The broad, long-term objectives of the applicant are to define the roles of fibrinogen and fibrin in angiogenesis, hemostasis, and thrombosis. This proposal addresses the issue of fibrin clot stability, which is a critical factor in the outcome of fibrinolytic therapy.
The specific aims of this proposal are to: I) Determine the role of the gamma' chain extension in modulating the stability of fibrin clots. The hypothesis is that the rate and extent of gamma and gamma' multimer formation and the incorporation of alpha2-plasmin inhibitor into gamma'-containing clots will be greater. In addition, the ability of a synthetic peptide corresponding to the gamma' extension, VRPEHPAETEYDSLYPEDDL, to inhibit clot lysis rates and inhibit gamma' fibrinogen/factor XIII complex formation will be tested. The stability of the factor XIII/gamma' fibrinogen complex will also be measured following thrombin activation to test the hypothesis that thrombin activation dissociates the complex. II) Identify the critical amino acids in the gamma' chain extension that mediate factor XIII binding. This will be accomplished by measuring the equilibrium association constant between factor XIII and recombinant gamma' fibrinogen mutants containing alanine-scanning substitutions. III) Identify the peptide sequence(s) in factor XIII b-subunit that bind to the gamma' chain extension. This will be accomplished by binding proteolyzed factor XIII subunits to a resin of immobilized gamma' peptide to identify the binding peptide fragment(s). IV) Test the hypothesis that alpha chain Lys residues alpha148 and alpha157 and gamma chain Lys residues gamma321, gamma338, gamma356, and gamma373 mediate t-PA-dependent plasminogen activation, t-PA binding, and plasminogen binding, using recombinant fibrinogens containing Lys to Ala mutations. The results from the experiments in this proposal should provide new knowledge about the process of fibrinolysis that may lead to ways of modulating fibrin clot stability and clot lysis in vivo.
