Although recently reported three-dimensional structures of fibrinogen and its core fragments have been very revealing, key regions of these molecules are inherently flexible and have not yielded to the X-ray approach. These mobile regions, which amount to almost one-third of the molecule, are extremely important in a functional sense, and ignorance of their whereabouts is a distinct limitation to understanding fibrin clot formation. The mobile regions include the amino-terminal segments of the alpha and beta chains where the thrombin-sensitive bonds occur, the carboxyl-terminal segment of the alpha chain where factor XIII-catalyzed gamma-gamma dimer formation takes place, and, finally, the entire carboxyl-terminal section of the alpha chain, commonly called the alphaC domain. The latter plays a poorly understood role in the association of protofibrils and also involves factor XIII-induced cross-links. The goal of the current project is to establish the whereabouts of the flexible regions in native fibrinogen by using conventional but carefully crafted biochemistry experiments. The experiments fall into two general realms, both employing the same general procedures. In one set of experiments the biochemistry is conducted directly on crystals of native fibrinogen (chicken) where the bulk of the molecule is constrained and only the regions of interest are able to move about and interact. Thrombin and other specific enzymes will be used to specifically remove the fibrinopeptides directly in the crystal, after which selective chemical cross-linking will be used to join the newly exposed A and/or B knobs within each molecule. Similar experiments will be conducted on fibrin (human), where the constraints are somewhat different. These studies are aimed at finding how far apart the mobile knobs are from each other, on the average. Other experiments will be performed that immobilize the a chain carboxyl regions in situ, again both in crystals of native fibrinogen and in fibrin clots, by taking advantage of easily reduced disulfide bonds. The goal here is to find how the alphaC domains are situated relative to each other. In a related study, hybrid fibrins prepared from fibrinogens from different species will be used to follow polymerization events, especially the interactions and cross-linking of gamma-chain carboxyl terminal segments on the one hand and alphaC domains on the other. These studies are aimed at determining the degree of specificity involved in the assembly of fibrin units and protofibrils as well as the relative locations of the regions involved.
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