Human platelet PAR4: novel activation, interindividual variation, and neutrophil interactions in vivo and in vitro

此申请是一份新的申请，将HL102482资助的先前研究扩展到2009年资助的 确定导致缺血性闭塞的血小板功能个体间差异的遗传基础 动脉。我们生成了一个人类参考的血小板转录组，开发了一个公开的、用户友好的交互 查询血小板功能和RNA-蛋白质关联的Web工具，并通过 蛋白水解酶激活受体-4(PAR4)凝血酶受体在黑人中大于白人。我们展示了 这种差异是由于PAR4 Ala120Thr替换具有种族差异的等位基因频率(Thr120： .63个黑人；.19个白人)。Thr120变异体对凝血酶的敏感性增加，并在体外显示 在动脉切应力作用下血栓形成，而不是静脉切应力作用下。&gt；12,000名患者进行了基因分型 Thr120等位基因与缺血性卒中风险增加和出血减少相关。最近的工作是 导致了与血小板PAR生物学的基础和临床方面相关的新数据，以及当前 应用于研究(1)PAR4与其他GPCRs和蛋白水解酶的相互作用以及(2)人类 PAR4(HPAR4)和Ala120Thr变异体在脑缺血/再灌注(I/R)损伤的体内模型中的作用。PAR4杆 比PAR1的信号动力学慢，我们的新数据表明PAR4与血小板共沉淀 前列腺素I2受体(IP)。与PAR4 Ala120相比，Thr120在存在的情况下相对抵抗脱敏 前列环素(PGI2)，活化后人血小板和原代巨核细胞产生的cAMP减少 (MKS)。我们假设IP与PAR4交叉对话，目标1将评估IP/Gas在PAR信令和 我们的新型人源化PAR4小鼠品系的基因改变的人巨噬细胞和血小板的脱敏作用。 中性粒细胞丝氨酸蛋白酶，组织蛋白酶G(cathepsin G，CATG)，是一种有效的血小板激动剂，可以激活血小板PAR4， 但不是PAR1，并且我们发现CATG比Ala120诱导更多的PAR4 Thr120的血小板激活。对于第一次 一次，我们在PAR4中鉴定了CATG酶切位点，其中之一产生了一种新的系链配体， SRALLLGWVPTR，可诱导人血小板aIIbb3活化、颗粒释放和聚集。目标2将 研究CATG与血小板PAR4的相互作用。血小板PAR4，而不是PAR1，对白细胞的募集、滚动和 黏附，我们的初步数据表明，我们的人源化PAR4小鼠系的小鼠脑I/R损伤是hPAR4- 中性粒细胞依赖型。HPAR4在脑I/R损伤后卒中和脑出血中的作用一直很差 目标3的目标是利用我们的hPAR4小鼠系来确定hPAR4是如何介导血小板的 和中性粒细胞活性，并评估Ala120Thr的药物遗传效应 HPAR4介导的梗塞、出血和血小板信号通路的变体。圆满完成 拟议的研究有望加强对个体间差异的分子基础的理解。 在人血小板生物学和PAR4表达组织中的表达，为个体化抗血小板作用奠定了基础 对有种族倾向的疾病的治疗。

This application is a new submission extending prior research funded by HL102482, initially funded in 2009 to identify the genetic basis for inter-individual variation in platelet function that contributes to ischemic occlusion of arteries. We generated a human reference platelet transcriptome, developed a public, user-friendly interactive web tool to query platelet function and RNA-protein associations, and discovered platelet aggregation through the protease activated receptor-4 (PAR4) thrombin receptor was greater in blacks than whites. We showed this difference was due to a PAR4 Ala120Thr substitution with racially divergent allele frequencies (Thr120: .63 blacks; .19 whites). The Thr120 variant had increased sensitivity to thrombin and demonstrated ex vivo thrombus formation under arterial, but not venous, shear stress. Genotyping >12,000 patients demonstrated the Thr120 allele was associated with an increased risk of ischemic stroke and less bleeding. More recent work has led to new data relevant to basic and clinical aspects of platelet PAR biology, and the overall goals of the current application are to study (1) PAR4 interactions with other GPCRs and proteases and (2) the effect of human PAR4 (hPAR4) and the Ala120Thr variant in an in vivo model of brain ischemia/reperfusion (I/R) injury. PAR4 has slower signaling kinetics than PAR1, and our new data shows that PAR4 co-immunoprecipitates with platelet PGI2 receptor (IP). Compared to PAR4 Ala120, Thr120 is relatively resistant to desensitization in the presence of prostacyclin (PGI2), and generates less cAMP after activation in human platelets and primary megakaryocytes (MKs). We hypothesize IP cross-talks with PAR4, and Aim 1 will assess the role of IP/Gas in PAR signaling and desensitization in genetically altered human MKs and platelets from our novel humanized PAR4 mouse lines. The neutrophil serine protease, cathepsin G (CatG), is a potent platelet agonist that activates platelet PAR4, but not PAR1, and we show CatG induces more platelet activation of PAR4 Thr120 than Ala120. For the first time, we identified CatG enzymatic cleavage sites in PAR4, one of which generates a novel tethered ligand, SRALLLGWVPTR, which induces human platelet aIIbb3 activation, granule release and aggregation. Aim 2 will study CatG-platelet PAR4 interactions. Platelet PAR4, not PAR1, is critical for leukocyte recruitment, rolling and adhesion, and our preliminary data show that murine brain I/R injury in our humanized PAR4 mouse line is hPAR4- and neutrophil-dependent. The role of hPAR4 in stroke and brain hemorrhage after I/R injury has been poorly studied, and the goal of Aim 3 is to utilize our hPAR4 mouse lines to determine how hPAR4 mediates platelet and neutrophil activities in a murine stroke model, and to assess the pharmacogenetic effect of the Ala120Thr variant on hPAR4-mediated infarct, hemorrhage and platelet signaling pathways. Successful completion of the proposed studies is expected to enhance the understanding of the molecular basis of inter-individual variation in human platelet biology and PAR4-expressing tissues, and provide groundwork for individualized anti-platelet therapies in disorders with racial predilections.