Thrombin regulation of Rap1 signaling in human platelet activity

凝血酶是最有效的血小板激活剂之一，通过激活G蛋白偶联来发挥作用 蛋白水解酶受体PAR1和PAR4在激活时导致RAP1活化和血小板增加 聚合。PAR信号系统被认为是抑制血小板活化的靶点，因为 阻断PAR信号被认为是降低其他抗血小板药物出血风险的关键。 治疗。这项研究提案的目的是确定凝血酶可能如何不同地调节 RAP1活性和随后的血小板激活。RAP1与凝血酶诱导的激活有关 可能是由内向外激活整合素受体的重要介质，此外 激活其他下游信号通路，如分泌、钙动员和聚集。我 建议研究凝血酶受体如何不同地传递RAP1活性的信号，如何在 信号转译为血小板激活的水平，以及PAR对Rap1的时间调节如何能够 从功能上确定人血小板内的信号转导，如以下聚集和分泌 凝血酶 在指导阶段，我将在Heidi Hamm的监督下与HAMM实验室密切合作 目的：通过PAR1和PAR4来充分认识凝血酶调节的血小板活性的差异。 此外，我将用这段时间完善与抑制各种G蛋白有关的关键检测方法 PAR激活下游的通路在调节RAP1及其后续过程中起重要作用 血小板活化和血栓形成。 在独立阶段，我将确定PAR1和PAR4如何调节Rap1的活性以及随后的 血小板被激活。目标1将专注于确定对Rap1激活重要的G蛋白信号 在刺激PAR1和PAR4之后。在目标2中，1将调查哪些RAP1激活剂(S)(RAPGEF)是 PAR介导的人血小板RAP1活化的重要性及其在RAP1-1中的不同作用 调节血小板活性。目标3将侧重于确定正反馈如何调节时间 RAP1的激活特性及其对血小板第一(可逆)和第二(不可逆)相的影响 活动。 这些研究将为RAP1如何介导血小板激活提供更深入的见解。另外， 它们将有助于阐明不同的PAR在激活RAP1中的贡献及其在 调节血小板活化。了解调节血小板激活的信号机制是一个 在试图确定抗血小板治疗的可能治疗靶点方面迈出了关键的一步。

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.