Two-substrate reaction model for the heparin-catalyzed bovine antithrombin/protease reaction

The kinetics of the heparin-dependent antithrombin/protease reaction were consistent with an ordered sequential two-substrate reaction model under all circumstances tested. In this model, heparin is the catalyst; while antithrombin is the first substrate and the protease is the second substrate. The first step in this reaction, the heparin-antithrombin interaction, has a K(D) of 25 nM but a diffusionally determined K(m) of about 150 nM regardless of protease substrate. The second step of the reaction, protease interaction with the heparin-antithrombin complex, was fast with a rate constant of 6.8 x 10⁶ M^-1.s^-1 for Factor X(a) and >8 x 10⁷ M^-1.s^-1 for thrombin. Differences between thrombin and Factor X(a) at low (nanomolar) concentrations of heparin were evident in this rate constant and the relative affinities for the heparin-antithrombin complex (K(m) for Factor X(a) = 100 nM; K(m) for thrombin ≤2 nM). In agreement with this difference in K(m), regardless of protease substrate, active site-blocked thrombin was a potent inhibitor of the antithrombin reaction; while active site-blocked Factor X(a) was an ineffective inhibitor. At high heparin concentrations (micromolar), the kinetic parameters for Factor X(a) were unchanged but the K(m) for thrombin increased dramatically to 100 nM. Other kinetic parameters were also estimated. Overall, the two-substrate reaction model provides a versatile approach for studying heparin function.