Antithrombin reactions with .alpha.- and .gamma.-thrombins

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Abstract
Human .alpha.-thrombin with high clotting activity and its proteolyzed derivative .gamma.-thrombin with virtually no clotting activity reacted in an essentially identical manner with antithrombin. The 2 enzyme forms bound proflavin with similar constants and showed identical behavior with small substrates. No significant differences were found for the antithrombin reactions (measured by proflavin displacement or active site titration) with respect to kinetics, extent of reaction or effect of added heparin. The enzyme-antithrombin complexes could not be dissociated with sodium dodecyl sulfate (NaDodSO4) but the NaDodSO4-denatured complexes were dissociated by hydroxylamine treatment. The .gamma.-thrombin-antithrombin complex had an approximate MW of 75,000 by disc gel electrophoresis as compared with 100,000 for the .alpha.-complex, consistent with the polypeptide structures of the 2 proteins. The .gamma.-thrombin-antithrombin complex did not inhibit clotting catalyzed by .alpha.-thrombin. Fibrinogen did not affect the reaction of .gamma.-thrombin with antithrombin or antithrombin-heparin. The antithrombin and antithrombin-heparin reactions did not involve the fibrinogen recognition sites which were destroyed by proteolytic conversion of .alpha.-thrombin to the noncoagulant .gamma. form.