The single most common cause of death in industrialized countries is cardiovascular disease, in which thrombosis plays a very prominent role. Many people are predisposed to development of thromboembolic complications through inheritance of defects in single genes, of which one of the most important is the gene for antithrombin III, since antithrombin III plays a critical role as an inhibitor of proteinases of blood coagulation. It has been estimated that from 2 to 5% of patients with thromboembolic disorders have such a deficiency. The long term goal of this proposal is to achieve an understanding of the molecular basis for defects in functioning of variant human antithrombins that result in thrombosis, through elucidation of the mechanisms of heparin activation and proteinase inhibition, and the determination of the ways in which mutations alter either or both of these processes. Such understanding is a prerequisite for rational design of therapeutic interventions. An expression system for glycosylated recombinant human antithrombin will be used to produce wild type and variant antithrombins. Structural studies will not be confined to one technique, but will utilize several parallel and complementary approaches including NMR, CD, FTIR, and fluorescence spectroscopies. Specific goals to be accomplished within the proposed grant period are: (i) Characterization of the heparin binding site in native antithrombin III, in which residues that constitute the heparin binding site(s) or are perturbed by heparin binding will be identified. (ii) Determination of the structure of the reactive center region of native antithrombin III. This will test whether the current model that this region is alpha-helical in the native state is correct and permit determination of subsequent changes upon perturbation by proteinases or heparin. (iii) Determination of the conformational changes transmitted to the reactive center region consequent to heparin binding, since such changes are a necessary part of antithrombin activation. (iv) Comparison of normal and naturally occurring pathological variants of antithrombin III. In this way alterations in structure and mechanism of the variant antithrombins will be revealed and can then be correlated with the manifestations of the dysfunction. (v) Growth of crystals of native antithrombin suitable for diffraction studies.
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