Verbatim): The overall goal of this project is to examine the role of fibrinogen in normal and pathologic hemostasis. Specifically, we will evaluate the structural details of fibrinogen that are critical to normal polymerization and fibrinolysis. During the previous grant period we examined several variant fibrinogens, generated by directed mutagenesis. Analyses of these fibrinogens suggest that the long-postulated two step model for fibrin polymerization, where fibrinopeptide A release mediates the first step and fibrinopeptide B release mediates the second, is incomplete. Furthermore, studies of clot formation in whole blood (Brummel et al. J Biol.Chem. 274:22862, 1999) challenge the two step model of thrombin-catalyzed fibrin polymerization because these clots formed with minimal FpB release. Nevertheless, recent structural studies (Everse et al. Biochemistiy 37:8637, 1998) support the two step model. The structural studies also suggest that calcium has a determining role in fibrin polymerization. Our proposed experiments will investigate the current models of polymerization and fibrinolysis. We will test four specific hypotheses and develop an assay to measure discrete protein-protein interactions.
Our specific aims are: 1) to determine whether the """"""""b"""""""" site located in the C-terminal domain of the beta chain has a significant role in polymerization, 2) to determine whether calcium has a role in modulating polymerization and (or) the """"""""A.a"""""""" interactions that promote protofibril formation; 3) to determine whether clot formation in the absence of FpB release can promote fibrinogen enhanced FXIII activation and intermolecular crosslink formation; 4) to determine whether plasminogen and tissue plasminogen activator each bind to a single specific site in fibrinogen; 5) to develop an assay, using surface plasmin resonance, to quantify discrete protein-protein interactions where one protein is monomeric fibrin. If our results are not consistent with current models, then we will propose alternative models that can be tested by analyses of both plasma and recombinant variant fibrinogen. With these in vitro data we will be able to envision which aspects of fibrin gel formation are critical to effective clot formation and clot lysis in vivo.
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