FUNCTIONAL CHARACTERIZATION OF PLATELET THROMBOSPONDIN

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Thrombospondin (TSP) is an adhesive protein which appears to be important in modulating cell growth and migration. The long-term objective of the proposed study is to determine the function of TSP in the formation and repair of tissue. Specific focus in this proposal will be directed toward the following areas: (1) The Interaction of Thrombospondin with Calcium High affinity (Kd - 100nM) and low affinity (Kd - 120 uM) cooperative binding of calcium to TSP has been detected using probes of protein structure. The amino acid sequence of thrombospondin includes a unique structural motif which appears to be composed of multiple calcium-binding sites that are immediately adjacent to each other.l The specific aims of this portion of the proposed study are to (1) quantitate the direct binding of 45 calcium to TSP and to synthetic peptides based on the type 3 repeats and (2) determine if a conformational change can be detected at the level of individual type 3 repeat. (2) Functional Domains of Thrombospondin TSP has the ability to bind to heparin, fibrinogen, fibronectin, type V collagen, laminin, plasminogen, histidine-rich glycoprotein, sulfated glycolipids and cell surface receptors. A specific aim of the proposed study is to identify the sites within the TSP molecule for these interactions through the production of (1) proteolytic fragments which retain the functional activity, (2) fusion proteins with functional activity, (3) polyclonal anti-fusion protein antibodies which block function, (4) monoclonal antibodies which block function, (5) variant forms of TSP in which specific sites have been mutated or deleted and (6) synthetic peptides which mimic the activity of TSP. (3) Thrombospondin in Growth and Development Recent data on the regulation of TSP synthesis by cells in vitro have led to the hypothesis that TSP is involved in the normal morphogenetic process and is a component of the cell's response to injury. This hypothesis would predict that thrombospondin would be an essential component of normal development. A specific aim of the proposed study is to determine the function of TSP in vivo by (1) the localization of TSP in the developing mouse embryo and (2) the generation of mice in which the TSP gene has been deleted or mutated.