Platelet activation occurs at the sites of injury, where extensive tissue remodeling takes place as part of the subsequent wound healing. Activated platelets secrete adhesive proteins that participate in formation of the extracellular matrix, and the nature of the matrix alters the adhesion, migration and proliferation of cells. This proposal is based on the hypothesis that protein disulfide isomerase is released by activated platelets and catalyzes the isomerization of disulfide bonds, leading to changes in protein conformation and to formation of disulfide-linked protein-protein complexes, thereby modifying the composition and the nature of the extracellular matrix. The hypothesis is based on the observations that when material secreted by activated platelets is incubated at 37 degree, thrombospondin shows isomerization of disulfide bonds, reduction of disulfide bonds, and formation f disulfide-linked multimers and disulfide- linked complexes with thrombin-serpin complexes. Each of these is similar to reactions catalyzed by protein disulfide isomerase. Preliminary experiments confirmed the presence of disulfide isomerase activity in the supernatant solution of activated platelets. Additional experiments will establish the disulfide isomerase specificity, the requirement for cofactors and the approximate mass of the enzyme. The disulfide isomerase will be purified, and antibodies will be prepared. The role of the disulfide isomerase will be tested in three ways: i) it will be added to combinations of purified proteins to determine which become disulfide linked, ii) antibodies will be used to immunoprecipitate the disulfide isomerase from the platelet supernatant solution to determine which reactions are inhibited, and iii) the ability of cells to adhere, spread and migrate on the matrix formed in the presence or absence of the disulfide isomerase will be tested. The specific conditions for disulfide linking of thrombin-serpin complexes to thrombospondin will be determined, and the thiols of thrombospondin will be characterized further.
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