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

Dissociation of the complexes formed during the reaction of 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. The results suggested that thrombin is released after reaction with antithrombin III via two 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 relatively low amounts. The results suggested that thrombin inhibition by antithrombin III does not 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. This further suggests that acyl bond formation is a secondary reaction that can occur during thrombin inhibition by antithrombin III but is not necessary for thrombin inhibition.