The aims of this resubmitted competitive renewal follow on results demonstrating that the ability of adherent platelets to assemble plasma fibronectin is determined by the ligands that mediate platelet adhesion. Thus, platelets adherent of fibrinogen, vitronectin, or von Willebrand factor are suppressed in their ability to assemble fibronectin whereas platelets adherent to fibrin, laminin, collagen, or fibronectin itself assemble fibronectin robustly. Further, assembly of fibronectin by platelets in an ex vivo flow system is a strong determinant of the extent of platelet thrombus formation on matrices of fibrin or collagen. The hypotheses are (i) platelet integrins and other cell surface proteins recognize critical features of adhesive ligands, initiating subtly different signaling pathways that support or suppress subsequent assembly of fibronectin by adherent platelets;(ii) as yet unappreciated differences in the microscopic and biochemical consequences of platelet adhesion correlate with the ability of adherent platelets to assemble fibronectin;and (iii) the N- terminal portion of fibronectin interacts specifically with unstructured stretches of certain proteins, among which are the cell surface molecules on adherent platelets and fibroblasts that drive fibronectin assembly.
Specific aims are to: 1. Identify features of fibronectin, fibrinogen/fibrin, vitronectin, and von Willebrand factor that account for their supportive or suppressive activity. 2. Characterize differences other than ability to assemble fibronectin that distinguish platelets adherent to supportive and suppressive ligands. 3. Discover the molecular features of fibronectin assembly sites present on adherent assembly- competent platelets and absent on adherent assembly-incompetent platelets. Methods to accomplish these aims include in vitro mutagenesis to dissect the structure/function of the supportive and suppressive adhesive ligands, microscopic and proteomic characterization of assembly- competent and assembly-incompetent adherent platelets, utilization of a reversible cross-linking strategy to identify the platelet surface proteins that interact with supportive and suppressive adhesive ligands, and development of activated blood coagulation Factor XIII as a tool to identify the platelet surface molecules that initiate fibronectin assembly.
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