Abstract
Because of the homology between factor IXa and factor Xa (f.IXa and f.Xa, respectively), and the critical upstream position of f.IXa in the coagulation cascade, the contribution of the heparin-derived pentasaccharide to antithrombin-mediated inhibition of f.IXa was investigated. Pentasaccharide promotes inhibition of both f.IXa and f.Xa generated in recalcified plasma. This result demonstrates that antithrombin is the predominant inhibitor of f.IXa in plasma, and that the activity of antithrombin is promoted by pentasaccharide. Kinetic experiments reveal that pentasaccharide increases the rates of antithrombin-mediated inhibition of both f.IXa and f.Xa by 2 orders of magnitude. These findings indicate that pentasaccharide-induced conformational changes in antithrombin enhance its capacity to inhibit both f.IXa and f.Xa. In the presence of Ca2+, full-length heparin produces an additional approximately 10-fold increase in the rates of inhibition of both enzymes, consistent with a template role of heparin. Heparin binding to f.Xa was previously shown to be promoted in the presence of Ca2+. Binding studies with f.IXa reveal a 10-fold higher affinity for heparin in the presence of Ca2+ compared with its absence. Thus, Ca2+ promotes heparin-catalyzed inhibition of f.IXa and f.Xa by antithrombin by augmenting the template mechanism. These results indicate that heparin-mediated catalysis of f.IXa inhibition by antithrombin reflects both pentasaccharide-induced conformational changes and heparin-mediated bridging of antithrombin to f.IXa. Furthermore, our data suggest that the efficacy of pentasaccharide for prevention and treatment of thrombotic disorders may reflect its action at two sites in the coagulation system.
Highlights
Heparin and low molecular weight heparin (LMWH)1 act as anticoagulants by catalyzing antithrombin, a serpin that inactivates thrombin, factor (f.) IXa, and f.Xa [1]
Ca2؉ promotes heparin-catalyzed inhibition of f.IXa and f.Xa by antithrombin by augmenting the template mechanism. These results indicate that heparin-mediated catalysis of f.IXa inhibition by antithrombin reflects both pentasaccharide-induced conformational changes and heparin-mediated bridging of antithrombin to f.IXa
Binding of heparin and LMWH to antithrombin is mediated by a unique pentasaccharide sequence found on one-third of heparin chains
Summary
Heparin and low molecular weight heparin (LMWH) act as anticoagulants by catalyzing antithrombin, a serpin that inactivates thrombin, factor (f.) IXa, and f.Xa [1]. Pentasaccharide-mediated binding of heparin or LMWH to antithrombin induces a conformational change in the reactive center loop of antithrombin that facilitates its interaction with f.Xa [3,4,5] Both agents increase the rate at which antithrombin inhibits f.Xa by 2 or 3 orders of magnitude [6, 7]. Ca2ϩ enhances the rate of f.Xa inhibition by antithrombin, presumably by inducing the exposure of a heparin-binding site on the protease, thereby allowing formation of a ternary heparin-antithrombin-f.Xa complex [27, 28] This concept is supported by surface plasmon resonance studies demonstrating that the binding of heparin to f.Xa is Ca2ϩ-dependent [29].
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