Platelets mediate the initial first-step in hemostasis through adhesive and aggregatory events, additionally providing the negatively charged phospholipid surface required for the contact phase and propagation of the coagulation cascade. A number of agonists cause platelet aggregation in vitro, although only strong agonists such as thrombin expose membrane phospholipid (PL). The molecular mechanisms that link thrombin activation to phospholipid exposure and the profound shape change that develops during cell activation (i.e. platelet adhesion and aggregation) remain poorly characterized, although they retain as their penultimate endpoint the process of actin polymerization. By using """"""""functional genomics,"""""""" we have identified and initially characterized a putative racl/cdc42 effector protein (IQGAP2) uniquely situated within the protease-activated gene cluster on chromosome 5q13. IQGAP2 and its related homologue IQGAP1 are expressed in human platelets, and linked to divergent GTPase pathways in thrombin-stimulated platelets; furthermore, murine platelets deficient in mIQGAP1 display a unique platelet phenotype manifest by dysfunctional calcium signaling and pathological platelet procoagulant activity (with microvesiculation). This competitive renewal of HL49141 will addresses the hypothesis that platelet IQGAPs represent key scaffolding proteins that integrate GTPase and intracytosolic calcium signals to regulate membrane events coordinating cytoskeletal actin reorganization, microvesiculation, and platelet procoagulant activity.
In specific aim 1, we will delineate the signals regulating the dysfunctional mIQGAP -/- platelet phenotype and dissect IQGAP1 and IQGAP2 functions using in vitro model systems;
in specific aim 2, we will determine the molecular mechanisms and phenotypic consequences of the procoagulant abnormality in mIQGAP 1-/- mice, specifically establishing the prothrombotic risk using in vivo models of arterial and microvascular thrombosis;
in specific aim 3, we will adapt a proteomics approach to identify and characterize multifunctional IQGAP complexes that coordinately regulate membrane events during platelet activation. These data will have considerable implications for understanding the molecular mechanisms linking intracellular events to a prothrombotic phenotype.
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