Dhg, a New Depolymerized Holothurian Glycosaminoglycan, Exerts an Antithrombotic Effect With Less Bleeding Than Unfractionated or Low Molecular Weight Heparin, in Rats

Kitazato, Kenji; Kitazato, Keiko T.; Nagase, Hideki; Minamiguchi, Kazuhisa

doi:10.1016/0049-3848(96)00166-1

Cited by 36 publications

(32 citation statements)

References 19 publications

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“…DHG demonstrates antithrombotic efficacy in models of thrombininduced pulmonary thromboembolism in the mouse, thrombininduced venous thrombosis in the rat, and dialysis during renal failure in the dog. [18][19][20][21] DHG has multiple 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. 22,23 In contrast to heparin, DHG lacks the antithrombindependent activities that contribute to increased bleeding risk.…”

Section: Introductionmentioning

confidence: 99%

“…[46][47][48][49][50] Likewise, DHG demonstrates significantly less prolongation of the rat tail bleeding time and reduced bleeding time and blood loss from a template incision of the dog ear, compared with equitherapeutic doses of UH or LMWH. [19][20][21] Because the factor IXa-A2 domain interaction is required for factor X activation by the intrinsic tenase complex, 51 disruption of this protein-protein interaction is expected to proportionally reduce plasma thrombin generation based on the rate-limiting nature of the intrinsic tenase complex. 13,37,40 This novel antithrombotic strategy should allow titration of thrombin generation while limiting the risk of bleeding at supratherapeutic doses.…”

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confidence: 99%

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Depolymerized holothurian glycosaminoglycan and heparin inhibit the intrinsic tenase complex by a common antithrombin-independent mechanism

Sheehan

Walke

2006

Blood

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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.

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Section: Introductionmentioning

confidence: 99%

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confidence: 99%

Depolymerized holothurian glycosaminoglycan and heparin inhibit the intrinsic tenase complex by a common antithrombin-independent mechanism

Sheehan

Walke

2006

Blood

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“…However, the native polysaccharide has side effects such as factor XII (FXII) activation, platelet aggregation, and serious bleeding (18). Nowadays, depolymerization is considered to be an effective method for reducing these adverse effects (19). For over 30 y since its discovery the detailed structures of native FG and its depolymerized products have not been elucidated, because these polysaccharides are heterogeneous, namely they are mixtures of isomers with different molecular weights, and because limitations exist in the available strategies for analyzing such molecules (17,20).…”

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confidence: 99%

Discovery of an intrinsic tenase complex inhibitor: Pure nonasaccharide from fucosylated glycosaminoglycan

Zhao

Xiao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

116

152

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Selective inhibition of the intrinsic coagulation pathway is a promising strategy for developing safer anticoagulants that do not cause serious bleeding. Intrinsic tenase, the final and rate-limiting enzyme complex in the intrinsic coagulation pathway, is an attractive but less explored target for anticoagulants due to the lack of a pure selective inhibitor. Fucosylated glycosaminoglycan (FG), which has a distinct but complicated and ill-defined structure, is a potent natural anticoagulant with nonselective and adverse activities. Herein we present a range of oligosaccharides prepared via the deacetylationdeaminative cleavage of FG. Analysis of these purified oligosaccharides reveals the precise structure of FG. Among these fragments, nonasaccharide is the minimum fragment that retains the potent selective inhibition of the intrinsic tenase while avoiding the adverse effects of native FG. In vivo, the nonasaccharide shows 97% inhibition of venous thrombus at a dose of 10 mg/kg in rats and has no obvious bleeding risk. This nonasaccharide may therefore serve as a novel promising anticoagulant.T hrombotic disease is seriously harmful to human health and is one of the major causes of death in modern society (1). Despite their long-term and widespread use as anticoagulants, heparin, low-molecular-weight heparin (LMWH), and coumarins still have a major unresolved issue: the risk of serious bleeding during therapy (1-3). It is generally recognized that the risk of bleeding associated with these agents is related to the nonselectivity of their anticoagulant activity. Therefore, selective inhibitors of human factor Xa (FXa) and thrombin (FIIa), such as dabigatran, rivaroxaban, and apixaban, which have predictable pharmacokinetics, have recently been developed; however, these agents have not effectively reduced the risk of bleeding in clinical applications (4-7).Components of the intrinsic coagulation pathway are promising targets for antithrombotic therapy because they are important for thrombosis but are not required for hemostasis (1,8). The development of new anticoagulant agents that inhibit components of the intrinsic pathway and that have a lower risk of causing bleeding has thus become a research focus (9-11). Factor IXa (FIXa), a serine protease, and factor VIIIa (FVIIIa), a protein cofactor, form a Ca 2+ -and phospholipid surface-dependent complex referred to as the intrinsic tenase complex, which efficiently converts zymogen factor X (FX) to FXa (1,12,13). Because the intrinsic tenase is the final and rate-limiting enzyme complex in the intrinsic pathway, the development of inhibitors of this enzyme complex is important for meeting clinical demands (1). However, limited progress has been achieved due to the unavailability of selective inhibitors with welldefined structures.Fucosylated glycosaminoglycan (FG; 1 in Fig. 1), which is a complex acidic polysaccharide isolated from sea cucumber, has recently attracted considerable attention because of its various bioactivities (14). Notably, FG has potent anticoagula...

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“…Since der matan sulfate hardly shows inhibitory activity toward the factor Xase complex, DHG is completely different from dermatan sulfate, too, as reported previously [18]. The dose of DHG for an efficient antithrombotic effect is more than 0.3 mg/kg in a thrombin-induced venous thrombosis model in the rat, and an intravenous bolus of 0.3 mg/kg of DHG ad ministered to rats prolonged the normal acti vated partial thromboplastin time (APTT) (16-17 s) to around 35 s [19], Based on an in vivo study, we suggested that DHG has an antithrombotic effect without any significant hemorrhagic effect in comparison with con ventional anticoagulants, i.e. UFH and LMWH [19], However, it may be necessary to neutralize DHG in emergency states, for ex ample if bleeding happens to occur during treatment with DHG or neutralization of DHG is urgently needed.…”

Section: Kaiuiermentioning

confidence: 62%

“…The dose of DHG for an efficient antithrombotic effect is more than 0.3 mg/kg in a thrombin-induced venous thrombosis model in the rat, and an intravenous bolus of 0.3 mg/kg of DHG ad ministered to rats prolonged the normal acti vated partial thromboplastin time (APTT) (16-17 s) to around 35 s [19], Based on an in vivo study, we suggested that DHG has an antithrombotic effect without any significant hemorrhagic effect in comparison with con ventional anticoagulants, i.e. UFH and LMWH [19], However, it may be necessary to neutralize DHG in emergency states, for ex ample if bleeding happens to occur during treatment with DHG or neutralization of DHG is urgently needed. On the other hand, we also confirmed that PF4 bound to a DHGconjugated cellulofine column as strongly as to a UFH column [20], Therefore, there is a possibility that PF4 modulates the anticoagu lant activity of DHG.…”

Section: Kaiuiermentioning

confidence: 99%

Neutralization of DHG, a New Depolymerized Holothurian Glycosaminoglycan, by Protamine Sulfate and Platelet Factor 4

Sasaki

Minamiguchi²,

Kitazato³

et al. 1997

Pathophysiol Haemos Thromb

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The neutralization of depolymerized holothurian glycosaminoglycan (DHG), unfractionated heparin (UFH), and low-molecular-weight heparin (LMWH) by protamine sulfate (PS) or platelet factor 4 (PF4) was studied. In in vitro studies, the prolongation of thrombin clotting time (TCT) by these glycos-aminoglycans was completely neutralized by PS, whereas activated partial thromboplastin time (APTT) was relatively resistant to neutralization. In rats, prolongation of bleeding time by DHG was neutralized by PS with concomitant normalization of TCT ex vivo. Heparin-cofactor-II-dependent antithrombin activity of DHG or UFH was neutralized by PF4 at a high concentration to the same extent; however, prolongation of the APTT by DHG was more resistant to neutralization by PF4 at a physiological plasma level than that by UFH. In conclusion, since the prolongation of bleeding time by DHG was neutralized by PS with concomitant normalization of TCT, and since PF4 neutralized the antithrombin activity of DHG, these proteins may be useful as antidotes for DHG to prevent bleeding in case of an overdose.

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Dhg, a New Depolymerized Holothurian Glycosaminoglycan, Exerts an Antithrombotic Effect With Less Bleeding Than Unfractionated or Low Molecular Weight Heparin, in Rats

Cited by 36 publications

References 19 publications

Depolymerized holothurian glycosaminoglycan and heparin inhibit the intrinsic tenase complex by a common antithrombin-independent mechanism

Depolymerized holothurian glycosaminoglycan and heparin inhibit the intrinsic tenase complex by a common antithrombin-independent mechanism

Discovery of an intrinsic tenase complex inhibitor: Pure nonasaccharide from fucosylated glycosaminoglycan

Neutralization of DHG, a New Depolymerized Holothurian Glycosaminoglycan, by Protamine Sulfate and Platelet Factor 4

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