Investigation of Platelet G-Protein Coupled Receptors

描述(申请人提供)：虽然ADP P2Y1和血栓素A2受体(TPR)的信号在血栓性疾病发病机制中的作用已有很好的文献记载，但目前还没有可用于临床使用的拮抗剂，可以针对这两条途径中的任何一条。这在一定程度上是因为缺乏关于受体信号和结构的知识。本申请提出了涉及血小板TPR和P2Y1受体的结构生物学和信号转导的基本方面的实验：1)P2Y1配体结合区(即第二细胞外环；EL2)拮抗在预防血栓性疾病中的生物学意义。我们的假设是，P2Y1的EL2在血小板活化中起重要作用。为了解决这一假设，我们的实验将评估一种新型的针对EL2的功能性抗体(简称EL2Ab)阻断ADP诱导的血小板激活的能力。随后的研究中，我们将调查EL2Ab和EL2多肽疫苗对出血时间和血栓形成的影响。2.TPR的G蛋白偶联结构域。我们的假设是TPR的细胞内(IL)结构域包含单独的区域，这些区域限制与特定G蛋白的偶联。在这方面，我们最有趣的结果之一是模拟TPR第一个IL的多肽(缩写为Myr-IL1pep)阻止了TPR介导的聚集，但不能阻止形状变化。这一发现表明，TPR的IL1结构域参与了GQ的受体偶联，而不是G13。同样，其他IL区域在G蛋白偶联中的作用将通过检测其对应的多肽对TPR触发的血小板聚集、形状变化和分泌的影响来确定。此外，还将研究任何具有生物活性的白介素肽对出血时间和血栓形成的影响。总的来说，这些研究的结果将提供关于TPR和P2Y1受体的生物学和结构的基本信息，并可能定义新的治疗靶点和/或辅助用于治疗血栓性疾病状态的有机衍生物/制剂的分子模拟研究预测。

DESCRIPTION (provided by applicant): While the involvement of signaling of the ADP P2Y1 and the thromboxane A2 receptors (TPR) in the pathogenesis of thrombotic diseases is well documented, there are no antagonists that are currently available for clinical use, and which target either of these pathways. This derives, in part, from lack of knowledge regarding receptor signaling and structure. The present application proposes experiments that address fundamental aspects of the structural biology and signaling of platelet TPR and P2Y1 receptors: 1) The biological significance of antagonism of P2Y1 ligand-binding region (i.e., the second extracellular loop; EL2 in the prevention of thrombotic diseases. Our hypothesis is that EL2 of P2Y1 plays an important role in platelet activation. To address this hypothesis, our experiments will evaluate the ability of a novel functional antibody targeting EL2 (abbreviated as EL2Ab) to block ADP-induced platelet activation. Subsequent studies we will investigate the effects of EL2Ab and an EL2 peptide-based "vaccine" on bleeding time, and thrombosis development. 2. The G-protein coupling domains of TPR. Our hypothesis is that the intracellular (IL) domains of TPR contain separate regions that confine coupling to a specific G-protein. In this regard, one of our most intriguing results is that a peptide mimicking the first IL of TPR (abbreviated Myr-IL1pep) blocked TPR-mediated aggregation but not shape change. This finding suggests that the IL1 domain of TPR participates in receptor coupling to Gq, but not G13. Similarly, the role of other IL regions in G-protein coupling will be determined by examining the effects of their corresponding peptides on TPR-triggered platelet aggregation, shape change and secretion. Also, the effects of any biologically-active IL peptides on bleeding time and thrombosis development will be investigated. Collectively, results obtained from these studies will provide fundamental information concerning TPR and P2Y1 receptor biology and structure, and may define new therapeutic targets and/or aid molecular modeling study predictions for organic derivatives/agents for treating thrombotic disease states.