Kinetic analysis of the heparin-enhanced antithrombin III/thrombin reaction. Reaction rate enhancement by heparin-thrombin association.

The distribution of heparin between thrombin and antithrombin III in solutions containing both proteins has been calculated using a heparin-thrombin dissociation constant value, KgssMT, of 1.5 X lo-’ M and a heparin-antithrombin III dissociation constant value, KEgsMAT, of 2.0 x lo-’ M. The enhancing effect of heparin on the antithrombin III/thrombin reaction rate appeared to correlate with the binding of heparin to thrombin, suggesting that this is the first step in the mechanism of action of heparin. A decreased effectiveness of heparin at concentrations which were high, relative to the thrombin concentration, was observed. This decrease appeared to correlate with the binding of a second heparin molecule to thrombin with a dissociation constant value of approximately 1.0 X lo-’ M. Changes in the antithrombin III/thrombin reaction system which would alter the distribution of heparin between the two proteins, e.g. adding active siteblocked thrombin or increasing the concentration of either protein, had an effect on the ability of heparin to enhance the reaction rate in accord with the binding of heparin to thrombin being the first step of the mechanism of reaction rate enhancement. Increasing the antithrombin III concentration while maintaining fixed thrombin and heparin concentrations inhibited the effectiveness of heparin in enhancing thrombin inactivation. This was consistent with the binding of heparin to the antithrombin III high affinity site, i.e. KgggMAT = 2.0 x 10e8 M, not affecting the rate of the antithrombin III/thrombin reaction other than by displacing heparin from thrombin. The results suggest that the binding of heparin to the high affinity binding site on antithrombin III does not directly enhance or inhibit the antithrombin III/thrombin reaction rate.