Function and dysfunction in human antithrombins

University Of Illinois At Chicago, Chicago IL

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Abstract

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