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激活剂之一，通过激活G蛋白偶联的蛋白酶受体PAR1和PAR4发挥作用，激活后导致RAP1的激活和血小板聚集的增加。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治疗中观察到的出血风险的关键。这项研究计划的目的是确定凝血酶如何不同地调节Rap1活性和随后的血小板激活。RAP1参与了凝血酶诱导的血小板活化，除了激活分泌、钙动员和聚集等下游信号通路外，还可能是整合素受体由内向外激活的重要中介。我建议研究凝血酶受体如何不同地发出RAP1活性的信号，信号的差异如何转化为血小板激活的水平，以及PAR对RAP1的时间调节如何能够在功能上决定人类血小板内的信号，如凝血酶后的聚集和分泌。 在指导阶段，我将在Heidi Hamm的监督下与HAMM实验室密切合作，以充分识别PAR1和PAR4在凝血酶调节血小板活性方面的差异。此外，我将用这段时间完善关键的分析方法，这些方法涉及抑制PAR激活下游的各种G蛋白途径，这些G蛋白途径在调节RAP1以及随后的血小板激活和血栓形成中发挥重要作用。 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激活。目标1将集中于确定在PAR1和PAR4刺激后对Rap1激活至关重要的G蛋白信号。在目标2中，我将研究哪些RAP1激活剂(S)(RAPGEF)在PAR介导的人血小板RAP1活化中起重要作用，以及它们在RAP1调节的血小板活性中的不同作用。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动的第一(可逆)和第二(不可逆)相的影响。 这些研究将为RAP1如何介导血小板激活提供更深入的见解。此外，它们将有助于阐明不同的PARs在RAP1激活中的作用及其在调节血小板激活中的作用。了解调节血小板活化的信号机制是试图确定抗血小板治疗的可能治疗靶点的关键一步。

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.