Depolymerized Holothurian Glycosaminoglycan and Heparin Inhibit the Intrinsic Tenase Complex by a Common Antithrombin-Independent Mechanism.
Authors: Sheehan John P. , Walke Erik N. 
Published: April 2020

Abstract
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&gt;K241A&gt;H92A&gt;R170A&gt;&gt;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 (&gt; 80 nM). Accordingly, DHG represents a lead compound for analysis of this novel antithrombotic mechanism in the absence of confounding antithrombin-dependent activities.