Calcium enhances heparin catalysis of the antithrombin-factor Xa reaction by promoting the assembly of an intermediate heparin-antithrombin-factor Xabridging complex. Demonstration by rapid kinetics studies

Heparin catalyzes the inhibition of factor Xa by antithrombin mainly throug h an allosteric activation of the serpin inhibitor, but an alternative hepa rin bridging mechanism has been suggested to enhance the catalysis in the p resence of physiologic calcium levels due to calcium interactions with the Gla domain exposing a heparin binding exosite in factor Xa. To provide dire ct evidence for this bridging mechanism, we studied the heparin-catalyzed r eaction of antithrombin with factor Xa, Gla-domainless factor Xa (GDFXa), a nd a heparin binding exosite mutant of GDFXa in the absence and presence of calcium using rapid kinetic methods. The pseudo-first-order rate constant for factor Xa inhibition by antithrombin complexed with a long-chain simila r to 70-saccharide heparin showed a saturable dependence on inhibitor conce ntration in the presence but not in the absence of 2.5 mM Ca2+, indicating the formation of an intermediate heparin-serpin-proteinase encounter comple x with a dissociation constant of similar to 90 nM prior to formation of th e stable serpin-proteinase complex with a rate constant of similar to 20 s( -1). Similar saturation kinetics were observed for the inhibition of GDFXa by the antithrombin-heparin complex, except that Ca2+ was not required for the effect. By contrast, no Ca2+-dependent saturation of the inhibition rat e constant was detectable over the same range of inhibitor concentrations f or reactions of either a short-chain similar to 26-saccharide high-affinity heparin-antithrombin complex with factor Xa or the long-chain heparin-anti thrombin complex with the heparin binding exosite mutant, GDFXa R240A. Thes e findings suggest that binding of full-length heparin chains to an exosite of factor Xa in the presence of Ca2+ produces a chain-length-dependent low ering of the dissociation constant for assembly of the intermediate heparin -antithrombin-factor Xa encounter complex, resulting in a several 100-fold rate enhancement by a heparin bridging mechanism.