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Delayed thrombin-induced platelet�fibrin clotgeneration by clopidogrel: A new dose-relatedeffect demonstrated by thrombelastography inpatients undergoing coronary artery stenting

Paul A. Gurbel a,*, Kevin P. Bliden a, Kirk Guyer b, Nikhil Aggarwal a,Udaya S. Tantry a

a Sinai Center for Thrombosis Research, Hoffberger Building, Suite 56, 2401 W. Belvedere Ave,Baltimore, MD 21215, USAb Department of Chemistry, Indiana University, South Bend, IN, USA

Received 25 January 2006; received in revised form 3 May 2006; accepted 9 May 2006

0049-3848/$ - see front matter D 200doi:10.1016/j.thromres.2006.05.006

* Corresponding author. Tel.: +1 410E-mail address: PGURBEL@LIFEBRID

KEYWORDSPlatelets;Clopidogrel;Thrombin;Clot;Aggregation

Abstract

Background: We have previously demonstrated that clopidogrel reduces plateletactivation and aggregation in patients undergoing stenting. However, the effect ofthe clopidogrel loading dose on the rate of thrombin-induced platelet—fibrin clotformation is unknown in this patient population.Methods and results: Using thrombelastography (TEGR) we measured the time toplatelet—fibrin clot formation (R), a marker of the speed of thrombin generation, in120 patients undergoing elective coronary artery stenting treated with standard andhigh loading doses of clopidogrel. Platelet reactivity to adenosine diphosphate (ADP)by light transmittance aggregometry (LTA) was determined simultaneously. Measure-ments were made immediately before and at 24 h after clopidogrel treatment.Clopidogrel produced a prolongation in R (4.4F1.4 min pre vs. 5.4F1.7 min post,p b0.001) that directly correlated with the change in platelet aggregation (r =0.65,p b0.0001). Prolongation in R was greatest in patients treated with a high loadingdose ( p =0.004).Conclusions: Delayed thrombin-induced platelet—fibrin clot formation as measuredby TEG is a newly reported dose-related effect of clopidogrel that may contribute tothe overall antithrombotic properties of the drug in patients undergoing stenting. Thiseffect was more marked in patients loaded with 600 mg, lending further mechanistic

ARTICLE IN PRESSThrombosis Research (2006) xx, xxx—xxx

6 Elsevier Ltd. All rights reserved.

601 9600; fax: +1 410 601 9601.GEHEALTH.ORG (P.A. Gurbel).

TR-02978; No of Pages 8

ARTICLE IN PRESSP.A. Gurbel et al.2

support for this dose of clopidogrel as a more effective antithrombotic regimen thanthe standard 300 mg dose. Measurement of R may serve as a new indicator ofclopidogrel responsiveness.D 2006 Elsevier Ltd. All rights reserved.

Introduction

Clopidogrel is a thienopyridine that selectivelyinhibits the platelet P2Y12 receptor and reducesischemic complications in patients with unstablecoronary syndrome. Thienopyridines also reducethe incidence of stent thrombosis [1—5]. Theprimary mechanism proposed for the beneficialeffects of clopidogrel is inhibition of ADP-inducedaggregation. The results of several studies havealso suggested that clopidogrel treatment may haveanti-inflammatory effects [6—8].

Our group and others have demonstrated thatclopidogrel attenuates platelet activation followingcoronary stenting andwe have also shown that a highloading dose produces greater platelet inhibition asmeasured by conventional light transmittanceaggregometry [9—11]. Since thrombin generationoccurs mainly on the surface of activated plateletsand platelet—monocyte aggregates, clopidogreltherapy may exert an antithrombotic effect throughattenuation of platelet activation with a resultantdecline in surface-generated thrombin and throm-bin-induced platelet—fibrin clot formation. Howev-er, the latter effect of clopidogrel has not beenextensively studied in patients following coronarystenting. Moreover, the effect of various clopidogrelloading doses on the rate of thrombin-induced clotformation is entirely unknown. Therefore, the aimsof our investigation were to measure the onset ofthrombin induced platelet—fibrin clot formation bythrombelastography, a marker of the speed ofthrombin generation, in patients undergoing coro-nary stenting treated with clopidogrel and analyzethe relation to clopidogrel loading dose and extentof platelet inhibition.

Methods

Patients

One hundred and twenty consecutive patientsundergoing non-emergent coronary stenting provid-ed informed consent prior to the procedure. TheInvestigational Review Board at Sinai Hospital ofBaltimore approved this study. All patients were N18years old and had to have been treated with a 81—325 mg daily dose of aspirin for at least 5 days prior

to the procedure. Exclusion criteria were a history ofbleeding diathesis, acute myocardial infarctionwithin 48 h, elevated cardiac markers (above upperlimits normal for the respective assay), cerebrovas-cular eventwithin 3months, chronic vessel occlusionor angiographically visible thrombus, illicit drug oralcohol abuse, prothrombin time greater than 1.5times control, platelet count b100,000/mm3, he-matocrit b30%, creatinine N4.0 mg/dl, glycoprotein(GP) IIb/IIIa inhibitor use, and thienopyridine useprior to the procedure. Patients treatedwith heparinor lowmolecular weight heparin prior to or after thecompletion of the procedure were also excluded.

Of the 120 patients, 73 were treated with astandard (300 mg) loading dose of clopidogrel and47 were treated with a high loading dose (600 mg)at discretion of the treating physician. Clopidogrelwas administered in the catheterization laborato-ry immediately after successful stent implanta-tion. All patients received unfractionated heparinas an intravenous bolus to achieve an activatedclotting time z300 s in the catheterizationlaboratory. Aspirin was administered at 325 mgon the day of the procedure and daily thereafter.The maintenance dose of clopidogrel was 75 mgdaily. Patients treated with a GPIIb/IIIa inhibitorin the catheterization laboratory were excludedfrom the analysis.

Blood sampling

Pre-treatment blood samples were obtained in thecatheterization laboratory before heparin adminis-tration through the indwelling femoral vesselsheath. The blood samples were transferred tovacutainer blood collecting tubes (Becton — Dick-inson) containing 3.8% trisodium citrate for lighttransmittance aggregation studies or 40 USP lithi-um heparin for TEG studies after discarding thefirst 2—3 ml of free flowing blood. Blood sampleswere also collected at 24 h post-stenting. Thevacutainer tubes were filled to capacity and gentlyinverted 3 to 5 times to ensure complete mixing ofthe anticoagulant.

Light transmittance aggregometry

Platelet aggregation was assessed as previouslydescribed [9—11]. Briefly, the blood-citrate tubes

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Figure 1 Schematic of TEG system. A torsion wire suspending a pin is immersed in blood. As the clot forms whilethe cup is rotated 458, the pin will rotate depending on the strength of the fibrin—platelet bonds. A signal isdischarged continuously that reflects the onset of clotting (reaction time, R) and the clot strength (MA).

Table 1 Patient demographics

300 mgclopidogrel(n =73)

600 mgclopidogrel(n =47)

p

Age (years) 66F12 67F12 NSRace (Caucasian) (%) 66 73 NSGender (male) (%) 61 70 NSBMI 30F8 28F5 NSRisk factors/past

medical history (%)Smoking 46 60 0.068Family history of CAD 40 48 NSHypertension 53 69 0.042Hyperlipidemia 65 77 0.082Diabetes 45 36 NSPrior myocardialinfarction

27 28 NS

Prior CABG 26 24 NSPrior PTCA 36 41 NS

Pretreatmentmedications (%)Beta blockers 72 86 0.037ACE inhibitors 56 59 NSCalcium blockers 20 27 NSLipid lowering agents

3A4 pathwaymetabolized 71 76 NS

Non 3A4 pathwaymetabolized 6 9 NS

Laboratory dataWBC (�1000/mm3) 7.8F2.6 7.3F2.1 NSPlatelets(�1000/mm3)

237F36 220F30 NS

Hemoglobin (g/dl) 15.4F2 18.4F2 NSCreatinine (g/dl) 1.1F0.8 1.2F0.4 NS

Data reported as meanFS.D.

ACE=angiotensin converting enzyme. BMI=body mass index.CABG=coronary artery bypass graft surgery. CAD=coronaryartery disease. PTCA=percutaneous coronary angioplasty.WBC=white blood cells. 3A4=hepatic cytochrome P 450 3A4.

Thrombelastography and platelet�fibrin clot generation by clopidogrel 3

were centrifuged at 120�g for 5 min to recoverplatelet rich plasma (PRP) and further centrifugedat 850�g for 10 min to recover platelet poorplasma (PPP). Platelets were stimulated with 5AM and 20 AM ADP and the aggregation was assessedusing a Chronolog Lumi-Aggregometer (Model 490-4D) with the Aggrolink software package (Chrono-log, Havertown, PA). Aggregation was expressed asthe maximum percent change in light transmit-tance from baseline using PPP as a reference.

Clot strength and fibrin generation time

The TEG Hemostasis System (Haemoscope Corpora-tion, Niles, IL, USA) with automated analyticalsoftware provides quantitative and qualitativemeasurements of the physical properties of a clot[12—14]. In essence, the TEG is a viscoelasticmonitor that measures the degree of platelet—fibrin mediated clot strength. The platelet—fibrinnetwork in the blood sample links a rotating samplecup with a stationary pin suspended by a torsionwire (Fig. 1). The torque of the rotating cup istransmitted to the immersed pin. Pin movement isconverted to an electrical signal by a transducerand is interpreted by the computer to create atracing. The degree of platelet contribution to theclot strength through platelet—fibrin bonding di-rectly influences the magnitude of pin movementand ultimately the amplitude of the tracing. In thepresent study, the time from the start of thesample run to the first significant levels of clotformation [reaction time (R) (min)] was measured(Fig. 1). The R parameter, a measure of time toinitial thrombin induced platelet—fibrin clot for-mation, has been correlated with the velocity ofthrombin generation [12].

About 1 mL of heparinized blood was transferredto a vial containing kaolin and mixed by inversion.500 AL of the activated blood were then transferred

to a vial containing heparinase and mixed toneutralize the heparin effect. The neutralizedblood (360 AL) was immediately added to a heparin-ase-coated cup and assayed in the TEG analyzer

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Table 2 Procedural characteristics

300 mgclopidogrel(n =73)

600 mgclopidogrel(n =47)

p

Length of procedure(min)

69F28 58F26 NS

Ejection fraction (%) 50F10 52F8 NSNumber of vesselstreated

1.2F0.5 1.3F0.6 NS

Lesion morphologyDe novo (%) 91 86 NS

Lesion location (%)LAD 39 29 NSCX 26 23 NSRCA 27 40 0.07SVG 8 8 NS

Stent types (%)Drug eluting 64 75 NSBare metal 27 23 NSPTCA only 9 2 NSReference vesseldiameter (mm)

3F0.5 3.1F0.4 NS

Total lesionlength (mm)

21F16 18F12 NS

Pre-stenosis (%) 86 85 NSPost-stenosis (%) 6 5 NSProcedural success(%)

96 95 NS

Data reported as meanFS.D.

CX=circumflex artery. LAD=left anterior descending artery.RCA=right coronary artery.

Figure 2 (A) Distribution of the absolute change inplatelet aggregation (5 AM ADP). The curve indicates thebest fit for a normal distribution as generated by theStatistica software. (B) Distribution of the absolutechange in platelet aggregation (20 AM ADP). The curveindicates the best fit for a normal distribution asgenerated by the Statistica software.

P.A. Gurbel et al.4

according to the manufacturer’s instructions toobtain the thrombin-induced clot.

Definitions

Absolute change in platelet aggregation

¼ Baseline platelet aggregation

� Post�treatment platelet aggregation:

Absolute change in R ¼ Baseline R

� Post�treatment R:

Statistical analysis

Comparisons were made between baseline andpost-clopidogrel measurements, and betweenpatients receiving 300 mg and 600 mg clopidogrelby t-tests (Statistica software). Based on thenormal distribution of data the meanFS.E.M. isreported. Pearson’s correlation coefficient wascalculated to describe the relation between theabsolute change in platelet aggregation at 24 hpost-stenting to the absolute change in R (Statisticasoftware).

Results

Patients and clinical outcomes

One hundred and twenty patients underwentcoronary stenting and had blood samples analyzed.The analyses were 100% complete for all measure-ments. The demographics and procedural charac-teristics of patients treated with 300 mg and600 mg clopidogrel are shown in Tables 1 and 2,respectively. There were no major differencesbetween the two groups except more patients in600 mg group were hypertensive (p =0.042) andreceiving beta blockers (p =0.037).

Platelet aggregation

5 and 20 AM ADP-induced aggregation were signif-icantly reduced by clopidogrel therapy in theoverall group (p b0.001 for both agonist concen-trations, Table 4). There was a wide variability in

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Figure 3 (A) Distribution of post-treatment plateletaggregation as measured by 5 AM ADP-induced aggrega-tion. The curve indicates the best fit for a normaldistribution as generated by the Statistica software. (B)Distribution of post-treatment platelet aggregation asmeasured by 20 AM ADP-induced aggregation. The curveindicates the best fit for a normal distribution asgenerated by the Statistica software.

Thrombelastography and platelet�fibrin clot generation by clopidogrel 5

the extent of the absolute change in aggregationand post-treatment aggregation as measured by 5AM and 20 AM platelet aggregation (Figs. 2A and Band 3A and B, respectively). The higher loadingdose produced lower post-treatment aggregation(p =0.03 for 5 AM ADP and p =0.009 for 20 AM ADP,Table 4) and greater absolute inhibition (p =0.002for 5 AM ADP and p =0.003 for 20 AM ADP, Table 3).

Reaction time

Clopidogrel therapy significantly prolonged R in theoverall group (p b0.001, Table 4) and the effect

Table 3 Effect of clopidogrel on absolute change in ADP-fibrin clot formation

Absolute change in platelet aggregation (5 AM ADP)Absolute change in platelet aggregation (20 AM ADP)Absolute change in time to platelet—fibrin clot formation (min)

was greater in patients treated with a 600 mgloading dose (p =0.004, Table 4). The absolutechange in R was also more pronounced in patientstreated with 600 mg clopidogrel (p =0.01, Table 3).

Relation of platelet aggregation to timeto platelet�fibrin clot formation

Strong concordance was demonstrated betweenthe absolute change in R and the absolute changein platelet aggregation after stimulation by both 5and 20 AM ADP (r =0.65, p b0.0001, Fig. 4A andr =0.62, p b0.0001, Fig. 4B, respectively).

Discussion

The current study demonstrates that clopidogreltherapy has an important inhibitory effect on therapidity of ex vivo fibrin—platelet clot formation, amarker of the speed of thrombin generation. Theabsolute change in the time to initial clot formationwas directly and strongly related to the degree ofplatelet inhibition. Patients treated with a higherloading dose of clopidogrel exhibited lower post-treatment aggregation and greater platelet inhibi-tion. In turn, these measurements directly corre-lated with a prolonged post-treatment R and agreater change in R. All of the above data suggestthat patients who are poorly responsive to clopi-dogrel as measured by light transmittance aggreg-ometry may also be at risk for thrombosis due torapid formation of a platelet—fibrin clot.

Platelets undergo activation-dependent process-es essential for the formation of a stable hemo-static plug during physiological conditions andstable thrombus generation during pathologicalprocesses. During the initial activation processes,platelets express phosphatidylserine, and P-selec-tin and CD-40L that facilitate the formation ofplatelet—monocytes aggregates. Both the phospha-tidylserine and platelet—monocytes aggregatesprovide the necessary environment for tissue factorexpression and thrombin generation [15,16]. Thus,

induced platelet aggregation and the time to platelet—

Totalgroup(n =120)

300 mgclopidogrel(n =73)

600 mgclopidogrel(n =47)

p-value(300 mg vs.600 mg)

21.0F1.6 16.4F1.8 26.2F1.6 0.00217.8F1.7 13.6F1.5 22.7F1.8 0.0030.91F0.17 0.73F0.15 1.42F0.14 0.01

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Table

4Effect

ofclopidogreltreatmentonpost-treatmentplateletag

grega

tionan

dthetimeto

platelet—

fibrinclotform

ation

Totalgroup

300mgclopidogrel

600mgclopidogrel

p-value

(300mgvs.600mg)

Baseline

Post-treatment

p-value

Baseline

Post-treatment

p-value

Baseline

Post-treatment

p-value

Baseline

Post-treatm

ent

5Am

ADP-induce

dag

grega

tion

59.8

F1.4

38.8

F1.4

b0.00

157

.1F1.4

41.7

F1.9

b0.00

162

.2F1.5

36.1

F2.1

b0.001

NS

0.03

20AM

ADP-induce

dag

grega

tion

74.5

F1.1

56.8

F1.5

b0.00

172

.5F1.1

60.2

F1.7

b0.00

176

.1F1.0

53.4

F2.3

b0.001

NS

0.009

Tim

eto

platelet—

fibrin

clotform

ation(m

in)

4.4F1.4

5.4F1.7

b0.00

14.3F1.4

5.0F0.2

b0.01

4.5F1.5

5.9F0.26

b0.001

NS

0.004

Figure 4 (A) Relation of absolute change in time toplatelet—fibrin clot formation (R) to absolute change inplatelet aggregation (5 AM ADP). (B) Relation of absolutechange in time to platelet—fibrin clot formation (R) toabsolute change in platelet aggregation (20 AM ADP).

P.A. Gurbel et al.6

activated platelets influence the rate of thrombingeneration that is critical for the formation of astable clot. In addition, P2Y receptors and espe-cially P2Y12 receptors, play important roles in theprocoagulant activity of platelets [17,18]. Howev-er, the role of antiplatelet therapy particularly,P2Y12 receptor antagonists, in attenuating plateletprocoagulant activity is unknown. Moreover, theeffect of a P2Y12 on the rate of thrombin-inducedplatelet—fibrin clot generation during percutane-ous intervention has not been previously investi-gated. However, antiplatelet therapy especially,treatment with clopidogrel, is known to attenuateplatelet activation, P-selectin expression andplatelet—monocytes aggregation in patients under-going stenting [6—11]. In an attempt to assess theantithrombotic effects of clopidogrel in patientsundergoing stenting, assays that measure plateletfunction, such as aggregometry or determine ADP-induced receptor expression, such as flow cytom-etry, have been most widely employed. However,the latter methods are limited because they do notassess the effects of clopidogrel on platelet—fibrininteractions. Our study was the first to usethrombelastography to measure the kinetics of exvivo fibrin—platelet clot generation in patientsundergoing coronary stenting with two different

ARTICLE IN PRESSThrombelastography and platelet�fibrin clot generation by clopidogrel 7

loading doses of clopidogrel. Previous studies havedemonstrated that the R is a direct indicator of thekinetics of thrombin generation [12]. Our dataprovide further evidence in support of a 600 mgloading dose of clopidogrel as a superior antith-rombotic regimen in patients undergoing electivecoronary artery stenting.

Our data are concordant with animal and humanstudies demonstrating that the P2Y12 receptorplays an important role in the initiation of coagu-lation [19—22]. Herault et al. showed that clopi-dogrel inhibited ex vivo thrombin generationstimulated by low concentrations of tissue factor[19]. Wegert et al., in human volunteers using anautomated endogenous thrombin potential (ETP)model, showed that clopidogrel reduced ETPgenerated by various agonists [20]. Leon et al.have shown that ex vivo inhibition of P2Y12 byclopidogrel reduced the expression of tissue factoron leukocytes after stimulation of whole blood withfibrillar collagen [21]. Our data are also concordantwith those of Sollier et al. who demonstrated thatclopidogrel reduced ex vivo thrombus cohesion; aneffect that was more marked in volunteers treatedwith a 600 mg loading dose as compared to a300 mg dose [22]. In contrast, clopidogrel treat-ment was not associated with decrease in thrombingeneration in a study by Eikelboom et al. Theseinvestigators suggested that early suppression ofcoagulation markers was related to the anticoag-ulant therapy [23]. However, recently by usingnovel thrombin generation experiments, it wasfound that suppression of thrombin generation isdependent on the platelet response to clopidogreland clopidogrel nonresponsive patients have sub-dued suppression of thrombin generation [24]. Inthe present study, the clopidogrel effect onplatelet—fibrin clot formation was measured at24 h when an anticoagulant effect of heparinshould not be present. It has been reported thatrebound effects of thrombin generation and ahypercoagulable state immediately after cessionof anticoagulant therapy can be attenuated byclopidogrel but not aspirin treatment [25].

Limitations

Thrombin generation and tissue factor expressionwere not directly determined in our study. How-ever, work by others has demonstrated the directrelation of R to thrombin generation [12]. Our studywas too small to correlate clinical events with themarkers we measured. Moreover, there are limiteddata to support a superior clinical effect from600 mg as compared to 300 mg [8,11,26]. A finallimitation is that our study was observational and

patients were not randomly assigned to 300 mg or600 mg clopidogrel treatment.

Conclusions

Clopidogrel strongly attenuated the time to ex vivoinitial platelet—fibrin clot generation (R) inpatients undergoing coronary stenting. This prop-erty directly correlated with the extent of inhibi-tion of platelet aggregation and was more markedin patients loaded with a 600 mg, lending furthersupport for this dose of clopidogrel as a moreeffective antithrombotic regimen than the stan-dard 300 mg. This effect was more marked inpatients loaded with 600 mg, lending furthermechanistic support. The contribution of attenuat-ed thrombin-induced platelet—fibrin clot formationto the overall clinical antithrombotic effect ofclopidogrel deserves further study. Measurementof R may serve as a new indicator of clopidogrelresponsiveness.

Acknowledgements

The study was supported by National Institute ofHealth (NIH) grant 5R44HL059753-03 and a grantfrom Haemoscope Corporation, Niles, IL.

Disclosure:Dr. Paul A Gurbel received research grants from

Haemoscope Corporation, Niles, IL and ScheringPlough Pharmaceuticals, Inc, Cambridge, MA.

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