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

项目摘要 血小板是止血的关键血液成分，即防止出血部位的失血。 血管损伤为了止血，血小板必须通过血小板膜上的小通道激活其初级整合素（aIIbb 3）。 GTP酶Rap 1和下游蛋白Talin。整合素活化对于血小板粘附和血小板活化是必不可少的。 聚合来活化的血小板也可以释放它们的颗粒内容物并进行磷脂混乱 以支持稳定止血塞的形成。血小板活化的机制必须紧密 控制低活性血小板导致长期出血，而高活性血小板与 血栓性疾病 经历磷脂混乱的磷脂暴露带负电荷的磷脂， 磷脂酰丝氨酸（PtdSer），其外膜表面。这些所谓的促凝血血小板 并激活凝血因子，导致凝血酶的产生和纤维蛋白的形成， 稳定止血塞的组分。PtdSer暴露或凝血因子缺失导致出血 并发症;相反，过量PtdSer暴露与血栓形成风险增加相关。尽管 与PtdSer调节异常相关的负面结果，在对PtdSer的认识方面存在差距。 调节血小板活化后PtdSer暴露的机制。 小的GTAP 1 Rap 1是血小板整合素活化和聚集的公认调节剂。 Rap 1在体外的缺失也会导致PtdSer暴露的减少;然而，在体内的机制和 Rap 1依赖性PtdSer暴露的意义是未知的。有趣的是，Rho家族GTP酶 (RhoA/Rac/Cdc 42）也调节血小板中的PtdSer暴露，并且在多种细胞类型中进行了研究，包括 血小板，已经证明了Rap 1和Rho GTP酶之间的串扰。因此，我假设 Rap 1依赖性PtdSer暴露通过与Rho家族小GTP酶的串扰发生， Rap 1依赖性促凝血活性在止血栓形成过程中至关重要。初步数据 RhoA信号抑制导致血小板缺乏PtdSer暴露增加，这支持了我的假设。 两种Rap 1亚型，Rap 1A和Rap 1B。我还建立了一个新的成像平台， 体内止血过程中血小板依赖性促凝反应研究。在目标1中，我将描述 血小板活化期间Rap 1和Rho家族GTP酶之间的串扰导致PtdSer暴露。在 目的2：我将使用我的新型4-D止血成像模型来表征Rap 1信号传导如何影响血小板， 体内凝血相互作用。这项工作将扩大我们对Rap 1介导的调控的理解。 血小板PtdSer暴露及其体内后果。

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.