Thrombin regulation of Rap1 signaling in human platelet activity
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.