Variation in platelet function: human PAR4 functional genomics

 描述(由申请人提供)：本申请是对HL102482“血小板功能变异：血小板基因表达的遗传学”的竞争性更新的重新提交。由这笔资金产生的工作包括发现(1)黑人受试者的血小板具有更大的PAR4介导的血小板对凝血酶的聚集，(2)一种新的血小板蛋白，磷脂酰胆碱转移蛋白(PC-TP)导致了这种种族差异，以及(3)microRNA miR-376c调节PC-TP水平(Edelstein等人，自然医学，2013)。我们对血小板聚集种族差异的分子遗传学基础的追求导致在F2RL3(编码PAR4)中发现了一个常见的种族差异变异，它诱导了Ala120Thr替换，约占PAR4反应活性种族差异的50%。PAR4 Thr120变异与PAR4-AP诱导的血小板聚集和IP在转基因细胞系中的生成有关。额外的初步数据表明，黑人种族赋予了 对COX和P2Y抑制的相对抗性为3，Thr120变异体通过PAR4增强凝血酶反应性，使其对FDA批准的12 PAR1抑制剂Vorapaxar具有相对抗性。此外，PAR4 Thr120阳性的血小板--尤其是纯合子(40%的黑人受试者)--对新型PAR4拮抗剂YD-3的抑制表现出抵抗力。这些数据表明，与患有心血管疾病的白人患者相比，黑人患者从FDA批准的大多数抗血小板药物中获得的好处可能较少。这一更新应用的目标是表征F2RL3变体的差异信号、药物遗传学和临床结果，并识别新的PAR4 Thr120拮抗剂。目的1将描述(A)PAR4Ala120Thr变异体凝血酶诱导的信号和剪切诱导的聚集的差异，以及(B)第二个PAR4Phe296Val变异体的显性-负性效应。目的研究在(A)抗血小板药物(阿司匹林、P2Y抑制剂和伏拉帕沙)12和(B)一类PAR4抑制剂临床或临床前研究中，PAR4变体对凝血酶诱导的血小板聚集的影响。AIM 3a将在约80,000名受试者的公共GWAS中检测F2RL3 SNPs作为缺血性心血管事件的危险因素。在AIM 3b中，我们将对示踪剂研究中的约6,000个样本进行基因分型，并测试F2RL3单核苷酸多态与接受Vorapaxar治疗的患者的心血管缺血和出血结果的相关性。由于目前的血小板信号传递模型可能主要基于使用白人献血者血液的实验，而且我们知道所有种族/民族的F2RL3等位基因频率，这些目标的成功完成将大大拓宽对血小板生物学的基本了解，开发变种特异性的PAR4抑制剂，可以改善PAR4Thr120阳性患者(约86%的非洲血统和36%的非非洲血统)的心血管预后，并可能为根据患者种族和/或F2RL3基因决定新的PAR1抑制剂的使用和/或剂量提供合理的基础。

 DESCRIPTION (provided by applicant): This application is a resubmission of a competitive renewal of HL102482 "Variation in platelet function: the genetics of platelet gene expression." The work resulting from this funding included discoveries that (1) platelets from black subjects have greater PAR4-mediated platelet aggregation to thrombin, (2) a novel platelet protein, phosphatidylcholine transfer protein (PC-TP) contributed to this racial difference, and (3) the microRNA miR- 376c regulated PC-TP levels (Edelstein et al., Nature Medicine, 2013). Our pursuit of the molecular genetic basis of the racial difference in platelet aggregation led to the discovery of a common, racially divergent variant in F2RL3 (encodes PAR4) that induces an Ala120Thr substitution, accounting for ~50% of the racial difference in PAR4 reactivity. The PAR4 Thr120 variant is associated with greater PAR4-AP-induced platelet aggregation and IP generation in transfected cell lines. Additional preliminary data indicates that black race confers relative 3 resistance to COX and P2Y inhibition, and the Thr120 variant confers relative resistance to the FDA-approved 12 PAR1 inhibitor, vorapaxar, via enhanced thrombin responsiveness through PAR4. Furthermore, PAR4 Thr120- positive platelets - and especially homozygotes (40% of black subjects) - show resistance to inhibition by a novel PAR4 antagonist, YD-3. These data suggest that compared to white patients with cardiovascular disease, black patients may receive less benefit from most FDA-approved anti-platelet agents. The goals of this renewal application are to characterize the differential signaling, pharmacogenetic and clinical outcomes of F2RL3 variants, and identify novel PAR4 Thr120 antagonists. Aim 1 will characterize (a) differences in PAR4 Ala120Thr variant thrombin-induced signaling and shear-induced aggregation, and (b) the dominant-negative effects of a second PAR4 Phe296Val variant. Aim 2 will characterize the effect of PAR4 variants on thrombin- induced platelet aggregation in the presence of (a) anti-platelet agents (aspirin, P2Y inhibitor and vorapaxar) 12 and (b) a class of PAR4 inhibitors in clinical or pre-clinical development. Aim 3a will test for F2RL3 SNPs as risk factors for ischemic cardiovascular events in public GWASs of ~80,000 subjects. In Aim 3b we will genotype ~6,000 samples from the TRACER study and test for associations between F2RL3 SNPs and both ischemic cardiovascular and bleeding outcomes in patients receiving vorapaxar. Because current models of platelet signaling are likely based primarily on experiments using blood from white donors, and because we know the F2RL3 allele frequencies in all races/ethnic groups, successful completion of these Aims will substantively broaden basic understanding of platelet biology, develop variant-specific PAR4 inhibitors that could improve cardiovascular outcomes for PAR4 Thr120-positive patients (~86% African ancestry and 36% non-African ancestry), and may provide a rational basis for decisions about use and/or dosing of new PAR1 inhibitors based on patient race and/or F2RL3 genotypes.