The goal of this proposal is to determine the molecular details of the fibrinogen A alpha chain which contribute to its role as a substrate for thrombin and a participant in fibrin polymerization. Directed mutagenesis will be used to explore the functional contribution of each relevant amino acid residute. Two vectors which express human A alpha polypeptides in E. coli have already been constructed. One expresses the complete A alpha chain while the other expresses the first 50 residues linked by a segment of collagen to the bacterial enzyme, beta-galactosidase. Both peptides are detected by protein blot analysis, with crossreactive bands at the predicted mobilities, and both are cleaved by thrombin. Initial experiments will use the beta-galactosidase fusion product because many of the functionally important residues lie within amino acids 1-50. In addition the fusion peptide can be readily purified based on its beta-galactosidase activity. The A alpha peptide can then be prepared in high purity by digesting away the beta-galactosidase with collagenase. The native A alpha peptide will be assayed as a substrate for thrombin and as an inhibitor of fibrin monomer polymerization. Then mutations will be construted from assembled oligonucleotides, and the activities of the expressed mutant peptides compared to the native activities. It has been proposed that cleavage by thrombin is dependent on the presence of a beta-turn structure at or near amino acids 13 and 14. Molecular modelling will be employed to suggest mutations which will disturb or support such a structure, and the predicted changes tested experimentally. The detailed structure of the polymerization domain will be explored by changing the charges on side chains and making polar/apolar substitutions. Once critical residues have been identified, more subtle changes will be explored and a model developed for the polymerization domain. Similar mutations will be studied in the complete A alpha chain in order to identify long range interactions. The results from the experiments will provide information which can serve as the basis for designing inhibitors of thrombin activity and fibrin polymerization. Such inhibitors could be clinically useful in treating or preventing pathological clot formation.
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