antiphospholipid syndrome.ppt

8/11/2019 antiphospholipid syndrome.ppt

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Uso de Modelos In Vitro e In Vivo en elDiseo de Nuevas Estrategias Teraputicas

Dirigidas a Blancos Moleculares EnEnfermedades Cardiovasculares.

SILVIA S. PIERANGELI, Ph.D.

Professor


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What is the Antiphospholipid Antibody Syndrome ?

An acquired autoimmune thrombophilia,characterized by:

a) vascular thrombosis.

b) recurrent pregnancy losses.

c) thrombocytopenia.

d) laboratory evidence for:-antibodies against phospholipids or

phospholipid-binding protein cofactors.


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Multiple Strokes

in a Young Woman

(Brain MRI)

Occlusion of Right Middle Cerebral Artery

In a 3 Years Old Child with

Severe Headache and Hemiparesis

With aCL Antibodies +


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Digital Necrosis and Gangrene


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Diagnostic Tests

Anticardiolipin Test

Lupus Anticoagulant Test


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ANTI-CARDIOLIPIN TEST

Advantages Overwhelming majority of APS patients are anti cardiolipin positive

Test can be performed reproducibly.

Clinicians and laboratories generally familiar with units ofmeasurement.

Disadvantages

Relatively nonspecific (particularly low positive, IgM positive).

Intra-laboratory and Inter-laboratory variability.

Problems with false positive results: aCL positive in a wide variety of

infectious diseases and in non-APS related autoimmune diseases.


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Predictive value of IgG aCL for thrombosis inpatients with SLE (Escalante et al)

IgG aCL levels below 21.4 = probability ofthrombosis 0.07

IgG aCL levels >21.4 and < 65.0 GPL =probability of thrombosis 0.20

IgG aCL levels >65.1 GPL units = probabilityof thrombosis 0.75


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Anti-2glycoprotein I

More specific than anticardiolipin test for diagnosis ofAntiphospholipid Syndrome (but not 100% specific)

Not as sensitive as anticardiolipin test (70-90% sensitivity) Efforts of standardization continuing

Useful in diagnosis of doubtful cases of APS. Some APSpatients negative for aCL and positive for anti-

2GPI.


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APhL ELISA - Principle

Based on observation that antiphospholipid antibodies cross-

react with negatively charged phospholipids but syphilis andother infectious diseases sera largely limited to cardiolipinbinding (no cross-reactivity)

Construction of a kit with negatively charged phospholipids

might eliminate non-specific binding.


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APhL ELISA k it

Antigen composed fo mixture of phospholipidsinstead of cardiolipin

Sensitivity of APS (greater than 90%)

More specific than anticardiolipin test and at leastas specific (or more) compared to anti-2GPI

Incorporation of an in-house positive control

Can be utilized for first line testing, and certainlyimportant in confirmation of APS


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Erkan and Lockshin, Rheum Dis Clin North Am

2006;32:129-48

Lim et al. JAMA 2006;295:1050-1057

Vascular Thrombosisprevention

Asymptomatic aPL No treatment or ASA?

Venous thrombosis Warfarin INR 2.0-3.0

Arterial thrombosis Warfarin INR 3.0

Recurrent thrombosis Warfarin INR 3.0-4.0+ASA

CAPS Anticoagulation +corticosteroid + IVIG orplasmapheresis

Treatment of thrombosis:Current Recommendations


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Erkan study

Aspirin for primary thrombosis prevention in theantiphospholipid syndrome: double-blind, placebo-controlledtrial in asymptomatic antiphospholipid antibody-positiveindividuals.

APLASA: multicenter, randomized, double-blind, placebo-controlled clinical trial

Observational parallel study.

Conclusions:

aPL-positive individuals do not benefit from low-dose aspirin forprimary thrombosis prophylaxis

have low overall annual incidence rate of acute thrombosis and

develop vascular events when additional thrombosis risk factorsare present.

(Erkan D et al. Arthritis Rheum 2007; 56: 2382-2391).


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Treatment of thrombosis in APS

Oral anticoagulation: problems

Bleeding

Frequent monitoring

Patient compliance with medication and diet


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Prevention of thrombosis in APS:Current problems

In patients with previous thrombotic event(s):

Oral anticoagulation at high INR vs. moderate INR ?? Most recommendations based on retrospective studies.

(Khamashta et al, NEJM, 1995; Krnic et al, Arch Intern Med, 1997

Prospetive studies: Crowther et al, NEJM, 2003; Finazzi et al, JTH,2005)

Patients with aPL and no thrombosis (low dose aspirin vs. no treatment?)


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Unresolved questions

Do patients with stroke require same level ofanticoagulation vs. those with DVT only?

Is aspirin or other anti-platelet agents alone,sufficient?

Do we discontinue oral anticoagulation in

some patients when an additional risk factoris no longer a problem (i.e. contraceptives)?


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There is a need for more safer and

efficacious modalities of treatment forthrombosis in APS.

Understanding the molecular andintracellular events triggered by

antiphospholipid antibodies is important

in designing new modalities of targetedtherapies for treatment of APS.


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Do aPL antibodiescause/inducethrombosis?


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Pierangeli, S. S. et al. Circulation 1996;94:1746-1751

Exposed femoral vein and fiber-optic lightpositioned under the vein in a mouse


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Pierangeli, S. S. et al. Circulation 1996;94:1746-1751

Photograph taken from the video monitor demonstrating themethod for analysis of thrombus size


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Thrombus formation


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METHODS

aPL-IgG (500g) or

Control IgG

0 hr 48 hr 72 hr

Thrombus

Dynamics

Analysis


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Antiphospholipid AntibodiesPromote Clot Formation in Mice


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Un Visitante Ilustre


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Micropoint laser to inducethrombogenic injury.


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How has the animal model of induced thrombosishelped us in understanding aPL pathogenic effects?

Induction of thrombosis in a mouse model by IgG, IgM and IgA immunoglobulins from patients with theAntiphospholipid Syndrome. Pierangeli et al.Thrombosis Haemost . 1995; 74: 1361-1367.

Generation and characterization of Monoclonal IgG Anticardiolipin Antibodies from a Patient with theAntiphospholipid Syndrome.Olee et al. Proc Nat Acad Sci. (USA).1996; 93: 8606-8611.

Identification of an Fc- receptor independent mechanism by which intravenous immunoglobulin (IVIG)ameliorates antiphospholipid antibody-induced thrombogenic phenotype. Pierangeli et al. ArthritisRheum 2001;44: 876-883.

Arginine residues are important in determining the binding of human monoclonal antiphospholipidantibodies to clinically relevant antigens. Giles et al. J Immunol2006; 177: 1729-1736.

A human monoclonal anti-prothrombin antibody is thrombogenic.in v ivoand upregulates expression oftissue factor and E-selectin on endothelial cells. Vega-Ostertag et al. Br. J. Haematology.2006;


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Do APL antibodies affect plateletactivation in v ivo?

Infusions of anti-GPIIb/IIIa (1B5)antibodies affect aPL-mediated enhanced thrombus formation in a mouse model ofthrombosis.

aPL-antibodies do not enhance thrombosis in 3-null mice.

Hydroxychloroquinediminishes platelet activation andthrombus formation induced by aPL antibodies.Edwards M et al.Circulation. 1997; 96:4380-4384.

Thrombogenicity of 2glycoprotein I-dependent antiphospholipidantibodies in a photochemically induced thrombosis model inthe hamster.Jankowski et al.Blood 2003; 101: 157-162.


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Hydroxychloroquine in APS

Yoon KH. Sufficient evidence to consider hydroxychloroquine as anadjunct therapy in antiphospholipid (Hughes) syndrome. J Rheumatol2002; 29: 1222-1226.

Wallace DJ. Does hydroxychloroquine protect against clot formation insystemic lupus erythematosus? Arthritis Rheum 1987; 30: 11435-1436.

Petri M. Hydroxychloroquine use in the Baltimore Lupus Cohort: effectson lipids, glucose and thrombosis. Lupus 1996; 5: S16-22.

McCarty GA and Cason TE. Use of hydroxychloroquine in

antiphospholipid antibody syndrome at three academic rheumatologyunits over two years: improvement in antibody titer and symptomsmanagement (abstract). 7th International Congress on SLE and Relatedcondictions. Abstracts Book, NY 2004.. pM17A.


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Question

aPL antibodies enhance platelet activation invitro and in vivo.

What are the in tracel lular pathways

invo lved in aPL-mediated p lateletact ivat ion?


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Intracellular events mediated byaPL on platelets.

APL induce platelet activation and thromboxaneformation and platelet-derived thromboxane urinary

metabolites.(Martinuzzo ME et al.Thromb Haemost 1993; 70:667-671 and Forastiero R et al.Thromb Haemost 1998; 79:42-45)

APL/anti-2GPI Abs induce production ofthromboxane A2 that is inhibited by cyclic-AMP

agonists.Indomethacine and phosphodiesteraseinhibitors such as theophylline inhibit TXA2.(RobbinsDL et al. J Rheumatol. 1998; 25: 51-56 and Opara E et al.2003; 30: 55-59).


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p38- P

p38

ERK1/ERK2-P

F(ab)2IgGNHS

F(ab)2aPL1

F(ab)2aPL2

F(ab)2aPL3

F(ab)2aPL4

F(ab)2a

PL5

PBS

PBS

+Thrombin 0.005 U/ml +Thrombin 1U/ml

43

43

43

Kda

A

B

C

Phosphorylation of p38MAPK and ERK1/ERK2 by aPL and thrombin

Vega-Ostertag M et al.Arthritis Rheum 2004; 50: 2911-2919


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Anti GPIIbIIIa antibody


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P38

cPLA2

AA

PKC

Fibrinogen

gp IIIagpIIb

Ca2+

PLC

PLC

DG IP3

Ca

2+

TXB2

(+)

TX Rc

Dimerized

2-glycoprotein I

Full activation

SB203580

Aspirin

Hydroxy-

chloroquine

(-)

(-)

Anti-GPIIbIIIa antibody

Receptor Antagonist

(-)

PS, PE

EXPOSITION

GPIb

(+)


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Receptor recognized by aPL onplatelets

APO ER2 Lutters BC et al.J Biol Chem 2003; 2778: 33831-33838.

Glycoprotein Ib/IX-V. Shi et al. Arthritis Rheum 2006; 54:

2558-2567.


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Dimers of 2GPI.

In previous studies, a chimericfusion protein was constructed bythe dimerization domain (apple 4) of2GPI .

As a control the monomeric protein

apple 2- 2GPIwhich is not able toform dimer - was constructed.

The authors demonstrated thatdimeric 2GPI mimics in vitro theeffects of 2GPI anti- 2GPIantibodies complexes. [Lutters, et al.JBiol Chemistry 2001; 276:5 , 3060-3067]


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Effects of B1 APOER2 on thrombus formation and platelet

aggregation in v ivo

Treatment Thrombus size Plateletaggregation

Dimer of 2GPI 3629 562 m2 56.3 % (n=3)

Monomer control 690 50 m2 10% (n=3)

B1D APOER2 532 147 m2 23% (n=3)


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Antiphospholipid Antibodiesand Endothelial Cells


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APL antibodies activate endothelialcells in v i t ro and in v ivo .

aPL or anti-2GPI antibodies upregulate EC adhesionmolecules and this effect is related to aPL binding to EC(Del Papa N et alArthritis Rheum 1997; 40: 551-561.

aPL-induced upregulation of ICAM-1, VCAM-1 and E-selectin on HUVEC and increased adhesion of monocytesto EC in the presence of 2GPI (Simantov R et al J Clin Invest 1995;96: 2211-2219).

Soluble levels of VCAM-1 significantly increased in plasmaof patients with APS and recurrent thrombosis (Kaplanski G etal 2000; 43: 55-64).


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APL antibodies enhance thrombus formation and thiscorrelates with activation of endothelial cells in v ivo

Antiphospholipid antibodies from patients with Antiphospholipid Syndrome activate

endothelial cells in v i t ro and in v ivo. Pierangeli et al Circulation,1999; 99:1997-2002.GDKV-induced antiphospholipid antibodies enhance thrombosis and activateendothelial cells in vivo. Gharaviet alJ Immunol 1999; 163: 2922-2927.

Functional analysis of patient-derived IgG monoclonal anticardiolipin antibodies usingin v ivothrombosis and in v ivo microcirculation Models.Pierangeli et al. ThrombosisHaemost2000; 84:388-395.

Thrombogenic effects of antiphospholipid (aPL) antibodies are mediated byintercellular cell adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1) and P-selectin. Pierangeli et al. Circ Res2001; 88: 245-250.

E-selectin mediates pathogenic effects of antiphospholipid antibodies.Espinola et alThromb Haemost.2003; 1:843-848.


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aPL antibodies upregulatetissue factor expression

Upregulation of tissue factor may account forarterial and venous thrombosis.

Increased expression of TF on monocytes by aPL.(Dobado-Barrios M et al Thromb Haemost 1999; 82: 1578-1582)

Inhibition of TF upregulation in monocytes bydilazep (Zhou H et al, Blood 2004; 104: 2353-2358).

Increased sTF and VEGF in plasma of patients withAPS (Williams FM et al Thromb Haemost 2000; 84: 742-746; ,Forastiero RR et alJ Thromb Haemost 2003; 10:2250-2251; CuadradoMJ et alJ Thromb Haemost 2006; 4:2461-2469)


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Upregulation of TF by aPL on ECSummary of our results

aPL increased TF expression on EC (1.8-4.2 fold increase)

The increase in TF expression was dependent on the dose ofantibody utilized.

TF function was increased by aPL (1.4 to 3.8 fold increase) TF function increase was dependent on the dose of antibody

utilized.

TF expression was inhibited by MG132(10-100%) and SB203580(50-100%).

TF function was inhibited by SB203580(34-54%).

aPL induce upregulation of IL-6 and IL-8.

Vega-Ostertag ME et alArthritis Rheum 2005; 52: 1545-1554.


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Effects of aPL onphosphorylation of p38 MAPK


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Effects of aPL on TF mRNA expression(RT-PCR) in EC)


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Conclusions

aPL induce phosphorylation of p38MAPK aPL induce iNOS

aPL induce transcription of TF mRNA and

this effect is inhibited by SB 203580 in adose-dependent fashion.(Vega Ostertag M, et al.Arthritis Rheum, 2005; 52: 5: 1545-1554)


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Experimental Design IIInjection schedule

IgG- APS (500g) i.p.

0 hr 48 hr 72 hr

1. Thrombus dynamics analysis2. Adhesion of WBC to EC in cremaster

3. TF activity in carotid artery homogenates

4. TF activity in mouse peritoneal macrophages

5. ACL activity

IgG- NHS(500g) i.p.

In some experiments, mice were

treated i.p. with 25 mg/Kg of SB

203580 or saline 30 min. before the

IgG injections


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THROMBOSIS MODEL IN MICE

Effects of a p38 MAPK inhibitor on aPL


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Effects of a p38 MAPK inhibitor on aPL-induced thrombosis in v ivo.

Vega-Ostertag ME et alJ Thromb Haemost 2007; 5: 1828-1834.


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Effects of a p38 MAPK inhibitor on aPL-mediated endothelial cell activation in v ivo.


D i i f TF i i i


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Determination of TF activity in mouseperitoneal macrophages

Procedure done in the animals immediately after the surgical procedures andafter they were sacrificed. Peritoneal macrophages obtained after lavage of theperitoneal cavity with 5 ml sterile PBS.

Two x 106 peritoneal cells were washed twice with PBS and resuspended in 1 ml

of Tris buffer saline-0.1% Triton X-100 pH 7.4 and centrifuged at 14,000 rpmduring 30 minutes. The cells were then washed twice and then resuspended in50 l TBS-0.1% Triton X-100 and sonicated.

The TF activity of peritoneal cells lysates determined using a commercialchromogenic assay (Actichrome TF, American Diagnostica, Stamford, CT) thatmeasures factor Xa after activation by the TF-Factor VII complex. The amount of

factor Xa generated is measured by its ability to cleave Spectrozyme Xa, a highlyspecific chromogenic substrate for factor Xa.

Results expressed in pM/100 g tissue.

Eff t f 38 MAPK i hibit PL i d d TF


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Effects of a p38 MAPK inhibitor on aPL-induced TFactivity in mouse peritoneal macrophages



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Determination of TF activity in carotid artery homogenates.

Pieces of approximately 5 mm of uninjured carotid arteries weredissected from both sides in each animal and were collected in a TBS-0.1% TritonX-100 buffer containing heparin as anticoagulant.

The samples were homogenized. Homogenates of pooled carotid artery

from four animals in each group were washed once with the same bufferand twice with TBS-0.1% TritonX-100. Finally the preparations wereresuspended in 50 mL of this buffer and sonicated.

The TF activity of lysates was determined using a commercialchromogenic assay (Actichrome TF, American Diagnostica, Stamford,CT).

Eff t f 38 MAPK i hibit PL i d d TF


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Effects of a p38 MAPK inhibitor on aPL-induced TFactivity in carotid artery homogenates of mice



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Platelet aggregation

Mouse blood was obtained in acid citrate dextrose anticoagulant (9/1volume/volume) by cardiac puncture . Platelet rich plasma (PRP) wasobtained by centrifugation for 20 min at 120g.

Aggregation of platelet in PRP was measured turbidimetrically using adual channel aggregometer (Minigator II) following calibration withplatelet-poor plasma (PPP) at a stirring speed of 800 rpm. PRP wasadjusted to 240,00 platelets/ mL with PPP.

Aliquots (250mL) of PRP were placed in cuvettes containing magnetic stirbars, warmed at 370C and stirred for 1 min to obtain a stable baseline.

Aggregation was induced by addition of 0.005 U/mL of thrombin in lighttransmission was recorded for 5 min.

Eff t f 38 MAPK i hibit PL i d d


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Effects of a p38 MAPK inhibitor on aPL-inducedplatelet aggregation



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Experimental Design

Ex vivoexperiments:

Determination of VCAM-1 in flat aorta preparations of mice using Qdotconjugates and dual photon confocal microscopy.

Mouse arteries were pressure-perfused with 10% formalin. After fixation, thearteries were washed three times with PBS.

The immunohistochemical procedure was done on a 24 well-plate. Blocking was

done with 2% BSA/5% goat serum for one hour. Primary antibodies wereincubated overnight at 4C. Washing steps were done to remove primary antibodyexcess. Qdot-conjugated secondary antibody (Qdot Corp) was incubated for onehour. Finally, nuclear visualization was done with Hoechst stain.

Image collection was done using a Zeiss LSM 510 Meta two-photon microscopeequipped with a near-infrared (NIR) titanium-sapphire femtosecond laser (Mira 900Ti:S Coherent) tuned and mode-locked at 750 nm. The separation of theemission signals was performed by acquisition of lambda stacks with posteriorselection of reference spectra using the META detector.

The following primary antibodies were used:, monoclonal rat anti-mouse VCAM-1IgG (1:50 dilution, BD Pharmingen) and a nonimmune primary used to address thecontribution of nonspecific Fc receptor-mediated binding (nonimmune purified ratIgG2a (BD Pharmingen). The following Qdot-bioconjugate was used in theexperiments: Qdot 655 goat F(ab')2 anti-rat IgG Conjugate

Effects of a p38 MAPK inhibitor on aPL induced VCAM 1 expression in


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Effects of a p38 MAPK inhibitor on aPL-induced VCAM-1 expression inaorta of mice ex vivo: nano crystalsQ dot conjugates and dual photon

confocal microscopy.


C l i


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Conclusions

A p38 MAPK inhibitor (SB 203580):Effectively diminished in vivoIgG-APS-

induced

thrombus formation endothelial cell activation

platelet aggregation

tissue factor activity (carotid EC andperitoneal macrophages)

VCAM-1 expression (aorta EC)

NF B


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NF-B

NF-B is a complex group of heterodimeric and homodimerictranscription factors that are trapped in the cytoplasm as an inactive

complex by I-B. NF-B involved in transcription of inflammatory genes such as: IL-6, IL-8,

TNF-and IL-1 and in induction of adhesion molecules on EC (VCAM-1, E-seland ICAM-1) and in recruitment of inflammatory cells to extravascular sites.

NF-B associated with rheumatoid arthritis and other autoimmune

diseases.

APL tib di d NF B


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APL antibodies and NF-B

Intracellular events in ECinduced by aPL antibodies:

aPL induce activation of

NF-B and correlateswith EC activation invitroand in vivoandwith thrombosis in vivo.

Espinola RG et al: J Thromb Haemost, 2003; 1:843-848.

Dunoyer-Geindre S. et al. Thromb Haemost. 2002;88: 851-857.

Bohgaki M, et al. Int Immunol. 2004; 16: 1632-1641.

MG 132


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MG 132

NF-B inhibitors used in RA and other autoimmune and inflammatorydiseases.

MG 132 = Carbobenzoxyl-leucinyl leucinylleucinal (Z-Leu-Leu-Leu-aldehyde; Z-LLL-CHO).

MG 132 is a potent 20 S proteasome inhibitor that has been shown effective in

suppressing NF-B activation in different cellular systems. Several studies have shown beneficial effects of MG132 on models of

rheumatoid arthritis, suggesting that this inhibitor may provide a new approach inthe treatment of this autoimmune disease.

Obj ti


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Objectives

Are NF-B inhibitors effective in reversingpro-inflammatory and pro-thrombotic effects

of aPL in vivo?


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Experimental Design

IgG -APS (500g) i.p

+ 10 M MG 132 or DMSO:PBS (60:40).

0 hr 48 hr 72 hr

1. Thrombus dynamics analysis2. Adhesion of WBC to EC in cremaster

3. TF activity in carotid artery homogenates

4. TF activity in mouse peritoneal macrophages

5. ACL activity

IgM - NHS(500g) i.p.

+ DMSO:PBS (60:40)


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Inhibition of aPL induced EC


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Inhibition of aPL-induced ECactivation in vivo by MG 132


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Effects of MG132 on TF activity on


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Effects of MG132 on TF activity onhomogenates of carotid artery of mice

treated with aPL Antibodies

Conclusions


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Conclusions

IgG-APS enhanced thrombosis, endothelialcell activation and tissue factor in vivoin

mice. These effects were significantly diminished

by pre-treatment of the mice with MG132.

Montiel-Manzano et al. NY Acad Med 2007; 1108: 540-553.


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Antiphospholipid Antibodies

and the statins

Pleiotropic effects of statins


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Pleiotropic effects of statins

-

TPA and

PA inhibitior-1 expression

-

Expression of adhesion molecules

-

Pro-inflammatory cytokines

-

Expression of tissue factor -

Thromboxane A2 synthesis and platelet reactivity

-Endothelin 1 synthesis

-NF-B activation

-MHC class II antigen expression

aPL antibodies and fluvastatin


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aPL antibodies and fluvastatin

Fluvastatin reduces aPL-induced adhesion of leukocytes and expressionof adhesion molecules on EC in vitro. (MeroniPL, et al.Arthritis and Rheum 2001;44:2870-2878).

Fluvastatin abrogated thrombogenic effects of aPL antibodies inv ivo(Ferrara DE et al, Arthritis Rheum, 2003; 48: 3272-3279)

Fluvastatin inhibited the effects of the IgG-APS on tissue factorexpression. The effect was dependent on the dose of fluvastatin.

Mevalonate abrogated the inhibitory effects of fluvastatin onexpression of TF by aPL on endothelial cells. (Ferrara DE. et al, J ThrombHaemost, 2004;2: 1558-1563).

Implications


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Implications

These f indings may have impo rtant

imp l ications in designing new modal it ies

of treatment and p revent ion o f recu rrentthrombosis in pat ients w i th APS.

Well designed c l inical tr ials are needed to

investigate and conf i rm these f ind ings inAPS patients

Objectives of the study


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Objectives of the study

To determine the effects of statins on pro-thrombotic and pro-inflammatory markers inpatients with aPL Abs

Patients


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Patients

Four groups of 20 patients each

A) PAPS

B) Asymptomatic APS with persistentlypositive aCL, LA and/or anti-2GPI tests.

C) SAPS

D) Asymptomatic SAPS with with persistentlypositive aCL, LA and/or anti-2GPItests.

Inclusion/Exclusion criteria


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Inclusion/Exclusion criteria

aCL titers > 40 GPL or MPL units

Anti-2GPI > 99thpercentile of normal controls.

>18 years of age.

No pregnant women.

Exclude if they are on statins or with pulse therapy withsteroids.

Not excluded if they are on HQ, aspirin, heparin, warfarin,low dose prednisone (5-10 mg/day).

Exclude patients with liver problems.

Study Intervention


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Study Intervention

Fluvastatin 40 mg/day for 5 months.

Blood will be collected at the screening visit,at one, three, six and seven months later.

At five months patients will be instructed to

stop fluvastatin.

Outcome measures


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Outcome measures

Determination of aCL, LA and anti-2GPItiters.

Determination of PCA and TF mRNA inPBMC.

Determination of VEGF, sTF, sVCAM-1, IL-6,IL-8 and TNF-.


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Does an anti-C5 MoAb prevent aPL-mediated thrombosis in


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mice?

Pierangeli SS etal. ArthritisRheum 2005;

52: 2120-2124.

Effects of aPL Abs on thrombosis in C5aR deficient mice


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Effects of aPL Abs on thrombosis in C5aR deficient mice.C5aR-/- + IgG-APS 3400 1681 108.9 33.4

C5aR-/- + IgG-NHS 777.3 270.4 0.8 0.4

C5aR +/+ + IgG-APS 3507 965 80.3 17.6

C5aR+/+ + IgG-NHS 1321 798 0.8 0.5

C5aR -/- + IgM-APS *676 690 96.4 30.8

C5aR-/- + IgM-NHS 958 388 0.0 0.0

C5aR +/+ + IgM-APS

3198 2361

99.8 31.4

C5aR+/+ + IgM-NHS 585 460 00.0 0.1

Romay-Penabad Z et al. NY Acad Sci 2007; 1108: 554-566.

CONCLUSIONS


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We demonstrated that complement activation is a centralmechanism contributing to aPL antibody-inducedthrombophilia using three approaches:

a specific complement inhibitor (Crry-Ig)

genetically deficient mice (C3-/- and C5-/-)

Using specific anti-C5 Monoclonal antibodies.

Using C5aR deficient mice.

CONCLUSIONS

Further evidence of complement


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Further evidence of complementinvolvement

Thrombus formation induced by antibodies to2glycoprotein I is complement dependentand requires a priming factor (Fischetti et alBlood2005; 106: 2340-2346).

IMPLICATIONS


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Pathogenic aPL antibodies, in addition to their direct effects on plateletand endothelial cell targets, induce complement activation, generatingcomplement split products which attract inflammatory cells that mayinduce then thrombosis and tissue injury

Activation of complement may be a critical proximal effector mechanism in aPL-associated thrombosis.

In APS patients, due to aPL IgG deposition targeted to the endothelium,complement activation is increased locally and overwhelms normallyadequate inhibitory mechanisms.

Therefore, inhibition of complement activation should ameliorateaPL-mediated vascular thrombosis.

IMPLICATIONS

APL antibodies in EC.


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APL antibodies in EC.

Receptor for aPL in EC?


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Receptor for aPL in EC?

Human 2GPI binds to

endothelial cells through a clusterof lysine residues that are criticalfor anionic phospholipids bindingand offers epitopes for anti-2GPIantibodies (Del Papa et al, 1997)

Effects of recombinant Domain I of


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Treatment aCL titer in-vivo

(GPLU)

Thrombus size

(mm2)

APS-IgG + rDI 90.742.4 568.4325.0

APS-IgG + control 70.118.3 7959.02647

Five mice per group

DI/control infused at 40mg i.v 30 minutes before thrombosisdynamics analysis

2glycoprotein I on aPL-mediated thrombosis

TIFI and aPL Abs


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TIFI and aPL Abs

TIFI is a a 20 amino acidsynthetic peptide (derived fromCMV) that shares similarity withthe PL- (membrane binding)region of 2GPI .

TIFI reduced aPL-mediatedthrombosis and EC activationin vivo.

TIFI reduced the binding ofFITC-

2GPI to target cells (EC,and monocytes).

(Vega-Ostertaget al. Lupus 2006;597: 247-256)


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Effects of various inhibitors on TF


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upregulation by 2GPI dimer.

Effects of various inhibitors on ICAM-1 expression induced by


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dimers of 2GPI


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Conclusions


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Conclusions

Animal models of thrombosis and endothelial cell activationin APS have been instrumental in

Elucidating the pathogenic mechanisms induced by aPLantibodies

Determining specific targets recognized by aPL antibodies.

Examining possible ways by which aPL antibodies areinduced.

Test new targeted therapies to prevent aPL-effects inv ivo .

WE NEED HUMAN STUDIES!

Animal models in APSNew Approaches to Prevention of Thrombosis in APS?


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New Approaches to Prevention of Thrombosis in APS?

Statins: Fluvastatin reversed EC activation and TF upregulation by aPL antibodies invitro and abrogated enhanced thrombus formation and EC in vivo. In mice

Hydroxychloroquine: Decreased platelet activation induced by aPL antibodies inv i t roand inhibited aPL-mediated thrombosis in mice in v ivo.

Antiplatelet agents: GPIIbIIIa inhibitors decreased aPL-mediated enhancement of

platelet activation and abrogated aPL-induced thrombus formation in mice. p38MAPK inhibitors: In vitro effects on aPL-induced TF upregulation in EC. Effects

on aPL-mediated thrombus formation, EC activation, TF upregulation and plateletactivation in v ivo.

NF-B inhibitors: In vitroeffects on aPL-mediated upregulation of TF. Significantdecrease in some aPL-enhanced thrombosis, TF upregulation,

Specific complement inhibitors: anti-C5 Monoclonal antibody decreased aPL-mediated thrombus formation.

Specific inhibitors/blocking agents to the receptor(s) in target cells: TLR-4inhibitors? Peptides that mimic regions of 2GPI?



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ACE inhibitors: Inhibit monocyte TF expression

Dilazep, dipyridamole: Adenosine uptake inhibitor; antiplatelet;inhibits monocyte TF expression.

LJP 1082: 2GPI-specific B cell toleragen; decreases anti-2GPI-specific B cell toleragen;decreases anti- 2GPI antibody levels.

Rituximab: Anti CD20.



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ACE inhibitors: Inhibit monocyte TF expression

Dilazep, dipyridamole: Adenosine uptake inhibitor; antiplatelet;inhibits monocyte TF expression.

LJP 1082: 2GPI-specific B cell toleragen; decreases anti-2GPI-specific B cell toleragen;decreases anti- 2GPI antibody levels.

Rituximab: Anti CD20.

Collaborators


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Mariano Vega-Ostertag, MS Morehouse School of Medicine

Zurina Romay-Penabad, PhD UTMB

Guadalupe Montiel, B.S. UTMB

Elizabeth Papalardo, B.S. UTMB

Dardo E. Ferrara, MD Morehouse School of Medicine

R.G. Espinola, MD Morehouse School of Medicine

X. Liu, MD Morehouse School of Medicine

Ian P. Giles University College London

Robert Swerlick, MD Emory University School of Medicine

Pier Luigi Meroni, MD University of Milan

Guillermina Girardi, PhD Hosp Spec Surgery

Jane Salmon, MD Hosp Spec Surgery

VM Holers, MD Univ Colorado,Denver

Philip deGroot Utrecht University.

The 13th International Congress on antiphospholipid antibodies:


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The 13 International Congress on antiphospholipid antibodies:Galveston, TX. Spring 2010.

Acknowledgements


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These studies were partially funded by aResearch Centers in Minority Institutions -National Institutes of Health grant # G12-RR03034 and a Minority BiomedicalResearch Support Grant from the NationalInstitutes of Health (GM58268-02) and a

multidisciplinary clinical research grant NIHgrant #: 2P60AR047785-06.

Documents

antiphospholipid syndrome.ppt