Verbatim): Platelet shape change is essential to seal damaged vascular surfaces. 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. Platelets circulate in blood as small discs that in response to tethered and soluble agonists convert into active forms which rapidly grow filopods and lamellipods from their surfaces. Process growth is dependent on actin assembly and on the spatial location of nucleating sites that initiate filament assembly and signaling pathways that lead to activation of these sites. We have shown that the signaling pathway for platelet shape change connects from PAR-1 through the small GTPases cdc42 and rac which are rapidly converted into their GTP forms following PAR-1 ligation. Dowstream of GTPases are phosphoinositide (ppl) kinases that synthesize ppls in bulk to inactivate capping proteins and expose barbed filament ends. The importance of barbed end capping proteins for platelet shape change has been demonstrated in gelsolin deficient mouse platelets that activate poorly. However, despite poor platelet spreading. mice show only minor bleeding defects. Recently, ppls and small GTPases have been linked to the Arp2/3 complex which may form some of the nucleation sites for platelet actin assembly. The proposal will define the pathway(s) leading to Arp2/3 activation in platelets and determine if, and how the Arp2/3 pathway links to filopodial and/or lamellar actin growth signaled by the PAR-1 receptor. Interconnections between small GTPases will be studied and related to filament uncapping or de novo nucleation and to the types of actin structure formed.
In Aim 1, we will establish the links between the small GTPases rac. cdc42. ralA and ARF6 in platelets, identify upstream activating factors (GEFS) and downstream targets of rac and P15-K I - that lead to barbed end exposure and determine how they regulate capping proteins and Arp2/3.
In Aim 2. we will explore the roles of the Arp2/3 nucleation system and filamin in G-protein coupled receptors compared to IgG-farnily and integrin receptors (vWFR, GPVI. Fc RIIa. GPIlb/IIIa). We will determine whether each pathway generates a unique actin cytoskeletal architecture and how Arp2/3 and filamin are involved in these structures. In the last aim, we will identify, purify and characterize factors that activate platelet Arp2/3 downstream of cdc42.
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