Thrombospondin (TSP) is a glycoprotein with broad tissue distribution. It is found in human platelet alpha granules, endothelial cells, fibroblasts and extracellular matrix. TSP interacts specifically with fibrinogen, fibronectin and collagen suggesting that it has multiple structural and functional domains. Preliminary data using an enzyme linked immunoadsorbant assay (ELISA) and rocket immunoelectrophoresis demonstrate complex formation between TSP and plasminogen (Plg) that is specific, saturable and mediated by the high affinity lysin binding sites of Plg. TSP will be purified from thrombin stimulated human platelets using gel filtration and heparin affinity chromatography. The affinity and stoichiometry of the Plg-TSP complex will be determined by ELISA and sucrose density ultracentrifugation. The specific TSP structural domains involved in the interaction will be determined using elastase digested peptide fragments of the purified protein separated by fast protein liquid chromatography. The functional significance of the Plg-TSP interaction in fibrinolysis will be investigated by characterizing the kinetics of Plg activation by tissue plasminogen activator (TPA) in the presence and absence of TSP. This will be done in the soluble phase using a synthetic chromogenic plasmin substrate and on a fibrin clot using 125I-fibrin plate assay. The effect of TSP on the specific binding of 125I-Plg to a fibrin clot will also be determined. To test whether surface bound TSP can support efficient Plg activation by TPA in the absence of fibrin, TPA activation of Plg complexed to TSP bound to Sepharose 4B beads and plastic plates will be characterized. These kinetic parameters will be compared to those of Plg activation by TPA in a soluble system and on a fibrin clot. The influence of TSP on the binding of Plg to cell surfaces will be studied. Binding of 125I-Plg to human platelets and mouse macrophages, both in the resting and stimulated states, will be quantified. To determine if TSP mediates the Plg binding to cell surfaces, monospecific anti-TSP Fab'2 will be used to inhibit Plg binding. The binding of 125I-TSP to resting and stimulated macrophages will also be investigated and the ability of TSP bound to resting cells to mediate TPA induced Plg activation will be studied. Regulation of Plg activation is important in the control of thrombosis and hemostasis, inflammation, tumor cell implantation and metastasis, cell-cell communication and organogenesis. The characterization of the TSP-Plg interaction and its functional consequence on TPS mediated Plg activation and proteolysis may thus provide important insights into these processes.
