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

小 GTP 酶 Rap1 在血小板-凝血相互作用中的作用

• 批准号: 10762329
• 负责人: Abigail Rene Ballard
• 金额: $ 3.92万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10762329
• 关键词: 3-Dimensional; 4D Imaging; Adhesions; Affect; Agonist; Biochemical; Biological; Blood; Blood Coagulation Factor; Blood Platelets; Cells; Charge; Coagulation Process; Communication; Complex; Cytoplasmic Granules; Data; Family; Fibrin; Generations; Guanosine Triphosphate Phosphohydrolases; Hemorrhage; Hemostatic Agents; Hemostatic function; Hyperactivity; Impairment; In Vitro; Integrins; Knowledge; Link; Mediating; Megakaryocytes; Membrane; Mitochondria; Modeling; Monomeric GTP-Binding Proteins; Mutation; Outcome; Patients; Permeability; Phosphatidylserines; Phospholipids; Phosphotransferases; Platelet Activation; Platelet aggregation; Prevention; Process; Protein Isoforms; Proteins; Proteomics; Regulation; Risk; Role; Signal Transduction; Site; Surface; Talin; Thrombin; Thrombosis; Time; Work; cell type; cyclophilin D; fibrous protein; imaging platform; in vivo; inhibitor; member; novel; platelet function; receptor; recruit; release of sequestered calcium ion into cytoplasm; response; rho; rho GTP-Binding Proteins; thrombotic; vascular injury

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.

项目概要 血小板是对止血至关重要的血液成分，即防止止血部位失血 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。整合素激活对于血小板粘附和 聚合。活化的血小板也可以释放其颗粒内容物并进行磷脂加扰 以支持形成稳定的止血塞。血小板活化机制必须严密 受到控制，因为血小板活性低下会导致长时间出血，而血小板活性过高则与 血栓性疾病。 经历磷脂加扰的血小板暴露出带负电的磷脂， 磷脂酰丝氨酸（PtdSer），到其外膜表面。这些所谓的促凝血小板招募 并激活凝血因子，导致凝血酶的产生和纤维蛋白的形成，纤维蛋白是不可或缺的 稳定止血塞的组成部分。 PtdSer 暴露或凝血因子的丧失导致出血 并发症；相反，过多的 PtdSer 暴露与血栓形成风险增加相关。尽管 与 PtdSer 失调相关的负面结果，在这方面存在知识空白 血小板活化后调节 PtdSer 暴露的机制。 小 GTPase Rap1 是公认的血小板整合素激活和聚集调节剂。 体外 Rap1 缺失也会导致 PtdSer 暴露量减少；然而，其机制和体内 Rap1 依赖性 PtdSer 暴露的意义尚不清楚。有趣的是，Rho 家族 GTPases (RhoA/Rac/Cdc42) 还调节血小板中的 PtdSer 暴露，以及多种细胞类型的研究，包括 血小板，已证明 Rap1 和 Rho GTPases 之间的串扰。因此，我假设 Rap1 依赖性 PtdSer 暴露是通过与 Rho 家族小 GTPase 的串扰发生的，并且 Rap1 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 支持我的假设，因为抑制 RhoA 信号传导会导致缺乏血小板的 PtdSer 暴露增加 Rap1 两种亚型：Rap1A 和 Rap1B。我还建立了一个新颖的成像平台，可以 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 血小板激活过程中 Rap1 和 Rho 家族 GTPases 之间的串扰导致 PtdSer 暴露。在 目标 2 我将使用我的新型止血 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 血小板 PtdSer 暴露及其体内后果。