Influence Of P2Y12and GPⅡb Genetic Polymorphisms On In Vitro Anti-platelet Activity Of Ticagrelor

Background:Ticagrelor is the first reversible oral P2Y12receptor antagonist, which can bind to the platelet P2Y12receptor reversibly and prevent platelet membrane glycoprotein Ⅱb/IIIa (GPbⅢb/Ⅲa) receptor binding to fibrinogen, thereby inhibits platelet aggregation. P2Y12recepter is the target of several anti-platelet agents and P2Y12genetic polymorphisms are reported to be associated with ADP-induced platelet aggregation, risk of peripheral arterial disease and coronary heart disease, and clopidogrel responsiveness as well. GPIIb/IIIa receptors bind different adhesive proteins, such as fibrinogen and vWF, and represent the final common pathway for platelet activation, adhesion and aggregation. GPⅡb/Ⅲa genetic polymorphisms are also observed to be associated with aspirin resistance and risk of ischemic stroke, myocardial infarction, and coronary heart disease. Recently, a Caucasian population-based study with a small population size revealed that the anti-platelet activity of ticagrelor is not affected by genetic polymorphisms in candidate gene. There is no literature regarding the genetic association of ticagrelor response in Chinese population presently.Objective:To investigate the impact of genetic polymorphisms at P2Y12and GPIIb gene loci on in vitro anti-platelet activity of ticagrelor in Chinese healthy subjects, and to explore the potential molecular mechanisms causing individual differences in ticagrelor efficacy.Methods:Optical aggregometry was performed at baseline and after in vitro addition of15and50μM ticagrelor to the platelet-rich plasma of196Chinese healthy male subjects. Genomic deoxyribonucleic acid (gDNA) samples were extracted from blood samples by using standard phenol/chloroform protocols. Ten candidate single nucleotide polymorphisms (SNPs) in P2Y12and GPⅡb were selected from both the HapMap database and literatures. All the subjects were genotyped for the10selected SNPs by methods of PCR-RFLP or direct PCR-products sequencing. Linkage disequilibrium (LD) analysis, haplotype and haplotype tag SNPs were inferred by the softwares Haploview4.2and PHASE2.1. The influences of genetic polymorphisms on platelet aggregation were analyzed by ANOVA followed by Least Significant Difference (LSD) test, or by Mann-Whitney U test. Analyses of associations between haplotypes and platelet aggregation parameters were performed by using the haplo.glm model in Haplo.Stats software.Result:The minor allele frequency for rs1907637C>T, rs79320243A>G, rs10935842A>T, rs6787801T>C, rs6801273A>G, rs16863323G>A, rs2046934T>C, rs6785930C>T, rs6809699G>T and rs5911T>G polymorphism were22.96%,32.91%,37.24%,37.24%,28.83,37.5%,21.43%,14.29%,15.82%, and38.52%, respectively, in our population. As compared with P2Y12rs6785930CC homozygotes, individuals with the rs6785930CT genotype exhibited significantly lower ADP-induced platelet aggregation values and significantly increased inhibition in platelet aggregation (IPA) after incubation with50μM ticagrelor (P<0.05, respectively). Individuals with the P2Y12rs16863323GA genotype also showed significantly increased IPA at both peak aggregation and late aggratation with50μM ticagrelor incubation as compared to rs16863323AA homozygotes (P<0.05, respectively). Individuals with GPIIb rs5911GT genotype also showed significantly increased IPA after incubation with50μM ticagrelor as compared to the rs5911GG homozygotes (P<0.05). On influence of other seven SNPs on either ADP-induced aggregation or the in vitro anti-platelet activity of ticagrelor was observed. When haplotypes were analyzed,9haplotypes with frequencies>1%were observed, and Haplotype9(H9) showed significantly higher IPA after15μM ticagrelor treatment (P<0.005) in comparison with the reference haplotype1(H1). No association between other eight haplotypes and the in vitro anti-platelet activity of ticagrelor was observed (P>0.05).Conclusion:Candidate SNPs in P2Y12and GPⅡb are common in Chinese population.P2Y12rs6785930C>T, rsl6863323G>A polymorphisms, Haplotype9and GPⅡb rs5911T>G polymorphism were associated with in vitro anti-platelet activity of ticagrelor in Chinese male subjects.