The plasma protein, antithrombin, and its glycosaminoglycan activator, heparin, act as essential anticoagulant regulators of blood clotting proteinases. Inherited mutations which cause a loss of antithrombin inhibitory function or which block heparin activation of this function are thus associated with an increased risk of thrombosis . Moreover, complete antithrombin deficiency in man is unknown and produces embryonic lethality in mice. The long term goal of this proposal is to provide a complete understanding of the anticoagulant function of antithrombin and heparin at the molecular level and elucidate their contribution to hemostasis. Our hypotheses are: i) antithrombin's specificity for inhibiting multiple procoagulant proteinases with different substrate specificities arises from secondary interaction sites or exosites outside of the main reactive loop region of the inhibitor used for binding proteinases; ii) three basic residues of antithrombin are principally responsible for binding heparin at an allosteric site through an induced-fit mechanism and the action of these residues is coupled to global conformational changes leading to inhibitor activation; iii) limited conformational changes in or near the 325-375 region which are not essential for inhibitor function are responsible for the expression of the anti-angiogenic activity of antithrombin. These hypotheses will be tested by the following specific aims: l) We will elucidate the structural determinants of antithrombin's specificity for different clotting proteinases and assess how these determinants are expressed upon heparin activation; 2) We will determine the relative importance of antithrombin residues responsible for binding heparin and how they cooperate to induce antithrombin into an activated state with high heparin affinity and rapid reactivity with clotting proteinases; and 3) We will characterize the conformational differences between antithrombin forms with and without anti-angiogenic activity and localize the serpin structure which mediates this activity. These studies will utilize site-directed mutagenesis together with thermodynamic and kinetic characterization of variant antithrombins to elucidate the relationship between antithrombin structure and function. These studies are expected to increase understanding of natural molecular defects in antithrombin and to provide a rational basis for the design of novel antithrombotic and antiangiogenic agents through the engineering of modified antithrombins or heparin mimetics.
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