The general thrust of the long range plans is to pursue crystallographically structural aspects of molecules of blood coagulation and fibrinolysis with the aim of relating them to functionality at the molecular level. Our research program has been exclusively devoted to the area for about the past 15 years and has made significant progress by determining the structures of all but one of the autonomous domains of these molecules (gamma-carboxyglutamic, kringle, epidermal growth factor, catalytic), along with many informative derivative studies involving them or combinations thereof. We will now extend the work to include additional catalytically active multidomain structures, which will also address domain-domain associations in a more general way. Since these molecules interact with macromolecular substrates, inhibitors and cofactors, the manner of such interactions will be revealed through the structure determinations of molecular complexes, including rationally designed mimetic molecules. Specific studies targeted include: (l) the inactive precursor of alpha-thrombin (prethrombin 2), (2) Serl95Ala anhydro thrombin and hirugen-anhydro thrombin to capture the active site conformational change accompanying fibrinogen recognition exosite binding, (3) a conformationally restricted non-peptide mimetic based on the unusual N-terminal hirudin interaction in the active site of thrombin, (4) the structure determination of Factor Xa, (a) in the presence of Ca+2 ions and (b) in complex with the second Kunitz domain of tissue factor pathway inhibitor and (5) the structure determination of the potent Factor Xa inhibitor tick anticoagulant protein. Crystallization searches are also underway with Factors VIIa and IX and protein C and activated protein C to extend the scope of the work to other areas of the blood coagulation cascade. Diffraction quality single crystals of many of the above have been grown and their X-ray diffraction patterns examined in a preliminary way and three intensity data sets have been measured that suggest a high probability of success.
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