The Role of the small GTPase Rap1 in Platelet-Coagulation Interplay

项目总结 血小板是一种对止血至关重要的血液成分，即防止部位失血。 血管损伤。为了止血，血小板必须通过小分子激活其初级整合素(AIIbb3)。 GTP酶RAP1和下游蛋白Talin。整合素活化对血小板黏附和 聚合。被激活的血小板也可以释放其颗粒内容物，并经历磷脂的扰乱 以支持形成稳定的止血栓子。血小板活化的机制必须紧密结合 控制为低活性的血小板会导致长时间的出血，而高活性的血小板与 血栓性疾病。 经过磷脂扰乱的血小板暴露出带负电荷的磷脂， 磷脂酰丝氨酸(PtdSer)结合到它们的外膜表面。这些所谓的促凝剂血小板 并激活凝血因子，导致凝血酶的生成和纤维蛋白的形成，这是一个完整的 稳定止血塞的组件。PtdSer暴露或凝血因子丢失会导致出血 并发症；相反，过量接触PtdSer会增加血栓形成的风险。尽管 与PtdSer调控失调相关的负面后果，对PtdSer的认识存在缺口 调节血小板活化后PtdSer暴露的机制。 小的GTPase Rap1是一种公认的血小板整合素激活和聚集的调节因子。 在体外失去Rap1也会导致PtdSer暴露的减少；然而，其机制和体内 依赖Rap1的PtdSer暴露的意义尚不清楚。有趣的是，Rho家族的GTP酶 (RhoA/RAC/CDC42)也调节血小板中PtdSer的暴露，并在多种细胞类型中进行研究，包括 血小板，已经显示了Rap1和Rho GTP酶之间的串扰。因此，我假设 依赖Rap1的PtdSer暴露通过与Rho家族小GTP酶的串扰而发生，并且 RAP1依赖的促凝血剂活性在止血栓形成过程中起着关键作用。初步数据 支持我的假设，因为抑制RhoA信号导致缺乏血小板的PtdSer暴露增加 两种Rap1亚型，Rap1A和Rap1B。我还建立了一个新的成像平台，允许 体内止血过程中血小板依赖性促凝血反应的研究。在目标1中，我将描述 在血小板激活导致PtdSer暴露期间，Rap1和Rho家族GTP酶之间的串扰。在……里面 目的2我将使用我的新的止血4-D成像模型来表征Rap1信号如何影响血小板- 体内凝血相互作用。这项工作将扩大我们对Rap1介导的调控的理解 体内血小板PtdSer暴露及其后果。

PROJECT SUMMARY Platelets are a blood component critical to hemostasis, i.e. the prevention of blood loss at sites of vascular injury. For hemostasis, platelets must activate their primary integrin (aIIbb3) through the small GTPases Rap1 and downstream protein Talin. Integrin activation is essential to platelet adhesion and aggregation. Activated platelets can also release their granule contents and undergo phospholipid scrambling to support the formation of a stable hemostatic plug. The mechanisms of platelet activation must be tightly controlled as hypoactive platelets cause prolonged bleeding while hyperactive platelets are associated with thrombotic disease. Platelets which undergo phospholipid scrambling expose a negatively charged phospholipid, phosphatidylserine (PtdSer), to their outer membrane surface. These so called procoagulant platelets recruit and activate coagulation factors resulting in the generation of thrombin and the formation of fibrin, an integral component of stable hemostatic plugs. Loss of PtdSer exposure or coagulation factors results in bleeding complications; conversely, excessive PtdSer exposure is associated with increased thrombotic risk. Despite the negative outcomes associated with dysregulation of PtdSer, there exists a gap in knowledge on the mechanisms regulating PtdSer exposure following platelet activation. The small GTPase Rap1 is a well-established regulator of platelet integrin activation and aggregation. Loss of Rap1 in vitro also results in decreased PtdSer exposure; however, the mechanism and in vivo significance of Rap1-dependent PtdSer exposure are unknown. Interestingly, Rho family GTPases (RhoA/Rac/Cdc42) also modulate PtdSer exposure in platelets, and studies in multiple cell types, including platelets, have demonstrated crosstalk between Rap1 and Rho GTPases. Therefore, I hypothesize that Rap1-dependent PtdSer exposure occurs through crosstalk with Rho family small GTPases, and that Rap1-dependent procoagulant activity is critical during hemostatic plug formation. Preliminary data supports my hypothesis as inhibition of RhoA signaling leads to increased PtdSer exposure in platelets lacking both Rap1 isoforms, Rap1A and Rap1B. I have also established a novel imaging platform which allows for the study of platelet-dependent procoagulant response during hemostasis in vivo. In aim 1 I will characterize the crosstalk between Rap1 and Rho Family GTPases during platelet activation leading to PtdSer exposure. In aim 2 I will use my novel 4-D imaging model of hemostasis to characterize how Rap1 signaling affects platelet- coagulation interplay in vivo. This work will expand our understanding of the regulation of Rap1 mediated platelet PtdSer exposure and its consequences in vivo.