Impact of CYP2C19 polymorphism on residual platelet reactivity in patients with coronary heart disease during antiplatelet therapy

Journal of Cardiology (2011) 57, 194—201

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Original article

Impact of CYP2C19 polymorphism on residual platelet reactivity in patients with coronary heart disease during antiplatelet therapy Koichiro Yamamoto (MD) a, Seiji Hokimoto (MD) a,∗, Tadasuke Chitose (MD) a, Kazunori Morita (MD) b, Takamichi Ono (MD) a, Koichi Kaikita (MD) a, Kenichi Tsujita (MD) a, Tomohide Abe (PhD) b, Mariko Deguchi (PhD) b, Haruna Miyagawa (PhD) b, Junji Saruwatari (PhD) b, Hitoshi Sumida (MD) a, Seigo Sugiyama (MD, FJCC) a, Kazuko Nakagawa (MD) b,c, Hisao Ogawa (MD, FJCC) a a

Department of Cardiovascular Medicine, Faculty of Life Sciences, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan b Division of Pharmacology and Therapeutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan c Center for Clinical Pharmaceutical Sciences, Kumamoto University, Kumamoto, Kumamoto, Japan Received 25 August 2010; received in revised form 12 October 2010; accepted 29 October 2010 Available online 17 December 2010

KEYWORDS Platelets; Genetics; Pharmacology; Coronary heart disease

∗

Summary Background and purpose: CYP2C19*2 loss-of-function allele in Caucasians may be associated with wide interindividual variability in platelet response to clopidogrel, and the incidence of gene mutation varies with racial differences, especially between Asians and Caucasians. The aim was to examine the impact of CYP2C19 genotype on the residual platelet reactivity in Japanese patients with coronary heart disease (CHD) during antiplatelet therapy. Methods and results: We measured the CYP2C19 genotype and platelet aggregation in 201 patients with stable CHD. Moreover, we examined the relation of CYP2C19 polymorphism to cardiovascular events in 98 patients treated with stent implantation. The distribution of CYP2C19 genotype was 37%, 33%, 11%, 11%, 7%, and 1% in CYP2C19*1/*1, *1/*2, *1/*3, *2/*2, *2/*3, and *3/*3, respectively. Residual platelet reactivity was lower in patients during dual antiplatelet therapy (DAT) than in those with aspirin (3975 ± 1569 aggregation units minute (AU min) vs 5850 ± 938 AU min, p < 0.05). In the DAT group, the platelet reactivity decreased

Corresponding author. Tel.: +81 96 373 5175; fax: +81 96 362 3256. E-mail address: [email protected] (S. Hokimoto).

0914-5087/$ — see front matter © 2011 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jjcc.2010.10.007

CYP2C19 and platelet reactivity

195 significantly in the wild-type homozygotes (CYP2C19*1/*1), subsequently in the *2, or *3 heterozygotes (*1/*2, *1/*3), and was not well inhibited in the *2, and/or *3 homozygotes (*2/*2, *2/*3, *3/*3; 3194 ± 1570 AU min, 4148 ± 1400 AU min, and 5088 ± 1080 AU min, respectively). However, when the duration of DAT was used to divide subjects into 2 groups, <7 days, and >7 days, patients carrying the variant allele showed significantly decreased platelet reactivities at >7 days compared with those at <7 days. Moreover, the incidence of cardiovascular events was higher in patients carrying at least one variant allele than in wild-type homozygotes. Conclusions: CYP2C19 polymorphism may be associated with high residual platelet reactivity and the occurrence of cardiovascular events. © 2011 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

Introduction Dual antiplatelet therapy with aspirin plus clopidogrel is currently recommended for the prevention of adverse cardiovascular events in coronary heart disease (CHD) patients treated with stent implantation [1,2]. Despite dual antiplatelet therapy, however, some patients do not achieve an adequate antiplatelet effect from clopidogrel and a substantial number of subsequent ischemic events, such as stent thrombosis, cardiovascular death, nonfatal myocardial infarction, and stroke, still occur [3,4]. This may be partially because of wide interindividual variability in the antiplatelet effect of clopidogrel. The mechanisms leading to a poor response to clopidogrel have not yet been fully elucidated and are most likely multifactorial. Besides some different clinical factors, such as age, renal failure, obesity, diabetes, high plasma fibrinogen, and lack of adherence [5—7], cytochrome P450 (CYP) polymorphisms are reported to be associated with non- or hypo-responsiveness to clopidogrel [8,9]. In Western countries, it has been demonstrated that patients carrying at least one CYP2C19*2 loss-of-function allele exhibit increased residual platelet aggregation and are at high risk of adverse cardiovascular events despite clopidogrel administration [10,11]. On the other hand, besides CYP2C19*2, a CYP2C19*3 variant allele has been reported in Asia, which generates the null-activity enzyme protein [12,13]. The relation between CYP2C19 polymorphism, including CYP2C19*3 as well as CYP2C19*2 allele, and the antiplatelet efficacy of clopidogrel is unknown in Japanese patients with CHD. To address this issue, we examined the distribution of CYP2C19 genotypes and platelet aggregation, and assessed the impact of CYP2C19 polymorphism on response to clopidogrel and clinical outcomes after stent implantation.

Methods Study population The study population consisted of 201 consecutive patients who underwent cardiac catheterization upon diagnosis with stable CHD. Patients receiving anticoagulants, or antiplatelet agents other than clopidogrel or aspirin, were excluded. The subjects were divided into 2 groups, (1) dual antiplatelet therapy (DAT) group (n = 123), 100 mg/day aspirin plus 75 mg/day clopidogrel as a maintenance dose after 300 mg of clopidogrel loading; and (2) aspirin group (n = 78), aspirin at 100 mg/day alone.

The study protocol was in agreement with the guidelines of the ethical committee of the institution and written informed consent was obtained from each patient or the family of the subject.

Genotyping Genomic DNA was extracted from whole blood using the DNA Extractor WB kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) using a modified protocol from Richards et al. [14]. Polymerase chain reaction restriction fragment length polymorphisms for CYP2C19*2 (681G > A) and CYP2C19*3 (636G > A) were performed as described previously [13,15]. CYP2C19*2 and *3 are considered to account for >99% of alleles generating the null-activity enzyme protein in the Japanese population [13]. Thus, CYP2C19 genotypes were classified into three phenotypes: (1) extensive metabolizer (EM) carrying normal function alleles (CYP2C19*1/*1); (2) intermediate metabolizer (IM) carrying one loss-of-function allele (*1/*2, *1/*3); and (3) poor metabolizer (PM) carrying two loss-of-function alleles (*2/*2, *2/*3, *3/*3).

Measurement of platelet aggregation Patients in the DAT group received a loading dose of clopidogrel of 300 mg, followed by a 75 mg daily maintenance dose. Platelet function test was performed at least 24 h after clopidogrel loading in the DAT group, and at least 7 days after aspirin at 100 mg/day as a maintenance dose in the aspirin alone group. In the DAT group, the time of testing from the clopidogrel loading was in 10 patients at 24 h, 40 at 36 h, 10 at 48 h, 9 at 60 h, 50 at 168 h, and 4 at 216 h, respectively. In short, platelet aggregation test was done in 69 patients at <7 days and 54 patients at >7 days. Platelet aggregation was measured as below. Whole samples of blood were obtained using a glass tube containing a solution of 0.38% sodium citrate. Platelet-rich plasma was prepared by centrifugation at 900 rpm at room temperature for 15 min and then, to separate platelet-poor plasma, centrifugation was also carried out at 3000 rpm at room temperature for 10 min. Aggregation in platelet-rich plasma induced by 20 ␮mol/L adenosine diphosphate (ADP; ChronoLog, Tokyo, Japan) was measured using a light transmission aggregometer (MCM HEMA TRACER 313; PAM12C, LMS Inc., Japan), where the degree of light transmission of plateletrich plasma was defined as 0% of the aggregation rate, and the cognitive platelet-poor plasma as 100%. Test time was 10 min. On treatment platelet reactivity was defined as the area under the platelet aggregation curve, which was used

196 Table 1A

K. Yamamoto et al. Patient characteristics.

Number Male Age (years) BMI (kg/m2 ) Diabetes Hypertension Dyslipidemia Current smoker Fibrinogen (mg/dl) Ejection fraction (%) eGFR (ml/min 1.73 m2 ) PPI use Statin use

DAT

Asp

p-Value

123 81(66%) 68.6 ± 10.0 24.0 ± 3.3 60(49%) 96(78%) 75(61%) 20(16%) 356.5 ± 104.5 58.1 ± 9.9 64.9 ± 16.2 32(26%) 105(85%)

78 53(68%) 68.7 ± 9.8 24.1 ± 3.7 30(38%) 55(71%) 44(56%) 14(18%) 341.8 ± 80.1 61.2 ± 8.3 68.2 ± 13.2 8(10%) 53(67%)

ns ns ns ns ns ns ns ns ns ns ns ns

DAT, dual antiplatelet therapy; Asp, aspirin; BMI, body mass index; eGFR, estimated glomerular filtration rate; PPI, proton pump inhibitor.

to express the aggregation response over the measured time (aggregation units minute; AU min).

Clinical outcomes Of the 123 patients receiving DAT scheduled for percutaneous coronary intervention (PCI), 98 underwent PCI due to severe coronary stenosis. Thus, the clinical outcomes of 98 patients treated with coronary stent implantation were evaluated except for 25 subjects. The patients were followed up every month at the outpatient department for one year or at end point. The end point was cardiovascular death, nonfatal myocardial infarction, or ischemic stroke.

Statistical analysis

ity showed a lower value in the DAT group than in the aspirin group as expected (3975 ± 1569 AU min vs 5850 ± 938 AU min, p < 0.05). However, values of platelet reactivity in the DAT group varied widely and these results indicate a wide variability in individual response to clopidogrel.

CYP2C19 genotype and phenotype The distribution of CYP2C19 genotype was 37%, 33%, 11%, 11%, 7%, and 1% in *1/*1, *1/*2, *1/*3, *2/*2, *2/*3, and *3/*3, respectively (Fig. 2). Patients were divided into 3 phenotypes of CYP2C19 polymorphism: (1) EM; (2) IM; and (3) AU*min

p<0.05

8000

Continuous variables are expressed as means ± SD. Categorical variables are expressed as frequencies and percentages. Statistical analysis was carried out exclusively by independent statistician. The comparison between groups was analyzed using Pearson’s Chi-square test for categorical variables and Kruskal—Wallis or Mann—Whitney U test for continuous variables. A p-value <0.05 was regarded as significant. The StatView 5.0 software (SAS Institute, Cary, NC, USA) was used for all statistical analyses.

Results

7000 6000 5000 4000 3000 2000 1000 0

Patient characteristics n

Clinical characteristics of each group (DAT and aspirin alone) are shown in Table 1A. Antiplatelet therapy was DAT in 123 cases (61%) and aspirin in 78 (39%). There were no significant differences in baseline characteristics between DAT and aspirin groups.

On-treatment platelet reactivity in all subjects Fig. 1 shows a comparison of platelet reactivity in the DAT and aspirin groups. On-treatment platelet reactiv-

DAT

Asp

123

78

Figure 1 Comparison of on-treatment platelet reactivity according to antiplatelet therapy. Platelet aggregation (AU min) induced by 20 ␮mol/L adenosine diphosphate was measured using a light transmission aggregometer. On-treatment platelet reactivity showed a lower value in the DAT group than in the aspirin group as expected (3975 ± 1569 vs 5850 ± 938 AU min, p < 0.05). On-treatment platelet reactivities were compared between two groups with Mann—Whitney U test. Asp, aspirin; AU, aggregation units; DAT, dual antiplatelet therapy (clopidogrel plus aspirin).

CYP2C19 and platelet reactivity

197

Figure 2 Distribution of CYP2C19 genotype (right) and phenotype (left) in patients with stable coronary heart disease. EM, extensive metabolizer, patients carrying normal function alleles (CYP2C19*1/*1); IM, intermediate metabolizer, patients carrying one loss-of-function allele (CYP2C19*1/*2, *1/*3); PM, poor metabolizer, patients carrying two loss-of-function alleles (CYP2C19*2/*2, *2/*3, *3/*3). Table 1B

Patient characteristics according to CYP2C19 phenotype in dual antiplatelet therapy group.

Number Male Age (years) BMI (kg/m2 ) Diabetes Hypertension Dyslipidemia Current smoker Previous MI Fibrinogen (mg/dl) Ejection fraction (%) eGFR (ml/min 1.73 m2 ) PPI use Statin use

EM

IM

PM

p-Value

47 31 (66%) 66.8 ± 10.8 24.9 ± 3.2 21 (45%) 34 (72%) 29 (61%) 8 (17%) 10 (21%) 368.3 ± 121.8 58.1 ± 10.4 63.9 ± 17.3 9 (19%) 38 (81%)

51 36 (73%) 68.6 ± 9.3 23.5 ± 3.3 26 (53%) 42 (82%) 30 (59%) 9 (18%) 10 (20%) 345.0 ± 97.2 59.3 ± 9.9 64.2 ± 18.2 15 (29%) 45 (88%)

25 14 (56%) 72.0 ± 9.1 23.5 ± 3.4 13 (52%) 20 (80%) 16 (64%) 6 (24%) 4 (16%) 356.0 ± 84.5 58.9 ± 11.9 64.9 ± 16.1 8 (32%) 22 (88%)

ns ns ns ns ns ns ns ns ns ns ns ns ns

EM, extensive metabolizer; IM, intermediate metabolizer; PM, poor metabolizer; BMI, body mass index; MI, myocardial infarction; eGFR, estimated glomerular filtration rate; PPI, proton pump inhibitor.

PM. The distribution of CYP2C19 phenotype was 37%, 44%, and 19% in EM, IM, and PM, respectively (Fig. 2). Table 1B lists the clinical characteristics according to CYP2C19 genotype in the DAT group. There were no significant differences in patient characteristics among the 3 phenotypes. As shown in Table 2, the proportions of CYP2C19 variant alleles in patients on DAT scheduled for PCI were 59%, 31%, and 10% in CYP2C19*1 (wild type), *2, and *3, respectively, and the similar distribution of CYP2C19 genotypes was seen compared with that of all CHD patients. Fig. 3 shows a comparison of on-clopidogrel platelet reactivity according to CYP2C19 genotypes in the DAT group. On-treatment platelet reactivity was significantly lower in the EM group than the values in

Table 2 Ratio of CYP2C19*2, or *3 variant alleles in 123 patients taking dual antiplatelet therapy. CYP2C19 Allele

*1

*2

*3

Number Ratio

145 58.9%

76 30.9%

25 10.2%

Total 246 100%

the IM and PM groups (EM, IM, and PM; 3194 ± 1570 AU min, 4148 ± 1400 AU min, and 5088 ± 1080 AU min, respectively). The average time of testing from the clopidogrel loading was 94 ± 70 h, 88 ± 69 h, and 105 ± 71 h, in EM, IM, and PM, respectively, and there was no difference in the measuring time of each group according to CYP2C19 genotypes.

On-treatment platelet reactivity according to time between clopidogrel loading and platelet function test among EM, IM, and PM groups As shown in Fig. 4, on-treatment platelet reactivity levels at <7 days decreased compared with the values at >7 days in the PM and IM groups. There were no differences in patient characteristics, such as age, sex, body mass index, diabetes, hypertension, smoking, plasma fibrinogen content, left ventricular ejection fraction, estimated glomerular filtration rate, proton pump inhibitor use, and statin use, between the two groups. However, the ratio of dyslipidemia was higher in >7 days after clopidogrel loading than in <7 days. There was a great difference among EM, IM, and PM at <7 days; how-

198

K. Yamamoto et al.

AU*min

Table 3A Cardiovascular events in patients with or without variant alleles of CYP2C19*2, or *3.

p<0.05

8000

p<0.05

7000

CYP2C19*2, or *3 allele

6000 5000

End point Cardiovascular death Myocardial infarction Stroke

4000 3000 2000

Non-carriers (n = 36)

Carriers (n = 62)

0

5 0 0 0

1 2 2

1000 0 EM

IM

(CYP2C19 *1/*1)

PM

(*1/*2, *1/*3)

(*2/*2, *2/*3, *3/*3)

n=51

n=25

n=47

Figure 3 Comparison of the on-treatment platelet reactivity according to CYP2C19 genotype in patients during dual antiplatelet therapy. On-clopidogrel platelet reactivity was significantly reduced in the EM group compared with those in the IM and PM groups. EM, 3194 ± 1570 AU min; IM, 4184 ± 1400 AU min; PM, 5088 ± 1080 AU min, at <7 days, and >7 days, respectively. Platelet aggregation values between groups were compared with Mann—Whitney U test. AU, aggregation units; EM, extensive metabolizer; IM, intermediate metabolizer; PM, poor metabolizer.

ever, the huge gap was reduced at 7 days (EM, 3186 ± 1595, 3007 ± 1541; IM, 4655 ± 1380, 3490 ± 1392; PM, 5663 ± 1385, 4674 ± 824; at <7 days, and >7 days, respectively). There was no significant difference in on-clopidogrel platelet reactivity at >7 days between the EM and IM groups, however, the value of platelet reactivity in the PM group was significantly higher than those in the EM and IM groups. There were no

significant differences in the time from clopidogrel loading to measuring among the EM, IM, and PM groups in the subgroups of < or >7 days (<7 days, 33 ± 12 h, 32 ± 13 h, and 40 ± 15 h in EM, IM, and PM; >7 days, 170 ± 10 h, 170 ± 10 h, and 176 ± 18 h in EM, IM, and PM, respectively).

Cardiovascular events and CYP2C19 variant alleles Clinical outcomes in patients undergoing PCI were as below: cardiovascular death, 1 patient; nonfatal myocardial infarction, 2; ischemic stroke, 2 (Table 3A). All of these patients were carrying at least one variant allele of CYP2C19; details of genotype were in three patients with CYP2C19*1/*2, one patient with *1/*3, and one with *2/*2 alleles, and the periods from PCI to cardiovascular events were 1, 10, 21, 74, and 340 days (Table 3B). Kaplan—Meier analysis based on with and without variant allele of CYP2C19*2, or *3 showed a tendency of increased risk of cardiovascular events in patients carrying at least one of the CYP2C19 loss-of-function variant alleles as compared with those carrying wild-type alleles, although the case number and rate of events were too small to be significant (Fig. 5).

p<0.05

AU*min

p<0.05

8000 7000

p<0.05

ns

6000 5000 4000 3000 2000 1000 0

EM (CYP2C19 *1/*1)

n=25

IM

PM

(*1/*2, *1/*3) (*2/*2, *2/*3, *3/*3)

n=32 < 7 days

n=12

EM

IM

(CYP2C19 *1/*1)

(*1/*2, *1/*3)

n=22

n=19

PM (*2/*2, *2/*3, *3/*3)

n=13

7 days

Figure 4 On-treatment platelet reactivity according to time between clopidogrel loading and platelet function test among extensive, intermediate, and poor metabolizer groups. EM, 3186 ± 1595 AU min, 3007 ± 1541; IM, 4655 ± 1380, 3490 ± 1392; PM, 5663 ± 1385, and 4674 ± 824; at <7 days, and >7 days, respectively. EM, extensive metabolizer; IM, intermediate metabolizer; PM, poor metabolizer. Data are shown as mean ± SD. Statistical analysis was performed with Mann—Whitney U test.

CYP2C19 and platelet reactivity Table 3B

199

Details of cardiovascular events.

Day(s)

Genotype

Event

1 10 21 74 340

*1/*2 *1/*2 *1/*2 *2/*2 *1/*3

Stroke Stroke Myocardial infarction Cardiovascular death Myocardial infarction

Discussion In this study, we measured ADP-induced platelet aggregation in patients with CHD during antiplatelet therapy, and defined the value of the area under the aggregation curve as one of the platelet functional values. We used on-treatment platelet reactivity directly, not inhibition of platelet aggregation calculated as the percentage decrease in the relative maximal platelet aggregation from the baseline. Observations of clopidogrel response that rely on baseline platelet reactivity have recently been appreciated to be less reliable predictors of ischemic risk than posttreatment platelet reactivity [16—18]. Moreover, the value of the area under the aggregation curve (AU min) is more sensitive and precise than that of maximal platelet aggregation calculated from a percentage of inhibition. Thus, we used the value of the area under the aggregation curve as on-treatment platelet reactivity during antiplatelet therapy. Although the impact of CYP2C19 polymorphism on the antiplatelet efficacy of clopidogrel in Japanese patients scheduled for coronary stent implantation has been reported [19], we enrolled a substantial number of consecutive patients undergoing cardiac catheterization for stable CHD and measured ontreatment platelet reactivity at different sampling times from clopidogrel loading to determine the effects according to CYP2C19 genotype. Moreover, CYP2C19 genotype was prone to be associated with the occurrence of major adverse cardiovascular events after stent implantation. As such, we consider this an important manuscript reporting the rela%

Cumulative cardiovascular events rate

20

15

Carrier

10

5

Non-carrier 0 0

100

200

300

400 Days

Figure 5 Cumulative event rate of cardiovascular death, nonfatal myocardial infarction, and stroke. There were cardiovascular events in 5 patients carrying at least one CYP2C19 variant alleles (Carrier), while none in patients carrying the wild-type, normal-function alleles (Non-carrier).

tion of CYP2C19 genomic variation to on-treatment platelet reactivity and to clinical outcomes in Japanese patients taking DAT. Recent studies have suggested that a wide interindividual variability of response to clopidogrel is associated with adverse cardiovascular events [16,20]. Different clinical factors and gene polymorphisms are considered to influence a persistent high platelet reactivity despite DAT with clopidogrel and aspirin [5]. Not only CYP2C19*2 but also *3 loss-of-function alleles were found in Japanese patients in contrast to Caucasians, and the ratio of PMs that have two loss-of-function alleles was 19%. The PM ratio is 2—3% in Western countries, so these results indicate a huge gap in the distribution of CYP2C19 polymorphism between Japanese and Caucasians. The ratio of patients exhibiting a high on-treatment platelet reactivity was increased owing to a high frequency of CYP2C19*2 and *3 variant alleles in the present study, however, the incidence of adverse cardiovascular events was low. Cardiovascular events including stent thrombosis are generally reported to be lower in Japan than in Western countries [21—23]. In spite of the high rate of PM in Japanese patients, the reason for the low risk of clinical outcomes remains unknown. Several studies in other countries have demonstrated that patients carrying at least one non-functional allele of CYP2C19*2 show an increased ontreatment platelet reactivity despite conventional DAT, and that it may be associated with an increased risk of adverse cardiovascular events following coronary stent placement [8,9,11]. Moreover, the risk of early but not late stent thrombosis is reported to increase in patients presenting with high residual platelet reactivity after stenting [24]. In this study, on-clopidogrel platelet reactivity was most decreased in the EM group compared with those in the IM and PM groups. However, when classified by time between clopidogrel loading and platelet function test, patients in both the PM and IM groups exhibited reduced on-clopidogrel platelet reactivity at >7 days compared with that at <7 days. Moreover, there was no significant difference in on-treatment platelet reactivity at >7 days between the EM and IM groups; however, the value of platelet reactivity even at >7 days in the PM group was significantly higher than in the EM and IM groups. The ratio of dyslipidemia was higher in >7 days after clopidogrel loading than in <7 days. Elevated plasma fibrinogen in the presence of diabetes and increased body mass index are reported to be associated with higher platelet aggregation in patients with CHD [5], thus we do not think that dyslipidemia may affect the platelet aggregation in this study. These results suggest that on-treatment platelet reactivity according to CYP2C19 genotype depends on the time between clopidogrel loading and platelet function test in Japanese CHD patients with variant alleles of not only CYP2C19*2 but also CYP2C19*3. In the present study, we evaluated the clinical outcomes in patients undergoing coronary stent implantation on DAT. The occurrence of cardiovascular events was detected in only patients carrying CYP2C19 variant alleles, and the periods from PCI to cardiovascular events were 1, 10, 21, 74, and 340 days. We do not have the data on residual platelet reactivity at the time of cardiovascular events, and the relation of the residual platelet reactivity on clopidogrel to clinical events is unknown. Although we speculate that the underlying mechanisms of cardiovascular events may not

200 account for the reduced antiplatelet effect of clopidogrel alone, sample number and cardiovascular event rate were too small in this study, thus, we cannot examine the possibility of speculation. Further studies using larger samples are needed to examine whether CYP2C19 genomic variations are related to an increased risk of clinical cardiovascular events in Japanese patients as well as Caucasians.

K. Yamamoto et al.

[4]

[5]

Limitations In the present study, values of platelet reactivity are not calculated from the values of changes between before and after the administration of clopidogrel; therefore, we do not know if the underlying platelet hyperreactivity per se accounts for a higher on-clopidogrel platelet reactivity. We did not measure plasma concentrations of the active metabolite of clopidogrel, thus, we cannot provide direct evidence of reduced antiplatelet efficacy of clopidogrel in patients carrying at least one CYP2C19*2 or *3 variant allele. In addition, we cannot exclude the effect of other drug metabolism enzymes, such as CYP1A2, 2B6, 3A, and 2C9, on clopidogrel response, besides CYP2C19. In this study, CYP2C19 genomic variations were not associated with an increased risk of cardiovascular events in carriers compared with noncarriers, however, the small sample size might have affected the power to detect such influence. Thus, further study is needed in the future.

[6]

[7]

[8]

[9]

[10]

Acknowledgments [11]

We wish to thank Dr. K. Matsui, Clinical Education Center, Yamaguchi University Hospital, for advice on statistical analysis and medical technologist, S. Iwashita, Kumamoto University Hospital, for measurement of platelet aggregation. This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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