Therapeutic heparin concentrations selectively inhibit the intrinsic tenase complex in an antithrombin-independent manner. To define the molecular target and mechanism for this inhibition, recombinant human factor IXa with alanine substituted for solvent-exposed basic residues (H92, R170, R233, K241) in the protease domain was characterized with regard to enzymatic activity, heparin affinity, and inhibition by low molecular weight heparin (LMWH). These mutations only had modest effects on chromogenic substrate hydrolysis and the kinetics of factor X activation by factor IXa. Likewise, factor IXa H92A and K241A showed factor IXa-factor VIIIa affinity similar to factor IXa wild type (WT). In contrast, factor IXa R170A demonstrated a 4-fold increase in apparent factor IXa-factor VIIIa affinity and dramatically increased coagulant activity relative to factor IXa WT. Factor IXa R233A demonstrated a 2.5-fold decrease in cofactor affinity and reduced ability to stabilize cofactor half-life relative to wild type, suggesting that interaction with the factor VIIIa A2 domain was disrupted. Markedly (R233A) or moderately (H92A, R170A, K241A) reduced binding to immobilized LMWH was observed for the mutant proteases. Solution competition demonstrated that the EC(50) for LMWH was increased less than 2-fold for factor IXa H92A and K241A but over 3.5-fold for factor IXa R170A, indicating that relative heparin affinity was WT > H92A/K241A > R170A >> R233A. Kinetic analysis of intrinsic tenase inhibition demonstrated that relative affinity for LMWH was WT > K241A > H92A > R170A >> R233A, correlating with heparin affinity. Thus, LMWH inhibits intrinsic tenase by interacting with the heparin-binding exosite in the factor IXa protease domain, which disrupts interaction with the factor VIIIa A2 domain.

Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chrondroitin sulfate that possesses antithrombin-independent antithrombotic properties and inhibits factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa). The mechanism and molecular target for intrinsic tenase inhibition were determined and compared with inhibition by low-molecular-weight heparin (LMWH). DHG inhibited factor X activation in a noncompetitive manner (reduced V(max(app))), with 50-fold higher apparent affinity than LMWH. DHG did not affect factor VIIIa half-life or chromogenic substrate cleavage by factor IXa-phospholipid but reduced the affinity of factor IXa for factor VIIIa. DHG competed factor IXa binding to immobilized LMWH with an EC(50) 35-fold lower than soluble LWMH. Analysis of intrinsic tenase inhibition, employing factor IXa with mutations in the heparin-binding exosite, demonstrated that relative affinity (K(i)) for DHG was as follows: wild type > K241A > H92A > R170A > > R233A, with partial rather than complete inhibition of the mutants. This rank order for DHG potency correlated with the effect of these mutations on factor IXa-LMWH affinity and the potency of LMWH for intrinsic tenase. DHG also accelerated decay of the intact intrinsic tenase complex. Thus, DHG binds to an exosite on factor IXa that overlaps with the binding sites for LMWH and factor VIIIa, disrupting critical factor IXa-factor VIIIa interactions.

Depolymerized holothurian glycosaminoglycan (DHG) is a low molecular weight form (M.W. 12,500) of fucosylated chondroitin sulfate isolated from the sea cucumber Stichopus japonicus . DHG demonstrates antithrombotic efficacy in models of thrombin-induced pulmonary thromboembolism in the mouse, venous thrombosis in the rat, and dialysis during renal failure in the dog. The in vitro anticoagulant activities and antithrombotic efficacy of DHG are antithrombin-independent, and associated with lower bleeding tendency compared to unfractionated or low molecular weight heparin (LMWH). DHG has several potential mechanisms of action including acceleration of thrombin inhibition by heparin cofactor II (HCII), inhibition of factor VIII activation by thrombin, and inhibition of factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa). DHG demonstrates significant affinity for both factor VIIIa and factor IXa, but the specific mechanism for inhibition of the intrinsic tenase complex (ITC) is undefined. We recently established the factor IXa heparin-binding exosite as the molecular target for antithrombin-independent inhibition of the ITC by LMWH (Yuan et al. Biochemistry44:3615–3625, 2005). The mechanism and molecular target for ITC inhibition by DHG was likewise determined, and compared to inhibition by LMWH. DHG completely inhibited factor X activation with a 50-fold higher apparent affinity (KI ~2 nM) than observed for partial inhibition by LMWH (KI ~111 nM). DHG reduced the Vmax(app) for factor X activation, without a significant effect on the KM(app), consistent with non-competitive inhibition. DHG did not affect the in vitro half-life of factor VIIIa activity, or inhibit chromogenic substrate cleavage by factor IXa-phospholipid. However, DHG reduced the affinity (KD(app)) of factor IXa for factor VIIIa in a dose dependent fashion, suggesting that the decreased Vmax(app) for factor X resulted from reduced complex assembly. DHG competed the binding of factor IXa to immobilized LMWH with an EC50 ~ 35-fold lower than soluble LWMH, suggesting that the binding sites for DHG and LMWH overlap on the protease. Likewise, the relative affinity of DHG for factor IXa compared to LMWH correlated with inhibitor potency. Kinetic analysis of ITC inhibition employing factor IXa with mutations in the heparin-binding exosite demonstrated that relative affinity for DHG (KI) was: wild type>K241A>H92A>R170A>>R233A; with partial rather than complete inhibition of the mutants. This rank order for DHG potency correlated with the effect of these mutations on factor IXa-LMWH affinity, and the potency of LMWH for the ITC. Submaximal inhibitory concentrations of DHG also accelerated decay of the ITC, under condition where the half-life is primarily dependent on dissociation of the factor VIIIa A2 domain. Thus, DHG binds to an exosite on factor IXa that overlaps with the binding sites for LMWH and factor VIIIa, disrupting critical factor IXa-factor VIIIa interaction(s). These structurally diverse glycosaminoglycans share a common mechanism for inhibition of factor X activation by the ITC. This inhibition occurs at DHG concentrations that are significantly lower (KI ~ 2 nM) than required for optimal acceleration of thrombin inhibition by HCII (~2.4 μM), or inhibition of factor VIII activation by thrombin (> 80 nM). Accordingly, DHG represents a lead compound for analysis of this novel antithrombotic mechanism in the absence of confounding antithrombin-dependent activities.

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Therapeutic heparin concentrations selectively inhibit the intrinsic tenase complex in an antithrombin-independent manner. To define the molecular target and mechanism for this inhibition, recombinant human factor IX (FIX) with alanine substituted for solvent-exposed basic residues (H92, R170, R233, K241) in the protease domain was expressed in HEK 293 cells, activated by FXIa, and characterized with regard to enzymatic activity, heparin affinity, and inhibition by low molecular weight heparin (LMWH). The recombinant FIX proteins were purified to homogeneity by SDS-PAGE analysis and exhibited indistinguishable chromatographic behavior when eluted from a Resource Q column with a calcium gradient. FIX was activated with human FXIa (150:1 molar ratio) at 4 °C for 2–6 hr, incubated with anti-FXIa polyclonal antisera crosslinked to Affi-gel, and FIXa active sites were determined by a modified antithrombin III titration. These mutations had only modest effects on chromogenic substrate hydrolysis and the kinetics of factor X activation by FIXa. The kcat/KM for factor X activation by FIXa-phospholipid (5 nM FIXa, 50 μM PC:PS vesicles, 30% ethylene glycol) was similar for all recombinant proteases except FIXa R233A, which was modestly lower due to a 1.4-fold increase in KM(app). In a functional binding assay, FIXa H92A and K241A exhibited apparent FIXa-FVIIIa affinity similar to FIXa wild type (WT) (KD(app)= 2.2, 1.9, and 1.7 nM, respectively). FIXa R170A had markedly increased FIXa-FVIIIa affinity (KD(app)= 0.4 nM), and consistent with previous results, dramatically increased coagulant activity (372%) relative to FIXa WT (J. Chang et al. JBC, 1998). FIXa R233A had significantly reduced cofactor affinity (KD(app)= 4.4 nM) and coagulant activity (59%). FIXa R233A also had reduced ability to stabilize the in vitro half-life of FVIIIa relative to FIXa WT (2-fold faster degradation), even at increased protease concentration. Thus, this mutation disrupts interaction with the A2 domain, suggesting that this critical cofactor interactive site extends from the established c165–170 α-helix to the proximal portion of the C-terminus α-helix on FIXa. Using KD(app) to calculate the concentration of FIXa-FVIIIa, no significant differences in the kcat/KM for factor X activation by FIXa-FVIIIa-phospholipid were observed between proteases(1–2 nM FVIIIa, 0.1 nM FIXa, 50 μM PC:PS vesicles). Relative heparin affinity of FIXa was assessed by direct and competition binding to immobilized LMWH, detected by surface plasmon resonance. Mutant FIXa (250 nM) demonstrated moderately reduced (H92A, R170A, K241A) or minimal (R233A) binding to immobilized LMWH, relative to FIXa WT. Solution competition demonstrated that the EC50 for LMWH was increased less than 2-fold for FIXa H92A and K241A, but over 3.5-fold for FIXa R170A, indicating that relative heparin affinity was WT>H92A/K241A>R170A>>R233A. Kinetic analysis of the inhibition of factor X activation by intrinsic tenase demonstrated that relative affinity for LMWH was WT>K241A>H92A> R170A>>R233A, correlating with heparin affinity. Notably, FIXa R233A demonstrated minimal binding to immobilized LMWH, and almost complete resistance to inhibition by LMWH in the intrinsic tenase complex. Thus, LMWH inhibits intrinsic tenase by interacting with the heparin-binding exosite on the FIXa protease domain. The extensive overlap between heparin and FVIIIa interactive sites on the protease domain suggests that oligosaccharide disrupts critical interactions with the cofactor A2 domain.