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 KD of 25 nM but a diffusionally determined Km 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 10(6) M-1.s-1 for Factor Xa and greater than 8 X 10(7) M-1.ls-1 for thrombin. Differences between thrombin and Factor Xa at low (nanomolar) concentrations of heparin were evident in this rate constant and the relative affinities for the heparin-antithrombin complex (Km for Factor Xa = 100 nM; Km for thrombin less than or equal to 2 nM). In agreement with this difference in Km, regardless of protease substrate, active site-blocked thrombin was a potent inhibitor of the antithrombin reaction; while active site-blocked Factor Xa was an ineffective inhibitor. At high heparin concentrations (micromolar), the kinetic parameters for Factor Xa were unchanged but the Km 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.