Thrombospondin (Tsp) is a 450 kDa protein with 3 disulfide-linked chains. It is secreted by platelets and certain cultured cells. It binds to cells and many different proteins; it may function as an adhesive or sub-cellular matrix protein. We have observed that Tsp i) has a conformation-sensitive thiol, ii) has an intrachain disulfide bond with a conformation-dependent stability, and iii) readily forms disulfide-linked complexes with other proteins and with other molecules of Tsp. I hypothesize that this reflects a dynamic function of Tsp as a covalent crosslinking protein. As a test of this hypothesis and for further characterization of Tsp, the number and locations of thiols and conformation-sensitive disulfide bonds will be determined, and the specificity for protein and for location of disulfide bonds for the thiol-disulfide exchange will be examined. The thiol-disulfide exchange was shown to have a pH optimum below 7 (8-10 would be predicted); this will be studied to determine if the pH dependence reflects a pH- dependent conformational change or the influence of another ionizable group on the dissociation of the thiol. Finally, attempts will be made to determine whether the thiol is necessary for Tsp binding to cells (including platelets), for its lectin activity or for platelet aggregation.
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