Thrombin regulation of Rap1 signaling in human platelet activity

描述（由申请人提供）： 凝血酶是最有效的血小板活化剂之一，通过活化G蛋白偶联蛋白酶受体PAR1和PAR4起作用，其在活化后导致Rap1活化和血小板聚集增加。PAR信号传导系统已被靶向作为抑制血小板活化的位点，因为阻断PAR信号传导被认为在降低其他抗血小板治疗中观察到的出血风险方面至关重要。这项研究的目的是确定凝血酶如何差异调节Rap1活性和随后的血小板活化。Rap1与凝血酶诱导的血小板活化有关，除了激活其他下游信号传导途径如分泌、钙动员和聚集外，Rap1可能是整合素受体由内而外活化的关键介质。我建议研究凝血酶受体的差异信号Rap1的活动，如何在信号的差异转化为血小板活化的水平，以及如何通过PAR的Rap1的时间调节是能够在功能上确定在人类血小板内的信号，如聚集和分泌凝血酶。 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，以充分确定通过PAR 1和PAR 4凝血酶调节的血小板活性的差异。此外，我将花这段时间完善参与PAR激活下游各种G蛋白通路抑制的关键测定，这些G蛋白通路在Rap1和随后的血小板激活和血栓形成的调节中发挥重要作用。 在独立阶段，我将确定PAR1和PAR4如何调节Rap1活性和随后的血小板活化。目的1将集中在确定G蛋白信号的Rap1激活重要的PAR 1和PAR 4刺激后。在目的2中，我将研究Rap1激活剂（S）（RapGEFs）是重要的PAR介导的Rap1激活在人类血小板和他们的差异作用Rap1调节血小板活性。目的3将集中于确定正反馈如何调节Rap1激活的时间特性及其对血小板活性的第一（可逆）和第二（不可逆）阶段的影响。 这些研究将为Rap1如何介导血小板活化提供更深入的了解。此外，它们将有助于阐明各种PAR在Rap1活化中的作用及其在调节血小板活化中的作用。了解调节血小板活化的信号传导机制是试图确定抗血小板疗法的可能治疗靶点的关键步骤。

DESCRIPTION (provided by applicant): 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, I 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.