We propose to use recombinant variant fibrinogens in structural and biochemical studies to identify and characterize the residues and domains that are critical for the conversion of soluble fibrinogen to an insoluble fibrin clot. Fibrinogen is converted to fibrin monomers by the serine protease thrombin. Fibrin monomers polymerize to form a fibrin clot. During the past grant period biochemical studies demonstrated that """"""""B:b"""""""" interactions have an unanticipated role in polymerization. To correlate the biochemical data with changes in structure, we determined X-ray crystal structures of variants that examined the """"""""B:b"""""""" interactions. These structure studies not only supported our biochemical findings, but also identified calcium-binding sites that likely modulate polymerization. We now propose to examine the role of """"""""B:b"""""""" interactions and calcium modulation in polymerization. We will test the hypotheses that: 1) both """"""""A:a"""""""" and """"""""B:b"""""""" interactions contribute to protofibril formation and 2) the beta1 calcium binding site modulates polymerization. Fibrin clots are stabilized and strengthened by the FXIIIa-catalyzed formation of isopeptide bonds between monomers. We propose to examine the interactions that are critical for normal crosslink formation and determine the interactions that mediate the strength of fibers and clots. We will test the hypotheses that: 3) the fibrin-enhanced thrombin-catalyzed activation of FXIII reflects the juxtaposition of fibrin-bound thrombin with fibrin-bound FXIII upon association of the D:D/E interface and 4) FXIIIa-catalyzed crosslinks are the molecular interactions that determine the mechanical properties of both fibrin fibers and fibrin clots. Our experiments are designed to provide a molecular analysis of the events that control fibrin clot structure and mechanical properties. Data obtained from these in vitro studies will lead to a more complete understanding of the events that are critical for effective clot formation and clot lysis in vivo.
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