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Effect of Cytochrome P450 Polymorphisms on Platelet ReactivityAfter Treatment With Clopidogrel in Acute Coronary Syndrome

Corinne Frere, MD, PhDa,b,c, Thomas Cuisset, MDd,*, Pierre-Emmanuel Morange, MD, PhDa,b,c,Jacques Quilici, MDd, Laurence Camoin-Jau, MD, PhDe, Noémie Saut, PhDa,b,c,

Dorothee Faille, PhDa,b,c, Marc Lambert, MDd, Irène Juhan-Vague, MD, PhDa,b,c,Jean-Louis Bonnet, MDd, and Marie-Christine Alessi, MD, PhDa,b,c

Genetic polymorphisms of cytochrome P450 (CYP) isoforms may promote variability inplatelet response to clopidogrel. This study was conducted to analyze, in 603 patients withnon–ST elevation acute coronary syndromes, the effect of CYP3A4, CYP3A5, andCYP2C19 gene polymorphisms on clopidogrel response and post-treatment platelet reac-tivity assessed by adenosine diphosphate (ADP)–induced platelet aggregation, vasodilator-stimulated phosphoprotein phosphorylation index, and ADP-induced P-selectin expres-sion. The CYP2C19*2 polymorphism was significantly associated with ADP-inducedplatelet aggregation, vasodilator-stimulated phosphoprotein phosphorylation index, andADP-induced P-selectin expression in recessive (p <0.01, p <0.007, and p <0.06, respec-tively) and codominant (p <0.08, p <0.0001, and p <0.009, respectively) models, but theCYP3A4*1B and CYP3A5*3 polymorphisms were not. The CYP2C19*2 allele carriersexhibited the highest platelet index levels in multivariate analysis (p � 0.03). Aftercovariate adjustment, the CYP2C19*2 allele was more frequent in clopidogrel nonre-sponders, defined by persistent high post-treatment platelet reactivity (ADP-induced plate-let aggregation >70%; p � 0.03). In conclusion, the present data suggest that theCYPC19*2 allele influences post-treatment platelet reactivity and clopidogrel response inpatients with non–ST elevation acute coronary syndromes. © 2008 Elsevier Inc. All rights

reserved. (Am J Cardiol 2008;101:1088–1093)

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lopidogrel is a thienopyridine that inhibits platelet activa-ion through an irreversible blockage of the platelet adeno-ine diphosphate (ADP) P2Y12 receptor. Clopidogrel is annactive prodrug that requires several biotransformationteps.1–3 After intestinal absorption, clopidogrel’s biotrans-ormation into its active metabolite is mediated mainly byepatic cytochrome P450 (CYP). Platelet inhibition withspirin and clopidogrel has significantly reduced recurrentschemic events after non–ST elevation acute coronary syn-romes.4,5 Numerous studies have reported interindividualariability in platelet response to clopidogrel,6–10 with clin-cal relevance.11–17 However, the mechanisms underlyinghe variability of response to clopidogrel remain unclear,nd genetic factors might be involved. Previous data haveemonstrated the contribution of hepatic CYP metabolicctivity to the phenomenon of “clopidogrel resistance.”18

ecause the metabolic activity of CYP3A4 is geneticallyegulated, variations in genes supporting the liver’s metab-lism of clopidogrel may be involved. Several functionalolymorphisms have been described in genes encodingYP. Previous studies reported that CYP3A4*1B and

aINSERM U626; bFaculté de Médecine; cLaboratoire d’Hématologiend dDepartement de Cardiologie, CHU Timone; and eLaboratoire’Hématologie, Hôpital Conception, Marseille, France. Manuscript re-eived September 17, 2007; revised manuscript received and acceptedovember 21, 2007.

*Corresponding author: Tel: 33-491385974; fax: 33-491254336.

lE-mail address: thomascuisset@voila.fr (T. Cuisset).

002-9149/08/$ – see front matter © 2008 Elsevier Inc. All rights reserved.oi:10.1016/j.amjcard.2007.11.065

YP3A5*3 polymorphisms did not explain the variabilityn the platelet inhibitory effects of clopidogrel.19,20 More-ver, recent data have shown that the CYP2C19*2 loss-of-unction allele is associated with a marked decrease inlatelet response to clopidogrel in young, healthy maleolunteers.21 Therefore, we conducted a prospective studyo assess the effect of those CYP polymorphisms on clopi-ogrel response and post-treatment platelet reactivity inatients with non–ST elevation acute coronary syndromes.

ethods

onsecutive patients admitted for non–ST elevation acuteoronary syndromes to the Department of Cardiology ofimone Hospital (Marseille, France) from October 2004 toune 2006 were eligible for this prospective study. Non–STlevation acute coronary syndromes were defined as clinicalymptoms compatible with acute myocardial ischemia �12ours before admission and �1 of the following: a newnding of ST-segment depression �0.05 mV, transient�20 minutes) ST-segment elevation �0.1 mV, T-wavenversion �0.3 mV in �2 leads, and elevated levels ofardiac markers. The exclusion criteria were a history ofleeding diathesis, persistent ST elevation acute coronaryyndromes, New York Heart Association class IV, percuta-eous coronary intervention or coronary bypass graftingithin 3 months, platelet count �100 G/L, creatinine clear-

nce �25 ml/min, and the use of a glycoprotein IIb/IIIantagonist before the procedure. Patients received 600-mg

oading doses of clopidogrel and aspirin 250 mg �12 hours

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1089Coronary Artery Disease/Cytochrome P450 Polymorphisms and Clopidogrel Response

efore coronary angiography. The study protocol was ap-roved by the ethics committee of our institution, and pa-ients gave written informed consent for participation.

Blood samples for testing platelet reactivity were drawn inhe catheterization laboratory from a 6Fr arterial sheath beforeoronary angiography. The blood was immediately collectedn Vacutainer tubes (Becton Dickson, Franklin Lakes, Newersey) containing 3.8% trisodium citrate, filled to capacity,nd sent immediately to the hemostasis laboratory.

To determine the vasodilator-stimulated phosphoproteinVASP) phosphorylation state of whole blood, we used atandardized flow cytometric assay (Platelet VASP; Diag-ostica Stago [Biocytex], Asnières, France), which is andaptation of the method of Schwarz et al22 previouslyescribed. Briefly, a citrated blood sample was incubatedith prostaglandin E1 (PGE1) or PGE1 and ADP 10 �mol/L

or 10 minutes and fixed with paraformaldehyde, afterhich the platelets were permeabilized with nonionic de-

ergent. The cells were labeled with a primary monoclonalntibody against serine 239-phosphorylated VASP (16C2),ollowed by a secondary fluorescein isothiocyanate-conju-ated polyclonal goat antimouse antibody. Analyses wereerformed using an EPICS XL-MCL flow cytometer (Beck-an Coultronics, Margency, France), the platelet populationas identified from its forward and side scatter distribution,

nd 10,000 platelets were gated. A platelet reactivity indexPRI) was calculated from the median fluorescence intensityMFI) of samples incubated with PGE1 or PGE1 and ADPccording to the formula PRI VASP � [(MFI �

able 1equence of primers and temperatures used for polymerase chain reaction

NP (Allele), dbSNPccession No.

Forward and Reverse Primers

YP3A4*1B (rs2740574) SS: Tgg AAg gAT gTg TAg gAg TgAS: TTg gAA gTT ggC AAA gAA

YP3A5*3 (rs776746) SS: gCA CTT gAT gAT TTA CCTAS: CAT ACC CCT AgT TgT ACg

YP2C19*2 (rs4244285) SS: CAg AgC TTg gCA TAT TgT AAS: TAT CgC AAg CAg TCA CAT

SNP � single nucleotide polymorphism; dbSNP � Single Nucleotide P

able 2linical characteristics of the whole population (n � 603)

haracteristic Value

ge (yrs) 64.7 � 12.2en 75.7%ypertension 56.3%iabetes mellitus 28%moker 44.1%ody mass index (kg/m2) 26.8 � 4.3tatins 56.4%blockers 45.8%

alcium antagonists 17%ngiotensin-converting enzyme inhibitors 41%ositive troponin (troponin I �0.04 ng/ml) 23%

Data are expressed as mean � SD or as percentages.

PGE1FIPGE1 � ADP)/MFIPGE1] � 100. W

For the assessment of ADP-induced platelet aggregationADP-Ag), the blood-citrate mixture was centrifuged at 120gor 5 minutes. The platelet count was determined in theesulting platelet-rich plasma sample and adjusted to 2.5 �08/ml with homologous platelet-poor plasma. Plateletsere stimulated with ADP (10 �mol/L), and aggregationas assessed using a PAP4 Aggregometer (Biodata Corpo-

ation, Wellcome, Paris, France). Aggregation was ex-ressed as the maximal percentage change in light transmit-ance from baseline, with platelet-poor plasma as theeference. Here we report data on the maximal intensity ofDP-Ag. The coefficient of variation of ADP-Ag was 6.5%.

Allele-Specific Forward and ReversePrimers or Restriction Enzyme

Temperature

SS: CCA TAg AgA CAA ggg CAA 64°CAS: ATT AAA TCg CCT CTC TCC TSS: gAg CTC TTT TgT CTT TCA G 60°CAS: CCA AAC Agg gAA gAg ATA TSma I 55°C

rphism Database.

able 3enetic distribution of the CYP3A4�1B, CYP3A5*3, and CYP2C19*2olymorphisms

Genetic Distribution, n (%)

olymorphism AA AG GGYP3A4*1B 537 (90%) 58 (9.7%) 2 (0.3%)YP3A5*3 8 (1.3%) 113 (18.8%) 481 (79.9%)YP2C19*2 23 (3.8%) 143 (23.8%) 435 (72.4%)

able 4elations among studied polymorphisms and platelet indexes

ADP-Ag (%) PRI VASP (%) ADP-PS (AU)

YP2C19*2AA 66.1 � 4 69.1 � 5.7 0.43 � 0.04AG 56.1 � 1.6 59.1 � 2.1 0.39 � 0.01GG 55.7 � 0.9 50.9 � 1.3 0.35 � 0.01p value 0.039 0.0001 0.035p value* 0.05 0.0001 0.030YP3A5*3AA 56.3 � 6.86 57.0 � 8.8 0.33 � 0.06AG 56.8 � 1.8 52.4 � 2.5 0.38 � 0.02GG 56.1 � 0.9 53.9 � 1.2 0.36 � 0.01p value 0.94 0.81 0.67YP3A4*1BAA 56.3 � 0.8 53.8 � 1.2 0.36 � 0.01AG 55.7 � 2.5 52.2 � 3.5 0.39 � 0.02GG 52.5 � 13.5 69.9 � 16.5 0.33 � 0.13p value 0.94 0.56 0.58

Data are expressed as mean � SEM.* Adjusted for age and gender.AU � arbitrary units.

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1090 The American Journal of Cardiology (www.AJConline.org)

g, and nonresponse to clopidogrel was defined by highost-treatment platelet reactivity (ADP-Ag �70%), as de-cribed previously by our group and others.13,16,17

The surface expression of the internal �-granule mem-rane protein P-selectin expressed on the surface of ADP-ctivated platelets (ADP-PS) was determined by flow cy-ometry. The following antibodies were used: anti-CD62PBeckman Coulter, Inc., Fullerton, California) and anti-ouse goat immunoglobulin G fluorescein isothiocyanate

Beckman Coulter, Inc.). Briefly, platelet-rich plasma wasiluted 1/10 in Tyrode’s albumin buffer (0.25 � 108/ml)nd gently mixed. Antibody anti-CD62P (10 �l of dilution/10) and ADP (final concentration 10 �mol/L) or Tyrode’slbumin buffer was added to 10 �l of diluted platelet-richlasma. After incubation, 10 �l of antimouse goat immu-oglobulin G fluorescein isothiocyanate (diluted 1/10) wasdded again. Scatter signals and fluorescence intensity werenalyzed using an EPICS XL-MCL flow cytometer. Theight-scattering properties projected on a scattergram iden-ified the platelet cluster. Fluorescence intensity was ex-ressed on individual cytohistograms, with the region ofnterest limited to the platelet cluster. The mean channeluorescence intensity was used as an index of antibodyinding and P-selectin surface expression.

Genomic deoxyribonucleic acid was extracted from periph-ral blood leukocytes by the salting-out method. Genotypingor CYP3A4*1B (rs2740574), CYP3A5*3 (rs776746), andYP2C19*2 (rs4244285) was performed by allele-specificolymerase chain reaction, 2 allele-specific primers by re-

igure 1. Distribution of ADP-Ag, PRI VASP, and P-selectin expressionrbitrary units.

ction, or restriction fragment length polymorphism. The d

olymerase chain reaction conditions were as follows (for 1eaction): 200 nmol/L of each primer (Invitrogen, Carlsbad,alifornia), 200 �mol/L of each deoxyribonucleotide tri-hosphate (Invitrogen), 60 ng of genomic deoxyribonucleiccid, and 0.3 U of Taq polymerase (Qbiotaq, Quantum; MPiomedicals, Solon, Ohio) with its supplied buffer (3.5mol/L MgCl2). The polymerase chain reaction productsere visualized on 2% agarose gels stained with 0.2 �g/ml

thidium bromide (Invitrogen). The primers used, the sizesf the different polymerase chain reaction products gener-ted, the restriction enzymes, and temperatures are listed inable 1.

Statistical analysis was performed using SAS version.01 (SAS Institute Inc., Cary, North Carolina). Continuousariables are expressed as mean � SD. Categorical vari-bles are expressed as frequencies and percentages. Forontinuous variables, mean levels were compared usingnalysis of variance, and for categorical variables, propor-ions were compared using chi-square tests (or Fisher’sxact tests as appropriate). Pearson’s correlation coeffi-ients were calculated to study the associations among vari-bles. Allele frequencies were estimated by gene counting,nd departure from Hardy-Weinberg equilibrium was testedsing a chi-square test. The effects of the polymorphisms onhe different variables studied were tested using a generalinear model, with the phenotypic variable (i.e., ADP-Ag,RI VASP, or ADP-PS) as the dependent variable and eacholymorphism as an independent variable. A second modeldjusting for age and gender was calculated. The power to

ing to the genotypic distribution of CYP2C19*2 polymorphism. AU �

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etect an effect of 1% of the CYP 2C19*2 polymorphism on

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1091Coronary Artery Disease/Cytochrome P450 Polymorphisms and Clopidogrel Response

DP-Ag at a risk of 5% was 69%. Values of p �0.05 wereonsidered statistically significant.

esults

ix hundred three unrelated participants were included in ourtudy. We assessed ADP-Ag, PRI VASP, and ADP-PS. PRIASP and ADP-PS were evaluated in 455 and 600 partici-ants, respectively. Table 2 lists the clinical characteristics ofarticipants. All platelet parameters studied were correlated.he associations between ADP-Ag and PRI VASP (r � 0.62,�0.0001) and between PRI VASP and ADP-PS (r � 0.52,�0.0001) appeared stronger than the association betweenDP-Ag and ADP-PS (r � 0.36, p �0.0001). In stepwise

inear regression models, the only clinical correlate of ADP-Agas body mass index. This covariate explained 5.4% of theariability in ADP-Ag levels (p �0.0001). Body mass indexas also the only clinical correlate of PRI-VASP and ADP-PS

evels and explained 5.2% and 1.2% of their variability, re-pectively (p �0.0001 and p � 0.0077, respectively). Geneticistributions of the CYP3A4*1B, CYP3A5*3, and CYP2C19*2olymorphisms are listed in Table 3 and were found to beimilar to those reported previously.22–24 The genotype fre-uencies for each allele considered separately were consistentith Hardy-Weinberg predictions. Neither the CYP3A4*1Bolymorphism nor the CYP3A5*3 polymorphism influencedither platelet response to clopidogrel or post-treatmentlatelet reactivity (Table 4), while CYP2C19*2 genotypesere significantly correlated with the 3 studied platelet

ndexes. The A-allele carriers exhibited the highest levels oflatelet indexes (Table 4, Figure 1). This significant asso-iation was obtained in recessive (p �0.01, p �0.007, and�0.06 for ADP-Ag, PRI-VASP, ADP-PS, respectively)

nd codominant (p �0.08, p �0.0001, and p �0.009 forDP-Ag, PRI-VASP, ADP-PS, respectively) genetic mod-

ls. After adjusting for variables found to be significantlyssociated with platelet response, associations remained sig-ificant for ADP-Ag (p �0.0001), PRI-VASP (p � 0.03)nd ADP-PS (p �0.01). Previous reports proposed to defineonresponse to clopidogrel by ADP-Ag �70%.13,16,17 Inccordance with our previous results,13,16 the rate of nonre-ponse to dual-antiplatelet therapy was 24.5% (n � 148).

e found that the nonresponders to clopidogrel had higherody mass indexes than responders (p � 0.03; Table 5) andere more likely to be carriers of the CYP2C19*2 loss-of-

able 5tudied parameters according to the occurrence of high post-treatmentlatelet reactivity (adenosine diphosphate–induced aggregation �70%)

ariable No HPPR HPPR p Value

ody mass index 26.66 � 0.21 27.61 � 0.39 0.03YP2C19*2AA 13 (3%) 10 (7%) 0.03*AG 115 (25%) 28 (19%) 0.03GG 325 (72%) 110 (74%) NS

Data are expressed as mean � SEM or number (percentage).* Assuming a recessive model.HPPR � high post-treatment platelet reactivity.

unction allele (p � 0.03; Table 5). C

iscussion

he results of this study of a large population suggest thatYP2C19 gene polymorphisms participate in the vari-bility of platelet response to clopidogrel in patients withon–ST elevation acute coronary syndromes treated withigh loading doses of clopidogrel. First, we found thatYP2C19*2 allele carriers had higher platelet reactivity

o ADP, whatever the studied platelet index. This result isonsistent with previous findings in healthy subjects fromulot et al.21 The association remained significant after

djustment for other clinical factors associated with plateleteactivity. Of these covariates, body mass index appearedtrongly positively associated with all 3 measured plateletndexes. Angiolillo et al23 also found that elevated body

ass index was the only independent predictor of subopti-al platelet response in patients who underwent coronary

tenting. Also, possession of the CYP2C19*2 allele wasore frequent in patients with high post-treatment platelet

eactivity (ADP-Ag �70 %), which has been associatedith an impaired prognosis.13,16,17

The active metabolite of clopidogrel, which irreversiblylocks platelet ADP P2Y12 receptors, arises from complexiochemical reactions involving several CYP isoforms.ariability in the catalytic activity of these isoforms may

herefore affect the pharmacodynamic action of clopidogrel.larke et al24 used genetically engineered microsomes con-

aining a single human P450 isozyme to test for their abilityo oxidize clopidogrel; they demonstrated that CYP3A4 andYP3A5 isoenzymes, which are also the most abundantYP isozymes in human liver, are predominantly responsi-le for the activation of clopidogrel in vivo. Moreover, Laut al18 reported that CYP3A4 metabolic activity was asso-iated with between-subjects variability in clopidogrel re-ponsiveness.

However, gene sequence variations of the CYP3A5 enzymen patients with coronary artery disease have not been associ-ted with variability in clopidogrel responsiveness.19,20 Thisbservation may be explained by the fact that CYP3A4 ishe dominant CYP3A enzyme in whites and therefore maye more important in clopidogrel metabolism. In support ofhis, the IVS10�12G�A polymorphism of the CYP3A4ene was found to modulate platelet activation in a steadyhase of clopidogrel therapy.19 In the present study, clopi-ogrel response was not modulated by the CYP3A4*1Bolymorphisms, as expected from previous studies.19,20

Another CYP isoenzyme, CYP2C19, is a major cause ofarge differences in the pharmacokinetics of a number oflinically important drugs. Nongenetic factors, such as en-yme inhibition and induction, old age, and liver cirrhosis,odulate CYP2C19 activity, but CYP2C19 catalytic effi-

iency is influenced mainly by the CYP2C19*2 polymor-hism in exon 5 (681G�A), which creates an aberrantplice site and results in a truncated and nonfunctionalnzyme.25 Several studies have established that CYP2C19enotyping identifies �90% of poor metabolizers, indicat-ng a good genotype-phenotype agreement.26,27

In accordance with previous published works on other

YP2C19 substrates, such as diazepam28 and omeprazole,29

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1092 The American Journal of Cardiology (www.AJConline.org)

ulot et al21 recently demonstrated that the CYP2C19*2oss-of-function allele is associated with a marked decreasen platelet responsiveness to clopidogrel in young, healthyale volunteers. However, a recent study by Brandt et al30

howed that loss-of-function polymorphisms of CYP2C19ere associated with decreased exposure to the active me-

abolite of clopidogrel but not prasugrel, suggesting that theatter could overcome this genetic variability of response tolopidogrel.

Decreased exposure to its active metabolite is associatedith a diminished pharmacodynamic response to clopi-ogrel. Our study confirmed these data, providing evidencehat the CYP2C19*2 mutant allele is a genetic determinantf the platelet response to a 600-mg loading dose of clopi-ogrel in patients with non–ST elevation acute coronaryyndromes. We found that in patients receiving 600-mgoading doses of clopidogrel before percutaneous coronaryntervention, the CYP2C19*2 allele was associated with aow platelet response to clopidogrel. Moreover, carriers ofYP2C19*2 were more prone to having high post-treatmentlatelet reactivity, which has been proposed as a risk factoror recurrent ischemic events.13,16,17 In addition, in subjectsarrying the defective CYP2C19*2 allele, we observed thatomozygotes for the CYP2C19*2 allele had higher plateletunction indexes than heterozygotes, showing a gene-doseffect with CYP2C19.

Our population was large compared with those in previ-usly published studies, diminishing referral biases andnabling multivariate analyses and genotyping and pheno-yping blinded to one another. It remains possible that thebserved association was due to linkage with another yetnknown polymorphism. The evidence that CYP2C19*2 isfunctional polymorphism using in vitro promoter assays

upports the hypothesis that there is a functional role for thisolymorphism in clopidogrel disposal.

1. Savi P, Herbert JM, Pflieger AM, Dol F, Delebassee D, Combalbert J,Defreyn G, Maffrand JP. Importance of hepatic metabolism in theantiaggregating activity of the thienopyridine clopidogrel. BiochemPharmacol 1992;44:527–532.

2. Savi P, Combalbert J, Gaich C, Rouchon MC, Maffrand JP, Berger Y,Herbert JM. The antiaggregating activity of clopidogrel is due to ametabolic activation by the hepatic cytochrome P450-1A. ThrombHaemost 1994;72:313–317.

3. Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrish-nan V, Yang RB, Nurden P, Nurden A, Julius D, Conley PB. Identi-fication of the platelet ADP receptor targeted by antithrombotic drugs.Nature 2001;409:202–207.

4. Yusuf S, Zhao F, Mehta SR, Chrolavicius S, Tognoni G, Fox KK;Clopidogrel in Unstable Angina to Prevent Recurrent Events TrialInvestigators. Effects of clopidogrel in addition to aspirin in patientswith acute coronary syndromes without ST-segment elevation. N EnglJ Med 2001;345:494–502.

5. Mehta SR, Yusuf S, Peters RJ, Bertrand ME, Lewis BS, NatarajanMK, Malmberg K, Rupprecht H, Zhao F, Chrolavicius S, et al;Clopidogrel in Unstable Angina to Prevent Recurrent Events TrialInvestigators. Effects of pretreatment with clopidogrel and aspirinfollowed by long-term therapy in patients undergoing percutaneouscoronary intervention: the PCI-CURE study. Lancet 2001;358:527–533.

6. Gurbel PA, Bliden KP, Hiatt BL, O’Connor CM. Clopidogrel forcoronary stenting: response variability, drug resistance, and theeffect of pretreatment platelet reactivity. Circulation 2003;107:

2908 –2913.

7. Angiolillo DJ, Fernandez-Ortiz A, Bernardo E, Ramírez C, Barrera-Ramirez C, Sabaté M, Hernández R, Moreno R, Escaned J, Alfonso F,Bañuelos C, et al. Identification of low responders to a 300-mg clo-pidogrel loading dose in patients undergoing coronary stenting.Thromb Res 2005;115:101–108.

8. Jaremo P, Lindahl TL, Fransson SG, Richter A. Individual variationsof platelet inhibition after loading doses of clopidogrel. J Intern Med2002;252:233–238.

9. Müller I, Besta F, Schulz C, Massberg S, Schönig A, Gawaz M.Prevalence of clopidogrel non-responders among patients with stableangina pectoris scheduled for elective coronary stent placement.Thromb Haemost 2003;89:783–787.

0. Serebruany VL, Steinhubl SR, Berger PB, Malinin AI, Bhatt DL,Topol EJ. Variability in platelet responsiveness to clopidogrel among544 individuals. J Am Coll Cardiol 2005;45:246–251.

1. Gurbel PA, Bliden KP, Guyer K, Cho PW, Zaman KA, Kreutz RP,Bassi AK, Tantry US. Platelet reactivity in patients and recurrentevents post-stenting: results of the PREPARE POSTSTENTINGstudy. J Am Coll Cardiol 2005;46:1820–1826.

2. Matetzky S, Shenkman B, Guetta V, Shechter M, Bienart R, Golden-berg I, Novikov I, Pres H, Savion N, Varon D, Hod H. Clopidogrelresistance is associated with increased risk of recurrent atherothrom-botic events in patients with acute myocardial infarction. Circulation2004;109:3171–3175.

3. Cuisset T, Frere C, Quilici J, Barbou F, Morange PE, Hovasse T,Bonnet JL, Alessi MC. High post-treatment platelet reactivity identi-fied low-responders to dual antiplatelet therapy at increased risk ofrecurrent cardiovascular events after stenting for acute coronary syn-drome. J Thromb Haemost 2006;4:542–549.

4. Geisler T, Langer H, Wydymus M, Göhring K, Zürn C, Bigalke B,Stellos K, May AE, Gawaz M. Low response to clopidogrel is asso-ciated with cardiovascular outcome after coronary stent implantation.Eur Heart J 2006;27:2420–2425.

5. Hochholzer W, Trenk D, Bestehorn HP, Fischer B, Valina CM, FerencM, Gick M, Caputo A, Büttner HJ, Neumann FJ. Impact of the degreeof peri-interventional platelet inhibition after loading with clopidogrelon early clinical outcome of elective coronary stent placement. J AmColl Cardiol 2006;48:1742–1750.

6. Cuisset T, Frere C, Quilici J, Morange PE, Nait-Saidi L, Mielot C, BaliL, Lambert M, Alessi MC, Bonnet JL. High post-treatment plateletreactivity is associated with high incidence of myonecrosis after stent-ing for non-ST elevation acute coronary syndromes. Thromb Haemost2007;97:282–287.

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9. Angiolillo DJ, Fernandez-Ortiz A, Bernardo E, Ramirez C, CavallariU, Trabetti E, Sabaté M, Hernandez-Antolin R, Moreno R, Escaned J,et al. Contribution of gene sequence variations of the hepatic cyto-chrome P450 3A4 enzyme to variability in individual responsivenessto clopidogrel. Arterioscler Thromb Vasc Biol 2006;26:1895–1900.

0. Smith SM, Judge HM, Peters G, Amstrong M, Fontana P, Gaussem P,Daly ME, Storey RF. Common sequence variations in the P2Y12 andCYP3A5 genes do not explain the variability in the inhibitory effectsof clopidogrel therapy. Platelets 2006;17:250–258.

1. Hulot JS, Bura A, Villard E, Azizi M, Remones V, Goyenvalle C,Aiach M, Lechat P, Gaussem P. Cytochrome P450 2C19 loss-of-function polymorphism is a major determinant of clopidogrel respon-siveness in healthy subjects. Blood 2006;108:2244–2247.

2. Schwarz UR, Geiger J, Walter U, Eigenthaler M. Flow cytometry analysisof intracellular VASP phosphorylation for the assessment of activatingand inhibitory signal transduction pathways in human platelets—defini-tion and detection of ticlopidine/clopidogrel effects. Thromb Haemost1999;82:1145–1152.

3. Angiolillo DJ, Fernandez-Ortiz A, Bernardo E, Barrera Ramírez C,

Sabaté M, Fernandez C, Hernández-Antolín R, Escaned J, Alfonso F,

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1093Coronary Artery Disease/Cytochrome P450 Polymorphisms and Clopidogrel Response

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