Mechanism of Catalysis of Inhibition of Factor IXa by Antithrombin in the Presence of Heparin or Pentasaccharide

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 Ca 2؉ , full-length heparin produces an additional ϳ10-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 Ca 2؉ . Binding studies with f.IXa reveal a 10-fold higher affinity for heparin in the presence of Ca 2؉ compared with its absence. Thus, Ca 2؉ 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.

Role of the Residues of the 39-Loop in Determining the Substrate and Inhibitor Specificity of Factor IXa

“…Surprisingly, in contrast to a dramatic cofactor-mediated improvement in the activity of FIXa toward FX, FVIIIa has a minimal cofactor effect on the reactivity of the protease with AT or its activity toward small synthetic substrates (12,13), suggesting that FVIIIa-mediated exosite interactions with FX, involving sites remote from the active-site pocket, play dominant roles in the catalytic reaction (14,15). In the case of the FIXa reaction with AT, the cofactor function of heparin-like glycosaminoglycans, similar to those lining the surface of the endothelium (16), is required to facilitate the protease recognition of the serpin (17)(18)(19).…”

“…Surprisingly, in contrast to a dramatic cofactor-mediated improvement in the activity of FIXa toward FX, FVIIIa has a minimal cofactor effect on the reactivity of the protease with AT or its activity toward small synthetic substrates (12, 13), suggesting that FVIIIa-mediated exosite interactions with FX, involving sites remote from the active-site pocket, play dominant roles in the catalytic reaction (14, 15). In the case of the FIXa reaction with AT, the cofactor function of heparin-like glycosaminoglycans, similar to those lining the surface of the endothelium (16), is required to facilitate the protease recognition of the serpin (17-19).Heparin accelerates the reactivity of AT with FIXa by two distinct mechanisms: a conformational activation of the serpin and a template mechanism in the presence of physiological levels of Ca 2ϩ (17)(18)(19). In the first mechanism, the binding of a unique pentasaccharide fragment of heparin to the basic D-helix of AT induces structural changes in the reactive center loop (RCL) and sites remote from the RCL (outside of P6 -P3Ј sites), thereby facilitating the exosite-dependent interaction of the serpin with the protease (20).…”

Antibodies against the Activated Coagulation Factor X (FXa) in the Antiphospholipid Syndrome That Interfere with the FXa Inactivation by Antithrombin

“…Binding of this pentasaccharide to AT induces a conformational change in the RCL of AT that facilitates its reaction with thrombin and FXa, thereby enhancing the rate of AT inactivation of thrombin and FXa by about two orders of magnitude (35)(36)(37). In addition, during the in vitro heparin-dependent AT inactivation of FXa, heparin binds simultaneously to FXa and AT in the presence of Ca 2ϩ , and thus bridges FXa and AT to form a ternary heparin-AT-FXa complex, leading to an additional ϳ10-fold increase in the rate of inhibition of FXa (30,38). Binding affinities of the above two FXa-reactive, AT-interfering mAb (Fig.…”

Novel Autoantibodies against the Activated Coagulation Factor IX (FIXa) in the Antiphospholipid Syndrome That Interpose the FIXa Regulation by Antithrombin

“…The effects of FIXa-reactive Ab on FIXa inactivation by AT were studied in a functional assay for the FIXa activity in the presence of AT and heparin, according to the report of Wiebe et al (38) with minor modifications. In particular, human AT (Enzyme Research Laboratories) was used at a concentration that was at least 10-fold higher than that of human FIXa, and the experiments were performed in the above buffer for the FIXa activity assay.…”