Variation in platelet function: human PAR4 functional genomics

 描述（由申请人提供）：本申请是HL 102482“血小板功能变异：血小板基因表达的遗传学”竞争性更新的再次提交。“这项资助产生的工作包括发现（1）来自黑人受试者的血小板具有更大的PAR 4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）促成了这种种族差异，以及（3）microRNA miR-376 c调节PC-TP水平（埃德尔斯坦et al.，Nature Medicine，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了F2 RL 3（编码PAR 4）中一种常见的种族差异变体的发现，该变体诱导了Ala 120 Thr置换，占PAR 4反应性种族差异的约50%。PAR 4 Thr 120变体与转染细胞系中更大的PAR 4-AP诱导的血小板聚集和IP生成相关。额外的初步数据表明， Thr 120变体对考克斯和P2 Y抑制具有相对抗性，Thr 120变体通过PAR 4增强的凝血酶反应性赋予对FDA批准的PAR 1抑制剂vorapaxar的相对抗性。此外，PAR 4 Thr 120阳性血小板-尤其是纯合子（40%的黑人受试者）-对新型PAR 4拮抗剂YD-3的抑制表现出抗性。这些数据表明，与患有心血管疾病的白色患者相比，黑人患者从大多数FDA批准的抗血小板药物中获益较少。此次更新申请的目的是表征F2 RL 3变体的差异信号传导、药物遗传学和临床结局，并鉴定新型PAR 4 Thr 120拮抗剂。目的1将表征（a）PAR 4 Ala 120 Thr变体凝血酶诱导的信号传导和剪切诱导的聚集的差异，以及（B）第二种PAR 4 Phe 296 Val变体的显性负效应。目的2将表征在（a）抗血小板剂（阿司匹林、P2 Y抑制剂和vorapaxar）12和（B）临床或临床前开发中的一类PAR 4抑制剂存在下PAR 4变体对凝血酶诱导的血小板聚集的影响。目标3a将在约80，000名受试者的公共GWAS中测试F2 RL 3 SNP作为缺血性心血管事件的风险因素。在目标3b中，我们将对来自TRACER研究的约6，000个样本进行基因分型，并在接受vorapaxar的患者中测试F2 RL 3 SNP与缺血性心血管和出血结局之间的相关性。由于目前的血小板信号传导模型可能主要基于使用来自白色供体的血液的实验，并且由于我们知道所有种族/民族中的F2 RL 3等位基因频率，因此这些目标的成功完成将大大拓宽对血小板生物学的基本理解，开发变体特异性PAR 4抑制剂，可改善PAR 4 Thr 120阳性患者的心血管结局（约86%为非洲血统，36%为非非洲血统），并可为基于患者人种和/或F2 RL 3基因型的新型PAR 1抑制剂的使用和/或给药决策提供合理依据。

 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.