Function and dysfunction in human antithrombins

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Inherited defects in single genes contribute significantly in many people to a predisposition to development of venous thrombosis, which in turn is a major contributor to the leading killer in industrialized countries, cardiovascular disease. One of the most important inherited defects is in the gene for antithrombin. Antithrombin is the principal inhibitor of the blood coagulation proteinases factor Xa and thrombin and is regulated by heparin. 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. This will be accomplished through elucidation first of the mechanisms of heparin activation and proteinase inhibition in normal antithrombin, and the ways in which mutations or changes in glycosylation alter either or both of these processes. The general hypotheses are (i) that the normal functioning of antithrombin can only be understood in terms of it being a serpin (member of the serine proteinase inhibitor superfamily) and of consequently being capable of undergoing necessary and dramatic conformational changes as part of both heparin binding and activation, and of proteinase inhibition and (ii) that, as a consequence of the need for antithrombin to fold as a metastable protein and to undergo conformational change as part of its function, it is prone to many more defects than other families of protein proteinase inhibitors which form simple lock-and-key type complexes. The specific areas are:- (1) To determine the gross structure of the thrombin-antithrombin complex. (2) To determine the conformational linkage between heparin binding and expulsion of residues of the reactive center of beta-sheet A. (3) To test whether the reactive center loop of antithrombin exists in an equilibrium between less reactive partially-inserted and more reactive fully loop expelled forms and that heparin activation results from a shift in this equilibrium. (4) To determine the role of basic residues in promoting the conformational change in the heparin binding site that results in expulsion of P15 and P14 residues of the reactive center loop. (5) To determine the basis for the dysfunction of naturally occurring human antithrombin variants. (6) To determine whether antithrombin is fucosylated in cancer and the functional consequences thereof. These specific aims will make extensive use of recombinant antithrombins expressed in mammalian cells that will be characterized by a combination of spectroscopic, thermodynamic and kinetic means. For antithrombins that have been activated by mutation, x-ray crystallography, through collaboration with Dr. Robin Carrell, will be used.