FIBRINOGEN--ROLE OF THE GAMMA VARIANT

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The broad, long-term objectives of the applicant are to define the roles of fibrinogen in hemostasis and thrombosis. This proposal addresses the issue of fibrin clot strength, a critical factor in the outcome of fibrinolytic therapy. Recent findings indicate that the minor gamma chain variant of fibrinogen binds directly to the zymogen form of factor XIII, a transglutaminase that stabilizes fibrin clots, and that the gamma variant serves as a carrier protein for factor XIII. This carrier function is likely to modulate the strength of the fibrin clot and its resistance to lysis by increasing the local concentration of factor XIII at the clot. The specific aims of this proposal are to: I) Determine the role of y fibrinogen in modulating the strength of fibrin clots. This will be accomplished by measuring the rate and extent of gamma and gamma~ multimer formation, and by measuring the incorporation of alpha2-plasmin inhibitor into the clot. In addition, the effect of a synthetic peptide corresponding to the gamma~ extension, VRPEHPAETEYDSLYPEDDL, on lot lysis rates will be measured, as well as the IC50 of the peptide on gamma~ fibrinogen/factor XIII complex formation. The binding of gamma~ fibrinogen to both activated and unactivated factor XIII will also be measured to see if thrombin activation affects factor XIII binding to gamma~ fibrinogen. II) Identify the critical amino acids in the gamma~ chain that mediate factor XIII binding. This will be accomplished by measuring the equilibrium association constant of recombinant gamma~ fibrinogen mutants with factor XIII, particularly mutants in the sulfated Tyr residues at positions gamma~418 and gamma~422. In addition, alanine-scanning substitutions will be introduced throughout the twenty amino acid gamma~ chain extension to map the critical binding residues. III) Identify the peptide sequences in factor XIII that bind to the gamma~ chain. This will be accomplished by binding proteolyzed factor XIII subunits to a resin of immobilized gamma~ peptide to identify the binding peptide fragment(s). The results from the experiments in this proposal should provide new knowledge about the process of blood coagulation that may lead to way of modulating fibrin clot stability and clot lysis in vivo.