Targeting Platelet Endocytosis and Exocytosis to Control Thrombosis

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World-wide, spurious thrombosis accounts for 1 of 4 non-communicable disease deaths. Cardiovascular disease is a leading killer of aging US veterans and is the major cause of death in older female veterans. Understanding how to modulate thrombosis will significantly aide the VA's mission to improve veterans' health. Normally platelets respond to vascular damage and secrete granule cargo that are essential for recruiting more platelets and for generating a thrombus. This releasate promotes normal sequelae but can also contribute to occlusive pathologies such as strokes and heart attacks. In platelets, VAMPs, SNAP-23, and Syntaxin-11 form a membrane-spanning complex that mediates exocytosis. Formation of this complex requires a host of SNARE-regulators, e.g., Munc18b, STXBP5/tomosyn-1, and granuphilin/SLP4; however, the mechanisms by which these proteins control the complexity of the platelet release reaction (its rate, extent, and content) is uncertain. Platelets are also capable of other cellular processes (i.e., RNA splicing, translation, glycosylation, autophagy); however, their effects on platelet function are still unknown. Our data suggest that endocytosis affects thrombus growth by modulating platelet spreading and platelet-platelet contacts. Our goal is to manipulate the membrane trafficking in platelets, both endocytosis and exocytosis, in order to modulate occlusive thrombosis with only modest effects on hemostasis. To reach this goal, we must probe the molecular mechanisms of exocytosis. Our specific focus will be on Syntaxin-11 regulators. We will also use an endocytosis defective mouse strain to define the roles of endocytosis in thrombosis and hemostasis. Two aims are proposed: 1) Define the network of protein-protein interactions that affect Syntaxin-11-mediated membrane fusion and granule cargo release; and 2) Determine the roles of platelet endocytosis in hemostasis. To complete these aims, we will employ biochemical assays to define the interactions between the SNARE regulators and in vitro and in vivo functional assays, using transgenic mice, to define the roles of the specific Syntaxin-11 regulators and of endocytosis in thrombosis and hemostasis. Our results will expand the understanding of the molecular requirements and the sequence of protein-protein interactions controlling platelet exocytosis. We will also expand the mechanistic understanding of what cellular processes platelets can perform (i.e., endocytosis) and why they are important. Our results will be significant to the field since they will provide the needed mechanistic insights to identify potential targets for therapeutic intervention and to evaluate the relevance of the increasing volume of gene/risk associations that are guiding patient treatment strategies. This will enhance the anti-thrombotic treatment options available for the care of aging veterans.