Thrombosis Research 130 (2012) e67–e72

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The impact of CYP3A5*1/*3, PIA1/A2 and T744C polymorphisms on clopidogrel andacetylsalicylic acid response variability in Mexican population

Irma Isordia-Salas a,⁎, Marcos Jaciel Olalde-Román b, David Santiago-Germán a,c,Norma Corona de la Peña a, Jesús Salvador Valencia-Sánchez b

a Unidad de Investigación Médica en Trombosis, Hemostasia y Aterogénesis, H.G.R. No 1. Dr. “Carlos Mac Gregor Sánchez Navarro” Instituto Mexicano del Seguro Social, México DF, Méxicob Unidad de Cuidados Intensivos Cardiovasculares, Hospital de Cardiología, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, México DF, Méxicoc Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México DF, México

⁎ Corresponding author at: Apartado Postal B 32, CoahCP 06703. Tel./fax: +52 556395822x20883.

E-mail address: irmaisordia@yahoo.com.mx (I. Isord

0049-3848/$ – see front matter © 2012 Elsevier Ltd. Alldoi:10.1016/j.thromres.2012.06.024

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 3 May 2012Received in revised form 18 June 2012Accepted 26 June 2012Available online 20 July 2012

Keywords:ClopidogrelAcetylsalicylicPlateletsPolymorphism

Introduction: Clopidogrel is recommended in addition to aspirin to prevent atherothrombotic events in pa-tients with acute coronary syndromes (ACS) and in those undergoing percutaneous coronary intervention(PCI). However, an interindividual variability in platelet inhibition response to clopidogrel has been demon-strated, and is associated with recurrent cardiovascular events. Multiple mechanisms have been associatedwith no response including genetics factors.Materials and methods: The present study enrolled 60 patients with ACS undergoing emergent PCI. Plateletaggregation to adenosine diphosphate and arachidonic acid was assessed by turbidimetric method at 24hours after dual administration of 300 mg of clopidogrel and 300 mg of acetylsalicylic acid loading dose.Clopidogrel or acetylsalicylic acid resistance was defined by persistence of Platelet Reactivity (PR=ADP-Ag>70% or PR=Arachidonic Acid-Ag>20%) respectively. The CYP3A51*/5*, PIA1/A2, and T744C polymor-

phisms were determined in all participants by PCR-RFLP.Results: The allelic frequencies were: CYP3A5*3 (71.65%), PIA2 (10.8%), and 744 C (15.0%). We founded highpercent of clopidogrel resistance (60.0%), compared with 8.3% of acetylsalicylic acid in those patients. The ge-notype frequencies of those polymorphisms were similar between responders and non responders definedby PR. There was a high percent of coronary adverse events.Conclusions: We identified a high percent of clopidogrel resistance in Mexican patients with ACS undergoingPCI. However, a normal platelet response to acetylsalicylic acid was observed in most of them. There was noassociation between CYP3A5*1/*3, PIA1/A2, and T744C polymorphisms and clopidogrel resistance. Morestudies are needed to determine the possible interaction between genetics factors, platelet response toclopidogrel and cardiovascular adverse events.

© 2012 Elsevier Ltd. All rights reserved.

Introduction

Platelet activation and aggregation play an important role inthe pathogenesis of arterial thrombosis leading to acute coronarysyndromes (ACS) and in thrombotic complication during and afterpercutaneous coronary interventions (PCI). One of the key moleculesin platelet aggregation is adenosine and is released from activatedplatelets. Clopidogrel is an inactive prodrug that requires severalbiotransformation steps. After intestinal absorption, clopidogrel bio-transformation into its active metabolite is mediated mainly byhepatic cytochrome P450 (CYP) system to generate its active thiolmetabolite which targets and irreversibly inhibits the ADP P2Y12receptor, and it is metabolized in the liver by different hepatic CYP

uila No. 5 México, DF, México.

ia-Salas).

rights reserved.

isoenzymes, including CYP3A5 [1] However, it has been reported aninterindividual variability in platelet response to clopidogrel rangesfrom 5% to 40% [2,3] and nearly 20% of patients undergoing PCI willexperience recurrent ischemic or thrombotic events [4]. This phe-nomenon is often referred to as a clopidogrel resistance [5]. Previousstudies demonstrated a significant percentage of patients display nodemonstrable antiplatelet effect after a 300-mg clopidogrel loadingdose [6]. It has been hypothesized that patients exhibiting clopidogrelnon responsiveness are inadequately protected and might be at in-creased risk for recurrent thrombotic events [7].

Several mechanisms have been proposed for this wide variabilityin platelet response to clopidogrel, including polymorphism in plate-let receptor genes. In a study, Kuehl et al. [8] demonstrated that onlypeople with at least one CYP3A5* 1 allele actually expressed CYP3A5protein. There is a single nucleotide polymorphism at 6986G allele(CYP3A5*3), let to alternative splicing and protein truncation andthus absence of CYP3A5 activity. Because CYP3A5 may represent up

B)A)

Fig. 1. Induced ADP and acetylsalicylic acid platelet aggregation inhibition (Figure, Aand B respectively) in patients with ACS undergoing emergent PCI.

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to 50% of the total hepatic CYP3A content in people expressing CY3A5,this gene may be the most important genetic contributor to inter-individual and interracial differences in CYP3A5 dependent drugclearance. Previous studies have been demonstrated that CYP3A5*3is associated with increased risk of atherothrombotic events [9]. Incontrast, other investigators have failed to confirm this finding [10].

The adenosine diphosphate (ADP) P2Y12 receptor plays a key rolein platelet function. Activation of the P2Y12 receptor leads to the acti-vation of the glycoprotein GPIIb/IIIa receptor, which results in en-hanced platelet activation. Recently polymorphisms of the P2Y12

receptor genes have been associated with different degrees of plateletaggregation, and have been suggested to modulate clopidogrel re-sponse. Two functional haplotypes (H1 and H2) were determinedby Fontana et al. [11]. The polymorphic variant is named H2 haplo-type, whereas the wild type is denoted as H1 haplotype. Accordingto Fontana et al., the H2 haplotype (a C in 744 position) is associatedwith higher maximal aggregation in response to ADP. The study dem-onstrated increased platelet aggregability in homo and heterozygouscarriers of the H2 allele in a gene dose dependent manner. Previously,it has been demonstrated an association between T744C polymor-phism and clopidogrel resistance [12]. However, it has not been con-firmed by others [13].

Previous study by Angiolillo et al. [14], carried out in 38 patientstreated with 300 mg loading dose of clopidogrel undergoing PCI, thePIA/A2 polymorphism was explore. They identified a 35% less inhibi-tion of platelet aggregation in patients carriers of PIA2/A2 genotypecompared with PIA1/A1.

Clopidogrel is administrated in addition to acetylsalicylic for patientswith ACS to reduce major adverse cardiovascular events or preventionof stent thrombosis in patients undergoing PCI [15]. Acetylsalicylic acidacts by irreversibly inhibiting the cyclooxygenase-1 (COX-1) enzymethrough acetylating the serine residue at position 529. COX-1 catalysesthe conversion of arachidonic acid to prostaglandins G2 and H2, whichare subsequently converted by thromboxane synthase to thromboxaneA2 (TXA2), a potent vasoconstrictor and activator of platelet aggregation[16] However, adequate antiplatelet effects are not achieved in 5% to 40%of patients taking clopidogrel and 4-30% of patients taking acetylsalicylicacid due to an interindividual variability [17]. This phenomenon is oftenreferred to as a clopidogrel [5] or acetylsalicylic acid resistance [17].Acetylsalicylic acid resistance is likely to be multifactorial in origin.Reduced absorption and /or increased metabolism of Acetylsalicylic acidmay contribute. A genetic aetiology to acetylsalicylic acid resistance hasalso been proposed [16].

Despite the great variability of response to clopidogrel betweenpatients, and the difference in genotype distribution of genes ineach population, the aim of this study was to assess the impact ofthe CYP3A5*1/*3, T744C and PIA1/A2 polymorphisms on platelet re-sponse to a 300 mg clopidogrel and 300 mg of acetylsalicylic acidloading dose, in Mexican patients with acute coronary syndrome un-dergoing percutaneous coronary intervention.

Materials and Methods

Study population

We prospectively enrolled 60 consecutive patients with all types ofACS undergoing to emergent PCI with stent implantation and whowere treated with acetyl salicylic acid and clopidogrel. All patients re-ceived a loading dose of 300 mg of acetyl salicylic acid followed by adaily regimen of 150 mg, and according to the recommendations ofthe Guidelines for the Management of patients with Unstable Angina/Non ST -Elevation Myocardial Infarction and ST-Elevation MyocardialInfarction, at least a loading dose of 300 mg of clopidogrel followed by75 mg daily, for at least 1 month and ideally up to 1 year. They wereadmitted to the emergency department of the Cardiology Hospital,Centro Médico Nacional Siglo XXI, in Mexico City, between April and

August 2011.Clinical and demographic data were recorded. Data con-cerning demographic parameters, coronary heart disease risk factorsand treatment were collected for each patient.

The study protocol was reviewed and approved by the HumanEthical Committee, and Medical Research Council of the InstitutoMexicano Del Seguro Social, and conforms to the ethical guidelinesof the 1975 Declaration of Helsinki. Informed written consent wasobtained from all subjects before enrollment

Blood Samples

Fasting venous blood samples were obtained for platelet functionassays and to determine genotypes. Samples were collected at 24 hrsafter 300 mg of loading dose of clopidogrel and 300 mg of acetylsalicylicacid. The initial sample was discarded to avoid platelet aggregation.Platelet count was assessed at all time points to ensure the degree ofplatelet reactivity was not biased by the number of platelets.

Platelet Aggregation

Platelet aggregation measurements were performed by the turbi-dimetric method using a Chrono-log aggregometer with agro-linkcomputer interfarce (Chronolog Corporation, Havertown, PA). Bloodsamples were collected in tubes containing 3.8% trisodium citrateand platelet aggregation was assessed with platelet-rich plasma(PRP) with platelet count adjusted to 250×103/mm. Light transmis-sion was adjusted to 0% with PRP and to 100% for PPP for each mea-surement. Platelet aggregation was induced by adding either ADP10 μM ADP or 0.5 mM arachidonic acid. The extent of aggregationwas defined as the maximal amount of light transmission reached≤6 min after addition of the agonist, with platelet-pool plasma usedas a reference. Clopidogrel or acetylsalicylic resistance was definedby persistence of Platelet Reactivity (PR=ADP-Ag >70% or PR=Ara-chidonic Acid-Ag>20%) respectively (Fig. 1).

Genotyping

Genomic DNA was isolated from frozen packed white blood cellsaccording to manufacturer's instructions using a commercial kitQIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany).

Table 2Distribution of T744C Genotypes and allelic frequency in patients with ACS undergoingPCI.

Patients (n=60)

Genotype distributionT/T n (%) 44 (73.3)C/T n (%) 14 (23.3)C/C n (%) 2 (3.4)

Allelic frequencyT n (%) 102 (85.0)C n (%) 18 (15.0)

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T744C Polymorphism of the P2Y12

The T744C polymorphism was obtained by the use of the follow-ing primers: sense primer 5’ TCTGGTGAAATAAAAAGATTACGTA-3’and the antisense primer 5’-GTCAGAAATGGCCTGTGTATATATG-3’. Po-lymerase chain reaction (PCR) included a hot start at 94 °C for 5’, 35cycles at 94 °C for 30”, 50 °C for 30”, 72 °C for 30” and a final exten-sion at 72 °C for 5’. After PCR and restriction with RsaI (Promega Cor-poration, Madison, WI, USA), two fragments of 196 bp and 24 bp forthe C allele were obtained, and a single fragment of 220 bp for the Tallele. Patients were divided into 3 groups: CC homozygotes, CT het-erozygotes and TT homozygotes). We assayed all samples by dupli-cate Negative controls were included with every set of amplifications.

CYP3A5*1/*3 Genetic Polymorphism

The CYP3A5*1/*3 was determined using PCR-restriction fragmentlength polymorphism (RFLP) analysis. The forward primer was: (5’CTT TAA AGA GCT CTT TG TCT CTC A-3’), and reverse (5´CCA GGAAGC CAG ACT TTG AT-3´). The polymerase chain reaction (PCR) am-plification protocol consisted initial denaturation at 95 °C for10 min, 37 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for30 s, followed by 72 °C for 5 min for final extension. After PCR ampli-fication, each PCR product was digested for a minimum of 4 h at 37 °Cwith DdeI restriction enzyme (Promega Corporation, Madison, WI,USA), before electrophoresis using a 3% agarose gel. We assayed allsamples by duplicate Negative controls were included with everyset of amplifications.

PIA1/A2 Genetic Polymorphism

To identify the PIA genotype the following primers were used:5’-TTC TGA TTG CTG GAC TTC TCT T-3´, and 5’-TCT CTC CCC ATGGCA AAG AGT-3’. The polymerase chain reaction (PCR) amplificationprotocol consisted of an initial denaturation segment of 94 °C for5 min. After this, each cycle consisted of three segments (1 minutedenaturation at 94 °C, 45 seconds of annealing at 57 °C, and 1 minuteextension at 72 °C). This cycle was repeated 35 times followed by an

Table 1Baseline Demographic and Clinical Characteristics of Patients.

Variable Patients (n=60)

Age (years, mean±SD) 63.7±11.8BMI 29.3±1.5Sex n (male %) 46 (76.7)Smoking n (%) 35 (58.3)Hypertension n (%) 31 (51.6)Diabetes Mellitus n (%) 27 (45.0)Dyslipidemia n (%) 28 (46.6)Family history of CAD n (%) 23 (38.3)

Initial clinical characteristicsPrevious MI n (%) 12 (20.0)Unstable Angina n (%) 10 (16.7)

ACS presentationSTEAMI n (%) 51 (85.0)Non STEAMI n (%) 7 (11.7)Unstable Angina n (%) 2 (3.3)

MedicationClopidogrel 300 mg n (%) 60 (100)Acetylsalicylic acid 300 mg (%) 60 (100)Statins n (%) 60 (100)ACE inhibitor n (%) 54 (90)Fibrates n (%) 24 (40)Beta Blockers n (%) 6 (10)

CAD=Coronary artery disease; STEAMI=ST-Elevation Acute Myocardial Infarction;Non STEAMI=Non ST-Elevation Myocardial Infarction, Values are mean±SD forquantitative variables and n(%) for qualitative variables.

additional extension cycle at 72 °C for 15 min. The 268 base pair frag-ment amplified by PCR was digested with MspI (Promega Corpora-tion, Madison, WI, USA). Polymorphic genotypes were identifiedafter electrophoresis of digestion products in 2.5% agarose gel electro-phoresis (BIO-RAD, Laboratories Hercules, CA), and visualized underultraviolet light following ethidium bromide staining (BIO-RAD, Lab-oratories Hercules, CA). We assayed all samples by duplicate. Nega-tive controls were included with every set of amplifications.

Statistical Analysis

Continuous data are expressed as the mean ± standard deviation(SD). Categorical variables are expressed with percentages. The sig-nificance of differences between continuous variables was deter-mined by Student's t test. Differences between categorical variables,was determined with the chi square test. A P valueb0.05 was consid-ered as statistically significant. All statistical analyses were performedusing SPSS (Statistical Package for the Social Sciences) statistical soft-ware package (version 16: SPSS Inc, Chicago, IL, USA).

Results

Baseline characteristics of patients and controls are shown in(Table 1). The T744C polymorphism in the P2Y12 receptor wasfounded with the following genotype distribution: homozygous TT(73.3%), heterozygous (23.3%), and CC homozygous (3.4%). The allelicfrequencies for T and C alleles were (85%) and (15%) respectively(Table 2). There was a higher frequency for the CYP3A5*3 allele(71.65%), (Table 3). The genotype distribution of CYP3A5*1/*3 poly-morphism is show in (Table 3). The genotype distribution for thePIA1/A2 polymorphism was as follow: homozygous A1/A1 (81.7%),A1/A2 (15%), and A2/A2 (3.3%). The frequency for the A2 allele was(10.8%), (Table 4). We found a high percent of clopidogrel resistance(60.0%) in this group of patients, (Fig. 2). Clopidogrel resistance wasdefined as 10 μmol/L ADP-induced platelet aggregation ≥70%. In con-trast, only 8.3% were no responders to inhibitory effect ofacetylsalicylic acid. Acetylsalicylic acid was defined as –induced plate-let agregation,(PR=Arachidonic Acid -Ag>20 %), (Fig. 3).

The T744C genotype distribution was similar between respondersand nonresponders to inhibitory platelet effect by clopidogrel (P=0.37). Also, there was no difference in PRmax between patients with

Table 3Genotype distribution and allele frequency of CYP3A5 *1/*3 polymorphism in patients.

Patients (n=60)

Genotype distribution*1/*1n (%) 4 (6.7)*1/*3 n (%) 26 (43.3)*3/*3 n (%) 30 (50.0)

Allelic frequency*1 n (%) 42 (28.35)*3 n (%) 78 (71.65)

Table 4Genotype distribution and allele frequency of PIA1/A2 polymorphism in the platelet re-ceptor IIIa of patients.

Patients (n=60)

Genotype distributionA1/A1 n (%) 49 (81.7)A1/A2 n (%) 9 (15.0)A2/A2 n (%) 2 (3.3)

Allelic frequencyA1 n (%) 107 (89.2)A2 n (%) 13 (10.8)

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91.7

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es (

%)

Arachidonic Acid-induced aggregation (%)

Fig. 3. Maximal arachidonic acid induced platelet aggregation (%) among AcetylsalicylicAcid Responders (AAR) versus Acetylsalicylic Acid Non Responders (AANR).

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CYP3A5*1 or CYP3A*3, (P=0.79) or PIA1 or PIA2 (P=0.54). Therewas a high percent of cardiovascular events during 1 to 30 dayspost PCI (48.3%). In some patients more than one complication postPCI was observed (Fig. 4). The most frequent recurrent adverse out-come was acute myocardial infarction in 5 patients (8.3%), followedby stent reestenosis in 4 patients (6.6%), major bleeding in1 (1.6%),and 1 death (1.6%), and they were probably associated with an inad-equate inhibitory platelet effect. In the present study, there was notsignificant association between those polymorphisms and recurrencecardiovascular events. Percutaneous coronary intervention and stentimplantation was performed in twenty one patients (35%). However,the in-stent thrombosis was identified in 4 of them, from 2 and4 week after successful PCI (19.4%).

Discussion

Clopidogrel is recommended in addition to aspirin in patients withACS to reduce major adverse cardiovascular events or prevention ofstent thrombosis in patients undergoing PCI with stenting [2]. How-ever, adequate antiplatelet effects are not achieved in 5% to 40% of pa-tients taking clopidogrel and 4-30% of patients taking aspirin due toan interindividual variability [2–6,16,17]. Several mechanism hasbeen associated with this phenomenon such; diabetes mellitus,smoking, obesity, and genetics [5].

To the best of our knowledge this is the first study to investigatethe impact of 3 different genetic polymorphisms involved in the re-sponse to inhibitory platelet effect of clopidogrel and acetylsalicylicacid 300 mg loading dose in Mexican patients with ACS undergoingto PCI and stent placement.

We identified a high percent of clopidogrel resistance (60%) in thisgroup of patients, as assessed by platelet aggregation. In contrast,only 8.3% had no response to acetylsalicylic acid antiplatelet effect

0

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50

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70

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>70 <70

60

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ADP-induced aggregation (%)

Fig. 2. Maximal ADP induced platelet aggregation (%) among Clopidogrel Responders(CR) versus Clopidogrel Non Responders (CNR).

determined also by induced arachidonic acid platelet aggregation. Al-though, the definition of clopidogrel resistance is arbitrary, the cutoffvalues used to define no response to clopidogrel and aspirin havebeen validated in several clinical studies [4,5]. Our results are in dis-agreement with those reported by Cuisset et al., with low rates ofClopidogrel non responders (25%), but higher percent of acetylsalicylicresistance (15.4%) in French population [18], and those reported by Levet al., with 10% of individuals insensitive to aspirin, and 24.2% were in-sensitive to clopidogrel [19].

We founded a nearly 20% of in-stent thrombosis from 2 and4 weeks after the PCI and stent implantation. There was a higherplatelet reactivity (PR=ADP-Ag>80) in this subgroup of patientswith recurrent coronary events of thrombosis. However, there was anormal antiplatelet response to acetylsalicylic acid.

Clopidogrel is an inactive prodrug that requires several biotrans-formation steps. After intestinal absorption, clopidogrel is metabo-lized mainly by hepatic cytochrome P450 (CYP) system to generateits active thiol metabolite which targets and irreversibly inhibits theADP P2Y12 receptor [1].Several genetic variants had been associatedwith diminished platelet inhibitory effect by clopidogrel in differentpopulations worldwide.

In our study, the polymorphism T744C of the P2Y12 receptor genewas examined. We found a high percent of the wild type allele (85%),

0

2

4

6

8

10

Fig. 4. Major Adverse Cardiovascular Events from 1 to 30-days post PCI. AVB=atrioven-tricular block, MI=myocardial infarction, LBBB=left branch bundle block, RBBB=rightbranch bundle block.

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and it is in accordance with the frequency found in other populations[12]. However, the 744 C allele was not associated with cardiovascularadverse outcomes or non response to clopidogrel in this study. Our dataare in contrast with those published by Fontana et al., whom demon-strated that carriers of the minor haplotype (H2 haplotype) had an in-creased ADP aggregation in a French population. However, thatassociation was not corroborated by Cuisset et al., [20] in 597 patientswith non- ST-elevation acute coronary syndrome, where it was notshown any influence of the T744C polymorphism of the P2Y12 receptorgene in response to clopidogrel in French population. Previous study byAngiolillo et al., failed to identified significant difference between TT ge-notype and thosewith for at least one C allele (CT and TT) in clopidogrelresponse in a Spanish population. Also, our results are similar to thosefound by Namazi et al., whom reported no association between the744 C allele and the interindividual variability to clopidogrel in Iranianpopulation [21]. However, they reported a higher percent of the TT ge-notype (92.9%) compared with our population (85%). Moreover, Levet al., reported no significant difference in P2Y12 genotypes frequenciesbetween drug- insensitive vs. drug-sensitive patients [19]. However,they found a higher frequency of 744 C allele (27.5%) compared withthe one reported in this study (15%).

Recent reports had demonstrated an association between P2Y12polymorphism and acetylsalicylic acid resistance. In the presentstudy, platelet inhibitory effect by acetylsalicylic acid was normal in95% of those patients, evaluated by arachidonic acid-induced lighttransmittance aggregometry.

Another genetic factor associated with diminished platelet re-sponse to clopidogrel evaluated was CYP3A5. This is the first studyto documented high frequency of *3/*3 genotype in Mexican patientwith ACS undergoing to PCI. We found a high percent of theCYP3A5*3 non expressor allele in this group of patients (71.65%),compared with other populations such: Chinese (73%), Korean(70%), and Caucasians (90%) [8].However, our results are in contrastwith those reported by Kuehl et al., in African American (60%) [8].

Several studies had investigated the influence of CYP3A5 polymor-phism on antiplatelet effect of clopidogrel. In previous study, Suh etal., demonstrated a positive association between the non-expressorgenotype and the high frequency of atherothrombotic events in aKorean population [9]. The CYP3A5*1 carriers shown more inhibitionon platelet aggregation than CYP3A5*3 after clopidogrel treatment,though it was no significant in that study [9]. Kim et al., in a studyin south Korea show that the presence of CYP3A5*3 allele was not afactor causing interindividual variability in the clopidogrel pharmaco-kinetics and its platelet effect [10].Also, similar results were obtainedby Frere et al., in a French population [22]. In their study, showed thatCYP3A5*3 polymorphism did not influence the post-treatment plate-let reactivity and the clopidogrel response [22]. Previous findingspublished by Nazimi et al., in one hundred and twelve patients whowere candidate to elective PCI, established no effect between P2Y12polymorphism and the inter patient variability in response toclopidogrel in Iranian population [21]. In the present study theCYP3A5*3 allele was not associated with cardiovascular adverseevents or a diminished platelet inhibition after clopidogrel 300 mgloading dose.

We have previously demonstrated that the PIA2 allele representan independent risk for STEMI in youngMexican population [23]. Pre-vious studies had shown that the PIA1/2 polymorphism is associatedwith the inhibitory platelet effect of clopidogrel or acetylsalicylic acidin some individuals [17]. In the present study the PIA2 was not asso-ciated with non response to clopidogrel 300 mg or acetylsalicylic acid300 mg loading dose.

In previous study, it has been demonstrated a significant associa-tion between environmental factors such diabetes mellitus or obesityand antiplatelet effect of clopidogrel. Although, modifiable factorswere highly present in this group of patients, we failed to find an as-sociation between modifiable risk factors and clopidogrel resistance.

Study Limitations

In the present study, we used a single assessment post treatmentwithout baseline reactivity data. However, a previous study hasshown the correlation between a low response to clopidogrel (differ-ence between pre and post treatment values) and highpost-treatment platelet reactivity. Another limitation was that singleassessment of platelet function and using optical aggregometry. How-ever the method remains the gold standard in assessment of plateletfunction while on clopidogrel treatment. Ex vivo measurements ofADP-induced maximum platelet aggregation by light transmittanceaggregometry (LTA) has been the most commonly used laboratorymethod to evaluate clopidogrel responsiveness and is consideredthe gold standard [3,5]. Recently, it was suggested that since anti-platelet drug especially clopidogrel induce platelet disaggregation,the response to clopidogrel would be better demonstrated by mea-suring late platelet aggregation at 6 min after stimulation with ADPrather than maximum aggregation [24]. However, it has been demon-strated that both measurements were equivalent in determining theprevalence of clopidogrel nonresponsiveness [5]. Finally, we tested arelatively small sample size. However, a similar percent of patientshas been used by other investigators [3,7,17,19].

Conclusions

The present study identifies a significant incidence of clopidogrelresistance in Mexican patients with acute coronary syndrome whohave undergone PCI and coronary stenting. In contrast, normal plate-let inhibition to acetylsalicylic acid loading dose was found in 96.6% ofthe patients. Also, we observed a high frequency of the CYP3A5*3 nonexpressor allele in this group of patients. Thus, this genetic variant incombination with others polymorphisms, may be influence the di-minished platelet response to clopidogrel and increased risk for re-current cardiovascular events. We need to search for new strategiesof inhibitory platelet treatment, to obtain an adequate inhibitoryplatelet effect in patients with ACS undergoing PCI. Therefore, morelarge prospective studies might be warranted to determine the possi-ble genotype-clopidogrel resistance interaction in our population.Early identification of vulnerable patients and definition of the roleof antiplatelet no response in the development of cardiovascular ad-verse events should be the focus of future diagnostic and therapeuticstrategies.

Conflict of Interest Statement

None declared.

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