The Role Of Heparin In Antithrombin III Proteolysis

Recent reports indicate that thrombin is capable of degrading part of the uncomplexed antithrombin III (AT III). Presence of heparin in the reaction of AT III and thrombin, in addition to enhancement of the binding rate, causes an unexplained, excessive decrease in the total AT III binding capacity. This suggests that heparin plays a major role in thrombin-catalyzed AT III proteolysis. Addition of heparin to reaction mixtures of human AT III and α- thrombin, conducted with a large excess of inhibitor, increased the ratio of AT III utilization by more than 50% and induced a release of inactive 50,000-dalton AT III fragment. This fragment, detectable by SDS-polyacrylamide gel electrophoresis with SDS concentrations not exceeding 0.1%, showed a decreased affinity for heparin. It was released from unbound AT III by small quantities of thrombin simultaneously with the formation of AT III-thrombin complex, if at least 1 μg/ml of polydispersed heparin participated in the reaction. To detect a similar degree of proteolysis in heparin-free reaction about 10 times more of α-thrombin was required. The extent of AT III proteolysis promoted by individual heparin fractions obtained in gel filtration correlated inversely with the anticoagulant activity of these fractions. Excessive decrease of residual inhibitory activity and changes in two-dimensional immunoelectrophoresis suggested that part of AT III in plasma is also subjected to proteolysis following addition of thrombin and heparin. These data indicate that the effect of heparin on AT III is more complex than generally recognized. On the one hand, heparin contributes to a rapid neutralization of the enzyme; on the other, heparin facilitates proteolytic degradation of unbound inhibitor even by small quantities of thrombin, causing reduction of the overall binding potential of AT III.