Introduction

Clopidogrel (Plavix®) is an irreversible P2Y12 receptor antagonist prescribed for the treatment of arteriosclerotic events in patients with recent stroke, myocardial infarction (MI) or established peripheral arterial disease1,2. Although clopidogrel is safe and effective in most patients, there is an inevitable variability in responsiveness among individuals3. Current investigations indicate that 4% to 30% of patients fail to attain platelet inhibition after clopidogrel therapy4,5,6, and this phenomenon is called clopidogrel resistance (CR). In fact, several clinical studies conducted in Chinese, Japanese and Korean patients reveal that the frequencies of clopidogrel resistance in Asians may vary from 20% to 65%, which highly exceeds the incidence reported in other ethnic groups7,8. The underlying mechanisms of clopidogrel resistance remain unknown. Previous studies indicate that genetic factors may greatly contribute to the variability of platelet activity8.

Because clopidogrel is a prodrug that requires a two-step oxidation process for activation in the liver, polymorphisms of genes encoding the metabolic enzymes involved in clopidogrel biotransformation may be the primary influential factors of clopidogrel responsiveness. CYP2C19 plays a substantial role in both of these steps by producing nearly 50% of the active CYP2C19 metabolites9. CYP3A4 also participates in hepatic clopidogrel metabolism, producing 39.8% of the active metabolites during the second oxidation step9. Pregnane X receptor (PXR), a member of the nuclear receptor subfamily 1 (NR1I2), is a transcriptional regulator of several metabolic enzymes, including CYP3A4 and CYP2C1910. It has been previously reported that the genetic polymorphisms in NR1I2 may account for the interindividual variation in drug metabolism and disposition in certain diseases11,12. In addition, the P2RY12 gene encodes the P2Y12 ADP receptor, and genetic variants of P2RY12 have been associated with alterations in the platelet inhibition response in patients13. Many studies have focused on the association between genetic polymorphisms and clopidogrel resistance14,15,16.

According to the clopidogrel black box warning released by the US FDA, CYP2C19 genotyping now allows clinicians to adjust the treatment therapy for an individual patient. The different CYP2C19 genotypes have a significant impact on the response of clopidogrel and the prognosis of patients with ischemic stroke17. Although the hepatic CYP2C19 LOF genotypes are associated with a lower clopidogrel responsiveness18,19, only 12% of the platelet response to clopidogrel can be explained by the presence of the CYP2C19*2 polymorphism, as suggested by an intervention (PAPI) study20,21. Thus, other potential genetic factors and mechanisms remain to be discovered, which together may contribute to the individual variability of clopidogrel efficacy22. Moreover, limited documents have been published concerning the relationship between genetic polymorphisms and clopidogrel resistance in Chinese ischemic stroke patients. The aim of this study was to investigate the association between the CYP2C19*2 (rs4244285), CYP3A4*1G (rs2242480), NR1I2 (rs13059232) and P2RY12 (rs2046934) polymorphisms and clopidogrel resistance in Chinese ischemic stroke patients.

Material and methods

Study population

This study was approved by the Ethics Committee of Guangdong Provincial Hospital of Traditional Chinese Medicine, China. It was conducted in accordance with the Declaration of Helsinki and was consistent with applicable guidelines for good clinical practice. In total, 191 ischemic stroke patients between 40 and 80 years of age were enrolled in the study from January 2014 to January 2015. The inclusion criteria were as follows: (1) clinical diagnosis of ischemic stroke according to the revised guideline of the 4th Cerebrovascular Disease Forum of China, as shown by CT or MRI; (2) treatment with clopidogrel for at least 5 d with complete laboratory examinations and clinical records; and (3) signed informed consent prior to the study. The exclusion criteria were as follows: (1) patients who were allergic to clopidogrel; (2) a platelet count greater than 450×109/L or less than 150×109/L; (3) anti-coagulation therapy with warfarin or heparin within 1 month of the study; (4) recent history of active bleeding; (5) ALT, AST or TB values 3-fold higher than the normal range; (6) serum creatinine 50% above the normal upper limit; and (7) major surgery within 1 month of the study. Depending on their condition, enrolled patients were treated with either a 300 mg loading dose of clopidogrel followed by a 75 mg maintenance dose per day or a 75 mg maintenance dose daily. Demographic characteristics, clinical information and medications were collected from patient electronic medical records at the Guangdong Provincial Hospital of Traditional Chinese Medicine.

Extraction of peripheral blood DNA

Whole blood samples were obtained from participants and collected in EDTA tubes and stored at −80 °C. The extraction method was performed based on a previous study with some modifications23. The concentration and purity of extracted DNA samples were calculated using a Nanodrop 2000 Spectrophotometer (Thermofisher, USA).

Genotype analysis

Four single nucleotide polymorphisms were selected for the study, including NR1I2 (rs13059232), CYP3A4*1G (rs2242480), CYP2C19*2 (rs4244285) and P2RY12 (rs2046934), and they were genotyped using Sequenom MassARRAY iPLEX technology (Sequenom, San Diego, CA, USA). Operations were based on the instructions for multiplexed genotyping analysis using iPLEX gold in a 96-well format for MassARRAY. DNA (10 ng) samples were amplified by multiplex PCRs, and the amplification products were subsequently used for iPLEX extension reactions. The final products were desalted and dispensed into a 96-pad SpectroCHIP. Allele detection was performed using MALDI-TOF mass spectrometry (Sequenom). MassARRAY Typer software was utilized to analyze the mass spectrums.

Platelet function assay

Platelet function was measured by the light transmission aggregometry method to evaluate antiplatelet responses. Before the next dose of clopidogrel was administered, peripheral venous blood samples were collected from patients. The platelet aggregation rate was measured within 3 h. The blood was centrifuged to separate the platelet-rich plasma (PRP) and the platelet-poor plasma (PPP), and the PRP sample was diluted to 2×109 to 3×109/L using the PPP sample. Next, both the PPP and PRP samples were heated to 37 °C for 3 min. To measure platelet aggregation, 10 μmol/L of ADP was added to the PRP sample to induce platelet aggregation, and the change in light transmission in the PRP sample was recorded until the response reached a plateau. The maximal platelet aggregation rate (MPAR) was calculated and recorded. After the baseline platelet aggregation measurements were obtained, follow-up platelet aggregation studies were repeated 5 d after the last dose of clopidogrel.

Definition of clopidogrel resistance

Clopidogrel resistance was defined as either less than a 10% change in MPAR on d 5 or the MPAR (d 5) was greater than 50% of the baseline magnitude (d 0)24,25. The platelet inhibition rate was quantified as follows:

Platelet inhibition rate (PIR)=PAR (d 0)–PAR (d 5).

PIR<10%×PAR (d 0) or PAR (d 5)>50% PAR (d 0) was defined as clopidogrel resistance. According to this standard, participants were divided into clopidogrel resistance (CR) or non-resistance (NCR) groups.

Statistical analysis

Data are presented as the mean±SD for continuous variables and as frequencies for categorical variables. The significant differences between groups were assessed using the Chi-square test (when variables were categorical) or the independent-samples t-test (when variables were continuous). Hardy-Weinberg equilibrium and allele frequency comparisons were predicted using the Chi-square test. A significance level of P<0.05 was based on the two-sided probability test. All statistical analysis was performed using SPSS version 21.0.

Results

Baseline characteristics of study participants

In total, 191 ischemic stroke patients with different disease statuses were enrolled. There were 110 males (58%) and 81 females (42%) included in our study. The average age of the participants was 67 years old. All of the patients were divided into the NCR or CR group according to the definitions specified in the methods section. The CR rate was 36% of the research population. In our study population, 131 subjects were in an acute phase, 11 subjects were in a recovery phase, 32 subjects were in a sequela phase and 17 subjects were suffering from transient ischemia. No discrepancies in the CR rate were found among these four categories of stroke patients. Other characteristics of the study population are presented in Table 1. No significant differences in the distribution of age, sex, diabetes, smoking, alcohol, medications, previous history, NIHSS score or biochemical indexes were found between the CR and NCR groups. Patients with hypertension exhibited a higher prevalence of CR (81% with hypertension in 69 patients).

Table 1 Baseline characteristics of the patients.
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Genotype distributions and allele frequencies of CYP2C19*2, CYP3A4*1G, NR1I2 and P2RY12

Table 2 shows the genotype distributions and allele frequencies of the four selected SNPs. Allele frequencies of all SNPs were in Hardy-Weinberg equilibrium (P>0.05). The frequencies of the mutant alleles and genotypes of CYP3A4*1G, CYP2C19*2 and P2RY12 (rs2046934) were similar to previously reported findings13,15,26. The genotype distribution and allele frequency of NR1I2 (rs13059232) was not documented well in research articles; therefore, we consulted the NCBI database and found that there was no significant difference between the results in the database and our data.

Table 2 Alleles and genotypes frequencies for the four SNPs in the research population.
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Association of CYP2C19, CYP3A4, NR1I2 and P2RY12 gene polymorphisms with clopidogrel resistance

The association of CYP2C19, CYP3A4, NR1I2 and P2RY12 gene polymorphisms with clopidogrel resistance are shown in Table 3. CYP3A4*1G was significantly associated with a lower rate of clopidogrel resistance (*1/*1 vs *1G/*1G+*1/*1G, OR: 2.360, 95% CI: 1.247–4.468, P=0.008), and the CR rate was higher in the *1/*1 genotype than in those carrying the variant genotypes *1G/*1G and *1/*1G (25.1% vs 14.3%). NR1I2 rs13059232 was moderately associated with CR (CC vs TT+TC, OR: 0.533, 95% CI: 0.286–0.991, P=0.046). Subjects with mutant homozygotes (CC) of NR1I2 rs13059232 had a lower rate of CR than those carrying the T allele (16.1% vs 21.0%). CYP2C19*2 allele carriers had a higher risk of CR compared with wt/wt carriers (21.1% vs 13.6%). P2RY12 rs2046934 was positively associated with CR (CC+TC vs TT, OR: 0.407, 95% CI: 0.191–0.867, P=0.018), and mutant allele (C) carriers exhibited a lower rate of CR compared with wild-type homozygotes (TT) (6.2% vs 29.4%).

Table 3 Association of four selected SNPs with clopidogrel resistance (CR) in ischemic stroke patients.
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Discussion

The prevalence of clopidogrel resistance in our study population was approximately 36% (n=69), which is within the previously reported range7,8. Previous studies have shown that the antiplatelet effect of clopidogrel is achieved after taking a daily dose of 75 mg for 3 to 7 d4,27. Michelson et al reported that clopidogrel resistance may be explained by a pre-existing difference in the platelet response to ADP, indicating that there may be interindividual variation in ADP-induced platelet aggregation at baseline before clopidogrel therapy28. Therefore, we compared the platelet aggregation rate on d 0 with the follow-up rate on d 5 (defined as the steady state of clopidogrel treatment) in each patient. No variation in MPAR (d 0) was detected between the CR and NCR groups, and we observed significantly decreased platelet activity on d 5.

CYP2C19*2 was an independent predictor of clopidogrel resistance in patients with cardiovascular and cerebrovascular diseases. In previous studies15,17, the CYP2C19*2 variant was widely accepted as a risk factor for clopidogrel resistance, and this was confirmed in our study (*2/*2+wt/*2 vs wt/wt, OR: 2.366, 95% CI: 1.180–4.741, P=0.014). The CR rate of subjects with the *2/*2 and wt/*2 genotypes was 7.6% higher than those with the wt/wt genotype, and the estimated CR risk of the CYP2C19*2 allele carriers was 2.366-fold higher than in the wt/wt carriers. Therefore, these results confirm that the CYP2C19*2 allele reduced the activity of CYP2C19, decreasing the production of active metabolites and thereby impairing clopidogrel-induced platelet inhibition.

CYP3A4*1G, a SNP in intron 10 of CYP3A4 and characterized by a G to A substitution at position 82266, was the most frequent mutant allele of CYP3A4 in Asians. The allele frequency of CYP3A4*1G was 0.22-0.37 in the Chinese population26,29. This mutant genotype was correlated with a higher CYP3A metabolic activity, thus increasing the formation of metabolites, particularly for prodrugs, which are activated through biotransformation via CYP3A30. In addition, there is evidence of an association between the presence of CYP3A4*1G and clopidogrel response variability in Spanish patients with stable coronary artery disease31. However, the relationship between CYP3A4*1G and clopidogrel resistance in Chinese patients with ischemic stroke had not been investigated previously. In this study, we found that the CYP3A4*1G variant had a protective effect on the clopidogrel resistance of ischemic stroke patients in China. The CYP3A4*1/*1 genotype added a 10.8% increased risk of CR, which resulted in a 2.360-fold higher estimated risk for *1/*1 carriers of CR compared with *1G allele carriers. The variant allele increased the activity of CYP3A4, leading to higher production of active metabolites and thereby increasing the antiplatelet efficacy of clopidogrel. Therefore, the CYP3A4*1G variant was determined to be a protective factor in clopidogrel resistance.

It is well known that the Pregnane X Receptor (NR1I2), an upstream regulator of CYP450s, is closely associated with the metabolism of xenobiotics and endogenous substances. Recently, many SNPs in the NR1I2 promoter and intron regions were studied in different races32,33. NR1I2 (rs13059232), a SNP in intron 1 of NR1I2, was consistently associated with CYP3A4 phenotypic measures. In our investigation, NR1I2 (rs13059232) was moderately associated with clopidogrel resistance (P=0.046, OR: 0.533). The estimated CR risk of subjects carrying the T allele was 1.876-fold higher than those carrying the mutant genotype (CC). Because NR1I2 (rs13059232) may influence the phenotype of CYP3A434,35 and the CYP3A4*1G polymorphism would affect the enzyme activity of CYP3A4, there is potential interaction between CYP3A4*1G and NR1I2 (rs13059232). In this study, we found a moderate association between NR1I2 (rs13059232) and CR in Chinese ischemic stroke patients, which had not been previously reported.

The P2RY12 gene encodes the P2Y12 receptor protein, which is the pharmacological target of clopidogrel. Genetic polymorphisms of the P2Y12 ADP receptor might alter the receptor activation induced by ADP or the response to platelet inhibitors in patients36. Goran et al reported that P2RY12 (rs2046934) was consistently associated with a higher platelet reactivity in patients on clopidogrel in the ADP-induced LTA and the VerifyNow P2Y12 assay; this may provide protection against atherothrombotic events16. Robert et al provided evidence for an association between the P2RY12 haplotype H2 (composed of dbSNP rs10935838, rs2046934, rs5853517 and rs6809699) and a lower risk of DVT/PE37. We found that the P2RY12 polymorphism was significantly associated with individual variation of clopidogrel sensitivity in ischemic stroke patients. In the current study, P2RY12 (rs2046934) mutant allele carriers (CC+TC) exhibited a lower risk of CR than TT carriers, with an OR of 0.407. Therefore, P2RY12 (rs2046934) was included as one of the variations in the P2RY12 gene that was examined and was found to contribute to interindividual variability during clopidogrel therapy. The P2RY12 (rs2046934) C allele also exhibited a protective effect on CR.

In the current study, hypertension was found as a non-genetic factor significantly associated with the antiplatelet effects of clopidogrel. A correlation between CR and hypertension was also documented in a recent study conducted on 303 ischemic cerebral infarction patients in China38. However, the relationship between hypertension and the incidence of clopidogrel resistance remains unclear. Although diabetes mellitus was previously suggested to influence the antiplatelet effects of clopidogrel39,40, we did not observe any correlation between CR and diabetes mellitus in our study population. In addition, sex, age, smoking, drinking or other biomedical indexes were not associated with CR in ischemic stroke patients. Concomitant use of lipophilic statins (atorvastatin, simvastatin) and calcium-channel blockers were suspected to be associated with a diminished antiplatelet response, which is likely due to a shared metabolic pathway via the CYP2C19 or CYP3A4 isoenzymes41,42. Therefore, we evaluated the association between CR and the co-administration of statins, anti-hypertensive drugs and other co-administered drugs that might affect the efficacy of clopidogrel according to previous reports43,44. Our results did not demonstrate a significant association of co-medication with CR in our research population. Several previous investigations similar to ours also did not find any obvious association between CR and potential drug-drug interactions45,46,47. Therefore, combination therapy might have an inconsequential impact and likely does not contribute to the influence of genetic factors on CR. Discrepancies in non-genetic factors influencing CR occurrence may vary among population and races. In addition, the impact of non-genetic factors may be masked by the effect of genetic factors.

In summary, our findings indicate that the CYP2C19*2 allele and hypertension are risk factors for CR, whereas the CYP3A4*1G allele, P2RY12 (rs2046934) C allele and NR1I2 (rs13059232) CC genotype are protective against CR in Chinese ischemic stroke patients. Because our study was limited by the sample size, further investigations should be conducted to verify the associated genetic and non-genetic factors with CR. The current results may be useful for predicting the development of clopidogrel resistance in different patient populations.

Abbreviations

MI, myocardial infarction; PAPI, pharmacogenomics of antiplatelet intervention; CT, computed tomography; MRI, magnetic resonance imaging; TB, total bilirubin; CR, clopidogrel resistance; NCR, non-clopidogrel resistance; OR, odds ratio; CI, confidence interval; MPAR, max platelet aggregation rate; EDTA, ethylenediaminetetraacetic acid; PD, pharmacodynamics; PK, pharmacokinetics; SNP, single nucleotide polymorphism; NIHSS, National Institutes of Health stroke scale; ADP, adenosine diphosphate; ALT, alanine transaminase; AST, aspartate aminotransferase; PLT, platelet; TC, total cholesterol; TG, triglyceride; LTA, light transmission aggregation; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; HWE, Hardy-Weinberg equilibrium; TIA, transient ischemic attack.

Author contribution

Jin JING, Min HUANG, and Rui LIU designed the study; Rui LIU, Zi-yi ZHOU, and Ye-feng CAI performed the research; Min ZHAO, Yuan-qi ZHAO, Wei-bang YU, and Yi-bei CHEN assisted with the research; Xin-meng CHEN contributed reagents; Rui LIU analyzed data; Rui LIU, Jia-li LI, and Jin JING wrote the paper.