The Genotypic Combination of Homozygous Carriers of Both H1 Haplotype and 34C Shows a Significantly Lower Responsiveness to ADP-Induced Platelet Aggregation Than Others in Healthy Volunteers.
Authors: Isobe Koji , Matsubara Yumiko , Takahashi Shinichi , Iwashita Miho , Komine Risa , Shimizu Aya , Uchida Toshihiro , Ishihara Hiroaki , Shibano Toshiro , Watanabe Gentaro , Ikeda Yasuo , Murata Mitsuru 
Published: April 2020

Abstract
BACKGROUND: The adenosine diphosphate (ADP) receptor, P2Y12, plays a central role in platelet activation and its blocker clopidogrel reduces the incidence of cardiovascular events. Recently, the polymorphisms of the P2Y12 gene (H1 and H2 haplotype) was shown to be associated with increased ADP-induced platelet aggregation in healthy volunteers. In addition, the polymorphism (C34T) of the P2Y12 gene was reportedly associated with higher numbers of cerebrovascular events in patients with PAD receiving clopidgrel therapy. However, it is totally unknown if there is any association between polymorphisms combination and platelet reactivity. In this study, we aimed to investigate in detail about the association between ADP-induced platelet aggregation and the polymorphisms of P2Y12 gene, haplotype and C34T, in healthy volunteers.
METHODS AND RESULTS: We determined P2Y12 genotype and platelet reactivity in 267 healty volunteers. Genotyping was performed using direct sequencing or a single nucleotide primer extension (SNuPE). In previous study, H1 and H2 haplotype were defined by 4 polymorphisms, C139T, T744C and insertion801A in intron1 and G52T in exon2, and there were in complete linkage disequilibrium. Result of genotyping of haplotype, however, G52T was found not to be in complete linkage disequilibrium. Allele frequencies of H1 and H2 were 0.83 and 0.17, respectively. For C34T, allele frequencies of 34C and 34T were 0.75 and 0.25, respectively. Platelet reactivity was evaluated by optical aggregometry in platelet-rich plasma. Aggregation was induced by 2 or 5 μmol/L ADP to evaluate maximal aggregation (%) and the area under the aggregate curve (AUC) for 6 min. Maximal aggregation induced by 2 μmol/L ADP was similar in homozygous carriers of H1 haplotype and carriers of H2 haplotype (p=0.262). AUC induced by 2 μmol/L ADP was lower in homozygous carriers of H1 haplotype than carriers of H2 haplotype (p=0.074). However, haplotype was not associated with platelet reactivity induced by 5 μmol/L ADP. For C34T, whereas, maximal aggregation and AUC induced by 2 or 5 μmol/L ADP was lower in homozygous carriers of 34C than carriers of 34T (p=0.009 and p=0.006 or p=0.002 and p=0.003, respectively). Furthermore, for the genotypic combination of haplotype and genotype (C34T), maximal aggregation and AUC induced by 2 or 5 μmol/L ADP was significantly lower in homozygous carriers of both H1 haplotype and 34C than others (p=0.003 and p&lt;0.001 or p=0.05 and p=0.007, respectively). When platelet aggregation was induced by 1 mg/mL Collagen, the same trends were observed.
CONCLUSIONS: We demonstrated that polymorphisms of the P2Y12 gene are associated with increased ADP-induced platelet aggregation, moreover, that group with genotypic combination of homozygous carriers of both H1 haplotype and 34C shows a significantly lower responsiveness to ADP-induced platelet aggregation than others in healthy volunteers. This suggests the possibility that polymorphisms of the P2Y12 gene are related to the receptor expression of P2Y12.