Mechanism of inactivation of trypsin by antithrombin

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Abstract
General aspects of the mechanism of antithrombin action were elucidated by a comparison of the inactivation of trypsin by antithrombin with the inactivation of coagulation proteinases by the inhibitor. Bovine antithrombin and bovine trypsin were shown to form an inactive equimolar complex. A noncomplexed, proteolytically modified form of antithrombin, electrophoretically identical with that formed in the reaction with coagulation proteinases, was also produced in the reaction with trypsin. In the absence of heparin, the inactivation of trypsin by antithrombin was 20 times faster than the inactivation of thrombin; the 2nd-order rate constant was 1.5 .times. 105 M-1 .cntdot. s-1 at 25.degree. C and pH 7.4. The inhibition of thrombin was accelerated .apprx. 30 times more efficiently by small amounts of heparin than was trypsin inhibition. Dissociation of the antithrombin-trypsin complex at pH 7.4 followed 1st-order kinetics with a half-life [t1/2] for the complex of .apprx. 80 h at 25.degree. C. The complex was rapidly and quantitatively dissociated at pH 11, resulting in the liberation of a modified 2-chain form of the inhibitor, cleaved at the same Arg-Ser bond as in modified antithrombin released from complexes with thrombin, factor Xa and factor IXa. This bond apparently is the active-site bond of antithrombin. Antisera specific for thrombin-modified antithrombin reacted with purified antithrombin-trypsin complex, indicating that the inhibitor was present in the complex in a form immunologically identical with thrombin-modified antithrombin. A common mechanism but different kinetics, for the inhibition of trypsin and coagulation proteinases by antithrombin are suggested.