? ? The goal of the proposal is characterization of thrombin-mediated signaling pathways in platelets, a crucial component of the regulation of thrombosis and vascular disease. Thrombin signaling is mediated by a family of G-protein-coupled protease-activated receptors (PARs). Human platelets have been shown to express all three known thrombin receptors - PAR1, PAR3 and PAR4. Genomic analysis of the PAR cluster on chromosome 5813 identified the gene encoding IQGAP2, a putative Rho GTPase-activating protein (GAP), in close proximity to the PAR1 gene. GAPs have been shown to be required for many actin-dependent cellular processes, such as lymphocyte and fibroblast adhesion, cell motility, contraction, and cytokinesis. Preliminary data demonstrated IQGAP2 expression in human and murine platelets. Fluorescent immunostaining showed IQGAP2 and actin co-localization in filopodia of activated platelets and lamellae of COS1 cells. Thrombin/TRAP activation of human platelets was shown to induce IQGAP/actin/arp2/3 complex assembly. This complex formation appeared to require GTP-bound GTPases racl or cdc42, and was not induced by collagen or ADP. Thus, the primary hypothesis to be tested in this proposal is that IQGAP2 is a unique effector protein, possibly functioning as a scaffolding protein involved in thrombin-mediated cytoskeletal actin reorganization. Completion of Specific Aim I will characterize efficiency of racl and cdc42 at binding IQGAP2 and the effects of GTP/GDP-charging on IQGAP2 interactions and activation. Also it will identify other IQGAP2-binding proteins involved in actin cytoskeletal assembly. The goal of Specific Aim II is dissection of the molecular mechanisms regulating IQGAP2 function in thrombin-mediated signaling, including evaluation of the roles of individual thrombin receptors (PART/PAR3/PAR4). Generation and characterization of an IQGAP2-deficient (IQGAP2 -/-) knockout mouse (Specific Aim III) will define the role of platelet IQGAP2 in vivo. Should a definitive role for IQGAP2 as a scaffolding protein involved in platelet thrombin-mediated signaling be established, this will be a first example of evolutionary developed functional genomic unit of structurally unrelated clustered genes in humans. A further understanding of the mechanisms involved in signaling pathways of platelet activation and aggregation may identify potential targets for intervention in thrombosis-associated disease processes, a common long-term complications in patients with diabetes. During the award period, the candidate will develop an increased knowledge of genetics and platelet biology in a laboratory that is well-equipped for the execution of this proposal. The candidate's long-term goal is the pursuit of a career in academic medical research.
