Tissue Factor Pathway Inhibitor on Circulating Microparticles in Acute

Blood Coagulation, Fibrinolysis and CellularHaemostasis

Tissue factor pathway inhibitoroncirculating microparticles in acute myocardial infarction

Birgit Steppich,Christoph Mattisek, DeanSobczyk, Adnan Kastrati, Albert Schömig,Ilka Ott Deutsches Herzzentrum und 1. MedizinischeKlinik der Te chnischen Universität München, Germany

Summary In acute myocardial infarction (AMI), increasedTissueFactor After thrombolysis increasedD-dimer and F1+2plasma con- (TF) expression on circulatingmonocytesand microparticles centrations indicated activation of fibrinolysis and coagulation. (MP) maycontributetothrombotic events. Because surface- Significance of MPTFPI forinhibitionofTFactivity was measured boundTissueFactor Pathway Inhibitor-1 (TFPI) inhibitsTFactiv- using inhibitoryTFPIantibodies.Membrane-associated TFPI in- ity on monocytes and endothelialcells decreasedTFPIex- hibitedTFactivity on circulatingMPs. After thrombolysis in- pression mayreinforce the procoagulant activity of circulating hibition of TF activitybyTFPIwas decreasedascompared to MP.Aim of the study was to analyze TFPI expression andTFac- stenting. Correlationofcirculating TF with F1+2onlyafter tivity afterstentingand thrombolysis inAMI.Thirty-nine patients thrombolysis,suggests arole forTF-induced activation of coagu- of arandomized study comparing intravenous thrombolysis (n = lationafter thrombolysis.Enhanced TF activityoncirculating 19) and stenting (n =20) were included. Before and after ther- MPs inAMIisinhibited by endogenous surface-boundTFPI.After apybloodsamples foranalysis of MPs,TF antigenand activity, thrombolysis butnot after stenting MPTFPI is degradedand may prothrombin fragment F1+2 and D-dimer were obtained.TFPI induce thrombin generation duetounopposedtissuefactor ac- expression on TF positive MPswas decreasedafter thromboly- tivity.Anti-TFtherapies during thrombolysis mayreduce throm- sisbut not after stenting.Incontrast, TF plasma levels and TF bin generation in AMI. positive MP remainedunchanged in bothtreatment groups.

Keywords Tissuefactor pathway inhibitor (TFPI), Ischaemic heartdisease, hypercoagulability ThrombHaemost 2005; 93: 35-9

Introduction aryintervention (4),decreased TFPI expression on monocytes mayalready be restored and,therefore,not alter thrombin In acutemyocardialinfarction (AMI) earlyreperfusion is the formation. treatment of choice. Theuse of thrombolytic agents restores cor- Increasedlevelsofcirculating TF have beenreported in pa- onaryarterypatencyonlyinabout twothirds of patients with tients with acutecoronary syndromesand maycontribute to sub- AMI. In contrast, mechanical reperfusion with primaryangio- sequent thromboticcomplications (5). plasty and stenting achieveshigherpatencyrates (1). Independently of monocytic TF,blood-borneTFmay playa Tissue Factor (TF)-mediated activation of the coagulation roleinthe propagation of thrombosis (6). CirculatingTF consists

cascade playsakeyrole in intravascularthrombus formation and of soluble alternatively, splicedforms (7) and procoagulant This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. is inhibited by tissue factor pathway inhibitor-1 (TFPI). On cell microparticles(MP) (8)that maysupportthrombus formation. membranes endogenousTFPI supports transient downregulation MPsare small membrane vesicles, releasedfrom blood cells or of the quaternaryTF-VIIa-Xa-TFPIcomplex(2). In aprevious endothelial cells on activation or during apoptosis (8) and sup- study we have shown adecreaseinthe anticoagulant activity of port coagulation by exposure of negatively charged phospholi- TFPI-1oncirculating monocytesafter thrombolysis (3). As TF pids and TF.Ifinhibition of TF activity by endogenous TFPI oc- expression on monocytes is increased not until 96 hafter coron- cursonprocoagulant MP earlyinAMI, degradation of TFPI by

Correspondence to: Received June 24, 2004 I. Ott Accepted after resubmission October 25, 2004 Deutsches Herzzentrum Lazarettstraße36 Financial support: 80636 München, Germany Supportedinpartbygrants from the Deutsche Forschungsgemeinschaft(Ot 158/4–1), Te l.: +49891218 3515, Fax: +49 89 1218 4013 the Bayerische Wissenschaftsministerium (Habilitationsförderpreis I.O.) and the Gesell- E-mail: [email protected] schaftfür Thrombose und Hämostase.

Prepublished onlineDecember 8, 2004 DOI:10.1160/TH04–06–0393

35 Steppich, et al.: TFPI on microparticlesinAMI thrombolysis might enhance TF activity,activate the coagulation Enzygnost D-dimer,Dade Behring,Marburg, Germany). The cascade and,thereby, contribute to reocclusion after thromboly- inter-assayvariability in the lowerassayrangewere <14%.Toin- sis in AMI. hibit endogenous TFPIsamples were preincubated for 60 min The aim of thisstudy,therefore,was to analyze in AMITF with 100 μg/ml of amonoclonalinhibitoryanti-TFPIantibody and TFPI expression on circulating MPsafter thrombolysis and (W.Ruf, San Diego, CA). stenting and to investigatethe role of circulatingTF for thrombin TF activity wasmeasured using aFXa generation assayinthe formation in vivo . absence and presenceof100 μ g/ml rabbit anti-TFPI-1 IgG (kindlyprovided by Dr.W.Ruf) or nonimmune rabbit IgG Methods (Sigma) as described(4). The inter-assayvariability was10%. TF activity in the presenceofanti-TFPImAbs represent thetotal Patientselection capacity of TF activity in the plasma because endogenous in- We included39patients from arandomized study thatcompares hibition is abolished. In the absence of TFPIantibodies TF activ- fibrinolysis with alteplase with stenting plus abxicimab (STO- ity is decreasedaccording to the amount of endogenous TFPI. PAMI I) (9). The study wasapprovedbythe institutional ethics The TF activitythat is regulated by endogenous TFPI is givenas committee for human subjects. Informed consent wasobtained apercentage value relativetothe total cellular TF,definedbythe from all patients. All patients received 500mgofaspirin and following formula: 100 x(TF activityinthe presenceofanti- 5,000Uofheparin intravenously in the emergency room. After TFPImAbs–TF activity in theabsenceofanti-TFPImAbs)/TF randomization patients receivedtreatment with alteplase (n = activity in thepresence of anti-TFPI mAbs. 19) or stenting with abxicimab and ticlopidine (n =20) as described (3).Serial venous blood samples were obtainedbefore Statisticalanalysis and 24–48hafter therapy. All blood samples were putonice and Differences between more than 2matched samples were tested processedimmediately,asindicated below. by Friedman'stestfollowedbyWilcoxon'smatched-pairs signed-ranks test, and differences between the groups by the Microparticleisolation Mann-Whitney-Wilcoxon rank sum test. Ap<0.05 value in the MP were isolatedbysequentialcentrifugation as described (10). two-tailedtestwas regarded as significant. Briefly, sodium citrateblood sampleswere centrifuged at 1,500 gfor 15 min, followedby1min decantation at 13,000 g.Aliquots Results of 20 μ lofthe platelet-free plasma were dilutedin180 μ loffil- tered PBS containing 1mMCaCl 2 . Clinical and angiographic data Patient groups did not differsignificantly in sex, age, risk factor Flowcytometry profile,medication or infarct size and location (Table 1).Stent- MP suspensions were incubatedwith the followingantibodies: ing wassuccessful in all patients restoring TIMI grade 3flow. Fluorescein isothiocynate (FITC)-conjugatedanti-TF,anti-TFPI mAb (American Diagnostica, Pfungstadt,Germany), washed and incubatedwith phycoerythrin (PE)-conjugated Fabfrag- Ta ble1:Baseline characteristics of study patients. ments (Dako) for indirect TFPI-1 labeling. Phosphatidylserine probing wasperformed using annexin VAlexa 568 in appropri- Thrombolysis Stent P atebuffer(Boehringer Mannheim). Incubation with irrelevant mAb wasusedasacontrol. To investigatethe cellular origin of (n=19) (n=20) TF on MP costaining with anti-P1H PE (Chemicon, Marseille, Gender(M/F) 15/4 16/4 0.7 France),anti-CD15 FITC, anti-CD14 PE and anti-CD62 FITC Age,y,(range) 61+16 60+11 0.9 (Beckman Coulter,Krehfeld, Germany) wasperformed (n =3). Active smokers, n(%) 9(47) 10 (50) 0.7 MP were identifiedaccording to their characteristic forward and Hypercholesterolemia,n(%)12(63) 14 (70) 0.7 side scatterand by their ability to bind annexin Vand cell-spe- This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. cific antibodies. Forquantification aknown number of latex Systemichypertension, n(%) 11 (58) 14 (70) 0.5 beads (LB30, Sigma-Aldrich, Schnelldorf,Germany)was added Diabetes mellitus, n(%)2(11)3(25)0.7 to the sample before analysis. Thediameterofthe beads (3 μm) 1-vessel disease 6(32) 9(45) 0.5 discriminated them from the MP population. Analysis was 2-vesseldisease 4(21) 7(35) 0.3 stoppedwhen 200,000 beads were counted and the amount of corresponding MP wasanalyzed. The number of TF-positiveor 3-vesseldisease 8(42) 4(20) 0.1 TF-TFPI-1-double-positive MP in 1 μ lofplasma wascalculated Target vessel: according the followingformula: (MP events/latexbeads events) LAD 6(32) 12 (60) 0.1 x(latexbeads per tube/sample volume). LCx5(16)3(15)0.6 TFPI-1 and D-dimerimmunoassay RCA6(32)5(25)0.9 Plasmaconcentrations of TF,TFPI-1, F1+2and D-dimer were peak CK, U/L (range) 1140(268–4630) 1383 (230–5962) 0.9 determinedbysandwich type immunoassay(IMMUBIND TF CK indicatescreatine kinase. LAD,leftanterior descending coronary artery; LCx, left circumflex and TFPI ELISA, AmericanDiagnostica; Enzygnost F1+2, coronaryartery; RCA, right coronaryartery.

36 Steppich, et al.: TFPI on microparticlesinAMI

From the 19 patients that received thrombolysis 16 underwent coronaryangiographywithin 2weeks and in 5patients occlusion of the coronaryarterywas foundcorresponding to the infarct area.Therefore,inthese patients non-successful thrombolysis maybeassumed.

TF andTFPI expressiononcirculating MP After thrombolysis asignificant decreaseofsurface-boundTFPI on TF positiveMPwas observed,whereas, no changes occurred after stenting. ArepresentativeFACSanalysis is shown in figure 1: Figure 1A shows the characteristic light scatter propertiesof MP`s and latexbeads thatwere used as an internal standard while figure 1B demonstrates an exampleofTF-positive MP.Infigure 1C-D the TFPIexpression on TF-positiveMP`safter thrombolysis and stenting inAMI are shown in representativehistograms. The effect of thrombolysis on the numberofTF-positive and the number of TF and TFPI-positive circulating MP`s wascom- pared to the effect of stenting. After thrombolysis asignificant decreaseinTFand TFPI-positiveMPwas found, whereas after stenting no significant changes occurred (Fig. 2A). Theamount of TF containing MP`s did not differinboth treatment groups either before nor after the treatment (Fig. 2B).

Thrombingeneration and fibrin degradation in vivo The extent of fibrinolysis in AMI wasmeasured by analysis of D-dimerplasma levels, activation of the coagulation cascade by analysis of prothrombin fragment F1+2. As expected after thrombolysis asignificant increase in circulating D-dimerplas- Figure1:Flowcytometricquantification ofTF positiveMP . ma levels wasfound,whereasafter stenting no changes occurred MP arediscriminatedbysize according to theforwardand side scatter. Quantificationwas performed using adefinednumberoflatex beads (Fig. 3A), documenting astrong activation of the fibrinolytic (A). Detection of TF positive MP after staining withanti-TF FITC anti- system following alteplase administration. Compared to stenting bodiesinrelationtothe side scatter properties (B). Representative thrombolysis wasassociatedwith higher prothrombinfragment histogramofTFpositiveMPfromsamplesobtainedbeforeand after F1+2plasma levels by trend (Fig. 3B).This, consequently, indi- thrombolysis (C) andhistogramofTFpositiveMPfromsamplesob- cates thrombin generation and mayreflect the prothrombotic ef- tainedbeforeand after stenting (D). fectsofthrombolytic treatment (19).

CirculatingTF antigenand activity To investigate, whether the amount of circulating TF is altered during thrombolysis or stenting, we measured plasma concentrations of TF.Noeffect of eithertherapywas found(Fig. 4A). Becauseendogenous TFPIassociatedwith MPs inhibits TF activity,weanalyzedtotal TF activity in the presenceand the ab- senceofanti-TFPIantibodies. We measured the residual TF ac-

tivity as the TF activity thatremains after inhibition of endogen- This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. ousTFPI.The difference betweentotal and the residual activity reflects the TF activity that is inhibited by TFPI and,therefore, reflectsthe functional significance of endogenous TFPI for the inhibition of TF activity.After thrombolysis the amount of TF thatwas inhibitedbyTFPI wassignificantly reduced,whereas after stenting no changes were found.These resultsdemonstrate the functionalsignificance of the TFPI decreaseoncirculating MP`s after thrombolysis. Figure2:TFPI expression on circulatingTF positiveMPisde- creased afterthrombolysisinAMI.The numberofcirculating TF and Relationship of circulatingTFand thrombingeneration TFPI positiveMP(A) or TF positive MP (B)weremeasured before and in vivo after thrombolysis andstenting in AMI. Shownare theindividual values from each patient.Pvaluesrevealstatisticalsignificance. To investigate, whether decreased TFPI on circulating MP`s in- deed results in aimbalancebetween the TF and TFPI pathway potentiallycontributing to thrombin generation in vivo we ana-

37 Steppich, et al.: TFPI on microparticlesinAMI

Discussion The major findings of our study are: 1.After thrombolysis inAMITFPIexpression onTF positiveMP is decreased,whereas,after stenting no changeswere observed. 2. Functional significance of this decreasedTFPI expression was shown by adiminished inhibition ofTF activity by TFPI after thrombolysis as compared to stenting. 3. The correlation of circulating TF with prothrombin fragment F1+2 after thrombolysis only, suggests arole for TF-induced activation of coagulation after thrombolysis.

Figure3:D-dimer and prothrombinfragmentF1+2 arein- Plasma TFPI circulatesinvarioustruncated forms mostly bound creased afterthrombolysisinAMI.Plasma concentrations of to lipoproteins. However, the majority ofTFPI is produced by en- D-dimer(A) or prothrombinfragment F1+2 (B)weremeasured before dothelialcells and remains associatedwith the endothelialsur- and after thrombolysis andstenting in AMI.Valuesare expressedas face (11).Cell-bound TFPI is believedtoplayacritical role in mean ±SEM. Pvaluesrevealstatisticalsignificance. regulating surface TF-FVIIa and FXaactivity by transient and reversibletranslocation of the quaternarycomplexTF-FVIIa- FXa-TFPI into glycosphingolipid-rich microdomains(2, 4). lyzed the association of circulating TF with plasma F1+2con- This mechanism occurs on the surfaceofendothelial cells as centrations.After thrombolysis there wasasignificant cor- well as on monocytes. Among others, MP derive from mono- relation with TF plasma levels and F1+2concentrations (Fig. 5). cytesand endothelial cells (10, 12). Thus, it seems conceivable No association wasfound in patients after stenting in AMI (R = thatsimilarregulatorymechanisms occur on the MP surface as –0.4, P=0.59). Furthermore,nocorrelation wasobservedbefore on intact cells. RecombinantTFPI hasbeen shown to be proteo- therapyinboth treatment groups.These data areanindication for lytically degraded by plasmin, elastase and matrix metalloprotei- an ongoing TF inducedclotting activation followingthromboly- nases(13–15). Moreover,inacell-basedsystem we and others sis in vivo . have shown thatTFPI is degraded on endothelialcells in vitro and on circulating monocytesafter thrombolysis in vivo (3,21, Relationship betweenTFPI degradationand failure of 22). Degradation of MP and cellassociatedTFPI after throm- thrombolysis bolysis may, therefore, attenuate an important pathway of vascu- To analyze if degradation of TFPI is associatedwith successful lar surface thromboresistance. Whether diminished TFPI con- thrombolysis patients thatangiographically showedanoccluded tributestothrombin formation, or whether decreased TFPI oc- vessel2weeks after acutemyocardialinfarction were compared cursasanepiphenomenon has not beenaddressed so far. Mono- with patients thatshowedanopen vessel(Table2). No signifi- cytic TF is induced during the causeofAMI butoccursonly96 cant differences in TF positive MP,TFand TFPIpositiveMP, TF hafter recanalization of the infarct-related coronaryarteryby antigen or TF activityregulated by TFPI were detected in both stenting (4,16). Thus, the earlythrombin generation after throm- groups after thrombolysis. Furthermore,activation of coagu- bolysis, observedinthis study,cannot be attributedtomonocytic lation and fibrinolysis as assessed by prothrombin fragment TF activity.Previousstudieshaveshown an increased number of F1+2 and D-dimer wassimilar. circulating procoagulant MP in acute coronarysyndromes(8). This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

Figure4:Decreased inhibitionofTFactivity by TFPI after thrombolysis in AMI .Plasma TF Antigen (A) and TFPI dependent in- hibitionofTFactivity (B) were measured before and after thrombolysis Figure5:Association of circulatingTF and plasma prothrombin andstenting in AMI.Valuesare expressedasmean±SEM.Pvaluesreveal fragmentF1+2 afterthrombolysis. Shown arethe individual values statisticalsignificance. from each patient.

38 Steppich, et al.: TFPI on microparticlesinAMI

Our resultsextend these observations showingthat TF positive Ta ble2:TFandTFPI positiveMP, D-dimer, Prothrombin frag- MP express TFPI. Moreover,degradation of TFPI on MP after ment F1+2,TFantigen and TF-dependentinhibition of TF activ- thrombolysis contributestothrombin formation due to unop- ityand outcomeofthrombolysis. Shown arethe valuesafter thrombolysis. Valuesrepresent mean +SEM. posed TF activity.Thus, inhibition of TF activity by TFPI occurs on MP and impedes thrombin generation. Onlydegradation of TFPI during thrombolysis allows circulating TF to add to throm- Successful Non-successful P bin formation. thrombolysis thrombolysis (n=11) (n=5) Recently,apathway for the propagation of blood coagulation after endothelial injuryhas been proposed thatinvolves the ac- TF positiveMP/μl 627+04 657+40 0.3 cumulation and localization of TF positive MP on the platelet TF and TFPIpositive 48+6 59+2 0.2 thrombus by amechanism involving CD15and P-selectin (6, MP /μl 17). Moreover,deliveryofcirculating TF into adeveloping D-Dimer [μg/l] 393+73 484+1700.9 thrombus occurred throughinteraction of plateletP-selectinand Prothrombinfragment 2.4+0.25 3.2+0.60.6 P-selectin glycoprotein ligand 1onmonocytic MP (18).These F1+2 [nmol/l] mechanisms provide aconcept, howunopposed TF activity after TF antigen [ng/l] 300+73 261+56 0.11 degradation of TFPI on MPs maycontribute to rethrombosis. TF-dependent in- 38+20 58+8 0.7 Procoagulant MP in AMI contributeonlypartiallytocircu- hibition of TF activity lating TF activity: Arecently identifiedsoluble, splicedvariant [%] of TF addstocirculating TF and maycontribute to aprocoagu- lant state (7). Bolusorcontinuous administration of heparin has been Althoughinour study with onlyalimitednumber of patients shown not to altersurfaceTFPI expression orTFPIplasma levels no relationshipbetween TFPI degradation on MP and failureof (4, 20,21). Therefore, continued heparin administration after thrombolysis wasfound,further studies with alarger study popu- thrombolysis maynot contribute to the observedchangesinTFPI lation are needed,toinvestigatethe clinicalrelevance of the ob- expression. Furthermore,medication between the studygroups servedmechanism. Significance in clinical studieswould pro- differed in respect to GPIIb/IIIa antagonistsand ticlopidine. Al- vide the rationale for novelstrategiestoimproveefficacyof though ticlopidine has beenshown to contribute to activation of thrombolytic therapy. Enhancement of endogenous TFPI activ- TF in vitro(24), we did not find anychangesinTFactivity before ity,administration of exogenous excessTFPI or anti-TF ther- and after ticlopidine or GPIIb/IIIa antagonistsinthe stented pa- apies is thenconceivable. tients in vivo .Inaddition, surfaceTFPI-1 wasnot affectedbythe interventional procedureitself (4). These result suggest that Acknowledgments changes in TFPI expression maybeconsideredasaresultoffi- We thank W. Ruffor the inhibitoryanti-TFPI antibody.Wealsothank Mrs brinolysis. B. Campbell, A. Stobbe and C. Bauer forinvaluable technical assistance.

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