Molecular Basis of Blood Coagulation Regulation

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The serpin family coagulation protease inhibitor, antithrombin and its glycosaminoglycan activators, heparin and heparan sulfate, comprise a key anticoagulant mechanism which is essential for maintaining hemostasis in vertebrates. Intriguingly, antithrombin has been recently shown to possess antiangiogenic and antitumor functions whose physiologic role remains to be determined. To advance our long-term goal of elucidating the molecular mechanisms by which antithrombin carries out its physiologically relevant functions, we propose to pursue three specific aims: 1) We have previously shown that the molecular determinants which mediate the specificity of heparin-activated antithrombin for blood clotting proteases reside both in an exposed reactive center loop of the serpin and in exosite regions outside the loop. We propose to map these exosite regions on antithrombin and on factor Xa and factor IXa and determine the mechanism by which the exosites mediate heparin rate enhancement of antithrombin reactions with the proteases; 2) Antithrombin circulates in an unusual low-activity conformation in which the reactive loop is partially buried in beta-sheet A, but is induced by heparin into a high-activity conformation resembling that of other serpins in which the reactive loop is expelled from the sheet. We propose to identify the molecular determinants of this reactive loop conformational switch and delineate the sequence of molecular events in the heparin binding site which trigger the conformational switch; 3) Antithrombin expresses a potent antiangiogenic activity when it undergoes conformational changes which occur spontaneously or are induced by cleavage in the reactive loop and which result in the burial of the reactive loop into beta-sheet A. We will determine the structural basis for the antiangiogenic activity of cleaved, latent and a novel prelatent form of antithrombin. The role of specific endothelial cell binding sites in this activity and whether these binding sites involve a protein receptor and/or specific domains of a heparan sulfate proteoglycan will be determined. The proposed studies are expected to provide increased understanding at the molecular level of the function of a key natural anticoagulant and antiangiogenic blood protein. This understanding is expected to facilitate the rational design of a new generation of anticoagulant and antitumor drugs for the treatment and prevention of cardiovascular diseases and cancer.