FIBRINOGEN CRYSTALLIZATION REQUIRES LIMITED PROTEOLYSIS

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A complete understanding of blood clotting and its malfunction in certain cardiovascular diseases requires detailed information about the structure and interactions of the fibrinogen molecule. A major goal of this laboratory is the determination of the three-dimensional structure of fibrinogen by X-ray crystallography. Despite extensive efforts, native fibrinogen has not yet been crystallized. Limited digestion of bovine fibrinogen with a crude protease extract from Pseudomonas aeruginosa, however, results in the production of crystals that diffract to about 6 A. The major objective of this proposal is to determine the modifications in the primary structure of the protease-modified fibrinogen that forms these crystals. In order to carry out this proposal, it will be necessary to isolate and characterize the Pseudomonas protease. Characterization of this novel enzyme will include determination of its amino acid composition, cleavage specificity, and susceptibility to enzymatic inhibitors. The advantages of modifying fibrinogen with a purified and characterized preparation of Ps-1 include greater control of digestion conditions, as well as reduction of cleavage heterogeneity. Once digestion conditions have been established, various solvent systems, ionic strengths, and temperature and pH ranges will be explored to find the combination which consistently results in a good supply of well-ordered crystals. Attempts will also be made to produce highly-ordered crystals of human fibrinogen after modification with the Pseudomonas protease. The major focus of this proposal will be the determination of the changes in the primary structure of fibrinogen required for the molecule to form crystals. A four-stage approach will be used: 1) isolation of fragments and peptides released by limited digestion with Ps-1, 2) amino acid analysis, 3) amino-terminal sequence determination, and 4) placement of the polypeptides within the known structure of fibrinogen. The primary structure of the modified fibrinogen in crystals will be deduced from an analysis of these data. Taken together, these studies are essential for obtaining and interpreting electron density maps of the modified fibrinogen crystals. Knowledge of the detailed structure of fibrinogen is required to establish the critical interactions that determine the packing of these molecules in the fibrin clot.