PEAR1信号通路基因多态性对替格瑞洛抗血小板疗效的影响及机制研究

Remarkable interindividual difference in the drug response and clinical efficacy of antiplatelet drugs are observed. Genetic factors are supposed to play an important role. Ticagrelor is a novel antiplatelet drug and exhibits superior clinical efficacy than clopidogrel. Its efficacy is not influenced by genetic variations of genes coding drug metabolizing enzymes or drug transporters. Platelet endothelial aggregation receptor (PEAR1) plays a critical role in P2Y12-independent platelet aggregation and thrombosis. Genetic variation in PEAR1 gene can influence drug therapy for aspirin. In our preliminary work, we observed that polymorphisms in PEAR1 and the downstream signal transduction pathway were associated with in vitro antiplatelet activity of ticagrelor, indicating the polymorphism of PEAR1 signal pathway may serve as an important factor that affects the clinical efficacy and prognosis of ticagrelor. This study is designed to make clear whether genetics polymorphisms in PEAR1 and the downstream pathway can affect the antiplatelet activity, clinical efficacy and prognosis of ticagrelor in healthy volunteers and in patients undergoing PCI. In addition, functional studies of the newly identified polymorphisms associated with ticagrelor will be carried out to elucidate the molecular mechanism. The main aims of the study include: to identify new genetic biomarkers that affect antiplatelet drugs including ticagrelor, and to provide theoretical basis for the personalized medicine for antiplatelet drugs in clinic.

抗血小板药物疗效存在显著的个体差异，遗传因素在其中起重要作用。替格瑞洛为新型抗血小板药物，临床疗效优于氯吡格雷，且不受CYP2C19等药物代谢及转运基因变异的影响。血小板内皮聚集受体1（PEAR1）在血小板聚集和血栓形成过程中起重要作用，其遗传变异可显著影响阿司匹林的临床疗效。本项目在前期基于健康志愿者的体外研究发现PEAR1及其下游信号通路蛋白编码基因遗传多态可影响替格瑞洛的抗血小板作用基础上，提出PEAR1及其下游信号通路基因多态性可能影响替格瑞洛临床疗效和预后。项目拟通过严格设计的临床试验，查明PEAR1信号通路基因遗传变异对健康志愿者及PCI术后服用替格瑞洛患者抗血小板效应及临床疗效和预后的影响；同时通过细胞水平的研究，探讨PEAR1信号通路基因多态性影响替格瑞洛抗血小板效应的分子机制。本项目旨在发现影响替格瑞洛反应性和疗效的遗传标记物，为该药及同类药物的个体化治疗提供理论依据。

本课题分别在体外和体内水平研究了血小板内皮聚集受体1（PEAR1）基因多态性对替格瑞洛抗血小板活性的作用，并探索了遗传变异对中国汉族人群替格瑞洛、氯吡格雷抗血小板疗效的影响。..在体外实验中，采集196名健康受试者静脉血，测定不同浓度替格瑞洛的最大血小板聚集率（MPA），发现15μM及50μM替格瑞洛均能显著抑制血小板聚集；同时对PEAR1基因进行Sanger测序分型，结果显示在15μM替格瑞洛孵育后，PEAR1 rs12041331 AA突变纯合子携带者的MPA显著低于G等位基因携带者，Rs12566888 TT突变纯合子携带者的MPA显著低于GT杂合子携带者，Rs4661012 T等位基因携带者的MPA显著高于GG野生纯合子携带者。..其次，分别给予18名健康受试者180mg口服替格瑞洛，测定PEAR1 rs12041331、rs4661012，SLCO1B1 rs113681054、rs4149056，CYP3A4 rs2242480及CYP3A5 rs776746基因多态性对替格瑞洛药代及药效动力学的影响。研究显示以上不同基因型间替格瑞洛药代动力学参数未见显著差异；PEAR1 rs12041331 AA突变纯合子携带者的AUEC高于GG野生纯合子携带者，rs12041331 AA突变纯合子携带者给予替格瑞洛后2h的血小板聚集抑制率（PRI）低于GG基因型携带者。提示PEAR1 rs12041331多态性可能影响中国汉族人群替格瑞洛的抗血小板活性。. 临床募集了403名冠心病患者，口服300mg氯吡格雷负荷剂量或75mg/d维持剂量至少5天后，检测患者的PRI；并使用MassARRAY对患者进行基因分型，发现CYP2C19*2、*3携带者PRI显著高于野生型患者，ABCB1 rs1045642 A等位基因、P2Y12 rs6809699 A等位基因可升高PRI，CRISPLD1 rs12115090 C等位基因可降低PRI。..以上研究在不同层面探索了基因多态性对替格瑞洛、氯吡格雷抗血小板活性的影响，后续研究包括进一步探索DNA甲基化水平、miRNA等因素对抗血小板药物疗效的作用，以及在细胞分子水平探索其作用机制。