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: 10537859
• 负责人: Abigail Rene Ballard
• 金额: $ 3.83万
• 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/10537859
• 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; 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.

项目摘要 血小板是对止血至关重要的血液成分 血管损伤。对于止血，血小板必须通过小型整联蛋白（AIIBB3）激活 GTPases Rap1和下游蛋白塔林。整联蛋白激活对于血小板粘附至关重要 聚合。活化的血小板还可以释放其颗粒含量并进行磷脂 支持稳定的止血塞的形成。血小板激活的机制必须紧密 受控作为低血小板的血小板会导致长时间出血，而多动态血小板与 血栓性疾病。 经历磷脂的血小板暴露了带负电的磷脂的血小板， 磷脂酰丝氨酸（PTDSER），到其外膜表面。这些所谓的Procagulant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（积分） 稳定止血塞的成分。 PTDSER暴露或凝结因子的丧失导致出血 并发症；相反，过度的PTDSER暴露与增加的血栓形成风险有关。尽管 与PTDSER失调相关的负面结果，有关该知识的差距 血小板激活后调节PTDSER暴露的机制。 小GTPase RAP1是一个完善的血小板整合素激活和聚集的调节剂。 体外RAP1的损失也导致PTDSER暴露降低；但是，机制和体内 RAP1依赖性PTDSER暴露的意义尚不清楚。有趣的是，Rho Family GTPase （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 血小板已显示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家族小型GTPases的串扰发生的，并且 在止血塞形成过程中，依赖RAP1的凝聚活性至关重要。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暴露量增加 Rap1同工型，RAP1A和RAP1B。我还建立了一个新颖的成像平台，该平台允许 研究在体内止血过程中血小板依赖性的凝凝胶反应的研究。在AIM 1中，我将表征 血小板激活期间Rap1和Rho家族GTPases之间的串扰导致PTDSER暴露。在 AIM 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 体内凝结相互作用。这项工作将扩大我们对RAP1中介的调节的理解 血小板PTDSER暴露及其在体内的后果。