Dilation-Dependent Activation of Platelets and Prothrombinin Human Thoracic Ascending Aortic Aneurysm

Ziad Touat, Laurent Lepage, Veronique Ollivier, Patrick Nataf, Ulrich Hvass, Julien Labreuche,Martine Jandrot-Perrus, Jean-Baptiste Michel, Guillaume Jondeau

Objectives—The purpose of this study was to investigate whether thoracic ascending aortic aneurysm (TAAA) inducesplatelet activation as mural thrombus participates in aortic dilatation in abdominal aortic aneurysm and TAAA areassociated with rheological factors favoring coagulation activation.

Methods and Results—We studied the relation between coagulation activation and aortic diameter in Marfan patients(MFS) with various aortic diameters (n�52). We then studied patients presenting large aneurysms associated withbicuspid aortic valve (BAV) and degenerative form. Lastly, we used immunochemistry and biochemistry to investigateprothrombin/thrombin retention within the aortic wall. Microparticles, sGPV, tissue factor, and TAT complexes wereincreased in plasma from MFS with large aneurysms (�45 mm) compared to MFS with limited aortic dilatation(�45 mm). Similar elevations were observed in all patients with large aortic aneurysms, regardless of the etiology, thesite of maximal aortic dilation, associated valvulopathy, risk factors, or treatments. P-selectin and platelet-boundfibrinogen were also increased, demonstrating platelet activation in large aneurysms. Significant increase in sCD146plasma concentration suggested alteration of endothelium.

Conclusions—Platelet activation occurs in patients with large aneurysms of the ascending aorta, is dependent on aorticdilation, and is associated with thrombin generation, part of which appears to be retained in mucoid degeneration areas.(Arterioscler Thromb Vasc Biol 2008;28:940-946)

Key Words: thoracic aortic aneurysm � coagulation � Marfan � platelets

Thoracic ascending aortic aneurysms (TAAA) are definedby an enlargement of the ascending aorta. TAAA can be

observed in old patients without clear etiologic factors, in whomit represents a primary degenerative pathology of the ascendingaorta.1 But it can also occur in the younger population; it is thenusually linked to a genetic or congenital disease. Marfan syn-drome represents the majority of these cases. This is usuallyrelated to mutations in fibrillin12 and rarely to mutations intransforming growth factor (TGF)�R2, which can also be respon-sible for Loeys-Dietz Syndrome and familial aortic aneurysm.3–5

Other molecular abnormalities have been implicated, such asmutations in myosin,6 TGF�R1,5 ACTA2,7 or other currentlyunknown genes.8 Moreover, bicuspid aortic valves (BAV),which may be familial,9 are associated with a high risk ofaneurysm of the ascending aorta.10

In contrast to their etiologic diversity, all localized aorticdilations share common rheological characteristics. In normalhuman aorta, the regular geometry (tubular shape and parallelwalls) results in laminar blood flow and in a homogeneous shearrate, maintaining a physiological steady-state among the bloodcomponents and between the circulating blood and arterial

wall.11 Changes in arterial geometry considerably modify rheo-logical parameters and therefore influence interactions betweenblood components.11,12 The modulations by shear stress ofplatelet interactions with von Willebrand factor (vWF) exem-plify the role of rheology in hemostasis.13,14 In this respect, ananeurysm of the ascending aorta induces rheological perturba-tions, characterized by vortices (low wall shear rate).15

Rheological disturbances have been suggested to play a rolein mural thrombus formation associated with cerebral artery andabdominal aortic aneurysms, and the luminal thrombus appearsto play a role in the progression of aortic dilation.16 In contrastwith this localization, TAAAs are characterized by the absenceof any mural thrombus formation1 (Figure 1) even in olderpatients with diffuse atherosclerosis. Therefore, TAAA offers aunique opportunity to test the hypothesis that platelets and thecoagulation cascade could be activated by dilation of theascending aorta, and could be associated with this pathologyindependently of a mural thrombus formation. There have beenno clinical studies focusing on the hemostasis cascade in patientswith TAAA. For this purpose, we explored markers of activationof both platelets and prothrombin in patients with TAAA. In a

Original received October 28, 2007; final version accepted February 6, 2008.From INSERM (Z.T., L.L., V.O., M.J.-P., J.-B.M., G.J.), U698, University Paris 7, Paris, France; AP-HP (L.L., P.N., U.H., J.-B.M., G.J.) Medical and

Surgical Cardiology Department, Bichat Hospital, AP-HP, Paris, France, University Paris VII Denis Diderot (Z.T., L.L., P.N., J.L., J.-B.M., G.J.), andthe Centre de reference Marfan et syndromes apparentes (G.J.), Bichat Hospital, AP-HP, Paris, France.

Correspondence to Pr Guillaume Jondeau, CHU Bichat, INSERM U698, 46, rue Henri Huchard, 75018 Paris, France. E-mail guillaume.jondeau@bch.aphp.fr

© 2008 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.107.158576

940

Clinical and Population Studies

first step, we compared Marfan patients with various aorticdiameters and matched controls. In a second step, we studiedplatelets and prothrombin activation in patients presentingTAAA of other aetiologies (BAV and degenerative).

Materials and MethodsPopulationOne hundred fifty-six patients were divided into 4 groups (Table 1):Healthy individuals (Controls, n�66); Patients fulfilling interna-tional criteria for Marfan syndrome (MFS).17,18 They were subdi-vided into 2 groups: MFS patients with limited dilation (maximalaortic diameter �45 mm) (n�28), and MFS patients with largeaneurysm19 (aortic diameter �45 mm) (n�24); TAAA patientsscheduled for surgery because of aortic dilatation. Here, largeaneurysms were associated with bicuspid aortic valves (BAV)(n�15), or were isolated with no sign of genetic disease (Degener-ative; n�23). For more details, please see the supplemental datasection, available online at http://atvb.ahajournals.org.

ResultsPatient GroupsClinical and echocardiographic data are summarized in Table1. MFS patients were significantly younger (P�0.01) thanthose of the BAV and degenerative groups. Patients from thedegenerative group were the oldest with, as expected, morecardiovascular risk factors (�2�10.5, P�0.01), mainly hyper-tension, a higher frequency of medications, and a higherincidence of significant aortic valve regurgitation (P�0.01).

Aortic valve stenosis was present more frequently in theBAV group (�2�7.8, P�0.01). In this group, maximal aorticdilation was observed above the sinotubular junction (�2�20,P�0.0001), whereas it was maximal at the level of theValsalva sinuses in MFS patients, and the patients withdegenerative aortic aneurysm.

Marfan and Controls: Relation of PlateletActivation to Aortic DiameterNo increase in plasma markers of platelet activation wasobserved in the group of MFS with limited dilatation (ie,maximal diameter �45 mm), when compared to age- andsex-matched control: microparticle count (Annexin-V posi-tive) and concentrations of sGPV, Tissue factor and TATwere similar in the 2 groups (Table 2). In contrast, all thesemarkers were significantly higher in plasma from MFSpatients with large aneurysms (�45 mm) (Table 2). Theincrease in microparticle concentration (Figure 1) was corre-lated to aortic diameter (P�0.05, r2�0.38) in the Marfangroup with and without large aneurysms. Similar results werefound for sGPV concentration (P�0.001, r2�0.56), and these2 markers of platelet activation were highly correlated in thewhole Marfan group (P�0.0001, r2�0.65).

Extension to Other Aetiologies of TAAA

MicroparticlesMicroparticle counts were increased in plasma of patientswith large aneurysms of all etiologies (Marfan, BAV-associated or degenerative) when compared to controls(P�0.0001) (Table 2). However, plasma levels of micropar-ticles were similar, regardless of etiology (Table 2) of largeaneurysms. 93.2�3.9% of microparticles were positive forCD41 antibody (integrin GPIIb/IIIa) in patients with largeaneurysms and 90.9�6.4 in controls (NS) demonstrating theirplatelet origin in both cases.

Plasma MarkersOther markers for platelet activation (sGPV, sCD40L) werealso significantly (P�0.0001) higher in plasma of patientswith large aneurysms than in that from controls, with no

Table 1. Clinical Data

DilationControl

NoMarfanLimited Marfan

BAV LargeAneurysm Degenerative P

No. 66 24 28 15 23

Men, % 74 46 54 87 87

Age, y 50�17 36�12 39�15** 53�9 59�10 �0.01

Max aortic dilation

Value, mm 35�5 41�4 53�4* 54�7 59�10 �0.05

At sinuses of valsalva, % � � � � � � 100 20*** 69 �0.0001

Risk factors

Smoking, % 20 21 14 33 30 �0.01

HTN, % 23 25 11 13 70

Dyslipidemia, % 15 13 11 20 26

Diabetes, % 0 0 0 7 0

Aortic valvulopathy

Stenosis, % 8 0 0 33 0 �0.01

Regurgitation, % 24 0 4 20 61

Treatment

Beta-blockers, % 15 54 68 33 30 �0.01

Aspirin, % 23 0 7 13 70

ACEI, % 18 0 14 20 35

Statins, % 17 0 11 7 48

Touat et al Coagulation and Ascending Aorta Aneurysm 941

difference between etiologies (Table 2). In contrast, theplasma concentration of sP-selectin was not increased inassociation with large aneurysms compared to controls(36.2�1.7 ng/mL versus 34.2�1.8, ns).

We observed no difference in these plasma markers whenpatients were under low doses of aspirin. Aspirin blocksthromboxane A2 synthesis and platelet recruitment by thissecreted TxA2. It thus limits the secondary progression ofplatelet activation, but does not block primary plateletactivation.20

Platelet MarkersPlatelet membrane expression of P-selectin, measured withFACs on whole blood, was low, but significantly higher inlarge aneurysms compared to controls (4.08�0.53% positiveversus 2.91�0.56% in controls, P�0.05). Similar but moresignificant results were obtained by analyzing the curve shiftinduced by the specific antibody as compared to a nonrel-evant antibody (Overton method) (large aneurysms versuscontrols: 14.6�2.1% versus 7.8�0.6%, P�0.01) (Figure 1Aand 1B). P-selectin expression on platelets did not differ withregard to etiology.

Conversely, platelet-bound fibrinogen was not signifi-cantly different in large aneurysm patients compared tocontrols when measured by the classical analysis method(0.96�0.29% versus 0.89�0.21%, ns). However, curve shiftanalysis (Overton) revealed a small significant shift of thesignal to the right in platelets originating from patients withlarge aneurysms (TAAA: 7.34�1.3% versus controls:3.9�0.5%, P�0.05, Figure 1B).

Markers of Prothrombin ActivationIn plasma of patients with large aneurysms, TF antigen concen-tration was significantly higher than in control plasma, regard-less of etiology (Table 2). The 1-step recalcification clotting timewas reduced for plasma of patients with large aneurysmscompared to controls (175.8�8.0 seconds versus 210.7�7.9

seconds, P�0.001; Figure 2). The involvement of TF activity inthe shortening of the clotting time was demonstrated by addinga blocking anti-TF antibody that prolonged the clotting time ofpatients with large aneurysms by 35.9�5.8%, to reach a valuesimilar to that of controls. Plasma thrombin generation, mea-sured using a global assay, showed an increased “endogenousthrombin potential” (ETP) for large aneurysms relative tocontrols (1902.3�74 nM.min�1 for TAAA versus 1712.8�63.7nM.min�1 for controls, P�0.05; Figure 2) and an increasedmaximal thrombin generation (peak height) for the large aneu-rysm group (335.3�13.2 nmol/L versus 284.5�10.6 nmol/L forcontrols, P�0.001; Figure 2). Interestingly, there was a signifi-cant correlation between sGPV and maximal thrombin genera-tion (r2�0.49, P�0.01) in plasma of patients with large aneu-rysms. Furthermore, TAT complexes were also slightly butsignificantly increased in the plasma of patients with largeaneurysms (P�0.001, Table 2). These results were statisticallysimilar for all aetiologies.

Table 2. Levels of Positive Markers for Haemostasis and Endothelial Activation Determined in Plasma of Patients WithLarge Aneurysms as Compared to Controls and MFS With Limited Dilatation of the Ascending Aorta

Palatelet Activation Thrombin Activation

Microparticles(No./�L)

sCD40L(pg/mL)

SGPV(ng/mL)

TF(pg/mL)

TAT(�g/L)

Endothelial MarkersCD146 (ng/mL)

Control 630�77 65.2�11.9 51.7�2.8 61.7�8.5 2.8�0.3 282.8�10.9

(n�66) ns ns ns ns

Limited dilatation 569�63 ND 61.9�3.7 44.5�3.7 3.0�0.5 ND

(n�24) *** *** *** *** * *

Large aneurysms 2217�283 222.7�34.9 84.7�9.4 142.4�17.3 5.2�1.6 325.3�15.3

(n�66)

Marfan 1946�281 282.3�96.9 96.0�7.8 141.8�25.7 3.9�0.4 306.4�31.0

(n�28)

BAV 1827�480 221.4�58.5 72.5�9.9 151�19.7 6.9�3.4 322.3�38.0

(n�15)

Degenerative 2542�453 188.1�43.0 85.5�10.5 134.4�16.6 4.9�0.9 338.1�19.0

(n�23) ns ns ns ns ns ns

P values reported are adjusted for the presence of cardiovascular risk factors and treatment.*P�0.05; ***P�0.0001.

≥45 mm<45 mmControls

Microparticles (number/µL)

Aortic diameter (mm)

0

250

500

750

1000

1250

1500

1750

2000

2250

<35 35-40 40-45 45-50 >50

** ** *

ns

Marfan:

Figure 1. Microparticles in relation to aortic diameter. Numberof circulating microparticles, in plasma from Marfan patientswith various degrees of dilation and matched healthy individu-als, was increased only when the dilation was superior to45 mm.

942 Arterioscler Thromb Vasc Biol May 2008

Endothelial Plasma MarkersSoluble vascular cell adhesion molecule (VCAM)-1 was notstatistically different in large aneurysm and control groups(377.6�48.4 ng/mL versus 293.3�36.1 ng/mL, P�0.08). Incontrast, sCD146 plasma levels were significantly higher inpatients with large aneurysm than in controls, with no significantdifferences whatever the etiology of the TAAA (Table 2).

Inflammatory MarkersFibrinogen and hsCRP levels were similar in large aneurysmpatients and controls (2.8�0.1 g/L versus 2.6�0.2 g/L and1.88�0.3 mg/L versus 1.47�0.2 mg/L, respectively). Thesemarkers were slightly, but not significantly, superior indegenerative group. This is consistent with greater age andthe presence of more risk factors in this group.

Aortic Tissue Samples and Conditioned MediaIn control aortic tissue, immunoreactivity for prothrombin/thrombin (II/IIa) was negative (Figure 3A). In contrast, aorticsections from patients with surgically removed large aneurysms,whatever the etiology, showed prominent immunoreactivity forfactor II and IIa in the mucoid areas, which were localized inserial sections stained with Alcian blue (Figure 3A). Theseresults were confirmed on conditioned media of aortic samples,where factor II and IIa were detected by immunoblotting (Figure3B). Conditioned media from large aneurysms expressed largerbands of prothrombin and thrombin than conditioned mediafrom controls. By ELISA, a higher concentration of TAT wasdetected in conditioned media of large aneurysms, regardless the

etiology as compared to controls, supporting the immunohisto-logical and immunoblot data (Figure 3C).

DiscussionOur main result is the presence of biological signs of in vivoactivation of both platelets and prothrombin in patients withthoracic ascending aortic aneurysm. This activation is onlypresent when maximal aortic diameter is greater than 45 mmin Marfan patients of young age, devoid or risk factors, butnot observed in MFS patients when maximal aortic diameteris less than 45 mm. It is also present in patients presentinglarge aneurysms of other aetiologies (BAV, Degenerative),and is not dependant on the location of the aneurysm(maximal dilation of the aortic root at the level of sinuses ofValsalva or maximal dilation of the ascending aorta above thesinotubular junction).

In contrast to abdominal aortic aneurysm (AAA), aneu-rysms of the ascending aorta are not associated with a muralthrombus, which could readily explain the presence of circu-lating markers of platelet and prothrombin activation.1 Alsoin contrast with AAA, inflammation, which predominates inthe adventitia of AAA,21 is absent in the aortic wall obtainedfrom TAAA, including the adventitia.1,22 Our peripheralmeasurements of plasma CRP and fibrinogen are in keepingwith this observation.

It is possible that activated platelets and coagulation factorsare more rapidly diluted and washed away by the particularflow conditions in the ascending aorta. Indeed, it appears thatthe triggering event is sufficient to initiate thrombin genera-

A Control Large aneurysm

Control Control Large aneurysm

Large aneurysm

Figure 2. Platelet activation markers. Plateletexpression of P-selectin (CD62P) determinedby flow cytometry using Overton statisticalanalysis. For details please see supplementalmaterials.

Touat et al Coagulation and Ascending Aorta Aneurysm 943

tion and platelet activation but insufficient to permit fibrino-gen clotting and platelet aggregation. Because elevation ofP-selectin, a highly procoagulant mediator,23 is only minimal,it could also limit the onset of coagulation.

The absence of any thrombus or sign of significant inflam-mation in TAAA raises the question of the origin of theprothrombotic markers. Our most clear-cut observation wasrelated to the activation of platelets and prothrombin. The maintrigger of the thrombin generation cascade is tissue factor(TF),24,25 which is constitutively expressed in tissues.26 PlasmaTF concentrations were found to be increased in patients withlarge aneurysms compared to controls. This signifies that higheramounts of active TF are circulating in blood from patients withlarge aneurysms than in blood from controls, but that TF activityis not sufficient to generate a clot, as there was no evidence ofmacroscopic or microscopic thrombus in TAAA patients. Theincreased level of TAT is in favor of in vivo thrombin genera-tion, which is further supported by the elevated level of sGPV,a marker of platelet activation by thrombin.27

The rate of prothrombin activation is dependent on theassembly of tenase and prothrombinase complexes, which inturn depends on the presence of surfaces enriched in negatively-

charged phospholipids.28 The increased number of micropar-ticles of platelet origin observed in association with largeaneurysms is likely to provide the phosphatidylserine-enrichedmembranes required to favor thrombin generation.29 Micropar-ticles are the main link between platelet activation and thrombingeneration.29,30 Elevated levels of sGPV and platelet-derivedmicroparticles in vivo indicate platelet activation by thrombin27

in TAAA. Elevated soluble CD40L (CD154), a transmembraneprotein released by proteolytic cleavage in response to variousstimuli,31 in the plasma of TAAA patients also suggests plateletactivation. sCD40L release is directly related to platelet activa-tion,32,33 depends on metalloproteinase activities34 and triggersTF expression on binding to CD40 on monocytes.35 Neverthe-less, because CD40L can also be released by leukocytes andendothelial cells, a nonplatelet origin of this marker cannot becompletely excluded in our patients. The slight but significantincrease in P-selectin expression on circulating platelets providesadditional evidence for a platelet activation state in TAAA.However, when compared to the situation in AAA patients,16 alower level of proteolytic shedding probably occurs in TAAApatients, because plasma levels of soluble P-selectin were notsignificantly different from control levels.

-2

0

2

4

6

8

10

12

14

16

18TAT (ng/g of wet tissue)

Control Large aneurysm

**

aneurysm (IHC) aneurysm, Alcian blue

Control (IHC)

A

C

B

IIacontrol Large aneurysm

Western-blotanti-prothombin (II) / thrombin (IIa)

1 2

3

IIa

IIFigure 3. Prothrombin/thrombin in the aorticwall. A, Immunohistochemistry of prothrom-bin/thrombin (control and pathological aorticmedia). B, Immunoblot of prothrombin/throm-bin (conditioned media). IIa positive controlof active thrombin. C, Level of thrombin-antithrombin III (TAT) complexes in condi-tioned media (**P�0.01). For details pleasesee supplemental materials.

944 Arterioscler Thromb Vasc Biol May 2008

In older patients (degenerative group) or BAV patientswith associated valve disease, the activation detected may notbe solely related to the ascending aortic aneurysm. Indeed, inthe absence of cases of ascending aortic aneurysm of diam-eter below 45 mm in these groups, we cannot definitively ruleout the presence of platelet activation even when aorticdilatation is limited. However, this activation was not ob-served in the control group, nor was it related to the presenceof risk factors in multivariate analysis. In contrast, Marfanpatients are young and without cardiovascular risk factors orvalve disease. Our MFS patients were divided according tothe maximal aortic diameter (�45 mm) in 2 subgroups ofsimilar age, sex, and both without risk factors or valvulopa-thy. The only difference between these 2 subgroups was thevalue of the dilation of the Valsalva sinuses, suggesting thatactivation of platelets and prothrombin was directly depen-dent on the magnitude of TAAA dilation.

The ascending aortic aneurysm location is interesting,because the normal dilation of Valsalva sinuses induces aparticular, but physiological, disturbed flow. A significantdilation in this area should strongly increase these perturba-tions. Vortices have also been observed in vivo by magneticresonance imaging in aneurysms of the human ascendingaorta.15,36 An abrupt increase in lumen dimension leads tolocalized vortex phenomena characterized by a stagnationpoint and a recirculation zone in vitro.11,37 Jou et al reportedthe correlation between changes in luminal geometry andhemodynamics in fusiform intracranial aneurysms.38

The importance of rheological factors in platelet biology andhemostatic status is now well established.11,39 It was alsodemonstrated earlier that dilation-induced vortical flow couldlead to platelet activation,14,37 via the interaction of GPIIbIIIaand fibrinogen.11,40 This phenomenon could participate in thepoststenotic accumulation of platelets.41 In addition to platelets,vortices also influence red blood cell morphology and aggregateformation.11,42 In particular, red blood cells enhance plateletreactivity43 and release ADP at low shear rates,44 which couldparticipate in platelet activation.45,46 Therefore, this relationshipbetween arterial wall dilation, rheological disturbance, andplatelet activation could account for the observed increase inplasma markers of platelet and prothrombin activation in pa-tients with TAAA, regardless of their localization.16,47

Thrombin is able to increase endothelial cell monolayerdysfunction and permeability.48 The elevation of solubleCD146, a protein involved in endothelial cell-cell interac-tions49 in patients with TAA, suggests endothelial alter-ation.50 This is compatible with an elevation of endothelialpermeability to which in vivo thrombin generation couldcontribute. The increase in endothelial permeability couldallow convection of plasma prothombin/thrombin into theaortic wall from the blood compartment.51 Actually, weevidenced the presence of thrombin inside the pathologicalaortic wall using different approaches. Indeed, TAAAs arecharacterized histopathologically by mucoid degenerationassociated with vacuole formation within the aortic media,whatever their etiology.1,52 The initial pathological processprobably associates smooth muscle cell disappearance, mu-coid degeneration, and extracellular matrix breakdown notlinked to atherosclerosis. The release of TAT into the condi-

tioned media indicated that active thrombin was presentwithin the aortic media (Figure 3) and could play a role inthoracic ascending aortic aneurysm pathology.

In conclusion, large aneurysms of the ascending aorta areassociated with a significant level of platelet activation andthrombin generation which can be detected via a peripheralapproach. This activation process is related to the magnitudeof the aortic dilation, but independent of the etiology of theaneurysm. The generation of thrombin could have a directeffect on endothelial cells and on smooth muscle cells,because we found thrombin within the ascending aorticmedial layer. Exploring the role of this protease in aneu-rysmal progression requires further studies, and may open upthe perspective of new therapeutic targets in patients withaneurysm of the ascending aorta.

AcknowledgmentsWe thank Mary Osborne-Pellegrin for editing this article.

Sources of FundingThis study was supported by grants from the French NationalResearch Agency (ANR), the French Society and Federation ofCardiology (SFC and FFC), the Leducq Foundation and an EU grant:No. 200647, “Fighting Aneurysmal Disease, FAD.”

DisclosuresNone.

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