Calcium enhances heparin catalysis of the antithrombin-factor Xa reaction by promoting the assembly of an intermediate heparin-antithrombin-factor Xa bridging complex. Demonstration by rapid kinetics studies.
Heparin catalyzes the inhibition of factor Xa by antithrombin mainly through an allosteric activation of the serpin inhibitor, but an alternative heparin bridging mechanism has been suggested to enhance the catalysis in the presence of physiologic calcium levels due to calcium interactions with the Gla domain exposing a heparin binding exosite in factor Xa. To provide direct evidence for this bridging mechanism, we studied the heparin-catalyzed reaction of antithrombin with factor Xa, Gla-domainless factor Xa (GDFXa), and 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 approximately 70-saccharide heparin showed a saturable dependence on inhibitor concentration in the presence but not in the absence of 2.5 mM Ca(2+), indicating the formation of an intermediate heparin-serpin-proteinase encounter complex with a dissociation constant of approximately 90 nM prior to formation of the stable serpin-proteinase complex with a rate constant of approximately 20 s(-1). Similar saturation kinetics were observed for the inhibition of GDFXa by the antithrombin-heparin complex, except that Ca(2+) was not required for the effect. By contrast, no Ca(2+)-dependent saturation of the inhibition rate constant was detectable over the same range of inhibitor concentrations for reactions of either a short-chain approximately 26-saccharide high-affinity heparin-antithrombin complex with factor Xa or the long-chain heparin-antithrombin complex with the heparin binding exosite mutant, GDFXa R240A. These findings suggest that binding of full-length heparin chains to an exosite of factor Xa in the presence of Ca(2+) produces a chain-length-dependent lowering 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.
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Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA. rezaiear@slu.edu
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D000991:Antithrombins / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000737:chemistry*; D002384:Catalysis; D004357:Drug Synergism; D004789:Enzyme Activation; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism*; D065427:Factor Xa Inhibitors; D006493:Heparin / Q000031:analogs & derivatives / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009844:Oligosaccharides / Q000737:chemistry; D013379:Substrate Specificity; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism
D000991:Antithrombins; D065427:Factor Xa Inhibitors; D046911:Macromolecular Substances; D009844:Oligosaccharides; D006493:Heparin; D013917:Thrombin; D015951:Factor Xa; D002118:Calcium