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The Purification and Mechanism of Action of Human Antithrombin-Heparin Cofactor

Open AccessPublished:September 25, 1973DOI:https://doi.org/10.1016/S0021-9258(19)43472-8
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      A procedure is presented for purifying antithrombin-heparin cofactor from human plasma. The final product is homogeneous as judged by disc gel electrophoresis, sodium dodecyl sulfate gel electrophoresis, and immunoelectrophoresis. The final yield averages 12%. A specific antibody directed against pure inhibitor preparations precipitates virtually all of both antithrombin and heparin cofactor activity from defibrinated plasma. The purification, the immunoprecipitation, and other data, indicate that both activities are properties of a single molecular species.
      The inhibitor and thrombin form a 1:1 stoichiometric complex which cannot be dissociated with denaturing and reducing agents. Addition of heparin, a widely used anticoagulant which specifically accelerates the action of our inhibitor, increases the rate of formation of this complex without altering its stoichiometry or its dissociability. Interaction of thrombin with antithrombin-heparin cofactor requires the presence of the active center serine of the enzyme and arginine residue(s) on the inhibitor, since chemical modification of either of these critical residues inhibits complex formation both in the presence and absence of heparin. We suggest that, in analogous fashion to trypsin-trypsin inhibitor systems, a specific interaction occurs between the active center serine of thrombin and a unique arginine-x reactive site on the antithrombin-heparin cofactor.
      Furthermore, lysyl residues of the inhibitor probably serve as a binding site for heparin, since chemical modification of these residues virtually eliminates heparin cofactor activity with only minimal reduction of antithrombin activity. We postulate that heparin binds to the inhibitor and causes a conformational change which results in a more favorable exposure of the arginine reactive site, allowing a rapid interaction with thrombin.

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