Regulation of Platelet Shape Changes

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DESCRIPTION (provided by applicant): Shape change is essential for the damage control functions of blood platelets that prevent bleeding and is dependent on the remodeling and assembly of actin filaments in response to signals generated by ligated receptors. Details in differences of platelet surface shape changes may have important ramifications for the layering of platelets accompanying hemostatic and thrombotic reactions in vivo and may provide targets for future antithrombotic therapies. We previously defined certain platelet signaling cascades that end in net actin assembly/disassembly and have focused on the central roles of calcium, GTPases, and phosphoinositide production in the regulation of shape change and on the downstream targets of these agents. We have also labored to put the actin assembly reactions within a structural framework by first having defined the organization of actin and the membrane skeleton of the resting platelet and then dissecting the changes in this architecture that are required to initiate and sustain the shape change reaction. The overall goal of this project is to understand how platelets spread using large lamellae and filopods and integrate this information into the signaling pathways and proteins that mediate platelet shape change. This proposal has three specific aims. In the first, we highlight the central roles of rac, ralA and calcium in lamellapodial assembly. We take a novel structural approach that combines selectively permeabilized cells spread on defined surfaces to which we can introduce exogenous molecules. In the second aim, we focus on the role of cdc42 in platelet filopodial elaboration. The third aim details a bead-based actin assembly system, dependent on GTPase activity, which we will use to begin to reconstruct lamellar and filopodial actin assembly with the goal of biochemical simplification and then reconstitution of the essential components.