Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Linkage of protease-inhibitor-heparin interactions in the reaction with thrombin.

Heparin (H) was previously shown to accelerate the inactivation of alpha-thrombin (T) by antithrombin III (AT) primarily by promoting the initial binding of thrombin and AT in a ternary T.AT.H complex intermediate without significantly influencing the subsequent product formation step (Olson, S. T., and Shore, J. D. (1982) J. Biol. Chem. 257, 14891-14895). In the present study, the protein-heparin interactions which contribute to the assembly of the ternary complex intermediate and their linkage were quantitated by equilibrium binding and stopped-flow kinetic studies at pH 7.4, I 0.3, 25 degrees C, using p-aminobenzamidine (P) as a fluorescence probe. Equilibrium binding studies of the AT.H and T.H binary complex interactions monitored by the 40% enhancement in AT fluorescence or the 16-18% quenching of thrombin-bound p-aminobenzamidine fluorescence, respectively, indicated a 100-fold greater affinity of AT for heparin (KAT,H 0.23 microM) as compared to thrombin for heparin (KT,H 35-42 microM). Consistent with this large difference in affinities, rapid kinetic studies indicated that assembly of the ternary complex occurred predominantly as a bimolecular association between the AT.H binary complex and free thrombin. Thus, under pseudo-first order conditions ([AT]o, [H]o much greater than [T]o much less than [P]o), the observed thrombin inactivation rate constant (kobs) exhibited a saturable dependence on [AT]o or [H]o when [H]o much less than KT,H, reflecting a KAT,H (0.25 microM) similar to that directly determined by equilibrium binding. Moreover, competitive inhibition of the reaction by T.H binary complexes was indicated from the hyperbolic decrease in kobs produced by heparin with either high or low affinity for AT or active-site blocked thrombin at concentrations comparable to KT,H. This behavior was consistent with values of KT,H (27-51 microM) similar to those determined directly from equilibrium binding measurements. Comparison of the affinities of the binary protein-heparin interactions with the affinity of thrombin for AT.H complex in the ternary complex measured previously, indicated that the affinity of either protein for heparin was enhanced about 10-fold by the prior binding of the other protein to heparin. This linkage of the protein-heparin interactions implies that the ternary complex will be assembled at thrombin, AT, and heparin concentrations considerably lower than those predicted from previous reaction models which fail to account for this linkage.