Platelet thrombospondin (TSP1) is a large trimeric glycoprotein that binds to cells, platelets, and numerous extracellular matrix and blood proteins including collagen, fibronectin, heparan sulfate proteoglycan, fibrinogen and fibrin, plasminogen, and transforming growth factor-beta. Recently, the gene for a second thrombospondin, TSP2, was found. Both TSP1 and TSP2 are highly regulated in vivo and in vitro and are thought to be important for embryogenesis, morphogenesis, wound healing, tumor cell growth, and related processes. The proposed research will expand on the unexpected discovery that TSP1 is a slow, tight-binding inhibitor of plasmin, trypsin, and probably other trypsin-like proteases.
Aims will be to: (1) Identify the structural features of TSP1 that account for the inhibitory activity.
This aim will be accomplished by studying proteolytic fragments of TSP1, recombinant pieces of human TSP1 expressed as truncated proteins or as chimeras with the gelatin-binding part of fibronectin, and recombinant proteins in which critical residues are altered by in vitro mutagenesis. We also will attempt to find monoclonal antibodies that block inhibitory activity. Such antibodies will support the identification of the active region of TSP1 and be useful in more biological studies. (2) Learn whether TSP2 also functions as a protease inhibitor. The motif or modules homologous to the inhibitory part of TSP1 will be expressed and tested against proteases of different specificities and mechanistic classes. One possible outcome of this experiment is that TSP2 does function as a protease inhibitor but against a different spectrum of proteases than are inhibited by TSP1. (3) Learn the biological and pathobiological importance of the inhibitory activity of human TSP1. Laboratory models of lysis of platelet-rich thrombi, degradation of extracellular matrix, and tumor cell invasion will be studied to assess the relative contributions of endogenous and/or exogenous TSP1 in regulation of cell-mediated proteolysis. Levels of active TSP1 in the experiments will be controlled by addition of TSP, use of loss-of-function monoclonal antibodies, and/or use of cells expressing and secreting different amounts of TSP1.
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