Thrombin regulation of Rap1 signaling in human platelet activity

凝血酶是血小板最有效的激活剂之一，通过激活G蛋白偶联的 蛋白酶受体PAR 1和PAR 4，其在活化后导致Rap 1活化和血小板聚集增加。 聚合来PAR信号系统已被靶向作为抑制血小板活化的位点， 阻断PAR信号传导被认为是降低其他抗血小板药物中观察到的出血风险的关键。 治疗这项研究的目的是确定凝血酶如何差异调节 Rap 1活性和随后的血小板活化。Rap 1与凝血酶诱导的激活有关 可能是一个重要的介体信号由内而外激活整合素受体，除了 激活其他下游信号传导途径，如分泌、钙动员和聚集。我 我建议研究凝血酶受体如何差异信号Rap 1活性， 信号传导转化为血小板活化水平，以及PAR对Rap 1的时间调节如何能够 在功能上决定人血小板内的信号传导，例如血小板聚集和分泌， 凝血酶 在指导阶段，我将在Heidi哈姆的监督下与哈姆实验室密切合作， 为了充分确定凝血酶通过PAR 1和PAR 4调节血小板活性的差异。 此外，我将花这段时间完善涉及抑制各种G蛋白的关键测定法， PAR激活下游途径在Rap 1及随后的调节中发挥重要作用 血小板活化和血栓形成。 在独立阶段，我将确定PAR 1和PAR 4如何调节Rap 1活性，以及随后的 血小板活化目的1将集中于确定Rap 1激活的重要G蛋白信号 在刺激PAR 1和PAR 4之后。在目标2中，我将研究哪些Rap 1激活剂（RapGEFs）是 重要的PAR介导的Rap 1激活在人类血小板和他们的不同作用Rap 1- 调节血小板活性。目标3将集中于确定正反馈如何调节时间 Rap 1激活的性质及其对血小板第一（可逆）和第二（不可逆）相的影响 活动 这些研究将为Rap 1如何介导血小板活化提供更深入的了解。此外，本发明还 它们将有助于阐明各种PAR在Rap 1激活中的作用及其在 调节血小板活化。了解调节血小板活化的信号机制是一个重要的研究课题。 这是试图确定抗血小板治疗可能的治疗靶点的关键一步。

Thrombin, one of the most potent activators of platelets, works through activation of G protein-coupled protease receptors PAR1 and PAR4, which upon activation lead to increases in Rap1 activation and platelet aggregation. The PAR signaling system has been targeted as a site for inhibiting platelet activation because blocking PAR signaling is thought to be crucial in decreasing the risk to bleeding observed in other antiplatelet therapies. The aim of this research proposal is to identify how thrombin may differentially regulate Rap1 activity and subsequent platelet activation. Rap1 has been implicated in thrombin-induced activation of platelets and may be a crucial mediator signaling inside-out activation of integrin receptors in addition to activating other downstream signaling pathways such as secretion, calcium mobilization, and aggregation. I propose to investigate how the thrombin receptors differentially signal Rap1 activity, how differences in signaling translate to the level of platelet activation, and how temporal regulation of Rap1 by PAR is able to functionally determine the signaling within the human platelet such as aggregation and secretion following thrombin In the mentored phase, I will work closely with the Hamm lab under the supervision of Heidi Hamm in order to fully identify the differences in thrombin-regulated platelet activity through PAR1 and PAR4. Additionally, I will spend this time perfecting the crucial assays involved in inhibition of the various G protein pathways downstream of PAR activation that play an important role in regulation of Rap1 and subsequent platelet activation and thrombosis. In the independent phase, I will determine how PAR1 and PAR4 regulate Rap1 activity and subsequent platelet activation. Aim 1 will focus on determining the G protein signals important for Rap1 activation following stimulation of PAR1 and PAR4. In Aim 2,1 will investigate which Rap1 activator(s) (RapGEFs) are important for PAR-mediated Rap1 activation in the human platelet and their differential roles in Rap1- regulated platelet activity. Aim 3 will focus on determining how positive feedback regulates the temporal properties of Rap1 activation and its effects on the 1st (reversible) and 2nd (irreversible) phases of platelet activity. These studies will provide deeper insight into how Rap1 mediates platelet activation. Additionally, they will help to elucidate the contribution of the various PARs in the activation of Rap1 and its role in regulating platelet activation. Understanding the signaling mechanisms regulating platelet activation is a critical step in trying to identify possible therapeutic targets for anti-platelet therapies.