The goal of the proposed experiments is to determine the structural details of fibrinogen that are critical for the formation of normal clot structure. For each case where an altered clot structure is detected, it will be determined whether or not the altered structure is associated with impaired fibrinolysis. Directed mutagenesis experiments are proposed to better define which features of fibrinogen are critical to generate a normal fibrin clot and which features of the fibrin clot are critical to normal fibrinolysis. It has long been postulated that the association of fibrin monomers into an insoluble fibrin clot occurs in two steps, protofibril formation and lateral aggregation. From the cumulative data of many previous investigators, the residues that participate in protofibril formation have been reasonably well defined, but the residues that promote lateral aggregation remain essentially unknown. The current application proposes a new approach to determine which residues of fibrinogen participate in lateral aggregation by constructing intact variant fibrinogen molecules with specified changes that will test the validity of several current models of lateral aggregation. Many of these variant fibrinogens will form altered fibrin clots. It is planned to determine which clot features influence clot lysis by analysis of these altered clots.
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