In this study, we evaluated the effects of genetic polymorphisms in the P2Y12 receptor-mediated signaling pathway and CYP2C19 on clopidogrel antiplatelet response in Chinese CHD patients. We observed that CYP2C19*2 and *3 and P2Y12 rs6809699 polymorphisms were associated with an increased risk of clopidogrel resistance indicated by platelet VASP-P level.
Clopidogrel is a prodrug that needs to be bioactivated in two sequential cytochrome P450-dependent steps before it exerts an inhibitory effect on ADP-induced platelet aggregation. CYP2C19 activity is reported to be crucial in the metabolism and efficacy of clopidogrel. CYP2C19 LOF alleles, including *2 and *3 can decrease the plasma concentration and AUC0−24h of the active metabolite of clopidogrel, which results in impaired antiplatelet effect of clopidogrel6, 23. A recent meta-analysis has concluded that CYP2C19 LOF is associated with increased risk of adverse clinical events in patients who underwent clopidogrel therapy despite differences in clinical significance according to ethnicity 7. In support of these previous reports, we observed that patients with the CYP2C19*1/*1 genotype showed significantly lower PRI than the CYP2C19*2 heterozygous and homozygous genotypes. Besides, we found that carriers of any of the *2 and *3 alleles showed increased clopidogrel resistance. Our findings further confirmed the pivotal role of CYP2C19*2 and *3 as pharmacogenomics markers for clopidogrel response. In the 2013 updated Clinical Pharmacogenetics Implementation Consortium Guidelines for CYP2C19 Genotype and Clopidogrel Therapy, CYP2C19 genotype-guided clopidogrel therapy was recommended to ACS patients underwent PCI25. Standard dosing of clopidogrel is warranted among ACS/PCI patients with a predicted CYP2C19 extensive metabolizer phenotype (*1/*1). If genotyping identifies a patient as a CYP2C19 weak metabolizer phenotype (*2/*2, *2/*3 and *3/*3), the use of an alternative antiplatelet agent (e.g., prasugrel or ticagrelor) is recommended if not clinically contraindicated.
ADP is an essential activator of platelet and acts via P2Y1 (Gq-coupled) and P2Y12 (Gi-coupled) receptors. The Gq-coupled P2Y1 receptor is vital in Ca2+ mediated platelet shape change, while the Gi-coupled P2Y12 receptor is required for ADP-induced platelet activation26. The active metabolite of clopidogrel binds to the P2Y12 receptor irreversibly and inhibits ADP-mediated platelet activation and aggregation. The role of the P2RY12 genetic polymorphisms in clopidogrel response has been assessed previously19, 20, 27–30. Evidence shows that the P2RY12 T744C (rs2046934) polymorphism is associated with enhanced platelet aggregation and increased risk of atherothrombosis19, 28. However, Thomas et al. failed to replicate this observation with platelet activity assessed by either ADP-Ag (P=0.39), or PRI VASP-P (P=0.97), or P-selectin expression (P=0.62) in 597 NSTE ACS patients27. Other studies also come to negative findings 29, 30. In agreement with the latter investigators, we did not find any association between the P2RY12 T744C and clopidogrel resistance either.
The P2RY12 G52T (rs6809699) was also shown to be associated with increased risk of clopidogrel resistance and cardiovascular events in Chinese ACS patients after PCI 20. In support of this report, we observed that CHD patients with the P2RY12 rs6809699 CA genotype or carriers of the rs6809699 A allele showed an increased risk for clopidogrel resistance with an OR of 1.729 and 2.017, respectively. After stratification by CYP2C19*2 and *3 carrying status, the P2RY12 rs6809699 polymorphism remained to be associated with increased platelet activity. As the rs6809699 polymorphism is a synonymous SNP (Gly12Gly) does not result in amino acid change, the exact function of this SNP deserved further investigation.
Abnormality in GPIIb/IIIa complex is reported in Glanzmann’s thrombasthenia patients with impaired platelet aggregation and increased bleeding 31. The ITGB3 PlA1/A2 polymorphism (rs5918) results in a leucine (PlA1) to proline (PlA2) substitution in exon2 was observed 32. This SNP has been extensively studied and is shown to be associated with both antiplatelet drug resistance and increased cardiovascular events33, 34. Because the prevalence of the PlA2 allele is low in the Chinese population, the SNP was not included in our study. Two other SNPs, including rs3785873 and rs58847127 at the ITGB3 locus were investigated in our study. However, no significant findings were obtained for these two SNPs. A healthy subjects study showed that ITGA2B rs3760364 were related to bleeding time17, but we failed to find the association between ITGA2B rs3760364 and platelet activity.
In our study, we also observed that the APBB11P rs11015149 A allele was significantly over-represented in CR than non-CR patients, but this difference was disappeared after adjusted for statins use and dyslipidemia. The other 6 selected SNPs in genes in the P2Y12-mediated signaling pathway (PIK3CA rs67562832 and rs67562832, RASGRP2 rs2230414, APBB1IP rs11015149, TLN1 rs2295795, and rs10814270) also showed no association with clopidogrel resistance. It remains unknown whether genetic factors in other alternative pathways playing compensatory roles in GPIIb/IIIa inside-out signaling could affect clopidogrel response.
Limitations of the study include a relatively small sample size. As exemplified by only 4 patients with P2RY12 rs6809699 mutant AA genotype in our study, further studies are warranted to verify the impact of P2RY12 rs6809699 polymorphisms on antiplatelet effects of clopidogrel. Secondly, platelet function testing was done with only a single assessment of platelet function, VASP-P assay, which may not be sufficient to fully reflect the response to antiplatelet therapy. Finally, follow-up data is warranted to understand the influence of the positively associated SNPs on the endpoint events and outcome of CHD patients with long-time clopidogrel therapy.