Dissociation of antithrombin III-thrombin complex. Formation of active and inactive antithrombin III

Abstract

Dissociation of the complexes formed during the reaction of [human] thrombin with antithrombin III was studied by determining the kinetics of release of thrombin and active antithrombin III from the complexes. The rate of thrombin release increased gradually between pH 7.0 and 8.5 and then sharply increased at pH 9.0. The rate of active antithrombin III release did not change significantly over the same pH range. Thrombin is released after reaction with antithrombin III via 2 pathways which are differentially affected by the pH of the solution. At pH 7.0 thrombin is released predominantly by dissociation of a non-acylated enzyme-inhibitor complex since active antithrombin III was released in nearly stoichiometric amounts. At pH 9.5 thrombin is released predominantly by the rapid deacylation of an acylated enzyme-inhibitor complex since active antithrombin III was released in relatively low amounts. Thrombin inhibition by antithrombin III does not apparently require the formation of an acyl bond between the active-site serine of thrombin and a carbonyl carbon of antithrombin III. The release of thrombin by acylation-deacylation appeared to be limited by the rate of enzyme acylation which was favored by more alkaline pH. Acyl bond formation may be a secondary reaction that can occur during thrombin inhibition by antithrombin III but is not necessary for thrombin inhibition.