex, with transmem-

brane receptors lead to the activation of intracellular signaling

machinery like MAP kinases. Active recombinant PAI 1 induces

uPA/uPAR dependent sustained activation of ERK1/2 in an other-

wise PAI-1 free system through LDLR family members. This

sustained ERK1/2 activation was accompanied by translocation of

ERK 1/2 into the nucleus and focal adhesions and led to increased

adhesion. Now we wanted to address the question whether

activation of the MAP Kinase pathway by RTK can also become

sustained by simultaneous LDLR signalling. Therefore we mim-

icked the PAI mediated sustained ERK1/2 activation by combining

a RTK type (EGF – EGFR) and a scavenger receptor type [Lac-

toferrin (LF) – LDLR] signalling. We found a RAP dependent

sustained activation of ERK 2, but not of ERK1, upon stimula-

tion with EGF & LF together in human fibrosarcoma cell line

HT-1080 in a time dependent manner leading to an increased

adhesion on Vitronectin through the redistribution of integrin b5into focal adhesions and it was happening through LRP1. This

happened by downregulating the expression DUSP6 in the cyto-

plasm & DUSP 1 in the nucleus with EGF/LF, via an increased

proteasomal degradation of the same DUSPs, in a RAP dependent

manner. Experiments with R3 monoclonal antibody against uPAR

showed that sustained ERK activation & DUSP downregulation

by EGF & LF is uPAR dependent. From these data we conclude

that LDLR signalling can in principle make MAP-Kinase signaling

induced by RTKs to become sustained but that uPAR-uPA-PAI-1

signaling is peculiar in so far as it induces sustained ERK1 activa-

tion that is in contrast to sutained ERK2 activation acompanied

by nuclear translocation of P-ERK and increased adhesion. We

find it promising as it may explain the mechanism of higher inci-

dence of cancer in hyperlipidemic patients or at the state of

inflammation.


12 ORAL PAPERS____________________________________________________________________________________

O5B-4uPAR/LDLR interaction: a novel target for efficientendothelial cell migrationNovotny R, Unseld M, Bukowsky C, Poettler M, Binder BR,Zielinski CC and Prager GWMedical University of Vienna, Vienna, Austria

The urokinase plasminogen activator receptor (uPAR) system is

known to play a pivotal role in angiogenic endothelial cell behavior.

Recently, we have shown that in endothelial cells uPAR is redistrib-

uted to the leading edge of migrating cells upon VEGF stimulation

via a coordinated internalization and recycling mechanism, which

involves uPA:PAI-1:uPAR complex formation and LDLR-family

member mediated complex internalization. There is increasing evi-

dence that uPAR directly interacts with LDLR family members,

thereby linking integrin redistribution to the LDLR internalization

receptor.

In this study we have characterized the LDLR binding motif on

uPAR domain 3 in uPAR deficient HEK 293 cells or endothelial

cells, and studied the functional and biological consequences of sus-

pended uPAR/LDLR interaction by expression of either wild type

uPAR (wt) or mutated uPAR (mutL3) lacking LDLR binding capac-

ities. Additionally, we used a competitive interfering peptide (P1)

mimicking the binding domain of uPAR as well as chaperone Recep-

tor Associated Protein (RAP)-mediated inhibition of ligand binding

to LDLR family members.

Suspended uPAR/LRP interaction thereby led to reduced uPAR as

well as uPAR-dependent integrin internalization and redistribution

during cell migration. This was consistent with a decrease in integrin-

induced signal transduction such as pY576 FAK phosphorylation

most likely due to reduced integrin redistribution. As a consequence,

uPAR (mutL3) expressing cells revealed impaired cell spreading and

cell migration. Soluble as well as GPI-anchored uPAR domain 3,

however, was able to restore LDLR-mediated integrin internalization

during endothelial cell migration.

From these results we conclude that uPAR/LDLR interaction is

essential for uPAR-mediated integrin redistribution to the leading

edge of migrating endothelial cells during angiogenesis, which is a

prerequisite for efficient integrin-induced signal transduction. The

localization of the uPAR/LDLR binding motif will give novel

insights into a potential target for affecting endothelial cell behavior

in angiogenesis-related diseases.

O5B-5Growth factor induced endothelial cell migrationrequires urokinase receptor (uPAR)-dependent integrinredistributionPrager GW, Novotny R, Unseld M, Marina M, Mihaly J,Binder BR and Zielinski CCMedical University of Vienna, Vienna, Austria

Tumor angiogenesis is induced when the net balance of pro- and an-

tiangiogenic molecules is tipped in favor of angiogenesis, the so called

‘angiogenic switch’. Recently, we described a mechanism which

explains how the major angiogenic growth factor VEGF induces pro-

uPA activation via change of integrin beta-1 activity, which led to a

ternary complex formation between uPA/PAI-1 and uPAR, which

co-internalizes beta-1 integrins into the endosomal compartment.

Thereby, uPAR plays a central role for VEGF-induced endothelial

cell migration. However, limited treatment results of anti-angiogenic

therapies, which mainly aim to inhibit VEGF in malignancies(Cassidy

et al., 2008; Saltz et al., 2008), might lie in the fact that pro-angio-

genic endothelial cell behavior is not only induced by VEGF, but

also by a variety of other growth factors(Carmeliet, 2005).

Here we describe that uPAR associated pro-uPA activation, fol-

lowed by internalization and redistribution of uPAR and integrins,

is not specific for VEGF-induced endothelial cell activation, but

represents a more general pro-angiogenic mechanism, which is also

induced by others growth factors. We found that fibroblast growth

factor-2 (FGF-2), hepatocyte growth factor (HGF) as well as epi-

dermal growth factor (EGF), but not the VEGFR-1 ligand pla-

centa-like growth factor (PlGF) led to a PI3kinase dependent

activation of pro-uPA when bound to uPAR. As a consequence,

uPAR and integrins became internalized in an LDLR-like protein

dependent manner, while PlGF had no effect on uPAR redistribu-

tion. We found that domain-3 of uPAR is responsible and sufficient

to link integrin adhesion receptors to LDLR-family of internaliza-

tion receptors. Consistently, interference with the uPAR / integrin

internalization affected endothelial cell migration induced either by

VEGF, HGF, FGF-2, or EGF, but had no effect on PlGF-induced

endothelial cell migration.

From these data we conclude that uPAR represents a central mole-

cule in growth factor-induced endothelial cell behavior, which might

open a new avenue for therapeutic intervention in (tumor-)angiogene-

sis.

O5B-6Characterization of breast cancer cell linesoverexpressing the urokinase receptor splice variantuPAR-del4/5 or the GTP binding protein rab31Grismayer B1, Sato S1, Beaufort N1, Schmitt M1, Luther T2,Baretton G3, Magdolen V1 and Kotzsch M3

1Technical University Munich, Munchen; 2Medical Laboratory

Unit, Bautzen; 3Dresden University of Technology, Dresden,

Germany

The splice variant uPAR-del4/5 is a urokinase receptor form which

lacks domain DII. uPAR-del4/5 mRNA has been shown to be an

independent prognostic marker for distant metastasis-free survival in

node-negative breast cancer patients. Using microarray analyses to

identify differentially expressed genes associated with high uPAR-

del4/5 mRNA levels in breast tumors, the gene encoding rab31 was

one of seven genes found to be strongly up-regulated. Indeed, high

rab31 mRNA values were significantly associated, independent from

uPAR-del4/5, with worse outcome in breast cancer. Thus, rab31, a

member of the Rab family of small GTP-binding proteins involved in

intracellular transport, signal transduction and receptor internaliza-

tion/recycling, and uPAR-del4/5 may be components of associated,

tumor-relevant signaling pathways.

To analyze effects of uPAR-del4/5 and rab31 overexpression, respec-

tively, two different breast cancer cell lines, MDA-MB-231 and

MDA-MB-435, were stably transfected with expression plasmids

encoding uPAR-del4/5 or rab31.

FACS and Western blot analyses of transfected cell lines showed

an overexpression of both proteins about 3–10 times compared to

the vector control. The stably transfected cell lines were analyzed

regarding proliferation, and cell adhesion on several extracellular

matrix proteins. Interestingly, in highly metastatic MDA-MB-231

cells, proliferation was affected neither by overexpression of uPAR-

del4/5 nor of rab31, whereas overexpression of either proteins in

the only moderately metastatic MDA-MB-435 cells resulted in

enhanced proliferation. Furthermore, cells overexpressing uPAR-

del4/5 displayed reduced adhesion on several extracellular matrix

proteins. Strinkingly, a similar behavior was observed for cells over-

expressing rab31. In addition, cells overexpressing uPAR-del4/5

exhibit a significant decrease of tumor growth and metastasis in

vivo in comparison to the vector-transfected control cells. The

obtained results, and further experiments with single or combined

overexpression of both proteins concerning its invasive and migra-

tory potential may contribute to the further understanding of the

function/relation of uPAR-del4/5 and rab31 in tumor invasion and

metastasis.


ORAL PAPERS 13____________________________________________________________________________________

O5B-7Matriptase induces formation of skin carcinomathrough activation of hepatocyte growth factor-dependent signaling pathwaySzabo R, Rasmussen AL, Moyer AB, Schafer JM, Molinolo AA,Gutkind JS and Bugge THNational Institutes of Health, Bethesda, USA

Matriptase is an epithelium-specific cell surface-associated serine pro-

tease that is overexpressed in a remarkable variety of human carcino-

mas. We have previously reported that dysregulated expression of

matriptase is sufficient to induce formation of highly invasive squa-

mous cell carcinoma (SCC) in mouse skin and also strongly potenti-

ates development of tumors induces by tobacco-related chemical

carcinogen DMBA (1). Here, we show that matriptase-induced

tumorigenesis is critically dependent on the proteolytic activation of

pro-hepatocyte growth factor (HGF), its receptor proto-oncogene

cMet, and the cMet-dependent signaling pathway that involves Akt

and mTor. Thus, we found that DMBA-induced tumor formation is

associated with the onset of matriptase expression in proliferating,

cMet-expressing keratinocytes of the basal layer of mouse skin and,

similarly, that matriptase and cMet colocalize in overwhelming

majority of human carcinomas of skin and head and heck region.

Genetic inactivation of cMet in mouse epidermis strongly suppressed

matriptase-induced hyperproliferation of epidermal keratinocytes, epi-

dermal hyperplasia, and carcinoma formation. Furthermore, matrip-

tase-dependent tumorigenesis was associated with overactivation of

Akt, one of the downstream targets of cMet signaling pathway, and

it could be completely prevented by the inhibition of an immediate

downstream target of Akt, mTOR. These data strongly suggest that

translocation of matriptase expression to proliferative compartment

of epidermis is linked to an increased activation of pro-hepatocyte

growth factor and an overactivation of cMet-dependent signaling

pathway, which critically contributes to the malignant transformation

of murine epidermis.

List, K., Szabo, R. et al., Genes Dev. 2005; 19: 1934–1950.


14 ORAL PAPERS____________________________________________________________________________________

Plasminogen Activation and Fibrinolysis

O6A-1Polyphosphate modulates fibrin structure altering itssusceptibility to lysisMutch NJ, Engel E, Uitte de Willige S, Philippou H andAriens RASUniversity of Leeds, Leeds, UK

Upon activation by thrombin or other agonists platelets secrete a

negatively charged polymer, polyphosphate (polyP). We explored the

interaction of polyP with fibrin(ogen) and its impact on fibrin struc-

ture and fibrinolysis. Electrophoretic mobility and binding assays

indicate that polyP interacts directly with fibrinogen and soluble

fibrin. Clots formed in the presence of polyP displayed reduced tur-

bidity and permeability suggesting the formation of a tighter fibrin

network. These changes were independent of both cross-linking and

fibrinopeptide release. Microscopy revealed an altered pattern of

fibrin distribution in clots formed with polyP; with formation of tight

aggregates of fibrin fibers interspaced with large pores in contrast to

homogenous fiber distribution in control clots. Lysis by tPA and

plasminogen or pre-formed plasmin was delayed in clots formed in

the presence of polyP. These changes were dependent on both the

concentration of activator and polyP but were independent of throm-

bin. Addition of polyP to the clot after fibrin formation or to

repolymerising soluble fibrin did not affect lysis indicating that

changes induced by polyP occur at the level of conversion of fibrino-

gen to fibrin. Surface plasmon resonance revealed that the presence

of polyP during fibrin formation interfered with the binding of both

plasminogen and tPA but only after these surfaces were subjected to

partial-lysis by plasmin. Our data indicate that polyP produces pro-

thrombotic fibrin structures with increased resistance to fibrinolysis.

These effects result from architectural differences in the clot that

occur during conversion of fibrinogen to fibrin. The altered fibrin

structure generated in the presence of polyP is less susceptible to plas-

min cleavage, therefore reducing exposure of lysine binding sites. This

in turn modulates the cofactor capacity of fibrin in tPA-mediated

plasminogen activation.

O6A-2Fibrin structure and the regulation of tissueplasminogen activator (tPA) binding and potencyWilliams SC1, Kolev K2, Szabo L3, Silva M1 and Longstaff C1

1NIBSC, Potters Bar, UK; 2Semmelweis University, Budapest;3Chemical Research Centre, Budapest, Hungary

Regulation of tPA activity is highly dependent on fibrin binding and

tPA binds primarily by finger (F) and Kringle2 (K2) domains. How-

ever, rates of fibrinolysis are also affected by fibrin structure. We

have investigated structure/function and regulation of tPA activity in

fibrin formed at 10 IU/mL thrombin [fibrin(10), fine fibre mesh, small

pores] and 0.5 IU/mL thrombin [fibrin(0.5), thick fibres, large pores]

using domain variants of tPA. Parallel rate measurements of plasmin-

ogen activation in fibrin and fibrinolysis following clot turbidity were

made using domain variants. Kinetics studies on wild type tPA, (F-

G-K1-K2-P), K1K1tPA (F-G-K1-K1-P) and delFtPA (G-K1-K2-P)

showed a consistent trend for enzyme potency of tPA > K1K1tPA

> delFtPA with delFtPA up to 66% less potent than tPA in

fibrin(0.5), but differences reduced at high (PA) and in Fibrin(10).

Fibrin(10) was a better surface for plasminogen activation but more

resistant to lysis than fibrin(0.5). Reorganisation of fibrin(0.5) struc-

ture complicated turbidity measurements and SEM studies tied these

changes to reformation of large protein agglomerates during lysis.

Confocal microscopy using orange fluorescent fibrin with jellyfish

green fluorescent protein (GFP)-labelled tPA variants showed these

agglomerates strongly bind tPA. Movies and enzyme kinetics studies

highlight major differences in behaviour of tPA-GFP variants in

fibrin(10) and fibrin(0.5). Initial binding in fibrin(0.5) requires F

domain and delFtPA-GFP was less efficiently bound to agglomerates

so diffused through the clot ahead of the lysis zone. Poor initial bind-

ing and lag in plasminogen activation with delFtPA can be partially

compensated for by enhanced diffusion and fibrinolysis. Fibrinolysis

may be divided into three stages: (i) initial binding of tPA and activa-

tion of plasminogen; (ii) enhancement due to C-terminal lysine gener-

ation; (iii) tPA focalisation on large protein agglomerates. Stages (i)

and (iii) are more finger dependent, (ii) is K2 dependent, but fibrin

structure affects the three stages in different ways.

O6A-3Intra-vital analyses of plasminogen binding toplatelet-rich micro-thrombusTanaka A, Suzuki Y and Urano THamamatsu University School of Medicine, Hamamatsu, Japan

Aim: To elucidate the precise mechanism of thrombolysis in the

vasculature, we analyzed the process of plasminogen-binding to laser-

induced microthrombus in mice using intra-vital fluorescence micros-

copy. The binding of Glu-plg on activated platelets’ surface was also

analyzed in in-vitro experiment.

Method: Mesenteric vein of green fluorescent protein expressing

transgenic mouse (GFP-mouse) was irradiated by laser-beam through

objective lens of fluorescence confocal microscopy and platelet-rich

micro-thrombus was formed. The accumulation of GFP expressing

platelets as well as Alexafluor 568 labeled plasminogen (Glu- or

mini-plg) on injured vessel wall was measured as an increase in the

corresponding fluorescent intensity with time course, and their distri-

butions were analyzed.

Results: (i) Glu-plg accumulated in a time-dependent manner to the

center of micro-thrombus, where phosphatidylserine (PS) was

exposed on platelets surface and fibrin was formed. (ii) The bindings

of Glu-plg in the presence of EACA (approximately 20 mM) or after

treatment by carboxy-peptidase (approximately 10 U/mL), and of

mini-plg were significantly less than that of Glu-plg alone. (iii) Glu-

plg appeared to bind to the surface of calcium-ionophore treated

GFP-platelets, at the timing when GFP disappeared from the plate-

lets, which was possibly initiated as a result of sustained-elevation of

intra-cellular calcium ion concentration.

Discussion: Glu-plg appeared to accumulate in the center of micro-

thrombus in a time-dependent manner by binding to either activated

platelets’ surface or formed fibirn in a lysine binding site dependent

mechanism.

O6A-4Fibrinolysis regulation: Interaction of a2-antiplasminwith fibrin(ogen) and localization of the binding sitesMedved L1, Tsurupa G1, Yakovlev S1 and Mckee P2

1University of Maryland School of Medicine, Baltimore;2University of Oklahoma Health Science Center, Oklahoma, OK,

USA

Covalent incorporation of plasmin inhibitor a2-antiplasmin (a2-AP)

into fibrin clot results in effective inhibition of fibrinolysis. It is well

established that such incorporation is mediated by Factor XIIIa,

which cross-links a2-AP to the fibrin(ogen) Aa chain Lys303 located

in the aC-connector. We hypothesized that a2-AP may interact non-

covalently with fibrin prior to its covalent cross-linking. To test this

hypothesis, we studied the binding of a2-AP to immobilized fibrin(o-

gen) and its fragments by Surface Plasmon Resonance (SPR) and


ORAL PAPERS 15____________________________________________________________________________________

ELISA. SPR experiments revealed that a2-AP binds to fibrin while

no binding was observed with fibrinogen. To localize the a2-AP bind-

ing site(s), we studied the interaction of a2-AP with fibrin(ogen) D-D

and E3 fragments and the recombinant aC region (Aa221–610), whichtogether encompass practically the whole fibrin(ogen) molecule, as

well as with the aC-connector (Aa221–391) and aC-domain (aA392–

610) composing the aC region. In ELISA, a2-AP bound only to

immobilized D-D (Kd = 94 nM), Aa221–610 (Kd = 150 nM), and

aC-domain (Kd = 131 nM). The binding to both D-D and aC was

Lys-independent and was not inhibited by tPA or plasminogen. Fur-

thermore, the affinity of a2-AP to D-D was significantly increased in

the presence of plasminogen while that to aC remained unaffected.

Altogether, these results indicate that: (i) the D regions and aC-domains contain high affinity a2-AP binding sites which are cryptic

in fibrinogen and exposed in fibrin; (ii) these binding sites do not

overlap with the Lys303 cross-linking site; (iii) the presence of plas-

minogen facilitates the non-covalent binding of a2-AP to the fibrin D

region. The results also suggest that the non-covalent interaction of

a2-AP with fibrin and spatial separation of the non-covalent binding

sites may provide initial binding of a2-AP to fibrin and proper orien-

tation of the cross-linking sites to facilitate the covalent stage of the

interaction.

O6A-5Synthetic nonadecapeptide (SAK22-40) enhancesplasminogen activation and thrombolysisOkada KKinki University School of Medicine, Osakasayama, Osaka,

Japan

As we reported previously, a new synthetic nonadecapeptide

(SAK22-40) corresponding to Glu22-Leu40 of SAK (staphylokinase)

molecule bound to plasminogen and enhanced the activation of plas-

minogen by SAK-plasmin complex. We further investigate the mech-

anism in detail. SAK22-40 enhanced activation of plasminogen by

tissue-type plasminogen activator (t-PA), and enhanced also activa-

tion of plasminogen by t-PA on human endothelial cell. Analysis of

IAsys resonant mirror biosensor showed that SAK22-40 bound to

human and mouse Glu-plasminogen. This binding was completely

inhibited by synthetic peptide corresponding to C-terminal region of

plasmin B-chain. SAK22-40 bound to human endothelial cell through

Glu-plasminogen. The near-ultraviolet circular dichroism (CD)

spectra of the complex between SAK22-40 and Glu-plasminogen sig-

nificantly differed from Glu-plasminogen in the presence of �-amino-

caproic acid or Glu-plasminogen alone. Therefore, SAK22-40 binds

to Glu-plasminogen, and induces structural changes of Glu-plasmino-

gen. When SAK22-40 was administrated in mouse thrombosis model,

earlier recanalization was observed than in mice with vehicle adminis-

tration, and showed dose-dependent manner. However, SAK22-40

did not show the recanalization in t-PA gene deficient mice. Thus,

SAK22-40 enhanced plasminogen activation and induced effective

thrombolysis.

O6A-6Stable and reversible fusion protein conjugated to redblood cells for drug delivery in thromboprophylaxisMarcos-Contreras OA1, Gonzalez de la Fuente M2, Cines DB3,Muzykantov VR4 and Murciano JC5

1Fundacion Centro Nacional de Investigaciones

Cardiovasculares; 2Departamento de Farmacologia, Facultad de

Medicina, Universidad Complutense, Madrid, Spain;3Department of Pathology and Laboratory Medicine, University

of Pennsylvania; 4IFEM, University of Pennsylvania School of

Medicine, Philadelphia, PA, USA; 5Internal Medicin Department,

Hospital Ramon y Cajal, Madrid, Spain

Introduction: We developed a new thromboprophylactic agent by the

conjugation of plasminogen activators (PAs) to red blood cells

(RBCs). In order to bring this agent to clinics, we formulated fusion

proteins (FP) that combined a mutated streptavidin (SA) core, with a

limited capacity to tetramerized, with a thrombin-activatable uroki-

nase (UK-T). This results in a reversible conjugation between FP and

RBC, thus offering a thromboprophylactic agent with a controllable

duration.

Methods: S2 cells were trasfected with the construct of SA subunit

(13 kDa) with a single aminoacid mutation (D117A) and UK-T

(33 kDa). Produced FP was purified by affinity column chromatogra-

phy and analysed by western blot (WB). Its activity was tested by

biotinylated RBC (bRBC) binding and fibrinolytic activity. Revers-

ible binding to RBC was confirmed by FP displacement form the

bRBC surface using post-incubation with native SA.

Results: WB confirms the presence of two bands at 46 kDa (mono-

merized FP, 85% densitometric analysis) and 180 kDa (tetramerized

FP, 15%). FP fibrinolytic activity was confirmed by addition of

thrombin pre-activated FP over 125I-fibrin clots, resulting in a

99.7.�0.86% of lysis at 6 h, while non pre-activated one showed

23.5 � 3.1% and PBS control 6.5 � 0.4%. 125I-FP binds in a dose

dependent fashion over bRBC, peaking at 75 590 � 9003 molecules

per bRBC (606 � 135 over naıve RBC). RBC-FP remains stably

associated over 48 h in vitro (92 � 1.6% at 24 h and 64.4 � 1.5% at

48 h). RBC-FP efficiently dissolves fibrin clots (4 h) from within,

100 � 1.4% vs. 5.4 � 5% of FP pre-incubated with naıve RBC.

Interestingly, native SA displaced RBC-125I-FP over 53.6 � 5%,

resulting in a lack of RBC-FP fibrinolytic capacity 24.4 � 0.4%.

Conclusion: These data demonstrated that FP (i) stays mainly in a

monomeric form, (ii) binds specifically to bRBC, (iii) remains conju-

gated over days, (iv) get activated in the presence of thrombin and

(v) reversibly dissociates from the conjugate.


16 ORAL PAPERS____________________________________________________________________________________

Serpins

O6B-1Highly sensitive ELISA method for the determinationof alpha2-plasmin inhibitor antigen levelOrosz A, Muszbek L and Katona EUniversity of Debrecen, Medical and Health Science Center,

Debrecen, Hungary

Background: a2-plasmin inhibitor (a2-PI; a2-antiplasmin) is the pri-

mary inhibitor of plasmin-mediated fibrinolysis that forms an irre-

versible inhibitory complex with plasmin (plasmin-antiplasmin, PAP

complex). a2-PI is secreted into the plasma as a approximately

66 kDa single polypeptid chain with Met as the amino-terminus. In

the plasma it undergoes proteolytic cleavage to yield a 12 amino acid

shorter form with Asn as amino-terminus. Asn1-a2-PI becomes cross-

linked to fibrin by FXIIIa 13-times faster than Met1-a2-PI, thereby it

is more effective in the inhibitopn of fibrinolysis. There are also

C-terminal isoforms differing in 27 amino acids that cleaved by an

unidentified protease.

Methods: In this study we developed a two-step ELISA for the deter-

mination of total a2-PI using biotinylated monoclonal antibody

reacting with an epitope present in all isoforms of the protein and

peroxydase-labeled polyclonal antibody for capture and detection,

respectively. Samples and standards were incubated together with the

capture antibody in strepavidin-coated microplate in the first step.

After washing the detection antibody was applied and TMB substrate

was used for the quantification of the reaction.

Results: The limit of quantitation was 0.70 ng/mL, the measuring

range was 0.7–100 ng/mL. This high sensitivity allowed the determi-

nation of a2-PI in 1:2000 dilution of plasma samples. The assay

showed good precision with within run variability C = 4.6% at nor-

mal level (60.8 � 4.0 mg/L plasma), CV = 5.3% in the pathological

range (19.5 � 1.6 mg/L) and CV = 9.1% at extreme low level

(1.98 � 0.35 mg/L). Inter-assay reproducibility was CV = 5.8%,

7.3% and 16.6%, respectively. In normal citrated plasma samples

(n = 40) 69.3 � 8.0 mg/L (mean � SD) was measured.

Conclusion: Our method is suitable for the measurement of total a2-PI antigen level both in normal and pathologic range with good pre-

cision. Its high sensitivity makes it possible to detect very low level of

a2-PI present in body fluids other than plasma.

O6B-2The influence of alpha2-antiplasmin polymorphismArg407Lys on fibrinolysisUitte de Willige S, Miedzak M, Philippou H, Carter AM,Grant PJ and Ariens RASUniversity of Leeds, Leeds, UK

The main inhibitor of plasmin, alpha-2-antiplasmin, is secreted into

plasma as a 70 kDa protein of 464 amino acids. As a result of post-

translational proteolysis at the N- and C-termini, alpha-2-antiplasmin

circulates in functionally different molecular forms. In our search for

the position of C-terminal cleavage, we found that polymorphism

Arg407Lys (R407K; rs1057335) is located close to a potential cleav-

age site. This made us hypothesize that C-terminal cleavage of alpha-

2-antiplasmin, and thereby the lysis times of plasma clots, may be

modulated by this polymorphism.

To investigate this, we genotyped a group of 180 control samples by

RFLP and set up two ELISAs to measure (i) total alpha-2-antiplas-

min level and (ii) C-terminally intact alpha-2-antiplasmin level. To

determine the level of C-terminal cleavage of alpha-2-antiplasmin, we

calculated the ratio of C-terminal intact alpha-2-antiplasmin over

total alpha-2-antiplasmin.

Genotype frequencies were RR:60%, RK:35.6% and KK:4.4%,

allele frequencies were R:77.8% and K:22.2%. R407K did not asso-

ciate with C-terminal cleavage, as we found similar C-terminally

intact/total alpha-2-antiplasmin ratios for all three genotypes

(RR:0.96, RK:0.92, KK:0.94). However, total alpha-2-antiplasmin

level (90.8%) was lower in homozygous K-carriers compared to

RR-carriers (100%) or RK-carriers (102.6%), indicating that the

R407K polymorphism could be associated with diminished alpha-2-

antiplasmin expression. This was reflected in the time to 50% lysis,

with a shorter lysis time (8.66 min) for K-homozygotes compared to

RR-carriers (10.64 min) or RK-carriers (11.04 min). Intriguingly,

R407K also associated with the lagphase of clotting; RR:7.14 min,

RK:6.43 min, KK:4.80 min; P = 0.001), indicating that this poly-

morphism may influence the onset of coagulation. We next aim to

genotype alpha-2-antiplasmin R407K and measure alpha-2-antiplas-

min levels in 288 age- and sex-matched myocardial infarction (MI)

patient samples to investigate if this polymorphism influences MI

risk.

These data indicate that the alpha-2-antiplasmin R407K polymor-

phism influences alpha-2-antiplasmin expression and lysis times of

plasma clots, and possibly also the onset of coagulation.

O6B-3The adipokine vaspin inhibits smooth musclemigration in vitro and the development of coronaryin-stent restenosis in vivoKastl SP1, Katsaros KM1, Krychtiuk K1, Speidl WS1, Maurer G1,Huber K2 and Wojta J11Medical University of Vienna; 23rd Medical Department

Wilhelminenhospital, Vienna, Austria

Percutaneous coronary intervention (PCI) represents the most

important treatment modality of coronary artery stenosis. In-stent

restenosis (ISR) is still a limitation for the long-term outcome

despite the introduction of drug eluting stents. Adipokines may

directly influence vessel wall homeostasis by influencing the function

of endothelial cells and arterial smooth muscle cells (SMC). Visceral

adipose tissue-derived serpin (vaspin) was recently identified as a

member of serine protease inhibitor family and serveral studies

could demonstrate a relation to metabolic diseases like diabetes.

The aim of this study was to investigate a role of vaspin in SMC

migration in vitro and development of ISR in vivo. Human coronary

artery smooth muscle cell (HCASMC) migration was analyzed by

an in-vitro migration assay with different concentrations (0.004 ng/

mL up to 40 ng/mL) of vaspin. The development of ISR was stud-

ied in 57 patients with stable coronary artery disease who under-

went elective and successful percutaneous coronary intervention

(PCI) with implatation of drug eluting stents. Blood samples were

taken directly before PCI. Vaspin plasma levels were measured by

specific elisa. ISR was evaluated 8 months later by coronary angio-

graphy. During the follow up period, 13 patients developed ISR.

Patients with ISR had significantly lower vaspin plasma levels com-

pared to patients without ISR (0.205 ng/mL vs. 0.349 ng/mL;

P = 0.004). Further we could demonstrate that vaspin nearly abol-

ishes serum induced SMC migration (100% vs. 7%; P < 0.001) in

a biphasic manner. We were able to show for the first time that the

adipokine vaspin inhibits SMC migration in vitro. In addition, the

occurrence of ISR after PCI with implantation of DES was signifi-

cantly associated with low vaspin plasma levels before intervention.

Determination of vaspin plasma levels before PCI might be helpful

in the identification of patients with high risk for development of

ISR after stent implantation.


ORAL PAPERS 17____________________________________________________________________________________

O6B-4PAI-1 as a furin convertase inhibitor: a potential rolein insulin resistanceBernot D, Canault M, Nalbone G, Alessi MC and Peiretti FINSERM, Marseille, France

Epidemiological studies showed that elevated plasma levels of SER-

PINE1/PAI-1 predict the risk of type 2 diabetes and metabolic syn-

drome. A direct role of PAI-1 in these pathologies has been

suggested, but not demonstrated. Among several hypotheses, inhibi-

tion by PAI-1 of furin convertase (a serine protease involved in the

processing of many potein precursors including insulin proreceptor)

has been proposed.

We studied this possibility, focusing on the consequences of furin

and PAI-1 overexpression on insulin proreceptor maturation and

insulin signaling.

We showed that PAI-1 efficiently associated with furin in a SDS-sta-

ble complex, in vitro and in vivo (transfected cells). Using a golgian

furin-activity reporter construct, we demonstrated intracellular furin

inhibition by PAI-1. Association between furin and PAI-1 most prob-

ably occurred intracellularly during transport/addressing, since a

PAI-1 lacking its signal peptide did not associate nor inhibit furin. In

furin-defective human LoVo cells (expressing low PAI-1 level), furin

overexpression induced insulin proreceptor maturation. Concomitant

overexpression of a stabilized form of PAI-1 inhibited this matura-

tion (-54.4%, significant), as well as insulin-stimulated phosphoryla-

tion of both insulin receptor and Akt. Analysis of hepatic tissue from

PAI-1 KO mice revealed an increase in insulin receptor mature form,

compared to wild type mice (+31.6%, significant).

In conclusion, our results show that PAI-1 potently inhibits furin

convertase, leading to a decrease in insulin receptor functionality that

can contribute to insulin resistance. In addition, we described an ori-

ginal intracellular effect of PAI-1 regulating serine protease activity.

O6B-5Metals exert an unexpected role in regulatingenzymes in the plasminogen activation cascadePeterson CB, Thompson LC and Goswami SUniversity of Tennessee, Knoxville, TN, USA

Proteases regulate many biological processes biology, including coag-

ulation and clot lysis, the immune response, and processes that remo-

del the extracellular matrix (ECM) during wound healing and cell

migration. Because of the importance of proteases, their activity must

be closely checked. This is accomplished in part by the serine prote-

ase inhibitors (serpins), which form inactive covalent complexes with

their target proteases. Plasminogen activator inhibitor-1 (PAI-1) is

the major inhibitor of both urokinase (uPA) and tissue plasminogen

activator (tPA) that lyse fibrin clots and also cleave ECM compo-

nents. The active conformation of PAI-1 is metastable and spontane-

ously converts to a latent form with a half-life of 1.1 h at 37 �C. Thehalf-life of the active conformation is extended to 1.5 h when PAI-1

is bound to the plasma protein vitronectin. Our recent work demon-

strates that the observed stabilization of PAI-1 by vitronectin is

metal-dependent. Human PAI-1 more rapidly converts to the latent

conformation in the presence of cobalt, nickel, and copper. Strik-

ingly, the half-life is much longer in the presence of vitronectin and

these metals. Steady-state binding measurements using surface plas-

mon resonance have been used to determine a dissociation constant

for the interaction with nickel. Stopped-flow measurements of

approach-to-equilibrium changes in intrinsic protein fluorescence

have been used in a complementary fashion to characterize binding

to a broader range of metals. These studies reveal distinct steps in

the mechanism for metal interaction with PAI-1, with a binding step

followed by a series of conformational changes. Measurements of the

observed rate constants of binding as a function of varying metal

concentrations allowed accurate determination of binding affinities

for cobalt, nickel, and copper, yielding dissociation constants of

about 40, 30, and 0.09 lM respectively. Identification of binding sites

for copper is being pursued using a structural informatics approach.

O6B-6Evaluation of a panel of plasminogen activatorinhibitor-1 inhibiting monoclonal antibodies in amouse pulmonary embolism modelvan de Craen B, Declerck PJ and Gils AKatholieke Universiteit Leuven, Leuven, Belgium

Introduction: Plasminogen activator inhibitor-1 (PAI-1) is an impor-

tant inhibitor of the plasminogen activation system. Elevated PAI-1

levels have been implicated in a wide variety of thrombotic and non-

thrombotic conditions. Pharmacological inhibition of PAI-1 activity

might therefore be an interesting therapeutic approach in different

pathologies.

Aim: The evaluation of a panel of inhibitory monoclonal antibodies

(MA) reacting with glycosylated mouse PAI-1 (mPAI-1) in a pulmo-

nary embolism model.

Methods and Results: Using hybridoma technology we were able to

generate MA that showed good reactivity towards glycosylated

mPAI-1 and inhibited at least 50% of PAI-1 activity at a 2-fold

molar excess of MA over PAI-1 in an in vitro enzymatic assay. These

antibodies were evaluated for their inhibitory potential in a mouse

pulmonary embolism model. In this model, 10 mg/kg of MA was

administered (i.v.) 5 min before i.v. injection of a suboptimal concen-

tration of t-PA (0.1 mg/kg). Pulmonary embolism was evoked by i.v.

injection of thromboplastin (3.3 mg/kg). Mice were evaluated after

15 min and gained a positive score if they displayed normal physical

activity. A negative score was obtained in case of paralysis of the

limbs or death.

For two of the evaluated antibodies, MA-MP6H6 and MA-MP26H2,

we could detect a statistically significant increase in the proportion of

mice showing normal physical activity compared to control mice to

which no monoclonal antibody was administered (Table 1).

Table 1 for O6B-6

MA Mice with normal physical activity (%) n

33% 103

MA-MP6H6 66%* 44

MA-MP26H2 60%* 30

MA-MP2D9** 34% 32

*P < 0.05 (Fisher’s exact test).

**Non-inhibitory MA reacting with glycosylated mPAI-1.

Conclusion: The increased survival rate obtained with MA-MP6H6

and MA-MP26H2 in this pulmonary embolism model indicates that

these PAI-1 inhibiting MA might be promising tools to further unra-

vel the complex role of PAI-1 in other thrombotic and non-throm-

botic mouse models.


18 ORAL PAPERS____________________________________________________________________________________


O7-1The biochemistry of TAFINesheim MEQueen’s University, Kingston, Canada

TAFI, or thrombin activatable fibrinolysis inhibitor, is a 60 kDa

plasma zymogen that circulates at a concentration of about 100 nM.

It is activated to the basic carboxypepidase TAFIa by a single prote-

olytic cleavage at arginine 92. TAFIa down regulates the process of

fibrinolysis by removing the carboxy terminal lysine residues that

appear in fibrin as it is degraded. Because TAFI was discovered inde-

pendently and approximately contemporaneously by several groups it

has acquired other names which include plasma procarboxypeptidase

B (for basic), procarboxypeptidase U (for unstable) and procarboxy-

peptidase R(for arginine). TAFI can be activated by thrombin and

plasmin. The reaction with thrombin is enhanced 1250-fold by throm-

bomodulin. The mechanism for this enhancement is not known in

detail. Residues adjacent to the cleavage site appear not to be respon-

sible for the thrombomodulin dependence, whereas other key posi-

tively charged residues do, among them being three tandem lysine

residues in the activation peptide. TAFIa down regulates fibrinolysis

by eliminating the positive feedback steps comprising plasmin-

enhancement of the tPA cofactor activity of fibrin and plasmin-

catalyzed conversion of Glu-plasmingen to Lys-plasminogen. It also

suppresses the protection by partially cleaved fibrin of plasmin and

tPA from antiplasmin and PAI-1, respectively. TAFIa is down regu-

lated by spontaneous decay, with a half-life about 8 min at body

temperature. The human TAFI gene is located on chromosome 13.

Two common polymorphisms in the coding region have been identi-

fied. These translate as Ala147Thr and Thr325Ile in the protein. The

former has no known functional significance, but isoleucine at posi-

tion 325 doubles the half-life of TAFI and increases its antifibinolytic

potential. Numerous clinical studies indicate pathophysiologic roles

for TAFI. In addition, studies in the TAFI knockout mouse model

indicate that the TAFI pathway is relevant in inflammation and

pathologies associated with it.

O7-2Haemostasis and innate immunityGinsburg DUniversity of Michigan, Ann Arbor, MI, USA

Hemostasis is a critical component of the host response to microbial

pathogens and may be an important evolutionary selective pressure

underlying the wide variation in plasma level for many blood coagu-

lation factors. Work from our lab characterizing the interaction of

the microbial plasminogen activator, streptokinase, with host plas-

minogen will be reviewed, including recent efforts to modulate this

pathway as a novel approach to the therapy of Group A streptococ-

cal infection. Studies exploring the genetic basis for variability in

plasma von Willebrand factor levels and the potential implications of

these findings for the interaction between hemostasis and microbial

pathogens will also be discussed.


ORAL PAPERS 19____________________________________________________________________________________

President’s Session: Young Investigators’ Presentations and the

D. Collen Young Investigators’ Awards

O8-1Allosteric activation of the DegS protease: designprinciples of stress-sensor proteasesSohn J, Grant R and Sauer RMassachusetts Institute of Technology, Cambridge, MA, USA

In the E. coli periplasm, disruption of protein folding is sensed by

the PDZ domains of the trimeric DegS protease, which bind C-termi-

nal peptides of misfolded outer-membrane porins (OMPs). This bind-

ing event triggers proteolytic cleavage of RseA, a transmembrane

regulator, and eventually leads to initiation of transcriptional activa-

tion of stress genes. We find that DegS is an allosteric enzyme.

OMP-peptide binding to the PDZ domain relieves inhibitory contacts

between the PDZ and protease domains, triggering reconfiguration of

the active-site oxyanion hole from inactive to active conformation.

Both RseA substrate and OMP peptides activate the protease by

binding preferentially but not exclusively to the active conformation

of DegS. Disrupting inhibitory interactions between the PDZ and

protease domains results in faster substrate cleavage and tighter

OMP-peptide binding. Likewise, stabilizing the active conformation

of the protease domain by mutagenesis results in the same phenotype.

Cocrystal structures of DegS in complex with different OMP peptides

and in multiple lattices also support the idea that the role of the

PDZ domain is inhibitory. Allostery is an intrinsic property of the

protease domain of DegS, as the isolated protease domain also equili-

brates between inactive and active trimeric conformations, similar to

those observed in the full-length enzyme. A set of conserved residues

in the protease domain mediates this allosteric transition. Mutations

on these residues result in substantially lower proteolytic activity, and

multiple crystal structures of these mutant DegS variants consistently

show the non-functional conformation. Overall, our biochemical and

structural results support a concerted two-state allosteric model,

which can be quantitatively modeled. This two-state mechanism is

biologically robust, allows rapid responses to protein-folding stress,

and allows DegS function to be precisely regulated by the affinity of

OMP peptides and substrate for its active and inactive conforma-

tions.

Reference: Sohn et al. Cell 2007; Mol. Cell 2009; Structure 2009.

O8-2TAFI regulates hepatocyte survival during liverregeneration independent from cell-associatedplasminogenOkumura NNihon University College of Bioresource Sciences, Fujisawa,

Japan

Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) is

plasma procarboxypeptidase B secreted from the liver. We have dem-

onstrated that the expression of TAFI is suppressed by growth pro-

moted conditions during liver regeneration, and this down-regulation

promotes hepatocyte proliferation through cell surface plasmin(ogen).

In this study, we further investigated the role of TAFI in liver regen-

eration focusing on hepatocyte survival and death using TAFI defi-

cient mice as well as TAFI-gene silenced primary hepatocytes.

Methods: TAFI knock out mice (ko) and wild type mice (wt) under-

went 70% partial hepatectomy. The proliferation rate and the extent

of liver injury were measured during liver regeneration. Hepatocytes

were isolated from rat liver by collagenase perfusion and primary he-

patocytes were transfected with TAFI siRNA. The morphological

change of nuclei was observed by Hoechst33258 staining, and apop-

totic and survival signals were analyzed by western blotting.

Results: Liver regeneration was accelerated in TAFI ko in comparison

with wt. Liver injury after hepatectomy was milder in TAFI ko than

wt. TAFI siRNA suppressed TAFI mRNA expression in primary he-

patocytes by 90% of that in control hepatocytes. Apoptosis was

induced in control hepatocytes during primary culture; however, it

was reduced in TAFI-silenced hepatocytes. Cleaved caspase-3, which

is an executioner at the down stream of apoptosis, was decreased in

TAFI-silenced hepatocytes in comparison with control-hepatocytes.

Conversely, phosphorylated-Akt suppressing apoptosis and promoting

cell survival and proliferation was increased in TAFI-silenced hepato-

cytes. Treatment of hepatocytes with tranexamic acid failed to inhibit

the events induced by TAFI-gene silencing, suggesting that cell surface

associated plasmino(gen) was not involved in the signaling.

Conclusion: These results suggest a novel role for TAFI in the regula-

tion of hepatocyte survival and proliferation during liver regeneration

independent from cell-associated plasminogen.

O8-3Binding characteristics of thrombin-activatablefibrinolysis inhibitor to streptococcal surface collagen-like proteins A and BValls Seron M1, Marx PF2, Marquart JA2, Herwald H3, deGroot PG4 and Meijers JCM2

1Academic Medical Center (AMC); 2Academic Medical Center,

Amsterdam, The Netherlands; 3Biomedical Center, Lund

University, Lund, Sweden; 4University Medical Center, Utrecht

University, Utrecht, The Netherlands

Streptococcus pyogenes is an important human Gram-positive patho-

gen that mainly causes throat and skin infections, such us pharyngi-

tis, impetigo and cellulitis. Although the majority of streptococcal

infections are superficial, some cases may progress into invasive and

life threatening diseases with an extremely rapid progression, such as

sepsis and necrotizing fasciitis. Thrombin-Activatable Fibrinolysis

inhibitor (TAFI) binds to the collagen-like proteins SclA and SclB

found at the surface of Streptococcus pyogenes. Activation of TAFI

at this surface is thought to redirect inflammation from a transient to

a chronic state by modulation of the kallikrein/kinin system. Here,

Fig. 1. Initiation of the envelope-stress response.


20 ORAL PAPERS____________________________________________________________________________________

we investigated the TAFI binding characteristics to SclA and SclB.

38 overlapping TAFI peptides of approximately 20 amino acids were

generated. With surface plasmon resonance, it was shown that two of

these peptides (P18, residues G205-S221, and P19, residues R214-

D232) specifically bound to SclA and SclB with high affinity. In a

plate binding assay, it was demonstrated that P18 and P19 specifi-

cally competed in a dose-dependent manner with TAFI for binding

to SclA and SclB. In another series of experiments we aimed to study

the binding properties of activated TAFI (TAFIa) to SclA and SclB.

A quadruple TAFI mutant (TAFI-IIYQ) was used that after activa-

tion is a 70-fold more stable enzyme than wild-type TAFIa. Surface

plasmon resonance-based affinity measurements indicated that TAFI

and TAFI-IIYQ bound to the bacterial proteins with similar affini-

ties. However, the rate of dissociation was very different between the

proenzyme (both TAFI and TAFI-IIYQ) and the stable enzyme TA-

FIa-IIYQ. Although TAFIa-IIYQ bound to SclA and SclB, it disso-

ciated rapidly. In conclusion, the bacterial proteins SclA and SclB

bind to a TAFI fragment at least encompassing residues R214-S221.

Binding of TAFI to the bacteria allows activation of TAFI, whereaf-

ter the enzyme can easily dissociate.

O8-4Staphylococcus aureus clumping factor A is acausative virulence factor in experimental murinesepsisSmeds E1, Ko YP1, Shi Z1, Foster TJ2 and Hook M1

1Texas A&M Health Science Center, Houston, TX, USA; 2Trinity

College, Dublin, Ireland

Staphylococcus aureus Clumping factor A (ClfA) is a member of a

group of molecules collectively known as microbial surface compo-

nents recognizing adhesive matrix molecules (MSCRAMMs). ClfA is

almost ubiquitously expressed on S. aureus clinical strains and is

known to bind to the C-terminal region of the fibrinogen (Fg) c-chainas well as to complement factor I. ClfA has emerged as an attractive

pharmaceutical target as well as a vaccine target.

Since S. aureus expresses many different virulence factors it can be

difficult to understand the role of a single virulence factor. We there-

fore used the non-pathogenic bacterium Lactococcus lactis as a host

to heterologously express ClfA on its surface.

When mice were infected i.v. with either L. lactis vector or L. lactis

ClfA, L. lactis ClfA infected mice had a dramatically lower survival

rate as compared to L. lactis vector infected mice. When the mice were

examined in detail, the L. lactis ClfA-infected mice had a rapid onset

of thrombocytopenia and gastrointestinal bleedings. The observed

virulence was not a general property of S. aureus MSCRAMMs, since

L. lactis SdrC and L. lactis SdrD did not induce virulence in the mice.

Using a non-Fg binding mutant strain of L. lactis ClfA, the virulence

was significantly reduced and resulted in lower bacterial load in the

blood. Interestingly, MyD88-/- mice were not resistant to the L. lactis

ClfA-induced virulence, suggesting that the innate immune system is

not mediating the pathology.

Fg cD5 mice lack the five most C-terminal amino acids in the Fg c-chain and as a consequence can not bind ClfA or platelet integrin

aIIbb3. Fg cD5 mice were resistant to the L. lactis ClfA induced viru-

lence as compared to wild-type mice, suggesting that the Fg-binding

of ClfA is mediating the virulence.

O8-53D virtual histology using MR-T1 mapping toquantify venous thrombus organisationSaha P, Blume U, Varma G, Wiethoff A, Schaeffter T, Evans C,Ahmad A, Patel A, Modarai B, Waltham M and Smith AKing’s College London, London, UK

Introduction: Novel thrombolytic delivery systems are changing the

paradigm of treatment for deep vein thrombosis (DVT), but only

fresh thrombi are suitable for these interventions. Current imaging

methods are unable to characterise the organisation of venous

thrombi, which would help direct therapy. A technique that identifies

patients in whom thrombolysis has the greatest potential would be

beneficial.

Methods: An MRI 3D T1-mapping sequence was optimised for small

animal imaging and applied to experimental venous thrombi induced

in the inferior vena cava (IVC) of BLAB/C mice (n = 27). T1-relaxa-

tion times were quantified after 7, 10, 14, 21 and 28 days using cus-

tom-made software implemented in MATLAB. Histological sections

of thrombi were processed for markers of organisation including red

cell and collagen content (MSB stain). Image analysis software was

used to calculate the percentage area of thrombus containing stain

and compared to corresponding MR slices. Three-blinded observers

validated results.

Results: Typical examples of MRI and histology are shown in fig 1.

Young thrombi have a short T1 relaxation time that increases with

thrombus age (R2 = 0.69, P < 0.0001). Collagen content, a marker

of organising thrombi, is directly proportional to T1 relaxation time.

Short T1 relaxation times were correlated with low amounts of colla-

gen (R2 = 0.80, P < 0.0001).

Conclusions: This is the first study to show that non-invasive, MR

T1-mapping can quantify venous thrombus organisation. This tech-

nique could be used to predict clinical outcome following deep vein

thrombosis; guide management; and assess the efficacy of novel

treatments.

Fig. 1. L. lactis ClfA is a virulence factor in mice.

Fig. 1. MRI images and corresponding histological sections. Venogra-

phy demonstrates recanalisation of the IVC between day 7 and 28.

MSB sections of thrombus show reduced red cells (yellow) and

increasing organisation (blue=collagen content) of thrombus (T) dur-

ing its resolution (· 200, Bar = 200 lm). 3D T1-mapping shows

short relaxation times in young, fresh thrombi (red) that increase

with thrombus age and organisation (green).


ORAL PAPERS 21____________________________________________________________________________________

D-dimer and Fibrinolytic Markers

O9A-2D-dimer: the lack of comparability of test results andpossibilities for harmonizationMeijer PECAT Foundation, Leiden, The Netherlands

The measurement of D-Dimer largely contributes to the diagnostic

work-up of venous thromboembolism (VTE). Today more than 30

different commercial available test are used by clinical laboratories.

The numerical test result may differ between methods due to the het-

erogeneity of the analyte in the patient sample, the variation in the

specificity of antibodies and the kind of calibrator used in the test.

In a recent survey of the external quality assessment programme of

the ECAT Foundation this was clearly demonstrated by the use of a

highly positive patient sample (4–5 mg FEU/L) and a dilution of this

sample to a level close to the cut-off level. The ratio between these

two samples varied from 6 to 11 between the 11 most frequently used

methods in this survey.

This lack of standardization of D-Dimer results may be confusing in

daily laboratory and clinical practice.

Since 1995 several approaches for standardization and harmonization

towards D-Dimer methods were conducted, mostly within the frame

work of the SSC subcommittee on Fibrinolysis of the SSC. An over-

view will be given of the different standardization and harmonization

approaches, including the pros and cons of these approaches. Special

attention will be given to a model for harmonization which relies on

the transformation of an assay-specific regression line to a reference

regression line (P. Meijer et al. Thromb Haemost 2006; 95: 567–572).

This approach forms the basis for further development of a harmoni-

zation model within the SSC framework of the ISTH. An update will

be given on the current status.

O9A-3Performance of the Vidas D-dimer assay to rule outvenous thromboembolism across age categories in1004 consecutive out-patientsToulon P1, Claessens Y2, Allo JC2 and Dhainaut JF2

1CHU Cimiez, Laboratoire Hmatologie, Nice Cedex 1; 2Service

d’Accueil et de Traitement des Urgences, Hopital Cochin, Paris,

France

D-dimer levels below a well defined cut-off level enable to safely rule

out VTE in patients with a low or intermediate pre-test probability

of VTE, with a negative predictive value (NPV) around 100%. As

ageing is associated with increased concentrations of coagulation

markers including D-dimers, the question was raised of their useful-

ness to rule out VTE in elderly patients. We prospectively evaluated

the performance of Vidas D-dimer assay (BioMerieux), in 1004 con-

secutive out-patients with a low or intermediate pre-test probability

(Wells’ probability score). Standardized diagnostic procedures were

applied to assess VTE, with a 3 month-follow up.

VTE were confirmed in 70 patients: D-dimers level was above the

cut-off value (500 ng/mL) in 67 patients and below that level in three

patients, leading to NPV = 99.4% and sensitivity = 95.8%. D-dimer

assay specificity dramatically decreased in an age-dependent manner.

However, sensitivity and NPV remained nearly maximal in each age

category including older patients.

The Vidas D-dimer assay was found to perform well in 1004 unse-

lected patients suspected of VTE with an overall NPV = 99.4%.

However, the negative correlation between test specificity and age,

leading to high percentage of increased D-dimer test results in elderly

patients particularly those above 80 years old, raised the question of

its usefulness in those very old patients.

Table 1 for O9A-3

Age

(year)

Patients

(n)

VTE

(n)

Sensitivity

(95% CI)

Specificity

(95% CI)

NPV

(95% CI)

< 30 91 5 80% (28.4–99.5) 83.5% (74.3–90.5) 98.7% (93.0–100)

30–39 121 5 80% (28.4–99.5) 75.6% (67.0–83.0) 98.9% (94.0–100)

40–49 151 7 100% (59.0–100) 78.7% (71.3–84.9) 100% (96.9–100)

50–59 155 6 83.3% (35.9–99.6) 69.2% (61.4–76.4) 99.1% (95.0–100)

60–69 142 11 100% (71.5–100) 56.0% (47.1–64.5) 100% (95.2–100)

70–79 165 14 100% (76.8–100) 33.3% (26.1–41.1) 100% (93.4–100)

80–89 134 18 100% (81.5–100) 10.1% (5.3–17.0) 100% (73.5–100)

> 90 45 4 100% (39.8–100) 7.3% (4.0–20.0) 100% (29.2–100)

Total 1,004 70 95.8% (88.0–99.0) 57.4% (54.2–60.5) 99.4% (98.4–99.9)

O9A-4Different cut-off values of quantitative D-dimer (DD)assays to establish duration of oral anticoagulationtreatment (OAT) after venous thromboembolism (VTE)Legnani C, Cosmi B, Cini M and Palareti GUniversity Hospital S. Orsola-Malpighi, Bologna, Italy

The PROLONG study showed that continuing OAT in patients with

abnormal DD (evaluated by a qualitative assay, Clearview Simplify

D-dimer) resulted in a significant reduction of VTE recurrence. Theo-

retically, if the DD measurement has to be used to stratify the VTE

recurrence risk, each DD assay should be independently assessed in a

management study as the PROLONG. Nevertheless, several clinicians

have already adopted the DD-based risk stratification and implemen-

tation of OAT based on the PROLONG results, using quantitative

DD assays in the absence of method-specific validated cut-off values

for this clinical application.

The present study retrospectively analyzed a subgroup of patients

enrolled in the PROLONG study to define cut-off values of six quan-

titative D-dimer methods to predict the risk of VTE recurrence.

We measured DD levels by the VIDAS D-dimer Exclusion (Bio-

merieux), Biopool AutoDimer (Biopool), HemosIL D-dimer and He-

mosIL D-dimer HS (Instrumentation Laboratory), Innovance D-

dimer (Siemens) and STA Liatest D-dimer (Diagnostica Stago) in fro-

zen plasma aliquots sampled 30 � 10 days after OAT cessation in

321 patients enrolled in the PROLONG study who did not resumed

OAT.

During follow-up (542 year), 25 (7.8%) pts had recurrent VTE. Since

DD levels increase with age and are significantly higher in females

than in males, we calculated method specific cut-off values according

to age and gender. The criteria used to select these cut-off values (see

Table) were: 1) % of patients with altered DD between 36–40%; 2)

% of VTE recurrence as low as possible in patients with normal DD.

These data suggest that different cut-off values according to methods,

patient’s age and gender are indicated to identify patients at higher

risk for VTE recurrence. Furthermore, they are different than those

used in diagnostic strategies to rule out acute VTE. These method

specific cut-off values are being evaluated in the ongoing prospective

management multicenter DULCIS study.


22 ORAL PAPERS____________________________________________________________________________________

Table 1 for O9A-4

Cut-off values in ng/ml

Males Females

£ 70 year > 70 year £ 70 year > 70 year

VIDAS D-dimer exclusion* 490 1050 600 1300

Biopool autodimer & 90 190 111 240

HemosIL D-dimer & 205 300 225 330

HemosIL D-dimer HS & 170 345 215 430

Innovance D-dimer* 500 950 550 1150

STA Liatest D-dimer* 340 700 450 1050

*Results expressed in FEU; & results expressed in D-dimer units.

O9A-5Inflammatory and thrombotic markers in patients withST-elevation myocardial infarction treated withfibrinolysis and early PCIHalvorsen S, Seljeflot I, Bøhmer E and Arnesen HOslo University Hospital, Oslo, Norway

Background: The optimal treatment following fibrinolysis in patients

with ST-elevation myocardial infarction (STEMI) is still debated, but

several studies have shown improved outcomes with early transfer for

percutaneous coronary intervention (PCI). The aim of this study was

to investigate whether circulating levels of inflammatory and throm-

botic markers differ in patients treated with early versus late invasive

strategy following fibrinolysis.

Materials and Methods: This is a substudy of the NORwegian study

on District treatment of ST-elevation Myocardial Infarction (NOR-

DISTEM). Patients with STEMI of < 6 h duration and expected

time delay to PCI > 90 min were treated with tenecteplase, aspirin,

clopidogrel and enoxaparin, and randomized to early invasive

(n = 134) or ischemia-guided (late invasive) strategy (n = 132). Fast-

ing blood samples were collected in the morning 3 days and 3 months

after onset of STEMI for determinations of CRP, TNFa, IL- 6, IL-

10, IL-18, MCP-1, D-dimer, soluble TF and soluble CD40L. Com-

mercial available ELISA kits were used.

Results: After 3 days, D-dimer levels were higher and soluble CD40L

levels lower in the early compared to the late invasive group

(P = 0.001 and P = 0.008, respectively). The differences were abol-

ished after 3 months. No differences in other variables were observed.

The median change over time did not differ between groups, except

for D-dimer levels which were significantly more reduced in the early

invasive compared to the late invasive group (42% vs. 24% reduc-

tion, P = 0.002).

Conclusions: In STEMI patients treated with fibrinolysis and early

invasive strategy, D-dimer levels were higher after 3 days compared

to late invasive strategy, possibly due to early discontinuation of anti-

coagulation. No differences between treatment strategies were found

in other inflammatory markers. The clinical consequences of early

discontinuation of anticoagulation in these patients should be deter-

mined.

O9A-6Tissue plasminogen activator, fibrin D-dimer and VonWillebrand factor antigens and risk of future coronaryheart diseaseThompson A1, Lowe GD2, Rumley A2, Eiriksdottir G3, Danesh J1

and Gudnason V3

1University of Cambridge, Cambridge; 2University of Glasgow,

Glasgow, UK; 3Icelandic Heart Association, Reykjavik, Iceland

Background: Activation of blood coagulation and fibrinolysis may be

associated with increased risk of coronary heart disease (CHD).

Objectives: To assess associations of long-term circulating levels of

tissue plasminogen activator (t-PA), fibrin D-dimer and von Wille-

brand factor (vWF) antigens with CHD risk.

Methods: Measurements were made in 1925 people who had a

first-ever nonfatal myocardial infarction or died of CHD during

follow-up (median 19 years), and in 3616 controls nested within

the prospective population-based Reykjavik Study. Correction for

within-person variability was made using serial measurements taken

several years apart. Meta-analyses of published reports put the new

findings in context.

Results: The year-to-year variability of t-PA and D-dimer concentra-

tions within individuals was relatively high, yielding regression dilu-

tion ratios of 0.36 (95% CI: 0.26–0.47) for t-PA, and 0.28 (0.20–0.36)

for D-dimer. vWF was about as variable as total cholesterol, with a

regression dilution ratio of 0.54 (0.45–0.63). Ignoring such variability,

the odds ratios (ORs) for CHD adjusted for several vascular risk fac-

tors per 2 standard deviation (SD) higher measured levels were 1.06

(0.90–1.25) with t-PA, 1.12 (0.97–1.30) with D-dimer, and 1.19 (1.03–

1.37) with vWF. After correction for within-person variability, the

corresponding ORs were 0.89 (0.64–1.25), 1.11 (0.89–1.39), and 1.17

(1.01–1.36) respectively. When combined with the results from previ-

ous prospective studies, overall adjusted ORs were 1.44 (1.16–1.78)

with t-PA (11 studies, 4535 CHD cases); 1.57 (1.31–1.88) with

D-dimer (12 studies, 5123 cases); and 1.34 (1.13–1.59) with vWF

(10 studies, 4115 cases) per 2-SD higher baseline level.

Conclusions: Circulating levels of t-PA, D-dimer and vWF may be

more modestly associated with risk of CHD than previously reported,

particularly over the longer term. More detailed analyses, perhaps

involving pooling of individual data from prospective studies are

required to clarify the relevance of such markers to CHD.

O9A-7Plasma levels of fibrinolytic proteins and the risk ofmyocardial infarction in menLisman T1, Meltzer ME2, Meijers JCM3, de Groot PG4,Doggen CJM2 and Rosendaal FR2

1University Medical Center Groningen, Groningen; 2LUMC,

Leiden; 3AMC, Amsterdam; 4UMCU, Utrecht, The Netherlands

Hypofibrinolysis as measured with overall clot lysis assays is associ-

ated with an increased risk of arterial thrombosis. Individual com-

ponents of the fibrinolytic system, however, have not been studied

extensively in relation to arterial disease or results of studies were

inconsistent. The relation between plasminogen and a2-antiplasmin

levels and cardiovascular risk factors and the association between

plasminogen, a2-antiplasmin, thrombin-activatable fibrinolysis inhibi-

tor (TAFI), tissue-plasminogen activator (t-PA), and plasminogen

activator inhibitor-1 (PAI-1) and risk of myocardial infarction was

investigated in the Study of Myocardial Infarctions Leiden (555

men with a first myocardial infarction and 635 controls). a2-Anti-

plasmin was associated with age and lipid levels while plasminogen

correlated with lipids, C-reactive protein and smoking. Increased

levels of all fibrinolytic factors were associated with myocardial

infarction, except for TAFI which protected against myocardial

infarction at high levels. Age-adjusted odds ratios (OR) (95% confi-

dence interval) for quartile 4 compared with 1 were 1.7 (1.2–2.3)

for plasminogen, 1.9 (1.3–2.6) for a2-antiplasmin, 0.4 (0.3–0.5) for

TAFI, 1.7 (1.2–2.3) for t-PA, and 1.7 (1.2–2.4) for PAI-1. After

adjusting for cardiovascular risk factors, only a2-antiplasmin (OR

1.4 (1.0–2.0)) and TAFI levels (OR 0.3 (0.2–0.4)) remained associ-

ated with risk (OR 1.4 (1.0–2.0)). t-PA and PAI-1 levels predomi-

nantly reflected lipid levels whereas plasminogen reflected the

inflammatory state. Concluding, elevated a2-antiplasmin and

decreased TAFI levels are independently associated with risk of

myocardial infarction. We hypothesize that the protective effect of

high TAFI levels relates to its anti-inflammatory properties. t-PA,

PAI-1, and plasminogen levels appear to reflect other cardiovascular

risk factors.


ORAL PAPERS 23____________________________________________________________________________________

Structure, Function and Regulation of the Plasminogen Activator

System

O9B-1Regulation of cell surface proteolysis and cellmigration by the novel plasminogen receptor, Plg-RKTMiles LA1, Lighvani S1, Baik N1, Diggs JE1, Parmer CM1,Kiosses WB1, Bai H2, Booth NA3, and Parmer RJ21The Scripps Research Institute; 2University of California San

Diego and Veterans Admin. San Diego Healthcare Sys., La Jolla,

USA; 3University of Aberdeen, Aberdeen, UK

The integral membrane plasminogen receptor, Plg-RKT, has a

unique cell surface orientation that facilitates cell surface proteoly-

sis. Plg-RKT exposes a C-terminal lysine on the cell surface allow-

ing interaction with the kringle domains of plasminogen. The C-

terminus of Plg-RKT also interacts, specifically, with t-PA. Further-

more, Plg-RKT is highly spatially co-localized with the uPAR as

revealed in confocal microscopy and co-immunoprecipitation stud-

ies. Cell surface-dependent plasminogen activation by either t-PA

or u-PA is markedly inhibited by antibodies directed against the C-

terminus of Plg-RKT. Differentiation of monocytes along the

monocyte/macrophage lineage results in the induction of Plg-RKT.

Therefore, we investigated the role of Plg-RKT in plasmin(ogen)-

dependent macrophage migration both in vitro and in vivo. U937

human myelomonocytic cells that express high levels of Plg-RKT

were used to study cell migration induced by monocyte chemoattr-

actant protein (MCP-1) in transwells. In the presence of plasmino-

gen, cell migration was markedly decreased by anti- Plg-RKT

mAb, compared to the isotype control. Anti-Plg-RKT mAb did

not significantly affect cell migration in the absence of plasmino-

gen. In a peritoneal inflammation model, C57BL/6 female mice

were injected intravenously with either anti-Plg-RKT mAb or iso-

type control prior to intraperitoneal injection with 3% thioglycol-

late, and a second intravenous injection of mAb. Cells were

harvested by peritoneal lavage. Anti-Plg-RKT mAb inhibited mac-

rophage recruitment in a dose-dependent manner [at the maximum

dose of 27 lg/g, 58 � 5.4% fewer macrophages were recovered

compared to the isotype control (P < 0.001, n = 5)]. Macrophage

recruitment was significantly decreased in plasminogen null mice

compared to wild type littermates, as previously reported. We

found no effect of anti-Plg-RKT mAb upon macrophage recruit-

ment in plasminogen null mice. These results suggest a major role

of Plg-RKT in plasmin(ogen)-dependent regulation of the inflam-

matory response.

O9B-2The novel plasminogen receptor, Plg-RKT, regulatescatecholaminergic functionParmer RJ1, Bai H2, Baik N3, Kiosses WB3, Krajewski S4 andMiles LA3

1University of California; 2Veterans Administration San Diego

Healthcare System, San Diego; 3The Scripps Research Institute;4Sanford/Burnham Medical Research Institute, La Jolla, CA, USA

Local generation of plasmin is markedly enhanced when plasmino-

gen is bound to the surface of catecholaminergic cells, and cate-

cholamine release by the sympathoadrenal system is negatively

regulated by prohormone cleavage products formed from plasmin-

mediated proteolysis. We recently identified a novel transmembrane

plasminogen receptor, Plg-RKT. Here, we investigated the expres-

sion and subcellular localization of Plg-RKT in catecholaminergic

cells and examined the role of Plg-RKT in catecholaminergic func-

tion. Prominent staining with anti-Plg-RKT mAb was observed in

adrenal medullary chromaffin cells of both human and murine tis-

sue. In western blotting, Plg-RKT was highly expressed in murine

adrenal gland, bovine adrenomedullary chromaffin cells, human

pheochromocytoma tissue, PC12 rat pheochromocytoma cells as

well as in purified rat hippocampal neurons. PC12 cells were

transfected with an expression vector encoding Plg-RKT fused in-

frame to GFP. Confocal analysis of the transfectants demonstrated

that the GFP signal was present at the cell membrane and

colocalized with wheat germ agglutinin, a surface marker

(79 � 5% colocalization). In phase partitioning experiments, Plg-

RKT partitioned to the detergent phase, consistent with Plg-RKT

behaving as an integral membrane protein. In the presence of anti-

Plg-RKT mAb, cell surface plasminogen activation was markedly

suppressed, compared to PC12 cells incubated with isotype control.

Correspondingly, cells stably overexpressing Plg-RKT exhibited

enhanced plasminogen binding and enhanced cell-surface plasmino-

gen activation, compared to cells stably expressing empty vector.

In functional secretion assays, cells overexpressing Plg-RKT exhib-

ited a marked decrease in nicotine-evoked 3H-norepinephrine

release (by 51 � 2%, P < 0.001), compared with control transfect-

ed cells. In conclusion, Plg-RKT is present on the surface of cate-

cholaminergic cells and functions to stimulate plasminogen

activation and modulate catecholamine release. This novel receptor

thus represents a new mechanism and novel control point for regu-

lating the interface between plasminogen activation and sympat-

hoadrenal function that may substantially influence

catecholaminergic stress responses.

O9B-3Effect of histone deacetylase inhibitors on tissue-typeplasminogen activator expression in humanendothelial cellsDunoyer-Geindre S and Kruithof EKOUniversity Hospital of Geneva, Geneva, Switzerland

Tissue-type plasminogen activator (t-PA) is produced by endothelial

cells (EC) and responsible for the removal of intravascular fibrin

deposits. We investigated the role of histone deacetylase (HDAC)

inhibitors on t-PA expression. HDAC are of major importance for

the epigenetic control of gene expression. Methylation analysis of

the proximal t-PA promoter revealed an island of unmethylated

CpG stretching from position -366 to +59, while upstream CpG’s

were all methylated. Acetylation of histones H3 and H4 by histone

acetyl transferases (HAT) is associated with active genes, whereas

removal of the acetyl groups by HDAC is associated with inactive

genes. Treatment of EC with HDAC inhibitors trichostatin (TSA)

and MS-275 resulted in a 7 and 13-fold increase of t-PA mRNA

expression, respectively, while expression of PAI-1 was increased

fourfold by MS-275, but not by TSA. As TSA inhibits both class I

and class II HDAC and MS-275 only class II HDAC, these results

imply a role for class I HDAC in the increased expression of t-PA

and PAI-1 and a role for a class II HDAC in PAI-1 reduction.

HDAC and HAT not only interact with histones but also with a

variety transcriptional regulators. To determine whether the effect

of the HDAC inhibitors on t-PA expression was related to changes

in histone acetylation we performed chromosome immunoprecipita-

tion analysis of the t-PA promoter using antibodies specific for acet-

ylated histone H3 or H4. We observed an increase in H3

acetylation of 10 � 3 and 44 � 14 -fold in EC treated with TSA or

MS-275, respectively, and in H4 acetylation of 7.7 � 1.4 and 16 � 3

- fold, respectively.

Thus, expression of t-PA by EC is suppressed by HDAC in a mecha-

nism that involves de-acetylation of histone H3 and H4. Reversal of

this epigenetic suppression increases t-PA expression by EC.


24 ORAL PAPERS____________________________________________________________________________________

O9B-4Allelic imbalance of tissue-type plasminogen activator(tPA) gene expression in human brain tissueHultman K1, Tjarnlund-Wolf A1, Curtis M2, Faull R2, Medcalf RL3

and Jern C1

1Institute of Neuroscience and Physiology, Gothenburg, Sweden;2Department of Anatomy with Radiology, University of

Auckland, Auckland, New Zealand; 3Australian Centre for Blood

Diseases, Department of Clinical Haematology, Melbourne,

Australia

We have identified a single nucleotide polymorphism (SNP) at the

tPA locus (-7351C>T), which is associated with vascular tPA release

rates in humans and the risk of myocardial infarction. In vitro studies

demonstrated that this SNP is functional at the level of transcription,

and affects tPA gene regulation in endothelial cells. In the brain, tPA

has been implicated both in physiological and pathophysiological

processes. The aim of the present study was to examine the effect of

this SNP on tPA gene regulation in human brain tissue under in vivo

conditions. Human brain tissue was obtained from the Neurological

Foundation of New Zealand Human Brain Bank. Allelic expression

was determined in heterozygous samples by TaqMan assay using the

coding tPA 20 099T>C SNP as a marker for the -7351C>T SNP.

Thus, allele-specific gene expression is analysed in a normally func-

tioning nucleus where the different alleles are subjected to the same

genetic and environmental conditions. Protein-DNA interactions were

studied by EMSA and ChIP. In human brain tissue, a significantly

higher number of tPA mRNA transcripts were generated from chro-

mosomes expressing the wild-type C allele compared to the mutant

T allele (ratio C vs. T; mean � SEM, hippocampus 1.35 � 0.14,

P = 0.033, n = 12; cortex 1.25 � 0.10, P = 0.037, n = 12). EMSA

showed reduced neuronal and astrocytic nuclear protein binding

affinity to the mutant T-allele probe. Supershift experiments identified

Sp1 and Sp3 as the major transcription factors binding to the poly-

morphic site. ChIP analyses confirmed that Sp1 indeed recognised

the polymorphic enhancer in intact cells. The present study demon-

strates that the -7351C>T SNP affects tPA gene expression in

human brain tissue. As neurons are considered to be the major

source of tPA in the CNS, allele-specific tPA expression in these cells

may have a clinical impact in neurological conditions associated with

changes in plasminogen activation.

O9B-5Identification of proteins that selectively misfoldwithin the acutely injured brain and acceleratetPA-mediated plasmin formation: implications forischemic strokeSamson A, Borg RJ, Sashindranath M, Au AEL, Niego B,Cody SH and Medcalf RLMonash University, Melbourne, Australia

In the vasculature, fibrin acts as both a cofactor for tPA-mediated

plasmin formation and as a substrate for plasmin. An analogous situ-

ation arises in the brain whereby neuronal injury causes the forma-

tion of misfolded proteins. Like fibrin, these misfolded proteins

specifically bind tPA and plasminogen and act as a macromolecular

cofactor to promote tPA-mediated plasminogen activation (Samson

et al., Blood 2009).We have identified the intracellular proteins that

are most prone to aggregation during acute neuronal injury. Of the

proteins identified, we find that tubulin and GAPDH exhibit an espe-

cially strong propensity to misfold following a variety of in vivo (e.g.

traumatic brain injury) and in vitro (e.g. glutamate-induced excitotox-

icity) neuronal insults. One characteristic of misfolded proteins is

detergent-insolubility. Accordingly, we show that both tubulin and

GAPDH become detergent-insoluble following neuronal injury. Inter-

estingly, only the detergent-insoluble form of tubulin, and not the

soluble form, acts as a cofactor/substrate for tPA-mediated plasmin

formation. Another hallmark of misfolded proteins is their deposition

into spheroid-like structures within cells. We show that neuronal

injury causes the redistribution of tubulin from the cytoskeleton into

numerous intracellular spheroids which also act as a specific cofac-

tor/substrate for tPA-mediated plasmin formation.This is the first

description that GAPDH and tubulin can misfold and facilitate plas-

minogen activation following acute brain injury. Agents which block

the binding of tPA to aggregated tubulin and GAPDH would be

expected to specifically reduce plasminogen activation within the

injured brain whilst leaving fibrinolysis unperturbed. Such an

approach will likely improve the safety and efficacy of thrombolytic

therapy during ischemic stroke – a clinical situation where intravascu-

lar plasmin generation on fibrin is wanted, but where excessive tPA-

mediated plasmin formation in the injured brain is harmful.

O9B-6New functions of t-PA and factor XII in amyloid andprotein metabolismGebbink MCrossbeta Biosciences, Utrecht, The Netherlands

We identified in factor XII and t-PA a common domain, the fibro-

nectin-type I (or finger) domain, that could be shown to interact with

non-native proteins, including those comprising complex beta-sheet

structures such as crossbeta structure in amyloid. The consequences

of these interactions are multiple.

Stimulation of t-PA plasminogen activation was found common for

peptides and proteins that formed amyloid aggregates such as aggre-

gates of endostatin, amyloidbeta peptide, islet amyloid polypeptide

and fibrin peptides and fragments, ovalbumin, etc. The as such gener-

ated plasmin can be involved in processing amyloid-bound neuroen-

docrine factors, notably Chromogranin B and the neurosecretory

protein VGF, on the one hand, and in degradation of proteins/pep-

tides, most notably extracellular matrix components, such as fibrin

and vitronectin on the other hand.

Stimulation of the contact system (factor XII) can occur via protein

binding to surfaces. Traditional activators, such as kaolin, ellagic acid

and dextran sulphate do not induce factor-dependent kallikrein for-

mation in the absence of sufficient protein. Adsorption of protein can

result in unfolding into a non-native state. In agreement, non-native

proteins, including amyloid-beta can induce factor-FXII mediated

kallikrein formation in the absence of such traditional activator. This

route of non-native protein dependent activation can be exclusive for

kallikrein formation and leaves coagulation activation via factor XI

untouched. This process is considered of relevance for local oedema

formation involving bradykinin formation.

It is expected that in vivo several of these processes run in parallel

and may cooperate. The most appealing question is for each stimula-

tor of t-PA and contact system what is the exact profile of subse-

quent activation pathways.

It is concluded that a systematic study of stimulators is required to

overview the consequences of the newly discovered pathways of acti-

vation of t-PA and factor XII.

O9B-7Engineering Kunitz 1 domain (KD1) of human tissuefactor pathway inhibitor-2 to selectively inhibitfibrinolysis: properties of KD1-L17R variantBajaj MS1, Ogueli I1, Schmidt E2, Shanker S1, Velander W3,Calcaterra J3 and Bajaj SP1

1UCLA, Los Angeles, CA; 2Washinton University, St. Louis, MO;3University of Nebraska, Lincoln, NE, USA

Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, kallik-

rein and factor VIIa/tissue factor; accordingly, it has been proposed

for use as an anticoagulant. Full-length TFPI-2 or its isolated first

Kunitz domain (KD1) also inhibits plasmin and has been proposed


ORAL PAPERS 25____________________________________________________________________________________

for use as an antifibrinolytic agent. However, the anticoagulant prop-

erties of KD1 would diminish its antifibrinolytic function. In this

report, structure based investigations and analysis of the serine prote-

ases profiles revealed that coagulation enzymes prefer a hydrophobic

residue at the P2’ position in their substrates/inhibitors, whereas plas-

min prefers a positively charged residue at the corresponding posi-

tion. Thus, we changed the P2’ residue Leu17 in KD1 to Arg (KD1-

L17R) and compared its inhibitory properties with the wild-type

KD1 (KD1-WT). Both WT and KD1-L17R were expressed in E.

Coli, folded and purified to homogeneity. Amino-terminal sequences

and mass-spectra revealed proper folding of both proteins. Compared

to KD1-WT, the KD1-L17R neither prolonged aPTT of normal

plasma nor did it inhibit factor XIa, kallikrein or VIIa/tissue factor.

Further, KD1-L17R inhibited plasmin with ~5-fold increased affinity.

In mouse liver laceration model and tail transection bleeding model,

KD1-L17R was, respectively, ~6-fold and ~2-fold more effective than

KD1-WT in preventing blood loss. Importantly, in these bleeding

models, KD1-L17R was more effective than aprotinin (bovine pan-

creatic trypsin inhibitor), which has been used as an antifibrinolytic

agent to prevent blood loss during major surgery/trauma. Further, as

compared to aprotinin, renal toxicity was not observed with KD1-

WT or KD1-L17R. In rotational thromboelastography experiments,

KD1-WT and aprotinin increased the clotting time and clot forma-

tion time of whole blood whereas KD1-L17R had no effect. Plasmin-

induced fibrinolysis in whole blood or a purified system measured by

clot strength was prevented by KD1-L17R and aprotinin but not by

KD1-WT. KD1-L17R may be a superior therapeutic agent to prevent

fibrinolysis and blood loss in major surgery/trauma.


26 ORAL PAPERS____________________________________________________________________________________

Fibrinolysis in Disease

O10A-1Plasminogen deficiency in mice causes periodontaldiseaseSulniute R1, Lindh T2, Li J1 and Ny T1

1Umea University, Umea; 2County Council of Vasterbotten, the

National Dental Service/Periodontics, Ume, Sweden

Periodontal disease involves bacterial infection, periodontium inflam-

mation, gum tissue degradation, and alveolar bone resorption leading

to teeth loss. In human, plasminogen deficiency causes ligneous peri-

odontitis. Therefore, we decided to examine oral health of plasmino-

gen deficient mice and their wild type littermates to establish a mouse

model for studying the roles of Plg activation system in periodontal

disease.

The periodontium in wild type mice was unaffected at all time points

studied whereas in plg deficient mice periodontitis progressed rapidly

within 20 weeks. Morphological studies revealed detachment of gingi-

val tissue, resorption of cementum layer, formation of necrotic tissue,

and severe alveolar bone degradation in plg deficient mice. The

immunohistochemical staining indicated a mass infiltration of neu-

trophils in the periodontal tissues. Interestingly, mice deficient in both

plasminogen activators, tPA and uPA, developed periodontal disease

similar to plg deficient mice, although mice lacking either of PAs

remained healthy in periodontium. Furthermore, supplementation

with human plasminogen for 10 days led to recovery of the periodon-

tium and removal of necrotic tissue in plg deficient mice. In addition,

quantification of alveolar bone revealed significant difference com-

pared to control mice, suggesting even possible alveolar bone

re-growth.

Our data demonstrated that plasminogen is essential to maintain

healthy periodontium and plays an important role against the sponta-

neous development of chronic periodontal disease. Moreover, revers-

ibility to the healthy status after supplementation suggests the

possibility of using plasminogen for a clinical therapy of periodontal

diseases.

O10A-2Biological variation in tPA-induced plasma clot lysistimeTalens S, Malfliet JJMC, Rudez G, de Maat MPM and Rijken DCErasmus Medical Center, Rotterdam, The Netherlands

Hypofibrinolysis is a risk factor for venous and arterial thrombosis,

and can be established by measuring the plasma fibrinolytic potential

in a turbidimetric tPA-induced clot lysis time assay. However, not

much is known about the biological variation in clot lysis time. It is

important to obtain information about the variation when clot lysis

time is tested as risk factor for thrombosis in future studies. Thus,

this study aimed to determine the analytical, within-subject and

between-subject variation. We collected blood samples from 40

healthy individuals throughout a period of 1 year (average 13 visits)

and determined the clot lysis time of each plasma sample in dupli-

cate. The mean (� SD) clot lysis time was 83.7 (� 10.9) min. The

coefficient of variations for total variation, analytical variation,

within-subject variation and between-subject variation were 13.0%,

3.7%, 8.0% and 10.0%, respectively. One measurement of clot lysis

time was sufficient to establish the habitual value which does not

deviate more than 20% from its true value with a probability of

95%. The contribution of analytical imprecision to the within-subject

variation was 10.2%, the index of individuality was 0.88 and the ref-

erence change value was 24.4%. We observed that the clot lysis time

was prolonged in the morning compared to the afternoon and was

slightly higher in older individuals compared to younger individuals.

There was no seasonal variation observed. This study provides

insight into the biological variation of clot lysis time, which can be

used in future studies using clot lysis time as a potential risk factor

for thrombosis.

O10A-3Plasma fibrinolytic capacity and the risk of arterialthrombosis in young womenSiegerink B1, Meltzer M1, de Groot PG2, Algra A2, Lisman JA3

and Rosendaal FR1

1LUMC, Leiden; 2UMC Utrecht, Utrecht; 3UMC Groningen,

Groningen, The Netherlands

Background: Recently, reduced overall fibrinolytic capacity has been

shown to increase the risk of arterial thrombosis, especially coronary

heart disease and myocardial infarction in young men. The role of

fibrinolysis in clinical ischaemic stroke is unclear. So far, studies on

fibrinolysis and atrial disease have mainly been focussed on men.

Objectives: With this study we aim to determine the risk of both

hyper- and hypofibrinolysis on the risk of myocardial infarction and

ischaemic stroke in young women.

Methods: The RATIO study is a population based case control study

including women (18–50 years) with myocardial infarction (MI;

N = 205), ischaemic stroke (IS; N = 175) and 638 healthy controls,

frequency matched on age, year of event and area of residence. Over-

all fibrinolytic potential was determined with a tissue factor and tis-

sue plasminogen activator induced clot lysis assay. Clot lysis time

(CLT) was divided into quintiles based on the levels found in the

control group (Q1–Q5), with the middle quintile (Q3) as reference.

Odds ratios, obtained from a logistic regression model, adjusted for

the matching factors and cardio vascular risk factors were calculated

as a measure of rate ratios.

Results: Hypofibrinolysis was associated with an increase in risk of

myocardial infarction (Q4, odds ratio 1.5; 95% CI 0.8–2.9 and Q5,

2.3; 1.2–4.4). Hyperfibrinolysis had no clear effect (Q2, 0.8; 0.4–1.8

and Q1, 1.8; 0.9–3.8). Hypofibrinolysis did not affect the risk of is-

chaemic stroke (Q4 0.8; 0.3–2.0 and Q5, 1.1; 0.5–2.5), whereas hyper-

fibrinolysis increased this risk fourfold (Q2 1.6; 95%CI 0.7–3.7 and

Q1, 3,9; 1.7–8.8).

Discussion: These results show that hypofibrinolysis is a risk factor

for myocardial infarction in young women, which is similar to find-

ings in men and mixed populations. Surprisingly, hyperfibrinolysis

increased the risk of ischaemic stroke fourfold.

O10A-4Venous thrombosis risk associated with plasmahypofibrinolysis is explained by elevated plasmalevels of TAFI and PAI-1Lisman T1, Meltzer ME2, de Groot PG3, Meijers JCM4, LeSessie S2, Doggen CJM2 and Rosendaal FR2

1University Medical Center Groningen, Groningen; 2LUMC,

Leiden; 3UMCU, Utrecht; 4AMC, Amsterdam, The Netherlands

Elevated plasma clot lysis time (CLT) as measured with a plasma-

based assay increases risk of venous and arterial thrombosis. It is,

however, unclear which fibrinolytic factors contribute to thrombosis

risk. In 743 healthy controls we investigated determinants of CLT.

By comparison with 770 patients with a first venous thrombosis we

assessed plasma levels of fibrinolytic proteins as risk factors for

thrombosis. Plasminogen activator inhibitor-1 (PAI-1) levels were the

main determinants of CLT, followed by plasminogen, thrombin acti-

vatable fibrinolysis inhibitor (TAFI), prothrombin, and a2-antiplas-min. Fibrinogen, factor VII, X, and XI contributed minimally. These


ORAL PAPERS 27____________________________________________________________________________________

proteins explained 77% of variation in CLT. Levels of these proteins

allowed accurate prediction of CLT. Levels of the fibrinolytic factors

were associated with thrombosis risk (odds ratios, highest quartile

versus lowest, adjusted for age, sex, and BMI: 1.6 for plasminogen,

1.2 for a2-antiplasmin, 1.6 for TAFI, 1.6 for PAI-1, 1.8 for tissue

plasminogen activator (t-PA)). Adjusting for acute-phase proteins

attenuated the risk associated with elevated plasminogen levels. The

risk associated with increased t-PA nearly disappeared after adjusting

for acute-phase proteins and endothelial activation. TAFI and PAI-1

remained associated with thrombosis after extensive adjustment. Con-

cluding, CLT reflects levels of all fibrinolytic factors except t-PA.

Plasminogen, TAFI, PAI-1, and t-PA are associated with venous

thrombosis. However, plasminogen and t-PA levels may reflect

underlying risk factors.

O10A-5Clot lysis time (CLT) of PRP and PPP inhypercholesterolemia. Effect of atorvastatin andrelationships with inflammatory markersMezzano D, Acevedo M, Pereira J, Matus V and Panes OSchool of Medicine, P. Universidad Catolica de Chile, Santiago,

Chile

The role of platelet fibrinolytic components in hemostatic balance is

largely ignored. A decrease in fibrinolysis is postulated in the patho-

genesis of arterial thrombosis in hypercholesterolemia and athero-

thrombosis, but studies reporting effect of statins on total fibrinolytic

capacity (FC) are controversial. Our aim was to study the contribu-

tion of platelets on clot lysis time in patients with hypercholesterol-

emia, before and after atorvastatin (80 mg/day, 1 month). We used

the CLT-PPP described by Lisman et al (Blood, 2005) and developed

an assay using PRP (CLT-PRP) to assess FC. In healthy controls,

(LDL-Chol = 108 � 27 mg/dL), lysis times with both assays were

essentially identical (25 � 12 min vs. 24 � 12 min, P = NS, n = 9),

denoting no influence of platelets in FC. In 7 hypercholesterolemic

patients (LDL-Chol = 193 � 46 mg/dL), FC was significantly

decreased, with prolongation of CLT’s in both assays. CLT-PRP val-

ues were higher than those of CLT-PPP (81 � 34 vs. 68 � 29 min,

respectively, P = 0.015), suggesting higher sensitivity of CLT-PRP

and enhanced platelet-derived hypofibrinolytic effect. CLT-PRP and

CLT-PPP were correlated with LDL-Chol (r = 0.62, P = 0.01), tri-

glycerides (r = 0.81, P = 0.0002) and PAI-1 concentration

(r = 0.74, P = 0.006, n = 16). CLT-PRP, but not for CLT-PPP,

was significantly correlated with acute-phase reactants (APhR): usC-

reactive protein (r = 0.81, P = 0.0002), fibrinogen (r = 0.55,

P = 0.029), VWF:Ag (r = 0.64, P = 0.014). After atorvastatin,

LDL-Chol decreased to 88 � 21 mg/dL. This was accompanied with

decreases, though not normalization, of CLT’s in both assays: CLT-

PPP to 44 � 22 min (P = 0.012) and CLT-PRP to 64 � 29 min

(P = NS). No significant changes in inflammatory markers resulted

from atorvastatin treatment.

Conclusions: (i) FC, assessed by CLT-PRP, is decreased in hypercho-

lesterolemia, and is closely related with plasma cholesterol, PAI-1

and inflammatory markers. (ii) FC improves after atorvastatin treat-

ment. (iii) CLT-PRP appears more sensitive than CLT-PPP to assess

the abnormal FC of hypercholesterolemia. (iv) Further studies will

determine the role of the platelet fibrinolysis components in states of

enhanced/decreased fibrinolysis.

O10A-6Leukocyte- and endothelial-derived microparticles: acirculating source for fibrinolysisLacroix R1, Plawinski L2, Robert S1, Doeuvre L3, Sabatier F1,Anfonso F1, Leroyer A1, Boulanger C1, Angles-Cano E3 andDignat-George F1

1INSERM U608, Marseille; 2CNRS; 3INSERM U919, Caen, France

Membrane microparticles (MPs) derived from activated platelets, leu-

kocytes and endothelial cells, are well described for their procoagu-

lant activity. We recently assigned a new fibrinolytic function to

endothelial-derived MPs (Blood 2007, 110: 2432). Since the relevance

of this novel mechanism of plasmin formation remains unsolved, we

investigated whether circulating MPs found in healthy subjects and in

pathological situations could be a source of fibrinolysis.

We first compared the ability of circulating MPs, isolated from blood

of patients (atherosclerotic and auto-immune diseases, thrombotic

thrombocytopenic purpura: TTP) and healthy subjects to generate

plasmin activity. Plasmin generation on circulating MPs was signifi-

cantly higher in patients compared to healthy controls. Circulating

MPs from patients with TTP generate a range of plasmin activity

indicating a variable content in either urokinase- (uPA) or tissue-type

plasminogen activator (tPA). Complexes between tPA and plasmino-

gen activator inhibitor-1 (PAI-1) were also identified. In all cases,

plasmin generation was consistent with a lysine-dependent mechanism

for plasminogen binding and activation.

To determine the origin of MPs bearing this fibrinolytic activity, we

generated MPs from circulating blood subpopulations and from dif-

ferent human endothelial cell types. Plasmin generation was detected

on endothelial- and leukocyte-derived MPs, but was absent on plate-

lets- or erythrocyte-derived MPs. These findings were confirmed by

depletion of target MPs from plasma of patients with TTP. Leuko-

cyte-derived MPs express uPA and its receptor uPAR, but not tPA.

By contrast, tPA and tPA/PAI-1 complexes were detected in endothe-

lial-derived MPs with variations according to their anatomical origin

while uPA remained undetectable.

In conclusion, our study suggests that the fibrinolytic activity in the

circulation is supported by MP-borne uPA or tPA, derived from leu-

kocyte or endothelial cells. The fibrinolytic activity of these MPs is

modulated in pathological settings.


28 ORAL PAPERS____________________________________________________________________________________

Fibrin(ogen) and Repair

O10B-1Two cases of dysfibrinogenemia associated withthrombosisKotlin R, Khaznadar T, Suttnar J, Riedel T, Salaj P and Dyr JEInstitute of Hematology and Blood Transfusion, Praha 2, Czech

Republic

Fibrinogen, a 340 kDa glycoprotein, is synthesized by hepatocytes

and secreted to the circulation at a level in the range 2–4 g/L. Fibrin-

ogen plays an important role in many physiological processes, espe-

cially in blood coagulation; but it is also known thrombotic risk

factor. After injury, fibrinogen undergoes conversion into fibrin clot.

Fibrin network, if not needed anymore, undergoes proteolytic diges-

tion called fibrinolysis. Fibrinolysis is catalysed by the serine protease

plasmin. Inherited mutations in fibrinogen, causing congenital dysfi-

brinogenemia, may affect fibrinolysis what may lead to thrombosis.

Fibrinogens of two unrelated patients, both with history of throm-

botic events, pulmonary embolism and indication of dysfibrinogen-

emia, were studied. No common thrombotic risk factors were found.

Both dysfibrinogens showed remarkably delayed fibrin polymeriza-

tion after addition of either thrombin or reptilase. tPA-mediated

fibrinolysis was significantly slower than control. The most noticeable

delay occurred during the final stages of what appeared as a diphasic

lysis. DNA sequencing showed the heterozygous mutations c Tyr363-

Asn in one patient and Aa Asn106Asp in the second one. Both

abnormal fibrinogens caused the decrease of platelet aggregation.

Morphologies of both clots, studied by electron microscopy, showed

abnormal fibrin network with narrower fibers and abrupt fibril termi-

nations. Mutation c Tyr363Asn is situated in polymerization pocket

‘a’ in loop III in the P-domain of the c-nodule and is highly con-

served across species. The mutation Aa Asn106Asp is situated in the

coiled-coil connector of fibrinogen and the importance of Aa Asn106

is strengthened by its conservation.

This work was supported by a grant of The Internal Grant Agency

of The Ministry of Health of the Czech Republic, number NS 9636-

3/2008; by a grant of The Ministry of Health of the Czech Republic,

number 2373601; and by a grant of The Academy of Sciences of the

Czech Republic, number KAN200670701.

O10B-2Biologically active human fibrinogen produced in themilk of transgenic cowsCalcaterra J1, Vancott KE1, Forsberg EJ2, Inan M1, Germano M3,van Veen HV3, Nelson K2 and Velander WH1

1University of Nebraska-Lincoln, Lincoln, NE; 2Pharming

Healthcare, Inc., Deforest, WI USA; 3Pharming Technologies

B.V., Leiden, The Netherlands

In the future, hemostatic therapies will likely include recombinant

human fibrinogen (r-F1) as a preferred alternative to human plasma-

derived (pd-) fibrinogen (pd-F1) products. Here we characterize r-F1

purified from the milk of cloned, transgenic cows producing 2 to

4-fold higher concentrations than the pd-F1 found in human plasma.

Three transgenes consisting of genomic A-alpha, B-beta and gamma

fibrinogen genes under the control of the bovine alpha s1-casein pro-

moter were co-inserted into a female somatic cell lineage which was

used to make founder animals by nuclear transfer. The stoichiometric

ratio and molecular weights of alpha, beta and gamma chains as

judged by mass spectroscopy and SDS-PAGE were similar for pd-F1

and r-F1. The phosphorylation content at Ser-3 in fibrinopeptide

FpB is complete in r-F1 whereas only 30% of Ser-3 is phosphory-

lated in pd-F1. Glycosylation of r-F1 features mostly neutral, milk

protein-like glycoforms relative to the more acidic pd-F1. In vitro

findings indicate that the molecular and viscoelastic properties of

r-F1 clot formation are similar to those of pd-F1 when treated with

pd-thrombin (FIIa) and Factor XIII (FXIII). No contaminating

transglutaminase or fibrinolytic activity was found in r-F1 prepara-

tions. Proteolytic cleavage of r-F1 alpha and beta chains by thrombin

was similar to pd-F1. Formation of gamma-gamma dimers in the

presence of FXIII was faster for r-F1 compared with pd-F1. Throm-

boelastographic (TEG) analyses of fibrin formation confirmed similar

formation kinetics and maximum amplitudes between r-F1 and

pd-F1. These molecular properties make r-F1 desirable for use in

fibrinogen replacement therapy and as fibrin sealants.

O10B-3The density of surface bound fibrinogen effects therelease of fibrinopeptidesDyr JE1, Medved L2, Riedel T1 and Suttnar J11Institute of Hematology and Blood Transfusion, Prague, Czech

Republic; 2University of Maryland School of Medicine,

Baltimore, MD, USA

Fibrinogen and fibrin play overlapping roles in blood clotting, fibri-

nolysis, cellular and matrix interactions, the inflammatory response,

wound healing, and neoplasia. Fibrin polymerization is initiated by

thrombin cleveage of the fibinopeptides A and B (FPA, FPB), con-

verting fibrinogen to fibrin monomer. During fibrin polymerization in

solution the release of FPA occurs very rapidly while the release of

FPB is delayed and reaches its maximum when fibrin formation is

almost completed. Numerous studies demonstrated that fibrinogen in

solution and fibrinogen adsorbed on various surfaces exhibit different

properties.We previously reported that fibrinogen adsorbs on artifical

surfaces in basically two different orientation, side-on and end-on.

While side-on orientation prevails during adsorption from low fibrin-

ogen concentrations, the other orientation, in which adsorbed fibrino-

gen is close-packed, occurs at high fibrinogen concentrations. The

adsorbed fibrinogen molecules in both cases have reduced accessibil-

ity for thrombin to the E region suggesting their thrombin binding

properties and fibrinopeptide release may also be altered. Therefore,

the major aim of the present study was to estimate fibrinopeptides

release from fibrinogen and growing fibrin layers at different surface

densities and modes.

We found that the side-on adsorbed fibrinogen released FPB with no

lag phase, and the final concentration of both fibrinopeptides (A, B)

was the same. The initial rate of FpB and FpA release from fibrino-

gen adsorbed at high concentration of Fbg was approximately the

same, however, the final amount of relased FpB was substantially

higher as compared with the amount of FpA. Fibrinopeptide B is

known as a potent chemoattractant, therefore its preferential release

may indicate the physiological meaning in the attraction of cells to

the site of injury.

This work was supported by Grants NS10633-3/2009 and MZ

02373601 from the Ministry of Health, Czech Republic, by Grant

KAN200670701 from the Academy of Sciences, Czech Republic.

O10B-4New evidence for fibrinogen-boundalpha2-antiplasmin in bloodMosesson M, Holyst T and Hernandez IBlood Center of Wisconsin, Milwaukee, WI, USA

Alpha2-plasmin inhibitor (a2AP) occurs mainly as a single-chain pro-

tein in blood, although existing evidence indicates that a2AP is also


ORAL PAPERS 29____________________________________________________________________________________

covalently linked to plasma fibrinogen. First, Mosesson and Finlay-

son identified a plasmin inhibitory activity in purified fibrinogen (JCI

42:747,1963). Years later a2AP was shown by immunoelectrophoresis

to be present in purified fibrinogen (BC&F 11:293,2000). More

recently, we developed an ELISA for estimating fibrinogen-bound

a2AP (JTH 6:1565,2008), but the technique was not applicable to

plasma. Thus, to develop evidence for fibrinogen-bound a2AP in

plasma, we prepared Western Blots using near-infrared fluorescent

dye-labeled secondary antibodies (LI-COR Biosciences) to identify

primary antibody-labeled antigens. We labeled PVDF-membrane

bound plasma a2AP or fibrinogen with goat anti-a2AP or rabbit

anti-human achain, respectively. Then we reacted (i) with anti-goat

IgG tagged with green fluorescent dye (IRdye800) or, (ii) with anti-

rabbit IgG tagged with red fluorescent dye (IRdye700). Non-reduced

plasma showed four IRdye800-labeled a2AP-reactive bands: (i) in the

cathodal region of the IRdye700-stained 340 kD fibrinogen band; (ii)

at ~120–125 kD co-migrating with an IRdye700-stained band, proba-

bly a fibrinogen a chain/a2AP heterodimer; (iii) one corresponding to

‘free’ a2AP (~55–60 kD); (iv) a band at ~25 kD of unknown origin.

The IRdye800/IRdye700-stained fibrinogen band was absent from

serum, but the other IRdye800-stained bands remained.

The IRdye800-fibrinogen band was absent from disulfide-bridge

reduced plasma samples, but the other IRdye800 bands remained: (i)

a less intense 120–125 kD a2AP-stained band, co-staining with IR-

dye700; (ii) an a2AP band overlapping red IRdye700-stained a chain

bands; (iii) the 25 kD band. These observations show that (i) a2AP is

covalently linked to the fibrinogen in plasma, (ii) a heterodimeric

a2AP/Aa polypeptide chain exists in plasma. The role that plasma

fibrinogen-bound a2AP plays in regulating the fibrinolytic process

should be investigated.

O10B-5Early factor XIII activation and delayed fibrinolysis inindividuals with Factor V Leiden; a novel mechanismcontributing to increased thrombosis riskBagoly Z, Koncz Z, Haramura G, Mezei ZA and Muszbek LUniversity of Debrecen, Medical and Health Science Center,

Debrecen, Hungary

Background: Factor V Leiden mutation (FVLeiden) increases the risk

of venous thromboembolism by enhancing thrombin formation. It

has been also shown that FVLeiden inhibits fibrinolysis, however,

underlying mechanisms have not been fully elucidated. We investi-

gated the effect of FVLeiden on factor XIII (FXIII) activation, on

the cross-linking of fibrin and a2 plasmin inhibitor (a2PI) and studied

the effect of cross-linking on the rate of clot-lysis.

Methods: Clotting was initiated in plasma samples of fifteen healthy

individuals with known FVLeiden genotypes and wild type for FXIII

A subunit (FXIII-A) Val34Leu polymorphism by recombinant

human tissue factor and phospholipids with/without recombinant

human thrombomodulin (rhTM). Clots were recovered and analyzed

by SDS-PAGE and Western blotting for FXIII-A and a2PI. The

extent of FXIII activation, the cross-linking of fibrin c-chains and the

incorporation of a2PI into the clot were evaluated by quantitative

densitometry. In-vitro clot-lysis experiments with/without rhTM were

also performed.

Results: The presence of rhTM significantly slowed down the activa-

tion of FXIII in the plasma of wild type individuals as compared to

FVLeiden carriers. Time required for half maximal FXIII activation

was approximately 1.5-fold prolonged in wild types (mean � SEM:

629 � 75.3 s) in the presence of rhTM as compared to carriers of

FVLeiden (mean � SEM: 437 � 43.6 s). The delay of FXIII activa-

tion caused by rhTM in wild type individuals was more than 4-fold

reduced in heterozygotes and more than 8-fold in homozygotes. The

inefficiency of rhTM in delaying FXIII activation in FVLeiden carri-

ers resulted in earlier cross-linking of fibrin c-chain and a2PI to

fibrin; the rate of clot lysis in the presence of rhTM was slowed down

in carriers of FVLeiden.

Conclusion: Earlier FXIII activation and, as a consequence, earlier

cross-linking reactions resulting in earlier inhibition of fibrinolysis

might represent a novel mechanism contributing to the increased

thrombosis risk in FVLeiden carriers.

O10B-6Global health risk associated with the use ofrecombinant streptokinase as a biosimilar forthrombolytic therapyThelwell CNIBSC, Potters Bar, UK

Worldwide, streptokinase (SK) remains the most used thrombolytic

agent for the treatment of myocardial infarction (MI). Recombinant

SK expressed in E. coli is increasingly used in developing countries as

a biosimilar of native SK, but potency assignments against the WHO

International Standard (IS) are highly variable with potentially dan-

gerous consequences. A proportion of recombinant SK appears to be

incompletely processed, retaining the N-terminal Met engineered for

E. coli expression. The N-terminal residue of secreted mature native

SK (Ile) is known to be critical in the ‘molecular sexuality’ mecha-

nism of action of SK. Other sequence variations have been reported

including products that appear to be derived from different strepto-

coccus groups. Commercial SK is derived from the H46A isolate of

S. equisimilis (Group C); strains of S. pyogenes (Group A) are

reported to have differing plasminogen activation characteristics. We

have cloned and expressed commercial SK in E. coli (rSK), con-

structed a novel variant with an N-terminal Met (rSK-Met), and

expressed rSK from the M1 strain of S. pyogenes (rSK-M1GAS).

SKs are expressed as N-terminal fusions with SUMOstar (Life Sen-

sors). Crucially SUMOstar1 protease cleaves the fusion tag without

leaving behind unwanted residues, providing complete control over

the SK N-terminus. The potency of each recombinant SK was deter-

mined relative to the WHO IS using a chromogenic solution assay,

and in a fibrin-based assay. In solution, relative to the IS, rSK and

rSK-Met potencies are equivalent, however in the presence of fibrin

rSK-Met loses ~80% of its potency compared to ~30% loss for rSK.

Conversely rSK-M1GAS has <10% of rSK activity in solution,

which increases ~3-fold in fibrin. This apparent difference in activity

and fibrin selectivity highlights dangers in the unregulated clinical use

of biosimilars, and has serious health implications. Improved under-

standing of the detailed mechanism of SK-fibrin specificity will help

improve global thrombolytic therapy.


30 ORAL PAPERS____________________________________________________________________________________


O11-1ISFP Prize Lecture 2010. From PAI-1 to PDGF in thebrain, a series of fortunate eventsLawrence DAUniversity of Michigan, Ann Arbor, MI, USA

Fibrinolysis has been studied for well over 100 years, and the enzy-

matic components of the plasminogen activator (PA) system have

been known since the 1940’s. However, it was only in the 1990’s that

it began to be appreciated that plasminogen activators might be act-

ing on substrates other than plasminogen, and that their regulation

of physiologic systems may occur through pathways other than fibri-

nolysis. Our work has focused on understanding the non-traditional

roles of plasminogen activators and their inhibitors in normal and

pathologic physiology. Studies have included characterization of the

inhibitory mechanism of Serpins and demonstration of how this

mechanism provides conformational control of both protease and

non-protease ligand interactions, which in-turn regulate complex cel-

lular and physiologic functions. Studies have also investigated plas-

minogen independent roles of PAs and their inhibitors in the central

nervous system (CNS). This work has demonstrated that tissue-type

PA (tPA) can directly regulate the blood brain barrier (BBB) through

interactions with the neurovascular unit (NVU). In vivo results indi-

cate that this occurs through tPA-mediated activation of latent plate-

let-derived growth factor CC (PDGF-CC). Specific proteolysis of

PDGF-CC by tPA generates active PDGF-CC capable of triggering

PDGF receptor alpha (PDGFRa) signaling in perivascular astrocytes

within the NVU, which in-turn controls BBB permeability. The

regulation of BBB integrity after stroke plays a critical role in stroke

progression and in hemorrhagic transformation. Understanding this

pathway may hold the key to developing better treatments for stroke

and potentially other CNS disorders where BBB integrity is

disrupted.

O11-2Clinical value of D-dimer testingPalareti GUniversity Hospital of Bologna, Bologna, Italy

Venous thromboembolism (VTE) requires prolonged treatment to

prevent recurrences. However, the optimal duration of anticoagula-

tion is still controversial. The more recent guidelines of the American

College of Chest Physicians recommend that patients with unpro-

voked VTE should receive at least 3 months of anticoagulation and

then should all be evaluated for the risk-benefit ratio of long-term

therapy. Our line of clinical research is aiming to find criteria to dis-

tinguish subjects at high or low risk of recurrence, to avoid an indefi-

nite anticoagulation in the latter. Prospective studies involving

patients with VTE have consistently shown that the D-dimer (Dd)

levels after anticoagulation have a high predictive value -positive or

negative – for VTE recurrence. These findings suggested a possible

role of Dd assay in regulating the duration of anticoagulation in

VTE patients. The PROLONG trial showed that patients with abnor-

mal Dd 1 month after VKA withdrawal for unprovoked VTE who

were randomized to not resume anticoagulation had a high rate of

recurrences (15.0%), while those with normal Dd had a lower rate of

recurrences (6.2%). It was however unknown whether Dd normal

1 month after VKA withdrawal may change subsequently and

whether this may be associated with an increased risk of recurrence.

The prospective multicenter PROLONG II study was then performed

to assess Dd time course in these patients and its relation with late

recurrences. Patients with normal Dd 1 month after stopping antico-

agulation repeated Dd testing every 2 months for 1 year. Patients in

whom Dd became abnormal at the third month and remained abnor-

mal afterward had a higher risk of recurrence (27% p-y) than

patients in whom Dd remained always normal during followup

(2.9% p-y; adjusted hazard ratio: 7.9; P < 0.002). Repeated D-d test-

ing after anticoagulation suspension for a first episode of unprovoked

VTE could help tailor the duration of treatment.


ORAL PAPERS 31____________________________________________________________________________________

Thrombin-activatable Fibrinolysis Inhibitor

O12A-1Carboxypeptidase U (TAFIa): a new drug target forfibrinolytic therapy?Hendriks DFUniversity of Antwerp, Antwerp, Belgium

Procarboxypeptidase U (TAFI) is a plasma procarboxypeptidase that

upon activation by thrombin or thrombin-thrombomodulin turns

into a potent antifibrinolytic enzyme once its activity surpasses a cer-

tain threshold value. Its prominent bridging function between coagu-

lation and fibrinolysis raised the interest of several research groups

and pharmaceutical industry to develop carboxypeptidase U inhibi-

tors as profibrinolytic agents. Several studies have been performed

with small synthetic and naturally occurring CPU inhibitors in differ-

ent animal models of thrombosis, and many of these studies show

beneficial effects leading to an improvement of endogenous fibrinoly-

sis. Moreover, treatment with an oral CPU inhibitor has also been

used in a proof-of-principle clinical study in patients with pulmonary

embolism, indicating an improved rate of resolution of pulmonary

emboli and increased endogenous fibrinolysis.

Another approach is the use of CPU inhibitors combined with t-PA

thereby increasing the efficiency of pharmacological thrombolysis

allowing lower dosing of t-PA and subsequently fewer bleeding com-

plications.

In this presentation we will focus on the benefits/risks of targeting

carboxypeptidase U for the treatment of thrombotic disorders. We

will also discuss the advantages and pittfals of measuring proCPU

and active CPU – not only as a risk factor for thrombotic disease,

but also as a possible biomarker for therapy – in patient plasma sam-

ples both with published assays and with several commercially avail-

able immuno- and activity based assays.

O12A-2The role of activation peptide residues Lys42, Lys43,and Lys44 of TAFI in its activation by thethrombin-thrombomodulin complexWu C1, Kim PY2, Foley JH1, Gils A3, Declerck P3 andNesheim ME1

1Queen’s University, Kingston; 2David Braley Cardiac, Vascular

& Stroke Research Institute, Hamilton, Canada; 3Katholieke

Universiteit Leuven, Leuven, Belgium

Thrombin-activable Fibrinolysis Inhibitor (TAFI) is activated to TA-

FIa by thrombin-thrombomodulin complex with a 1250-fold higher

catalytic efficiency than thrombin alone. TAFIa is a potent antifibrin-

olytic enzyme, which significantly prolongs clot lysis time in vitro.

TAFI has three exclusive consecutive lysine residues at positions 42,

43, and 44, in its activation peptide that are not found in rattus,

bovine or human tissue procarboxypeptidases A and B. We previ-

ously reported an 8-fold decrease in catalytic efficiency for TAFI acti-

vation when the three lysine residues were all mutated to alanine

(K42/43/44A).

We constructed and expressed combinations of mutations of the three

lysine residues in order to identify which one (s) is key for TAFI acti-

vation. We activated TAFI wild-type and mutants with thrombin for

10 min in the presence or absence of TM and then added the syn-

thetic TAFIa substrate AAFR to quantify levels of TAFIa formed.

The activation rates were used to determine kinetic parameters of

TAFI activation. We also tested the antifibrinolytic potential of each

mutant activated in situ using an in vitro plasma clot lysis assay. The

time to 50% clot lysis was determined as the midpoint between maxi-

mum turbidity of a clot and the minimum turbidity when a clot was

completely lysed.

kcat values (1/s) ranged from 1.09 � 0.25 (WT) to 8.53 � 4.58 (K43/

44A). Km values (lM) ranged from 0.58 � 0.05 (WT) to

19.87 � 14.08 (K43/44A). Compared to wild-type, the catalytic effi-

ciencies for TAFI activation decreased by 2.3-fold (K43A) to 4.1-fold

(K43/44A). The TAFI concentration required to achieve the half-

maximal effect on attenuation of clot lysis increased from 2.2-fold

(K43A) to 14.8-fold (K42/43/44A).

Our data show that each of the three lysine residues contributes to the

efficiency of activation of TAFI. The increases in kcat suggest that

product (TAFIa) release may be rate limiting in TAFI activation.

O12A-3Identification and characterization of monoclonalantibodies that impair the activation of human TAFIthrough different mechanismsMishra N, Vercauteren EB, Develter J, Bammens R, Declerck PJand Gils AKatholieke Universiteit Leuven, Leuven, Belgium

Introduction: Thrombin activatable fibrinolysis inhibitor (TAFI)

forms a molecular link between coagulation and fibrinolysis and

therefore it is a promising target to develop profibrinolytic drugs.

Objective: To characterize functional properties of three monoclonal

antibodies (MA) raised against a stable TAFI variant (TAFI-AI-

CIYQ; Ceresa et al. JTH, 2007; 5:418–420) with the focus on their

inhibitory properties and mode of action and to elucidate their

respective epitopes.

Methods and Results: Out of a panel of MA raised against a stable

TAFI variant, MA-TCK11A9, MA-TCK22G2 and MA-TCK27A4

revealed high affinity towards human TAFI-TI-wt, but no affinity

towards either mouse or rat TAFI-wt. Even at a 16-fold molar excess

of MA over TAFI, none of these MA was able to reduce TAFIa

activity (Table 1). However, using an 8-fold molar excess of MA over

TAFI, MA-TCK11A9 was able to inhibit plasmin-mediated activa-

tion, MA-TCK22G2 inhibited plasmin- and thrombin-mediated

TAFI activation and MA-TCK27A4 inhibited TAFI activation by

plasmin, thrombin and thrombin/thrombomodulin (Table 1). Using

an 8-fold molar excess of MA over TAFI, all three MA were able to

reduce clot lysis time significantly (Table 1). Phe113 was identified as

the major residue interacting with MA-TCK27A4. Using a sandwich-

type ELISA, we could deduce that this epitope is distinct from the

epitopes of MA-TCK11A9 and MA-TCK22G2, which are currently

being elucidated.

Conclusion: We identified three MA that impair the activation of

TAFI through different mechanisms and demonstrated that MA-

TCK11A9 which exclusively impairs plasmin-mediated TAFI activa-

tion can also significantly reduce clot lysis time in vitro.

Table 1 for O12A-3

MA

% Reduction

on TAFIa

activity

% Reduction on

TAFI activation

% Reduction on

clot-lysis time

By

T/TM

By

plasmin

By

T

Expressed relative

to reduction

by PTCI

TCK11A9 < 2% < 2% 64 � 4.3 < 2% 84 � 27*

TCK22G2 < 2% < 2% 58 � 4.0 41 � 7.0 99 � 15*

TCK27A4 < 2% 98 � 2.0 98 � 4.0 97 � 2.0 128 � 8.0*

Mean � SD, n ‡ 3; *P < 0.05 versus no TAFI inhibitor.


32 ORAL PAPERS____________________________________________________________________________________

O12A-4The role of TAFI and Factor XI in platelet-mediatedresistance to fibrinolysisColucci M, Carrieri C, Galasso R, Incampo F, Semeraro F,Ammollo CT and Semeraro NUniversity of Bari, Bari, Italy

While it is well documented that platelets make thrombi resistant to

fibrinolysis through clot retraction and PAI-1 release, little is known

about the involvement of TAFI in platelet-mediated resistance to

fibrinolysis. We investigated the contribution of TAFI to the antifi-

brinolytic effect of platelets in whole blood by thromboelastography.

Platelet-poor (PP-WB, < 40 · 103/lL) and platelet-rich (PR-WB,

> 400 · 103/lL) blood samples were obtained from normal blood

(N-WB, 150–220 · 103/lL). Clot lysis time was measured by throm-

boelastography (Haemoscope), using recalcified human blood supple-

mented with t-PA (100 ng/mL) and tissue factor (1/1000 diluted

Recombiplastin).

t-PA-induced lysis time increased proportionally to platelet concen-

tration (from 21.5 � 8.9 to 60.1 � 18.4 min, in PP-WB and PR-WB,

respectively). Neutralization of TAFIa activity (by PTCI) or inhibi-

tion of thrombin-mediated TAFI activation (by a specific monoclonal

antibody) shortened lysis time by 52.3% in PR-WB and by 6.3% in

PP-WB, suggesting that the contribution of TAFI was substantial

only in the presence of platelets. Qualitatively similar results were

obtained when clot retraction was prevented by cytochalasin D or

when PAI-1 was inhibited by a neutralizing antibody. Assay of pro-

thrombin F1 + 2 and TAFIa generation during clot lysis confirmed

that platelets enhanced both thrombin generation and TAFI activa-

tion. Addition of a neutralizing anti-FXI antibody had little effect on

clot formation but shortened the lysis time by 38% in PR-WB and

by < 5% in PP-WB. A significant shortening of lysis time (27%)

was also observed when N-WB collected on CTI (corn trypsin inhibi-

tor, an inhibitor of FXIIa) was supplemented with the anti-FXI, but

not with an anti-FXII antibody. Our data indicate that TAFI activa-

tion is one major mechanism whereby platelets make clots resistant

to fibrinolysis and suggest that FXII-independent activation of FXI

plays an important role in platelet-mediated enhancement of throm-

bin and TAFIa generation.

O12A-5Internalization of thrombin-activatable fibrinolysisinhibitor in keratinocytes by receptor-mediatedendocytosisVerkleij CJN, Reits EAJ, Plug T, Marx PF and Meijers JCMAcademic Medical Center, University of Amsterdam,

Amsterdam, The Netherlands

Introduction: Mice that are deficient for thrombin-activatable fibrino-

lysis inhibitor (TAFI) have delayed wound healing due to impaired

keratinocyte migration. We hypothesized that TAFI may affect

wound healing via direct interactions with keratinocytes. The aim of

this study was to elucidate interactions between TAFI and keratino-

cytes by studying endogenous TAFI in, and internalization of TAFI

by these cells.

Methods: The presence of TAFI in the skin was studied by immuno-

histochemical staining of skin tissue. Direct interactions of TAFI

with keratinocytes were determined in HaCaT cells, a keratinocyte

cell-line. Endogenously generated TAFI was studied at protein level,

whereas TAFI internalization was studied using fluorescently labeled

TAFI.

Results: Immunohistochemical staining for TAFI indicated interac-

tions of TAFI with keratinocytes, while endogenous TAFI was

detected in keratinocytes on protein level, which was dependent on

the phase of the cell cycle. Live-cell imaging showed that keratino-

cytes internalize TAFI via receptor-mediated endocytosis.

Conclusions: This study shows that TAFI can bind to and becomes

internalized in keratinocytes. The enhanced endogenous TAFI pro-

tein levels in mitotic cells suggest that TAFI is involved keratinocyte

proliferation.


ORAL PAPERS 33____________________________________________________________________________________

Proteases in the Brain

O12B-1Antibodies based immunotherapy for strokeVivien D, Ali C and Macrez RINSERM, Caen, France

Tissue-type plasminogen activator (tPA, endogenous and exogenous)

has two faces in acute ischemic stroke: beneficial fibrinolysis in the

vascular bed, but damaging effects on the neurovascular unit and the

brain parenchyma. To improve this profile, we used our knowledge

of the underlying molecular (patho)physiology to develop a novel

treatment strategy for stroke, relying on antibodies targeting the pro-

neurotoxic effects of tPA. Based on a dedicated model of murine

thrombo-embolic stroke, we demonstrate the efficiency of immuno-

therapy in a complete pre-clinical screen: after a single administration

(alone or with late thrombolysis), antibodies dramatically reduce

ischemic brain injuries, improve long-term neurological outcome and,

in parallel, attenuate critical ischemic events including blood-brain

barrier leakage and activation of MMP-3, MMP-9, and the PDGF-

CC pathway. Thus, the prospect of this immunotherapy is an exten-

sion of the range of treatable patients, whether used as a monothera-

py or, in combinations, to extend the therapeutic window for

thrombolysis.

O12B-2Role of tPA in the rewarding effect of abused drugsNagai TNagoya University Graducate School of Medicine, Nagoya,

Japan

Activation of the mesocorticolimbic dopamine system, which origi-

nates in the midbrain ventral tegmental area and projects to the

nucleus accumbens (NAc) has been implicated in the positive rein-

forcing (rewarding) effects of nicotine and other drugs of abuse.

Recently, it has been proposed that activity-dependent synaptic plas-

ticity and remodeling of the mesolimbic dopaminergic system play a

crucial role in the development of drug dependence. Tissue plasmino-

gen activator (tPA) is a serine protease that catalyzes the conversion

of plasminogen to plasmin. In our previous study, it has been demon-

strated that the tPA-plasmin system participates in the rewarding

effect of opiate and psychostimulant. Here we show that the tPA-

plasmin system regulates nicotine-induced reward and dopamine

release. In vivo microdialysis revealed that microinjection of either

tPA or plasmin into the NAc significantly potentiated whereas plas-

minogen activator inhibitor-1 reduced the nicotine-induced dopamine

release in the NAc in a dose-dependent manner. Nicotine-induced

dopamine release was markedly diminished in tPA-deficient (tPA-/-)

mice, and the defect of dopamine release in tPA-/- mice was restored

by microinjection of either exogenous tPA or plasmin into the NAc.

Nicotine increased tPA protein levels and promoted the release of

tPA into the extracellular space in the NAc. Activation of postsynap-

tic dopamine D1 receptors in the NAc leads to an increase in extra-

cellular tPA activity via PKA signaling. Immunohistochemistry

revealed that protease activated receptor-1 (PAR1)-immunoreactivity

was localized to the nerve terminals positive for tyrosine hydroxylase

in the NAc. Furthermore, we demonstrated that nicotine-induced

conditioned place preference and dopamine release were diminished

in PAR1-deficient (PAR1-/-) mice. Our findings suggest that the tPA-

plasmin-PAR1 system plays a crucial role in the rewarding effects of

nicotine.

O12B-3MMP-10: a new fibrinolytic agent in ischemic stroke?Paramo JA, Orbe JO and Rodriguez JACIMA (University of Navarra), Pamplona (Navarra), Spain

The fibrinolytic and metalloprotease (MMP) systems act in concert to

degrade extracellular matrix proteins and also fibrin, a main structural

component of most thrombi. We have investigated the pathophysio-

logical relevance of vascular MMP-10 (stromelysin-2) expression and

our recent results lead us to postulate a new role for MMP-10 in fibri-

nolysis. After testing different pro-atherosclerotic/proinflammatory

molecules, we found that thrombin strongly induced endothelial

MMP-10 expression in vitro and in vivo. This led us to assess the

effects of MMP-10 on thrombosis and fibrinolysis, and we observed

that MMP-10 significantly enhanced t-PA-induced fibrinolysis in vitro,

either in turbidimetric-based plasma tests, on fibrin plates, and in

whole blood by thromboelastography, while MMP-10 inhibition pre-

vented t-PA-induced fibrinolysis. These results were confirmed in vivo

in the MMP-10 KO mouse. We first assessed thrombus formation

and lysis in the carotid artery after rose bengal infusion and laser

injury, and found that MMP-10 KO exhibited faster thrombus devel-

opment and delayed fibrinolysis compared with wild type mice, sug-

gesting an important role of MMP-10 in thrombus formation and

lysis in vivo. In a murine model of stroke by thrombin microinjection

in the middle cerebral artery, spontaneous reperfusion was severely

impaired in MMP-10 KO animals. We also used this model of stroke

to show in wild type mice that MMP-10 administration significantly

reduced brain lesion volume as compared to controls (P < 0.01), with

no increase in bleeding. Finally, we found that MMP-10 exerts its

profibrinolytic effect at least in part by cleaving thrombin activatable

fibrinolysis inhibitor (TAFI), thus preventing its activation.

We propose that MMP-10 administration, alone or in combination

with t-PA, can become a new profibrinolytic strategy in arterial

thrombosis, particularly useful in the treatment of ischemic stroke.

O12B-4Roles of urokinase receptor (uPAR) on ischemic strokeNagai N1, Okada K1, Kawao N1, Ishida C1, Ueshima S2 andMatsuo O1

1Kinki Univ School of Med, Osaka; 2Kinki Univ School of

Agriculture, Nara, Japan

Urokinase-type plasminogen activator receptor (uPAR) is a key com-

ponent of the plasminogen activation system at the cell surface. Recent

studies showed that uPAR is expressed in the ischemic damaged brain,

suggesting its involvement in brain damage. To evaluate the roles of

uPAR, we evaluated the role of uPAR in ischemic brain damage

induced by permanent middle cerebral artery (MCA) occlusion in mice

with genetic deficiency of uPAR (uPAR-/-) or of uPA (uPA-/-). Brain

damage on day 3 was smaller in uPAR-/- mice (4.5 � 1.0 mm3) than

in littermate wild-type mice (uPAR+/+) (9.1 � 1.8 mm3, P < 0.05),

whereas it was comparable in uPA-/- (8.0 � 4.1 mm3) and uPA+/+

(6.9 � 2.6 mm3) mice. uPAR expression was upregulated in the ipsilat-

eral cerebral cortex of control mice within 12 h, and remained elevated

for up to 3 days. On day 1 or 2 after MCA occlusion, uPAR expres-

sion was selectively localized in vessels at the border of the damaged

area. It was also found that uPAR-/- mice showed less blood-brain bar-

rier disruption than uPAR+/+ on day 1. In bEnd.3, an endothelial cell

line derived from mice brain, uPAR expression was upregulated at

24 h after 2 h of oxygen-glucose deprivation mimicking ischemia or

after 24 h serum deprivation, indicating that both hypoxic stress and

starvation of trophic factors by ischemia induced uPAR on endothelial

cells. These findings suggest that uPAR expressed by endothelial cells

augments of the ischemic brain damage via failure of endothelial cell

function by uPA-independent mechanism.


34 ORAL PAPERS____________________________________________________________________________________

Clinical and Basic Aspects of Thrombolysis

O13A-1Clinical thrombolysisVerheugt FWAOnze Lieve Vrouwe Gasthuis, Amsterdam, The Netherlands

Reperfusion therapy for ST-elevation myocardial infarction (STEMI)

aims at early and complete recanalization of the infarct-related artery

in order to salvage myocardium and improve both early and late clin-

ical outcomes. The benefit rises exponentially the earlier therapy is

initiated. Both coronary fibrinolysis (formerly called coronary throm-

bolysis) and primary coronary angioplasty can achieve full reperfu-

sion. Angioplasty is more successful, but has severe logistic

constraints, whereas fibrinolysis can be administered everywhere.

Time to treatment can be shortened by prehospital diagnosis and

treatment. The optimal site for STEMI diagnosis is the patient’s home

or place where the infarction occurs. Prehospital ECG diagnosis has

shown to increase the number of patients treated earlier in hospital by

either fibrinolysis or primary coronary angioplasty (1). Furthermore,

prehospital initiation of fibrinolysis shows a gain in time to treatment

of about one hour and results in 15% relative risk reduction of early

mortality (2). Compared to primary angioplasty prehospital fibrino-

lytic therapy may be not inferior to primary angioplasty because of

this gain in time to treatment (3). Finally, prehospital diagnosis fol-

lowed by direct transfer to a hospital with PCI facilities rather than

to a hospital without reduces time-to-treatment with at least 1 h in

Denmark. Four Administration of fibrinolytic therapy in the emer-

gency room is superior to starting fibrinolysis in the Coronary care

Unit (5). In patients transferred for primary angioplasty direct trans-

fer to the catheterization laboratory saves time to treatment in com-

parison to admission to the emergency department or Coronary Care

Unit (6). The ideal strategy is prehospital triage with direct transfer to

the catheterization laboratory (7, 8). Thus, ambulance diagnosis and

triage as well as direct transfer to a catheterization laboratory are the

major leaps forward in the reduction in time-to-treatment in STEMI.

The several advantages and disadvantages of fibrinolysis and primary

coronary intervention are summarized in the table.

References

1 Curtis JP, Portnay EL, McNamara EL, et al. The pre-hospital elec-

trocardiogram and time to reperfusion in patients with acute myo-

cardial infarction, 2000–2002: findings from the National Registry

of Myocardial Infarction-4. J Am Coll Cardiol. 2006; 17:1544–1552.

2 Morrison LJ, Verbeek PR, McDonald AC, Sawadsky BV, Cook

DJ. Mortality and prehospital thrombolysis for acute myocardial

infarction. JAMA. 2000; 283:2686–2692.

3 Steg PG, Bonnefoy E, Chabaud S, et al. Impact of time to treat-

ment on mortality after prehospital fibrinolysis or primary angio-

plasty: data from the CAPTIM randomized clinical trial.

Circulation 2003; 103:2851–2856.

4 Terkelsen CJ, Lassen JF, Norgaard BL, et al. Reduction of treat-

ment delay in patients with ST-elevation myocardial infarction:

impact of pre-hospital diagnosis and direct referral to primary per-

cutaneous coronary intervention. Eur Heart J. 2005; 26:770–777.

5 Verheugt FWA, Funke Kupper AJ, Sterkman LGW, Meijer A,

Peels CH, Roos JP. Emergency room infusion of intravenous strep-

tokinase in acute myocardial infarction: feasibility, safety end he-

modynamic consequences. Am Heart J. 1989; 117:1018–1022.

6 Bradley EH, Herrin J, Wang Y, et al. Strategies for reducing the

door-to-balloon time in acute myocardial infarction. N Engl J

Med. 2006; 355:2308–2320.

7 Vermeulen RP, Jaarsma T, Hanenburg FGA, Nannenberg, Jessu-

run GAJ, Zijlstra F. Prehospital diagnosis in STEMI patients trea-

ted by primary PCI: the key to rapid reperfusion. Neth Heart J.

2008; 16:5–9.

8 Liem S, Dieker H, Keuper W, Clappers N, Brouwer MA, Aeng-

evaeren WRM, Verheugt FWA. The impact of ambulance-based

triage for primary angioplasty.on treatment intervals and clinical

outcome. J Am Coll Cardiol Int. 2010 in press.

O13A-2Optimizing thrombolytic efficacy and safety bypreventing non-specific plasminogen activationGurewich V and Pannell RBeth Israel Deaconess Medical Center, Cambridge, MA, USA

The shortcomings of available thrombolytics include their limited effi-

cacy and bleeding risk. Consequently, thrombolysis is being replaced

by angioplasty (AMI) or used with reluctance (stroke). The maximum

efficacy of all plasminogen activators is a function of the rate of

fibrin degradation by plasmin. This rate, however, is unattainable

clinically because of dose-dependent hemorrhagic complications due

to non-specific plasminogen activation or direct lysis of hemostatic

fibrin. At safer fibrin specific doses, the clot lysis rate is only ~50%

of the maximum with either tPA or prouPA. These activators have

distinctly different properties. TPA is promoted by intact fibrin (D-

domain), which corresponds to hemostatic fibrin, and thereby can

induce bleeding directly by fibrinolysis. ProuPA is promoted by the

fibrin E-domain, which is absent in hemostatic fibrin. Unfortunately,

at therapeutic concentrations, prouPA proved vulnerable to activa-

tion by plasmin with loss of fibrin-specificity (a reaction amplified by

positive feedback) leading to a bleeding diathesis, which deterred

EMEA from granting approval. Mutant prouPA, M5, is more stable

in plasma due both to a lower intrinsic activity and an unusual inhi-

bition of two-chain M5 by plasma C1-inhibitor (C1I), a negligible

inhibitor of tPA or tcuPA. When plasma was enriched with C1I in vi-

tro, stability of M5 was further enhanced, enabling the maximum rate

of lysis with fibrin-specificity. C1I selectively inhibited only non-spe-

cific plasminogen activation. Other serpins (PAI-1, AT, a2AP) inhib-

ited both fibrin-specific and non-specific plasminogen activation. This

M5/C1I phenomenon was tested in vivo in dogs with thromboembo-

lism given high-dose M5 � C1I. Lysis and bleeding from hemostatic

injury sites were quantitated. Equivalent, rapid lysis (100% in

30 min) occurred in both groups, but extensive bleeding was inhibited

only with C1I supplementation.

C1I is already an approved drug. Used as adjunctive therapy with

M5, C1I can make therapeutic thrombolysis optimally effective and

safe for the first time.


ORAL PAPERS 35____________________________________________________________________________________

O13A-3Exploring the sensitivity of fibrinolysis rates toinhibition of tissue plasminogen activator (tPA)-fibrinbinding and plasminogen-fibrin bindingSilva M, Thelwell C and Longstaff CNIBSC-HPA, Potters Bar, UK

As part of our efforts to model the kinetics of plasminogen activation

and fibrinolysis we have investigated plasminogen- fibrin-tPA ternary

complex formation and the mechanism of stimulation of fibrinolysis

by C-terminal lysines. Generation of C-terminal lysines by plasmin in

fibrin upregulates binding of plasminogen and tPA (via Kringle

domains) and stimulates further generation of plasmin. This positive

feedback can be damped by carboxypeptidases (e.g. CPB and TAFI)

and binding can be inhibited by lysine analogues such as tranexamic

acid. To dissect out the relative importance of C-terminal lysine bind-

ing for tPA and plasminogen we have compared tPA (F-G-K1-K2-P)

and a domain-switched variant K1K1tPA (F-G-K1-K1-P) that lacks

the K2 lysine binding domain but retains normal tPA structure. Pre-

cise measurements using high throughput methods of plasminogen

activation in fibrin and of fibrinolysis show that tranexamic acid

affects rates with both variant enzymes across a range of conditions.

In fibrin, 50% inhibition of plasminogen activation and fibrinolysis

was achieved by tranexamic acid at 0.3 or 0.075 mM, for lys- and

glu-plasminogen, respectively. Fibrinolysis rates with tPA were only

up to 10% more affected than K1K1tPA, indicating tranexamic acid

acts on plasminogen binding, rather than tPA. Clots incorporating

dose ranges of CPB behaved in a similar way. At 2 U/mL CPB, inhi-

bition of plasminogen activation and fibrinolysis was around 25%

and 70% for lys- and glu-plasminogen, respectively. Assays with tPA

were maximally only 10% more inhibited than K1K1tPA assays, by

CPB, indicating the importance of C-terminal lysine-plasminogen

binding in regulating fibrinolysis. Significant inhibition of fibrinolysis

using an inactive ser-> ala active site tPA variant was not observed

at concentrations up to 200 nM in the clot, a 5000-fold excess over

active tPA. All these results highlight the greater sensitivity of fibri-

nolysis rates to blocking plasminogen (substrate) binding to fibrin,

over tPA binding.

O13A-4A multi-scale mathematical model of fibrinolysis:from fiber cross sections to whole clotsBannish BE, Fogelson AL and Keener JPUniversity of Utah, Salt Lake City, UT, USA

Mathematical models can be very useful for making predictions

about biological processes that are difficult to quantify experimen-

tally. Models give insight to underlying mechanisms and provide

explanations for phenomena seen in experimental results. We study

fibrinolysis using a multi-scale mathematical model intended to

answer the following question: Why do coarse clots composed of

thick fibers lyse more quickly than fine clots composed of thin fibers,

despite the fact that individual thin fibers lyse more quickly than

individual thick fibers? We use stochastic methods to model lytic pro-

cesses on scales ranging from individual fiber cross section to whole

clot. The biochemistry is contained in the fiber cross-section model,

which we use to predict the average time it takes plasmin to cut

through single fibers of varying diameter, the average amount of

plasmin produced by a single tPA molecule, and the spatial and tem-

poral concentration profiles of fibrin-bound plasminogen and plas-

min. This information is used in a fibrin clot model to investigate the

pattern and speed of lysis induced by tPA added to the surrounding

plasma. Concentration profiles of the lytic enzymes are obtained in

different regions of the clot, and the lysis of coarse and fine clots are

contrasted. An advantage of studying fibrinolysis mathematically is

the ability to cheaply and (relatively) easily change the experiment of

interest. We can test hypothesized clinical therapies by adding the

effect of a proposed drug into our system, for example. In the future,

we intend to build upon the model to address questions arising from

current research.

O13A-5Lysis of thrombi formed in the Chandler loop orBadimon chamberMostefai HA1, Baeten KM1, Langrish JP2, Lucking AJ2,Newby DE2 and Booth NA1

1University of Aberdeen, Aberdeen; 2University of Edinburgh,

Edinburgh, UK

Thrombus stability is central to stroke, heart attack and deep venous

thrombosis. Some thrombi are removed by naturally-occurring fibri-

nolytic activity, and we explored the potential of endogenous fibrino-

lysis, caused by tissue-plasminogen activator (tPA) released from the

endothelium. Model thrombi were formed from blood in vitro in

Chandler loops and ex vivo using the Badimon chamber, with FITC

labelled fibrinogen as a tracer for quantitative analysis of lysis. In

addition, tPA was introduced to blood at final concentrations of 0–

50 ng/mL, accurately mimicking stimulated endogenous t-PA release.

Added tPA was successfully incorporated into the forming Chandler

model thrombus over the entire concentration range. Zymography

showed that all tPA and plasminogen activator inhibitor type 1 (PAI-

1) incorporated in the thrombus was free, whereas tPA in serum was

predominantly in complex with inhibitors. When preformed tPA-

PAI-1 complex was added to the blood, this was only recovered in

the serum and not in extracts of model thrombi, showing that it was

not incorporated into the forming thrombus. Addition of increasing

concentrations of tPA resulted in marked increases in thrombus lysis,

as shown by fluorescence release and D-dimer measurements. Similar

data were generated from thrombi formed in the Chandler loop and

those formed in the Badimon chamber at high or low shear rates.

Incorporated tPA was much more efficient than tPA added to bath-

ing fluid, to the extent that some lysis already occurred during forma-

tion, such that the rotation time for formation of Chandler model

thrombi was decreased from 90 min, as published, to 30 min. In con-

clusion, tPA at physiological concentrations was incorporated during

thrombus formation in a free and active form, despite the presence of

PAI-1 and other inhibitors. Once incorporated within the thrombus,

tPA was effective in lysis, confirming the importance of endogenous

tPA localized inside the thrombus.


36 ORAL PAPERS____________________________________________________________________________________

Tissue Remodeling and Matrix Metalloproteinases

O13B-1Tissue remodeling and matrix metalloproteinasesLijnen HRCenter for Molecular and Vascular Biology, Leuven, Belgium

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIM-

Ps) play a role in most biological processes requiring tissue remodel-

ing. In general, the net proteolytic activity is determined by a delicate

balance between MMP and TIMP activity. TIMP-1 is a main physio-

logical inhibitor of most MMPs. In murine atherosclerosis models,

TIMP-1 deficiency is associated with reduced plaque area but

enhanced occurrence and severity of aneurysms. MMP activity (e.g.

MMP-3, -9, -12, -13) develops via activation of proMMPs by plasmin

that is generated via plasminogen activation by macrophage derived

urokinase. Extracellular matrix proteolysis is also required for expan-

sion of adipose tissue. We have used a nutritionally induced obesity

model in transgenic mice to study the role of the MMP system. In

this model, stromelysin-1 (MMP-3) or stromelysin-3 (MMP-11) but

not stromelysin-2 (MMP-10) deficiency promoted adipose tissue

development, whereas deficiency of gelatinase A (MMP-2) but not

gelatinase B (MMP-9) impaired it by contributing to adipocyte hypo-

trophy. Deficiency of TIMP-1 induced impaired adipose tissue devel-

opment associated with adipocyte hypotrophy. However, adenoviral

overexpression of TIMP-1 in mice had no significant effect on body

weight or developing adipose tissue mass. Administration to wild-

type mice kept on a high fat diet of broad-spectrum MMP inhibitors

or of relatively gelatinase-specific inhibitors resulted in moderate to

significant reduction of adipose tissue weight. Thus, studies in trans-

genic mouse models as well as pharmacologic studies support a role

of the MMP system in adipogenesis and obesity; the role of specific

members of this family, however, remains to be determined in more

detail.

O13B-2Plasminogen-dependent mobilization of hematopoieticstem cells-induced by G-CSF requires twoindependent pathways, activation of MMP-9 and SDF-1/CXCR-4 regulationHoover-Plow JL and Gong YLerner Research Institute Cleveland Clinic, Cleveland, OH, USA

Ischemic heart diseases including myocardial infarction (MI) are the

leading causes of death in the USA. Use of hematopoietic stem cells

(HSC) to improve recovery of the injured heart after MI is an impor-

tant emerging therapeutic strategy. The plasminogen (Plg) system, an

inflammatory mediator, is critical for tissue repair after injuries. Plg

deficiency (Plg-/-) significantly decreases HSC mobilization from bone

marrow (BM) to the circulation induced by granulocyte colony-stim-

ulating factor (G-CSF). After G-CSF treatment, there was no differ-

ence in proMMP-9 between Plg+/+ and Plg deficient mice, but

actMMP-9 was higher in Plg+/+ mice than Plg-/- mice. Reconstitu-

tion of actMMP-9 activity, but not proMMP-9, rescued HSC mobili-

zation in Plg-deficient mice. Thus, Plg-dependent HSC requires

MMP-9 activation. SDF-1 is a key chemoattractant for HSC mobili-

zation from BM, and G-CSF decreases SDF-1 in BM of Plg+/+

mice creating a gradient of SDF-1 between BM and blood. Our data

show that Plg deficiency abolishes the gradient of SDF-1 and pro-

motes the expression of CXCR-4, a major receptor of SDF-1 on

HSC, and suggests that Plg may regulate stem cell mobilization by

modulating SDF-1/CXCR-4 signal. In MMP-9 deficient mice, SDF-1

was not different compared to MMP-9+/+ after G-CSF treatment

indicating a distinct and novel mechanism by which Plg regulates

HSC mobilization through SDF-1. In addition, our data show that

Plg deficiency inhibits G-CSF-induced neovascularization in the

infarcted area, a key step of cardiac repair. Taken together, these

data suggest that Plg may enhance HSC recruitment by regulating

both MMP-9 activation and BM SDF-1/CXCR-4 function. Those

pathway cascades may also contribute to cardiac repair after MI.

This pathway cascade may also be involved in the cardiac repair after

MI.

O13B-3Activated protein C prevents neuronal apoptosis atexcitotoxicity via inhibition of p53Gorbacheva L1, Savinkova I1, Pinelis V2, Ishiwata S3, Reiser G4

and Strukova S1

1Lomonosov Moscow State University; 2Scientific Centre for

Children‘s Health, Moscow, Russian Federation; 3School of

Science and Engineering, Waseda University, Tokyo, Japan;4Institute for Neurobiochemistry, Otto-von-Guericke University,

Magdeburg, Germany

Brain ischemic stroke led to glutamate (Glu)-induced toxicity which

can initiate the cascade of mitochondrial dysfunctions and induction

of apoptosis. It was shown that one of the key molecular regulators

of neuronal survival and death can be the tumor suppressor protein

p53. Activated protein C (APC) exerts the direct cytoprotective

effects on neurons via EPCR activation of protease activated receptor

1. However, the molecular mechanisms of APC neuroprotective

effects at Glu-toxicity have not been elucidated. We suggested that

APC can suppress the molecular cascade responsible for neuronal

apoptosis at Glu-induced toxicity via inhibition of p53 or blocked the

activation of caspases and NF-kB. This is the focus of the current

investigation.

Studies were performed using primary culture of neurons from cortex

or hippocampus of brain of Wistar rat pups. Cell death was deter-

mined by biochemical (LDH) and morphological methods (Hoe-

chst33342, Ethidium bromide and Syto-3). By immunostaining and

Western blot the nucleus and cytoplasm levels of pro-apoptotic pro-

teins (p53, AIF and caspase-3) and NF-kBp65 were assessed during

Glu-toxicity and incubation with APC.

APC at low concentrations was shown to have the protective effect

on neurons survival at Glu-toxicity. Hippocampal neurons sensitivity

to APC was higher than one to cortical neurons. In hippocampal

neurons APC inhibits Glu-induced caspase-3 activity with maximum

effect 24 h after treatment. APC inhibits AIF translocation into

nucleus. Incubation of neurons with APC blocks the increase of

nuclear level of p53 as well as 4 h and 24 h after exposure cells to

Glu. APC protects neurons from Glu-toxicity due to blockage of

both caspase-dependent and -independent apoptosis. APC reduces of

the nuclear level of NF-jBp65 at Glu-toxicity.

Thus APC can suppress the molecular cascade responsible for

neuronal apoptosis at Glu-induced toxicity via inhibition of p53 or

blockage the activation of caspases and NF-kB. (RFBR grant 08-04-

01123a)

O13B-4Tissue-type plasminogen activator (tPA) andplasmin(ogen) participate in the removal of dead cellsBorg RJ, Samson A, Schlozen A, Plebanski M and Medcalf RLMonash University, Melbourne, Australia

Fibrin is the classic cofactor that promotes tPA-mediated plasmin

formation, which in turn leads to fibrinolysis. We have shown that


ORAL PAPERS 37____________________________________________________________________________________

dead cells, regardless of their background lineage, also provide a co-

factor for tPA-mediated plasmin formation (Samson et al. Blood

2009) and we considered the possibility that tPA-mediated plasmin

formation participates in the proteolytic clearance of dead cells. We

show that a brief treatment of dead cells with both tPA and plas-

minogen leads to their proteolytic degradation (n = 4) suggesting

that tPA and plasminogen indeed participate in dead cell break-

down and clearance. Phagocytosis is also a key element of dead cell

clearance. Dendritic cells (DCs) are antigen presenting phagocytes

that link the innate and adaptive immune system. We show that

tPA-mediated plasmin formation induces profound morphological

changes in monocyte-derived DCs (MoDCs), whereby MoDCs

become more adherent and develop long extended appenditures.

These plasmin-induced changes in MoDC adherence and morphol-

ogy were rapid (‡ 3 h; n = 3) and persistent (£ 48 h; n = 3) and

were blocked by aprotinin, indicating a dependency on plasmin for-

mation. No effect was observed when MoDCs were treated with the

closely-related protease thrombin (n = 3). Cell-surface marker stud-

ies showed that plasmin treatment did not induce MoDC matura-

tion (n = 4). These findings align with those of Li and co-workers

(2010) who showed that plasmin induces MoDC chemotaxis. Collec-

tively, plasmin influence dead cell removal via direct proteolysis of

dead cell proteins and may also engage dendritic cells. We are cur-

rently determining whether plasmin promotes DC-induced phagocy-

tosis and conducting experiments to assess the effect of tPA-

mediated plasmin formation in systems where dead cell proteolytic

degradation, phagocytosis, and DC activation/migration occur con-

currently.

O13B-5Plasminogen as a novel therapeutic agent to treattympanic membrane perforationsShen Y1, Guo Y1, Li J1, Wilczynska M1, Hellstrom S2 and Ny T1

1Umea University, Umea; 2Karolinska Universitetssjukhuset,

Stockholm, Sweden

Tympanic membrane (TM) perforations are commonly seen in clini-

cal practice, e.g. after trauma to the ear or during episodes of acute

otitis media. So far there is no effective treatment to TM perforation

except tympanoplasty, a surgical procedure. It has been shown previ-

ously that healing of TM perforation is completely arrested in plas-

minogen-deficient mice. Inflammatory cells were recruited to the

wounded area but there were no signs of tissue debridement. In addi-

tion, removal of fibrin, keratinocyte migration and in-growth of con-

nective tissue were impaired. Here we show that topical application

of purified human plasminogen in plasminogen-deficient mice success-

fully restores the normal TM perforation healing in these mice and

speeds up the healing of a hydrocortisone-induced chronic TM perfo-

ration model in rats. The healing is dependent on the concentration

of the plasminogen solution used as well as the time length of appli-

cation. Also by local injection, purified human plasminogen shows

sufficient healing effect. In conclusion these results indicate that puri-

fied human plasminogen can be locally used as a novel therapeutic

agent to treat TM perforations. Future clinical studies will elucidate

whether local treatment by plasminogen can replace surgical tympa-

noplasty.


38 ORAL PAPERS____________________________________________________________________________________

Educational Workshop on Technologies

O14A-1An in silico model for haemostasis: computationalmethods and algorithmsBakker B, van den Ham R and van Ooijen HPhillips Research, Eindhoven, The Netherlands

A correct haemostatic balance is essential for human health. A vast

and increasing number of people require support in maintaining this

balance through anti-coagulants like Warfarin on the one hand and

monitoring of their current haemostatic balance on the other. The

current standard tool for such monitoring is the INR (International

Normalized Ratio) test which is based on prothrombin time. This

tool is sensitive for only a small subsection of the full haemostatic

system and is for example unaffected by influences from fibrinolysis,

platelet reactions or the endothelium. Improvement on the accuracy

of the INR, the use of new anti-coagulants and a number of specific

clinical questions require the development of a more comprehensive

test panel which encompasses all relevant aspects of haemostasis.

To identify the components of such a test panel, Philips Research has

started the development of a computer model that describes (within

reason) all the aspects of haemostasis. The finished model will feature

modules on the coagulation cascade, fibrin network formation and

fibrinolysis, endothelial and platelet reactions, blood flow and to

some extent also inflammation. The qualitative part of such a model

is obtained from literature. Examples of such qualitative knowledge

are the many protein-protein reactions that feature in the coagulation

cascade, fibrinolysis and on the platelet and endothelial surface. The

quantitative part, e.g. the rate constants involved in the aforemen-

tioned reactions, is less well known. We have developed a number of

methods to identify important model parameters, set up in vitro

experiments and estimate model parameters based on experimental

observations.

I will describe our approach in building a computer model for hae-

mostasis from literature and in vitro experiments, the estimation of

model parameters and the identification of the most important model

parts. I will show examples of these techniques applied to the coagu-

lation cascade part of the model and demonstrate the results in the

form of an on-line demo.

O14A-2Global fibrinolysis assaysColucci MUniversity of Bari, Bari, Italy

Intravascular fibrinolysis is a complex process and is heavily influ-

enced by thrombin generation which reduces clot lysability directly

(by altering clot architecture) and indirectly (by activating FXIII and

TAFI). It is therefore practically impossible to judge the overall func-

tion of the system from the concentration of single components.

Attempts to develop global assays date back to the 50s. Because

in vitro a plasma or blood clot will not lyse spontaneously due to the

excess of inhibitors, earlier methods either removed the inhibitors

(euglobulin lysis time) or reduced their effect (diluted blood clot lysis

time). Obviously, none of these methods reflected the physiological

condition. A more recent and widely adopted alternative is to chal-

lenge plasma with a fixed concentration of exogenous plasminogen

activator (generally t-PA) prior to clot induction and to measure the

‘lysis time’ by the changes in optical density. The method is quite

simple and is sensitive to the main fibrinolytic components and to

thrombin generation. It can be regarded as an estimate of the fibrino-

lytic ‘potential’, assuming that in vivo, at sites of vessel occlusion,

enough t-PA is released to overcome PAI-1. This method has been

successfully used for basic research and clinical studies. Each labora-

tory, however, uses its own version of the assay so that there are

many variations on the theme. This is a critical point because analyti-

cal conditions have a great impact on the results and may even com-

promise the test’s ability to unmask some fibrinolytic alterations. To

better approximate the in vivo condition, the assay can be performed

on platelet-rich plasma and may include elements of the vessel wall

(e.g. thrombomodulin). Assays in whole blood, in which fibrinolysis

is monitored by thromboelastography or through the release of

D-dimer, have also been proposed. The pros and the cons of each

method will be discussed.

O14A-3Cell culture technology and stem cells in research:emphasis on proteolysisvan Hinsbergh VWM and Koolwijk PVU University Medical Center, Amsterdam, The Netherlands

The use of cultured cells has facilitated previous studies on the regu-

lation of fibrinolysis and proteolysis. Cells provide a versatile tool to

study the effect of many stresses on the gene and activity regulation

of proteases, their inhibitors and their receptors. These stresses

include inflammation, hypoxia, ageing, metabolic stresses, angiogenic

factors and inhibitors, and others. They can be studied in single cells,

but the three-dimensional embedding of the cell has major effects on

their regulation. In addition to the classical assays of mRNAs, pro-

tein antigens and protein activities and regulation parameters such as

transcription factors and micro-RNAs, cultured cells provide a plat-

form for deletion and overexpression of genes and factors. Sophisti-

cated imaging techniques provide new clues about the cellular

localization of specific proteases as well as that of activities of specific

regulators within the cell. The availability of in vivo models enables

to verify the in vitro observations so that misinterpretations based on

vitro artifacts can be avoided. This is discussed on the basis of differ-

ent types of endothelial cells, various types of cells indicated as endo-

thelial progenitors cells, and stem cells that can be obtained in

different ways. The latter provide a versatile system to investigate

differentiation and to monitor the adaptive responses in a variety of

settings.


ORAL PAPERS 39____________________________________________________________________________________

Neurovascular Dysfunction

O14B-1Neurovascular-derived microparticles: potentialbiomarkers and fibrinolytic messenger in strokeAngles-Cano E1, Doeuvre L2, Plawinski L3, Dejouvencel T2,Nicole O3, Goux D4, Lacroix R5, Dignat-George F5 andBraeckmans K6

1Inserm; 2Inserm U919; 3CNRS UMR6232; 4CMABIO Universite

de Caen, Caen; 5Inserm U608, Marseille, France6University of

Ghent, Ghent, Belgium

Cells of the neurovascular unit and circulating leukocytes express

plasminogen activators and generate plasmin at their membrane.

Membrane microparticles (MPs) released upon stimulation/activation

of these cells may therefore convey plasminogen activators and the

plasmin generated at their surface (Blood 2007, 110: 2432). MPs are

vesicles of 100–1000 nm size released by activated cells under the

influence of a variety of stimuli including proteases. Data presented

here indicate that plasmin formed at the surface of neurons by the

tPA they express may also elicit the release of MPs. The plasmin

formed rapidly induces a calcium influx that could be inhibited by a

selective PAR-1 antagonist. This is an important feature preceding

membrane blebbing and followed by the release of membrane MPs

of about 300 nm in diameter (shown by electron microscopy and

fluorescent single particle tracking). These MPs as those of endothe-

lial and leukocyte origin bear plasminogen activators and have the

potential to generate plasmin de novo when in contact with plasmin-

ogen. The proteolytic activity they convey may participate in fibrino-

lysis, matrix remodeling and cell migration or angiogenesis. Indeed, it

has been recently shown that endothelial- or leukocyte-derived MPs

bearing uPA activate plasminogen bound to fibrin or to platelets

unveiling thereby a new mechanism for plasmin formation: the fibri-

nolytic cross-talk (Blood 2010, 115: 2048).

The in vivo pathophysiological relevance of these phenomena is under

study. Since MPs display molecular signatures from the parent cells it

is possible to identify their origin within the neurovascular complex.

Furthermore, beyond their well-characterized procoagulant activity it

is now possible to characterize their fibrinolytic/proteolytic activity as

well as other biomolecules. MPs are therefore evolving as characteris-

tic biomarkers of affected distinct cell subpopulations and could be

considered a direct message from a specific tissue undergoing activa-

tion or damage, e.g. ischemic stroke.

O14B-2Tissue-type plasminogen activation, the blood brainbarrier and traumatic brain injuryMedcalf RL, Sashindranath M, Sales E and Niego BMonash University, Melbourne, Australia

The human plasminogen activating (PA) system is well known for its

role in the turnover of the extracellular matrix and in the removal of

blood clots. However, over the past 15 years, there has been an

explosion of information linking this enzyme cascade with important

proteolytic and non-proteolytic events within the central nervous sys-

tem. Tissue-type plasminogen activator (t-PA) is arguably the most

relevant member of the PA system in this context. While a positive

role for t-PA has been linked with learning and memory under nor-

mal conditions, a deleterious role for t-PA is now well established in

brain pathology, notably following ischaemic stroke and traumatic

brain injury (TBI). While much effort has been devoted to under-

standing the molecular basis for these effects, a pattern is emerging

to suggest that a major component of these unwanted effects of t-PA

is linked to its effect on the blood brain barrier (BBB). We have

established in vitro models of the BBB to explore the means by which

t-PA modulates permeability across the BBB and if this effect is

unique to t-PA or shared by other plasminogen activators. We have

also established a reliable mouse model of TBI where we have com-

pared the degree of protein extravasation, lesion volume and motor

function post-TBI in mice under- or over-expressing t-PA in neurons.

Results from these in vivo experiments provide unambiguous evidence

that the degree of injury severity post-TBI is positively correlated

with the level of endogenous neuronal t-PA and its downstream

effects on BBB permeability. These results further suggest that thera-

pies directed at blocking the ability of t-PA to modulate the BBB

without compromising its classical fibrinolytic role will be of benefit

in TBI patients as well as patients with ischaemic stroke.

O14B-3Quantitation of net tissue-type plasminogen activatoractivity following CNS stimulation and injury using arapid and reliable amidolytic assaySashindranath M1, Samson AL1, Abdella E1, Lawrence AJ2,Tarlac V1, Downes CE3, Crack PJ3 and Medcalf RL1

1Monash University; 2Howard Florey Institute, Melbourne;3Department of Pharmacology, University of Melbourne,

Melbourne, Australia

Tissue-type plasminogen activator (tPA) is best known as an initiator

of fibrinolysis via its cleavage of plasminogen into active plasmin.

tPA is also widely expressed in the central nervous system where it

mediates numerous physiological and pathological processes. Most

studies to date have only used in situ and gel-based zymography

assays to monitor in vivo changes in neural tPA activity. Here we

describe an amidolytic assay which we have termed the ‘FAStPA’

assay (fibrin-accelerated S2251 hydrolysis by tPA) to quantitate

changes in net tPA proteolytic activity in brain tissues. Using tPA-/-

and wildtype mice we show that this assay can be used selectively to

detect changes in net tPA activity in the brain. The FAStPA assay

offers several advantages over zymography in that it is fully quantita-

tive, rapid and high-throughput, whilst maintaining a high degree of

sensitivity. We used this assay to quantitate alterations in net tPA

activity in various brain structures including the cortex, sub-cortical

structures and cerebella following systemic morphine treatment and

subsequent to a controlled cortical impact model of traumatic brain

injury and middle cerebral artery occlusion (MCAo)-mediated ische-

mic stroke. A role for tPA has been described in each of these

instances, and significant and compartment-specific alterations in net

tPA activity (up to 30%) were observed using the FAStPA assay

(P < 0.05). Finally, we also show that net tPA activity is increased

in both the cortex (P < 0.05) and cerebellum (P = 0.07) of mice

expressing a pathogenic form of Ataxin-1, the mutant protein respon-

sible for spinocerebellar ataxia type-1 (SCA1). This finding corrobo-

rates previous reports linking increased cerebellar tPA activity to

degeneration and dysfunction. In summary, we describe a practical

and accurate method for monitoring in vivo changes in net tPA activ-

ity in brain tissue. Our results shed further light on tPA as a complex

mediator of CNS function and dysfunction.

O14B-4Brain plasminogen systemDoeuvre L1, Chimienty G2, Orset C1, Mezzapesa A2, Mata S3,Pepe G2 and Angles-Cano E1

1Inserm, Caen, France; 2Department of Biochemistry and

Molecular Biology, University of Bari, Bari; 3Department of

Neurology, Careggi Hospital, Florence, Italy

Endothelial cells, glial and neuronal cells, representing the princi-

pal sources of plasminogen activator (tPA and uPA), are in close


40 ORAL PAPERS____________________________________________________________________________________

interaction in the neurovascular unit. Formation of plasmin at the

surface of endothelial cells and neurons has important consequences

on these cells (apoptosis of endothelial cells and detachment/aggrega-

tion of neurons). The plasminogen activation system is also known to

participate in various inflammatory conditions of the central nervous

system. In such pathologies, beyond circulating plasminogen, the ori-

gin of plasminogen is still a matter of debate.

First, we have investigated the presence of plasminogen in human

cerebro-spinal fluids. The presence of plasminogen and the activity of

plasmin, tPA and uPA in inflammatory diseases (GBS, Guillain Barre

Syndrome patients, n = 14; MS, Multiple sclerosis, n = 9) and also

in non-inflammatory diseases (n = 13) were studied. Western blot-

ting, zymography and chromogenic detection were used to evaluate

antigens and activity of plasmin(ogen), uPA and tPA.

In human, plasminogen was detectable in both inflammatory (66%)

and non-inflammatory (65%) patients. Plasminogen was found in

larger concentration in inflammatory diseases (4.6 nM in GBS,

6.5 nM in MS and 2.2 nM in non inflammatory diseases). Active

plasmin was detected in GBS and MS patients (3.55 nM vs.

2.6 nM). uPA was detectable in a minority of patients (15% of

GBS, 20% if MS and 7% of non-inflammatory diseases), and tPA

was not detect.

To further investigate the origin of plasminogen in the central ner-

vous system, we are currently exploring the presence of plasminogen

in mouse parenchyma in physiological and inflammatory conditions

by immuno-histochemistry.

In conclusion, we have shown that a plasminogen activation system

is detectable in CSF of patient with inflammatory and non-inflamma-

tory diseases. The role of these proteolytic messengers in diseases out-

come remains to be determined.


ORAL PAPERS 41____________________________________________________________________________________

Thrombolytic Therapy

O15A-1Combination of intra-arterial thrombolysis andmechanical treatment for acute ischemic strokeDippel DWJErasmus MC University Medical Center, Rotterdam, The

Netherlands

Acute ischemic stroke leads to death or disability in more than half

of all patients. National and international guidelines recommend

thrombolysis with intravenous rtPA in patients who can be treated

within 4.5 h and have no contra-indications. In the one fifth of

patients who have a proximal occlusion of an intracranial artery,

symptoms are often more severe and outcome is even less favorable.

Treatment with i.v. thrombolysis leads to recanalization in a minority

of these patients. Three small randomized clinical trials suggest that

local intra-arterial thrombolysis with delivery of the thrombolytic

agent through a microcatheter leads to better results: recanalization

in more than half of the patients. Mechanical treatment with a local

device may consist of aspiration, stenting, or thrombectomy with or

without ultrasound enhancement. Uncontrolled studies suggest that

these approaches may lead to recanalization in more than 60% of

treated patients. Complications of i.a. thrombolysis consist of reper-

fusion edema and hemorrhage, often leading to neurological deterio-

ration or death. Mechanical treatment is associated with similar and

with device-related complications such as dissection, vessel wall rup-

ture and local ischemia.

Prompt recanalization does not ensure improved neurological out-

come. The frequent complications cast doubt on the overall effect of

this treatment. The challenge for the coming years will be to assess

the effect of intra-arterial thrombolysis and mechanical treatment on

functional outcome after stroke, and to assess its safety in various

combinations, dosages and time windows. Large ongoing clinical tri-

als are the Interventional Management of Stroke trial 3 (IMS3) and

the Multicenter CLinical trial of Endovascular treatment for Acute

ischemic stroke in the Netherlands (MR CLEAN).

We will describe state of the art in intra-arterial treatment for acute

ischemic stroke, and discuss the results of published studies, as well

as the ethics and design of new and ongoing clinical trials.

O15A-2Improving stroke thrombolysis (tPA) safety throughthe use of plasma biomarkersMontaner JVall d’Hebron Hospital, Barcelona, Spain

The use of blood biomarkers is getting increasingly popular in the

field of cerebrovascular diseases, since biomarkers might aid physi-

cians in several steps of stroke evaluation. Although thrombolytic

therapy using tissue plasminogen activator (t-PA) in acute stroke is

effective since it accelerates clot lyses and earlier restoration of blood

flow, up to 40–50% of treated patients do not recanalize or do it too

late, and between 6% and 15% suffer hemorrhagic transformations

with high death rates. In the context of the neurovascular unit, t-PA

may degrade extracellular matrix integrity and increase risks of neu-

rovascular cell death, blood-brain barrier leakage, edema and hemor-

rhage. We have shown that t-PA treatment increases and activates

MMP-9 in human stroke, our data suggest that neutrophils are good

candidates to be the main source of MMP-9 following t-PA stroke

treatment and, in consequence, partially responsible of brain bleed-

ings. In humans, biomarkers such as matrix metalloproteinase-9

(MMP-9), vascular adhesion protein-1 (VAP-1) or fibronectin, which

might be used to select patients at higher risk of hemorrhagic trans-

formation, and high plasminogen activator inhibitor-1 (PAI-1) inter-

fering with tPA-induced recanalization, thus predicting clot-lyses

resistance and poor outcome, have been recently identified. More-

over, high levels of MMP-9 and MMP-13 are involved in DWI lesion

growth in spite of thrombolytic therapy suggesting its ultra-early role

in brain injury. Other biomarkers such as C-reactive protein may

accurately predict stroke mortality following reperfusion therapies.

Finally, we will also show that genetic background of stroke patients

may condition plasma levels of some of these biomarkers and influ-

ence therapeutic response in t-PA-treated patients.

O15A-3Thromboprophylaxis by cell-bound thrombolyticsMuzykantov VRUniversity of Pennsylvania, Philadelphia, PA, USA

Efficacy and safety of current thromboprophylaxis are limited, espe-

cially in acute settings associated with a short-term propensity for

thrombosis in post-surgical patients, among other potentially eligible

cohorts. In theory, timely delivery of thrombolytics into the interior

of nascent pathological clots could provide their rapid dissolution

preventing occlusion, if such a maneuver can be performed without

damaging hemostatic mural clots formed shortly after surgery. How-

ever, plasminogen activators have insufficient time of circulation,

attack preexisting hemostatic clots and penetrate into tissues includ-

ing the CNS where they exert adverse effects. Coupling plasminogen

activators to carrier red blood cells (RBC) does not affect negatively

RBC, yet prolongs tPA circulation time by several orders of magni-

tude, as well as prevents dissolution of hemostatic clots and side

effects in tissues. RBC glycocalyx protects RBC-bound tPA from

plasma inhibitors. Further, recombinant proteins fusing mutant

thrombin-activated plasminogen activators with a single chain frag-

ment of a monoclonal antibody to glycophorin A safely bind to cir-

culating RBC after injection in animals, circulate for many hours and

prevent formation of occlusive venous and arterial clots in animal

models. Results of recent and ongoing studies in models of cerebral

and peripheral thrombosis and ischemia in mice, rats and pigs indi-

cate that using RBC as a carrier for prophylactic delivery of fibrino-

lytics provides immediate and safe short-term thromboprophylaxis,

unattainable with free tPA causing severe side effects.


42 ORAL PAPERS____________________________________________________________________________________

New Factors, Functions, Mechanisms

O15B-1Epithelial development and homeostasis requirecomplex interactions between matriptase and theKunitz-type transmembrane serine protease inhibitors,HAI-1 and HAI-2Bugge TH1, Szabo R1, Kosa P1 and List K2

1NIH, Bethesda, MD; 2Wayne State University, Detroit, MI, USA

Matriptase (a.k.a., MT-SP1 and epithin) is an autoactivating type II

transmembrane serine protease that is constitutively expressed in

many developing and adult epithelia. We show that postnatal abla-

tion of matriptase from epithelia of diverse origin and function leads

to a spectrum of abnormalities ranging from impaired barrier func-

tion and loss of secretory function to complete de-differentiation and

malignant transformation. This epithelial demise was often preceded

by loss of tight junction function, increased paracellular permeability,

and mislocation of tight junction-associated proteins. Hepatocyte

growth factor activator inhibitor (HAI)-1 and HAI-2 are Kunitz-type

transmembrane serine protease inhibitors that display potent inhibi-

tory activity towards matriptase in vitro. We found that both HAI-1

and HAI-2 display near-ubiquitous co-localization with matriptase in

embryonic as well as adult epithelial tissues. Disruption of either the

HAI-1 or the HAI-2 gene in mice led to embryonic lethality, which

was associated with loss of epithelial cell polarity. However, simulta-

neous genetic inactivation of matriptase in either HAI-1- or HAI-2-

deficient embryos completely rescued this embryonic lethality, identi-

fying matriptase as the primary developmental protease target for

both protease inhibitors. Moreover, HAI-1-deficient mice with low

matriptase levels (1–15%) not only completed embryonic develop-

ment, but displayed normal long-term survival. Combined heterozy-

gosity for HAI-1 and HAI-2 also caused embryonic lethality

(nonallelic noncomplementation), which could be rescued by the loss

of just a single matriptase allele. Taken together, these data show

that epithelial development and homeostasis is enabled by complex

interactions between the membrane serine protease, matriptase, and

the Kunitz-type transmembrane serine protease inhibitors, HAI-1

and HAI-2.

O15B-2Protease signaling contributes to neural tube closureCamerer E1, Barker A2 and Coughlin SR2

1Inserm U970, Paris Cardiovascular Research Center, Paris,

France; 2Cardiovascular Research Institute, University of

California San Francisco, San Francisco, CA, USA

Protease-activated receptors (PARs) are G protein-coupled receptors

that mediate cellular responses to proteases. Mice with single deficien-

cies in PARs displayed defects in vascular development and hemosta-

sis, most likely reflecting a disruption in the ability of mutant

endothelial cells and platelets to sense active coagulation proteases.

To identify roles for protease signaling beyond the vasculature and

seek evidence for additional physiological PAR agonists, we gener-

ated mice with combined deficiencies in all PARs. We thus revealed

an unexpected role for protease signaling in neural tube closure and

formation of the central nervous system. Mouse embryos lacking

PARs 1&2 showed defective hindbrain and posterior neuropore clo-

sure and developed exencephaly and spina bifida. Par1 and Par2 were

expressed in surface ectoderm, Par2 selectively along the line of clo-

sure. Ablation of Gi/z and Rac1 function in these Par2-expressing

cells disrupted neural tube closure, further implicating G protein-cou-

pled receptors and identifying a likely effector pathway. Cluster anal-

ysis of protease and Par2 expression patterns revealed a group of

membrane-tethered proteases often co-expressed with Par2. Among

these, matriptase activated Par2 with picomolar potency, and hepsin

and prostasin activated matriptase. Together, our results suggest a

role for protease-activated receptor signaling in neural tube closure

and identify a local protease network that may trigger Par2 signaling

and monitor and regulate epithelial integrity in this context.

O15B-3Proteolysis of human Factor IX and FIXa:gla-dependant conformational specificity in itsinactivation by plasminSutton A1, Fatemi M1, Vancott KE1, Bajaj SP2 and Velander WH1

1University of Nebraska-Lincoln, Lincoln, NE; 2University of

California Los Angeles, Los Angeles, CA, USA

The biosynthesis of recombinant human Factor IX (rhFIX) creates

major subpopulations that differ in post-translational modification

(PTM). We have compared differences in PTM between subpopula-

tions of rhFIX made in transgenic pig milk (tg-rhFIX) and rhFIX

made in CHO cells (CHO-rhFIX) to human plasma-derived Factor

IX (pd-hFIX). Differences occur in the content of gamma-carboxy-

glutamic acid (gla) that affect procoagulation activity. Interestingly,

similar proteolytic degradation products were found in rhFIX and

pd-FIX preparations. The proteolysis pattern of tg-rhFIX is consis-

tent with plasmin’s established role as a predominant milk protease.

This is the first report of gla-dependant conformational effects upon

plasmin cleavage of Factor IX. in vitro treatment by plasmin of

CHO-rhFIX, pd-hFIX, and tg-rhFIX subpopulations having different

gla contents showed contrasting degradation in the presence of cal-

cium or EDTA. Higher gla content rhFIX or pd-FIX species (11 or

12 gla) showed a bias towards plasmin mediated proteolysis to form

rFIX-alpha in the presence of Ca2+. This indicates a gla-dependant

presentation of the Arg 145 cleavage site on the NH2-terminus of

activation peptide. In contrast, lower gla content (average nine) spe-

cies showed increased production of rhFIX-gamma indicating

increased accessibility of Arg 318 cleavage site in the catalytic domain

of Factor IX relative to Arg 145. While rhFIX- or pd-FIX-alpha

retain procoagulation activity, their gamma proteolysis products are

inactive due to Factor X exosite disruption by plasmin cleavage.

Fully activated Factor IX exhibited resistance to hydrolysis by plas-

min in either Ca2+ or EDTA indicating that it less efficiently pre-

sents the Arg 318 cleavage site than does Factor IX zymogen and

Factor IX alpha. Based upon this data, we hypothesize that Factor

IX activity consumption in blood circulation by plasmin is affected

by Factor IX gla-content. For example, a more rapid inactivation of

lower gla content Factor IX by plasmin could arise from warfarin

therapy.

O15B-4Thresholding behaviour in prothrombin activation byprothrombinaseCook PM, Colizza K and Nesheim MEQueen’s University, Kingston, Canada

In systems that are initiated by stimuli and have both positive feed-

back and inhibition, thresholding can exist, depending on the relative

kinetics of feedback and inhibition. Two manifestations of threshold-

ing are steep dose-response curves and ‘all or none’ responses. These

studies were carried out to determine whether prothrombin activation

exhibits thresholding, when the dose is defined as the input factor Xa

concentration and the response is defined as the area under the

thrombin concentration time course (thrombin potential). Factor Xa

was added to prothrombin, factor V, antithrombin, phosphatidylcho-


ORAL PAPERS 43____________________________________________________________________________________

line and phosphatidylserine vesicles, CaCl2, and the fluorogenic

thrombin substrate Z-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-

GGR-AMC). The free thrombin concentration over time was calcu-

lated from rates of Z-GGR-AMC hydrolysis. In the above condi-

tions, the thrombin potential was nearly zero from 0 pM to 0.5 pM

factor Xa, increased approximately linearly from 0.5 pM to 3 pM

factor Xa, and plateaued thereafter. This relationship demonstrates

the existence of thresholding with respect to the dose of factor Xa in

prothrombin activation. Activated protein C addition (0–5 nM)

raised the threshold factor Xa dose from 0.5 pM to 10 pM. Similarly,

heparin (0–2 lg/mL), enoxaparin (0–100 nM), and fondaparinux (0–

100 nM) increased the threshold factor Xa dose from 0.5 pM to 50,

150, and 200 pM, respectively. The thrombin time courses were fit to

a computer model of prothrombin activation which includes inhibi-

tion of thrombin and factor Xa by antithrombin and antithrombin-

heparin, positive feedback activation of factor V by thrombin and

factor Xa, and factor Va inactivation by activated protein C. The

model predicted the experimental data well, which suggests that

thresholding is a property intrinsic to this system of components. The

experimental data and model both show that prothrombin activation

is threshold dependent and the threshold dose of factor Xa changes

with changes in the kinetics of feedback and inhibition.

O15B-5Reduced thromboxane production in morbidly obesesubjects: another facet of the ‘obesity paradox’Cialdella P1, Graziani F1, Biasucci LM1, Giubilato S1, Liuzzo G1,Della Bona R1, Pulcinelli FM2, Iaconelli A1, Mingrone G1 andCrea F1

1Universita Cattolica del Sacro Cuore; 2Universita La Sapienza,

Roma, Italy

Purpose: Post-mortem studies have demonstrated that morbidly

obese subjects (MO) surprisingly show less coronary atherosclerosis

than obese subjects (O), but the reasons for this apparent protection

from atherosclerosis are not clear yet. Thromboxane A2 (TxA2), a

marker of platelet activation, is higher in O compared to lean sub-

jects (L) and this might represent a clue to their increased cardiovas-

cular risk. However, data on TxA2 in MO are still lacking, therefore

we hypothesized that lower levels of TxA2 in MO might play a role

in their lower atherosclerotic burden.

Methods: We measured serum levels of Thromboxane B2(TxB2), a

stable metabolite of TxA2, high-sensitivity C-reactive protein(hs-

CRP)and leptin in 17 L (BMI 22.9 � 1.6), 25 O (BMI 32.6 � 2.4)

and 23 MO (BMI 48.6 � 7.1), without insulin resistance, diabetes

and overt cardiovascular diseases.

Results: Serum TxB2 levels were lower in L versus O (P = 0.046),

and in MO versus L and O (P = 0.03 and P < 0.001 respectively).

In contrast, hs-CRP and leptin levels were higher in O versus L (hs-

CRP P < 0.001; leptin P = 0.002) and MO versus L (P < 0.001 for

both). Leptin was also higher in MO versus O (P < 0.001). TxB2

negatively correlated with leptin and BMI. Hs-CRP correlated with

leptin, and both of them were also correlated with waist circumfer-

ence, BMI and HOMA-IR.

Conclusions: Insulin sensitive MO show lower levels of TxB2 as com-

pared with O and L, suggesting that a reduced platelet activation

could play a role in the paradoxical protection of MO from athero-

sclerosis, despite higher levels of leptin.


44 ORAL PAPERS____________________________________________________________________________________

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