Effect of heparin on thrombin inactivation by antithrombin-III

Abstract

Inactivation of [bovine] thrombin by heat and by its physiological inhibitor, [human] antithrombin-III, showed different dependence on heparin concentration. Heparin at 250 .mu.g/ml protected thrombin against heat inactivation, and thrombin behaved heterogeneously in this reaction. Without heparin, the thermodynamic activation parameters changed with temperature .**GRAPHIC**. [activation enthalpy] = 733 kJ/mol and 210kJ/mol at 50 and 58.degree. C, respectively). When heparin was present, heat inactivation of the protected thrombin species proceeded with .**GRAPHIC**. = 88 kJ/mol and was independent of temperature in the same range. Heparin at 0.125-2.5 .mu.g/ml accelerated the thrombin-antithrombin-III reaction. Thrombin did not show heterogeneity in this reaction and the time courses at any heparin concentration and any temperature between 0-37.degree. C appeared to follow 1st-order kinetics. Activation enthalpy was independent of heparin concentration or temperature, .**GRAPHIC**. = 82-101 kJ/mol, varying slightly with antithrombin-III concentration and thrombin-specific activity. Heparin seemed to exert its effect by increasing activation entropy. A mechanism of action of heparin in the thrombin-antithrombin-III reaction is suggested which accounts for all the important features of the latter and seems to unify the different hypotheses that have been advanced.