Platelets play a critical role in mediating a variety of biological processes, including cell proliferation, cell adhesion, immunoregulation, thrombosis and fibrinolysis; many of these events are directly influenced by proteins secreted from the platelet alpha-granule. The central hypothesis of this proposal is that at least two such proteins, namely thrombospondin and platelet factor 4, participate in such regulatory mechanisms through their interaction with specific ligands and, furthermore, that this interaction is the function of unique and specific peptide domains, both within a given ligand and the platelet proteins. Specifically, the requisite thrombospondin binding domains within the Aalpha- an Bbeta-fibrinogen chains will be identified. Synthetic peptide analogues to each of these domains will be synthesized and evaluated for their ability to modulate the secretion-dependent phase of platelet aggregation. Utilizing a blot-binding assay, the region of thrombospondin which binds fibrinogen will also be identified, the requisite peptide domain(s) characterized, synthesized, and assessed for their ability to regulate thrombospondin-fibrinogen interaction and platelet aggregation. The COOH-terminal 18 kDa domain of thrombospondin has been purified in quantities which will permit a direct assessment of its ability to bind to non-activated and activated platelets. These studies will be conducted using fluorescent-labeled 18 kDa domain as well as polyclonal and monoclonal antibody probes. The binding of thrombospondin to specific kringle regions of plasminogen, particularly kringle 5, will be determined. The ability of peptide analogues to this region will be probed for their potency in regulating plasminogen activators' kinetics effect on plasmin generation. The plasmin generation protocols will also be assessed when histidine rich glycoprotein, and the thrombospondin binding domain regions from that protein, are included in the trimolecular complex activation of thrombospondin-histidine rich glycoprotein-plasminogen. Further studies on the interaction of thrombospondin and the 29 kDa domain of fibronectin will also be performed. Monoclonal antibodies against thrombospondin will be utilized to define which regions within the thrombospondin molecule are """"""""decorated"""""""" by specific protein ligands. These studies should provide a better understanding of the role of thrombospondin in platelet aggregation, fibrin formation and fibrinolysis and permit the development of synthetic peptides which prevent or attenuate these processes.
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