Depolymerized Holothurian Glycosaminoglycan Research Articles

Fucosylated chondroitin sulfate inhibits plasma thrombin generation via targeting of the factor IXa heparin-binding exosite

Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chondroitin sulfate with antithrombin-independent antithrombotic properties. Heparin cofactor II (HCII)-dependent and -independent mechanisms for DHG inhibition of plasma thrombin generation were evaluated. When thrombin generation was initiated with 0.2 pM tissue factor (TF), the half maximal effective concentration (EC(50)) for DHG inhibition was identical in mock- or HCII-depleted plasma, suggesting a serpin-independent mechanism. In the presence of excess TF, the EC(50) for DHG was increased 13- to 27-fold, suggesting inhibition was dependent on intrinsic tenase (factor IXa-factor VIIIa) components. In factor VIII-deficient plasma supplemented with 700 pM factor VIII or VIIIa, and factor IX-deficient plasma supplemented with plasma-derived factor IX or 100 pM factor IXa, the EC(50) for DHG was similar. Thus, cofactor and zymogen activation did not contribute to DHG inhibition of thrombin generation. Factor IX-deficient plasma supplemented with mutant factor IX(a) proteins demonstrated resistance to DHG inhibition of thrombin generation [factor IX(a) R233A > R170A > WT] that inversely correlated with protease-heparin affinity. These results replicate the effect of these mutations with purified intrinsic tenase components, and establish the factor IXa heparin-binding exosite as the relevant molecular target for inhibition by DHG. Glycosaminoglycan-mediated intrinsic tenase inhibition is a novel antithrombotic mechanism with physiologic and therapeutic applications.

Depolymerized Holothurian Glycosaminoglycan Inhibits Plasma Thrombin Generation Via Interaction with the Factor IXa Heparin-Binding Exosite

Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chrondroitin sulfate that possesses antithrombin-independent antithrombotic properties in rodent thrombosis and dog hemodialysis models. DHG demonstrates significantly less bleeding in template or tail transection assays than therapeutically equivalent doses of heparins. Several potential in vitro mechanisms have been described for DHG, 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). The relevant mechanism(s) for inhibition of tissue factor (TF) induced plasma thrombin generation by DHG were examined in HCII or mock-immunodepleted, and factor-deficient human plasmas, using selected recombinant factor IX(a) with mutations in the heparin-binding exosite. Plasma thrombin generation was detected by fluorogenic substrate cleavage in the presence of corn trypin inhibitor to block contact activation, and compared to a standard curve generated with α2-macroglobulin-thrombin complex. The dose-dependent decrease in velocity index, a parameter reflecting the rate of thrombin generation between lag phase and peak thrombin concentration, was used to compare DHG potency. When triggered by 0.2 pM TF, the EC50 for inhibition of thrombin generation by DHG was 0.16 ± 0.01 μM in both HCII-depleted and mock-depleted plasma, suggesting that DHG acts independently of HCII. When triggered by excess (4 pM) TF, plasma thrombin generation was independent of factors VIII and IX. Under these conditions, the EC50 for DHG inhibition of thrombin generation was increased 13-fold in mock-depleted plasma (2.02 ± 0.09 μM) and 28-fold in HCII-depleted plasma (4.31 ± 0.23 μM). These results suggest that components of the intrinsic tenase complex contribute to inhibition of plasma thrombin generation by DHG, and HCII contributes only at high tissue factor concentrations. In the presence of 0.2 pM TF, Western blotting under nonreducing conditions showed preservation of the prothrombin/meizothrombin band and delayed/reduced thrombin generation in the presence of 0.5 μM DHG, confirming that the inhibition involves reduced prothrombin activation rather than accelerated thrombin inhibition. When triggered by 0.2 pM TF in factor VIII-deficient plasma supplemented with 700 pM factor VIII or thrombin-activated factor VIIIa, the EC50 for inhibition by DHG was 0.41 ± 0.02 μM and 0.44 ± 0.05 μM, respectively. Similarly, the EC50 for DHG inhibition of thrombin generation in factor IX deficient plasma supplemented with 0.2 pM TF and 100% plasma-derived factor IX (90 nM), or 100 pM plasma-derived factor IXa alone, was 0.36 ± 0.01 μM and 0.34 ± 0.02 μM, respectively. Thus, activation of factors VIII and IX do not contribute significantly to the inhibition mechanism for DHG. The contribution of intrinsic tenase activity to DHG inhibition of plasma thrombin generation was assessed using recombinant factor IX(a) mutants with moderate (R170A) or marked (R233A) reductions in heparin affinity. Factor IX deficient plasma was supplemented with 0.2 pM TF and 100% recombinant factor IX, or 100 pM factor IXa, with increasing concentrations of DHG. Similar to plasma-derived factor IX(a), DHG demonstrated an EC50 of 0.38 ± 0.01 μM for inhibition of thrombin generation in the presence of factor IX(a) wild type (WT) zymogen or protease. In the presence of factor IX(a) R170A, the EC50 for DHG was 0.86 ± 0.06 μM and 1.02 ± 0.02 μM, respectively, a 2–3 fold increase relative to WT (P ≤ 0.01). For factor IX(a) R233A, the EC50 for DHG was 3.55 ± 0.47 μM for zymogen and 2.98 ± 0.64 μM for protease, an 8–9 fold increase relative to WT (P ≤ 0.01). Thus, mutations in the factor IXa heparin-binding exosite induced resistance to DHG inhibition of thrombin generation as follows: factor IX(a) R233A> R170A> WT. These findings are consistent with the common mechanism for intrinsic tenase inhibition demonstrated for heparin and DHG in purified systems, and establish the factor IXa heparin-binding exosite as the relevant molecular target for inhibition of plasma thrombin generation by DHG. This antithrombin-independent mechanism likely mediates the antithrombotic efficacy of DHG and related glycosaminoglycans, and may represent a novel therapeutic target with lower bleeding risk.

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

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 and Heparin Inhibit the Intrinsic Tenase Complex by a Common Antithrombin-Independent Mechanism.

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. Biochemistry 44:3615–3625, 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.

Depolymerized holothurian glycosaminoglycan (DHG), a novel alternative anticoagulant for hemodialysis, is safe and effective in a dog renal failure model

Depolymerized holothurian glycosaminoglycan (DHG) is a new agent with anticoagulant properties quite different from those of unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) in terms of antithrombin III-dependency, and exerts an antithrombotic effect with less bleeding than UFH and LMWH in vivo. In this study, the anticoagulant and hemorrhagic effects of DHG were investigated on hemodialysis in a dog model of renal failure and compared with those of UFH, LMWH, and nafamostat mesilate (FUT). The dog renal failure model was prepared by 7/8 renal artery ligation. Effectiveness was based on completion of 3-hour hemodialysis, no marked clot deposition in the extracorporeal circuit, and permeability of blood urea nitrogen (BUN) and creatinine. Template bleeding was measured by determining the hemoglobin content of the blood from the wound. DHG induced no major bleeding or clot formation during 3-hour hemodialysis, in contrast to UFH and LMWH, each of which induced marked bleeding. These glycosaminoglycans (GAGs) were equally effective in decreasing plasma levels of BUN and creatinine. On the other hand, dogs treated with FUT failed to complete 3-hour hemodialysis. These anticoagulants prolonged activated partial thromboplastin time (APTT) to different extents and GAGs prolonged thrombin clotting time markedly but FUT did not. Our findings suggest that thrombin clotting time prolongation can contribute to prevention of clot formation in extracorporeal circuits, and the non-antithrombin III-dependent activities of DHG may be related to its low risk of hemorrhage for hemodialysis. DHG appears to be promising as an alternative anticoagulant with low risk of hemorrhage for hemodialysis.

Prolonged bleeding time induced by anticoagulant glycosaminoglycans in dogs is associated with the inhibition of thrombin-induced platelet aggregation

Introduction: The clinical use of unfractionated heparin (UFH) is complicated by hemorrhage. This has led to a search for safer alternatives, one of which, the recently identified depolymerized holothurian glycosaminoglycan (DHG), causes less bleeding and exhibits a better antithrombotic–hemorrhagic ratio in rats and dogs than UFH and low-molecular-weight heparin (LMWH). In contrast to UFH and LMWH, which exert their anticoagulant effects by inhibiting thrombin in the presence of antithrombin III (AT), DHG exerts its anticoagulant effect by inhibiting the intrinsic factor Xase complex and thrombin in the presence of heparin cofactor II (HCII). Materials and Methods: The hemorrhagic effect of DHG was compared with those of UFH and LMWH in healthy dogs, and the mechanism responsible for prolonging bleeding time was examined both in dogs and with human platelets. Results: DHG prolonged template-bleeding time in dogs less than UFH and LMWH do. Although the maximum noneffective concentrations of each glycosaminoglycan (GAG) that prolong the bleeding time are almost the same as the concentrations that inhibit thrombin-induced platelet aggregation, they are not related to those that inhibit ADP-induced platelet aggregation. Results of experiments on gel-filtered platelets from humans indicate that the inhibition of thrombin-induced platelet aggregation caused by UFH and LMWH in the presence of AT is more prominent than that caused by DHG with HCII. Conclusions: These results suggest that the prolongation of bleeding time caused by GAGs are associated with the inhibition of thrombin-induced platelet aggregation, and DHG may cause less bleeding than UFH and LMWH because of its different thrombin inhibition mechanism in platelet-rich plasma (PRP).

Chiral resolution of basic drugs by capillary electrophoresis with new glycosaminoglycans

New glycosaminoglycans, fucose-containing glycosaminoglycan (FGAG) and depolymerized holothurian glycosaminoglycan (DHG), were investigated as chiral additives for the separation of drug enantiomers by capillary electrophoresis. The average molecular masses of FGAG and DHG were estimated to be about 59 000 and 14 000, respectively. A variety of basic drug enantiomers were resolved using 10 m M phosphate buffer, pH 5.0, containing 3% FGAG or DHG. Since chiral recognition properties of FGAG and DHG are different, some drug enantiomers were only separated by using FGAG or DHG. With regard to comparison of chiral recognition abilities of FGAG and DHG with other chiral selectors, tolperisone and eperisone enantiomers were not separated with α- or β-cyclodextrin, or heparin as the chiral additives, but were separated with FGAG and DHG. The results obtained reveal that FGAG and DHG are useful as the chiral selectors for separations of drug enantiomers by CE, and that they could be complementarily used with other chiral additives.

Oral Absorption and Clearance of Partially Depolymerized Fucosyl Chondroitin Sulfate from Sea Cucumber

Partially depolymerized holothurian glycosaminoglycan (DHG), a fucosyl chondroitin sulfate chains isolated from sea cucumber, was administered by the intravenous and oral routes to experimental animal. After intravenous injection, clearance of DHG, as measured by the postcolumn HPLC, displayed complex kinetics that were not dose dependent. DHG was excreted unchanged in the urine. No degradation products of DHG were detected by either gel filtration or anion exchange HPLC at any time in plasma, indicating that administered DHG is not catabolyzed by mammalian. Anion exchange chromatographic behavior of DHG excreted into urine after oral administration showed that partial desulfation might occur through intestinal absorption. After oral administration of DHG (50 mg/kg), 0.1% of the dose was found in urine collected for 24 hours. More than 5% of intravenously administered DHG (1 mg/kg) was excreted into urine in 24 hours. These results are suggesting that orally administered macromolecules such as DHG are absorbed in the gastrointestinal tract.

Depolymerized holothurian glycosaminoglycan (DHG) prevents neointimal formation in balloon-injured rat carotid artery

The in vivo activity of depolymerized holothurian glycosaminoglycan (DHG), a newly developed polysaccharide anticoagulant, on neointimal formation induced by a balloon catheter in the left common carotid artery of rats was investigated. In every Sprague-Dawley rat weighing approximately 400 g, a Forgaty 2Fr balloon catheter was inserted from the left femoral artery to the left common carotid artery, and was passed through three times in order to denude the endothelium of the artery. These rats were divided into four groups by the following treatment protocols; DHG was given to rats by daily subcutaneous injection into their abdomens at a dose of 3 mg/kg or 10 mg/kg (D3 or D10 group). For controls, 250 μl saline was injected daily (C group). Furthermore, 1 mg/kg of unfractionated heparin was also injected daily as a comparison to DHG (H group). Each treatment was performed in six rats, and the injections were continued for two weeks after the catheterization. The area ratio of thickened intima/media ( I/ M ratio) treated with DHG decreased in a dose-dependent manner compared to the control. In addition, the ratio in the D10 group was significantly lower than in the control ( P<0.01). However, the ratio in the H group did not decrease. By anti-a smooth muscle actin antibody staining the intimal thickening layers were seen to be completely occupied by proliferated smooth muscle cells, and their amount in these layers was attenuated by the DHG treatment. This indicated that DHG has an inhibitory effect on intimal thickening induced by balloon catheterization, and that this might be due to the inactivation of aberrant smooth muscle cells by this agent.

Effect of Depolymerized Holothurian Glycosaminoglycan (DHG) on Tissue Factor Pathway Inhibitor: In Vitro and In Vivo Studies

Depolymerized holothurian glycosaminoglycan (DHG) is a glycosaminoglycan extracted from the sea cucumber Stichopus japonicus Selenka. In previous studies, we demonstrated that DHG has antithrombotic and anticoagulant activities that are distinguishable from those of heparin and dermatan sulfate. In the present study, we examined the effect of DHG on the tissue factor pathway inhibitor (TFPI), which inhibits the initial reaction of the tissue factor (TF)-mediated coagulation pathway. We first examined the effect of DHG on factor Xa inhibition by TFPI and the inhibition of TF-factor VIIa by TFPI-factor Xa in in vitro experiments using human purified proteins. DHG increased the rate of factor Xa inhibition by TFPI, which was abolished either with a synthetic C-terminal peptide or with a synthetic K3 domain peptide of TFPI. In contrast, DHG reduced the rate of TF-factor VIIa inhibition by TFPI-factor Xa. Therefore, the effect of DHG on in vitro activity of TFPI appears to be contradictory. We then examined the effect of DHG on TFPI in cynomolgus monkeys and compared it with that of unfractionated heparin. DHG induced an increase in the circulating level of free-form TFPI in plasma about 20-fold when administered i.v. at 1 mg/kg. The prothrombin time (PT) in monkey plasma after DHG administration was longer than that estimated from the plasma concentrations of DHG. Therefore, free-form TFPI released by DHG seems to play an additive role in the anticoagulant mechanisms of DHG through the extrinsic pathway in vivo. From the results shown in the present work and in previous studies, we conclude that DHG shows anticoagulant activity at various stages of coagulation reactions, i.e., by inhibiting the initial reaction of the extrinsic pathway, by inhibiting the intrinsic Xase, and by inhibiting thrombin.

Antithrombin III-Independent Effect of Depolymerized Holothurian Glycosaminoglycan (DHG) on Acute Thromboembolism in Mice

A previous study in this laboratory showed that depolymerized holothurian glycosaminoglycan (DHG) has two different antithrombin III (ATIII)-independent inhibitory effects on the in vitro blood coagulation system: heparin cofactor II (HCII)-dependent inhibition of thrombin, and ATIII- and HCII-independent inhibition of factor X activation by factor IXa-factor VIIIa complex (Nagase et al. Blood 85, 1527-1534, 1995). In the present study, we compared the antithrombotic effects of DHG in normal and in ATIII-deficient mice with those of unfractionated heparin (UFH) and low molecular weight heparin (LMWH). DHG, unlike UFH and LMWH, exerted an in vivo antithrombotic effect even in mice with decreased plasma ATIII activity (about 30% of normal). We then compared the anticoagulant and antithrombotic effects of DHG in mice with those of high molecular weight (HMW)-DHG, low molecular weight (LMW)-DHG, and dermatan sulfate (DS). In terms of in vitro anticoagulant activity assessed by use of purified human components, DHGs (DHG, HMW-DHG, and LMW-DHG) had different anti-thrombin activity in the presence of HCII and anti-factor Xase activities, which differences were dependent on the molecular weight. With respect to in vivo antithrombotic activity, DHG, HMW-DHG, and LMW-DHG showed almost the same inhibitory effect on acute thromboembolism in mice (minimum effective dose [MED]: > 0.3 mg/kg). Since the antithrombotic activities of DHGs were not correlated with the anticoagulant-specific activities, the contribution of the two anticoagulant activities to the in vivo antithrombotic effect of DHGs remains unknown. However, DHG was more effective against acute thromboembolism in mice than DS (MED > 1 or > 3 mg/kg), which showed no inhibitory activity toward factor Xase. Therefore, it seems that factor Xase inhibition contributes greatly to the antithrombotic effect of DHG and that DHG exerts this effect in mice mainly by inhibiting factor Xase.

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

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 glycosaminoglycans 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.

The Anticoagulant and Hemorrhagic Effects of DHG, a New Depolymerized Holothurian Glycosaminoglycan, on Experimental Hemodialysis in Dogs

We studied the use of depolymerized holothurian glycosaminoglycan (DHG) as an anticoagulant in experimental beagle-dog hemodialysis using a hollow-fiber dialyzer compared to that using unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), and nafamostat mesilate (FUT). Effectiveness was based on 5 h hemodialysis and no marked clot deposition in the extracorporeal circuit. At effective doses, UFH and LMWH significantly prolonged template bleeding time, in sharp contrast to FUT and DHG, which scarcely prolonged bleeding time during hemodialysis. DHG prolonged activated partial thromboplastin time (APTT) about 6 times that of normal plasma and prolonged thrombin clotting time (TCT) markedly; FUT showed marked APTT prolongation but hardly prolonged TCT in the hemodialysis circuit at the effective dose. The anticoagulant profile of DHG thus differs completely from that of FUT. These results suggest that DHG may be useful as anticoagulant for hemodialysis with low hemorrhagic risk.

DHG, A NEW DEPOLYMERIZED HOLOTHURIAN GLYCOSAMINOGLYCAN, EXERTS AN ANTITHROMBOTIC EFFECT WITH LESS BLEEDING THAN UNFRACTIONATED OR LOW MOLECULAR WEIGHT HEPARIN, IN RATS

We characterized the antithrombotic, haemorrhagic, and ex vivo anticoagulant effects of a recently identified depolymerized holothurian glycosaminoglycan (DHG), and compared these effects with those of unfractionated heparin (UFH), low molecular weight heparin (LMWH), and dermatan sulfate (DS). In thrombin-induced venous thrombus formation in rats, DHG had a significant preventive effect at 0.3 mg/kg or more at 5 min after i.v. administration. UFH, LMWH, and DS also showed a significant antithrombotic effect at 0.3, 0.3, and 1 mg/kg, respectively, under the same experimental conditions. After rat tail transection, DHG, UFH, LMWH, and DS prolonged the bleeding time significantly at 10, 1, 1, and 10 mg/kg, respectively, at 5 min after i.v. injection. Therefore, DHG exerts its antithrombotic effect with less bleeding than UFH and LMWH in experimental animals. DHG prolonged the activated partial thromboplastin time in a dose-dependent manner at 0.3 − 3 mg/kg, at which dose an antithrombotic effect was exhibited without any significant haemorrhagic effect. All of these glycosaminoglycans prolonged thrombin clotting time markedly at their haemorrhagic doses. Copyright © 1996 Elsevier Science Ltd

INTERACTION OF A NEW DEPOLYMERIZED HOLOTHURIAN GLYCOSAMINOGLYCAN WITH PROTEINS IN HUMAN PLASMA

In a rat template bleeding model, depolymerized holothurian glycosaminoglycan (DHG) prolonged bleeding time at 30 mg/kg i.v. but unfractionated heparin (UFH) had the same effect at 1 mg/kg i.v., indicating that DHG is much less bleeding than UFH. To characterize this difference, we examined the affinity of DHG for plasma proteins by means of a glycosaminoglycan-conjugated cellulofine column in comparison with that of UFH. The DHG column strongly bound factor V, factor IX, protein S, histidine-rich glycoprotein, platelet factor 4 (PF4), β-thromboglobulin, von Willebrand factor, fibronectin, and heparin cofactor II, but did not bind fibrinogen, prothrombin, factor VII, protein C, antithrombin III (ATIII), plasminogen or α 2-plasmin inhibitor. The profile of protein binding to the UFH column was almost the same as that of the DHG column except that ATIII showed affinity for UFH. One of the reasons why DHG caused much less bleeding than UFH is thus suggested to be the differences in their affinity for ATIII in plasma.

Effect of Depolymerized Holothurian Glycosaminoglycan (DHG) on the Activation of Factor VIII and Factor V by Thrombin

Our previous study has shown that depolymerized holothurian glycosaminoglycan (DHG) has two different inhibitory activities in the blood coagulation cascade: heparin cofactor II-dependent thrombin inhibition; and antithrombin III- and heparin cofactor II-independent inhibition of the intrinsic factor Xase complex [Nagase et al. (1995) Blood 85, 1527-1534]. In the present study, the effect of DHG on the activation of factor VIII and factor V by thrombin was examined with purified human components. DHG inhibited the activation of factor VIII by thrombin at concentrations exceeding 80 nM, but not the activation of factor V by thrombin at concentrations of up to 8 mu M. On Western blot analysis, DHG inhibited the cleavage of factor VIII light chain at concentrations exceeding 0.8 mu M. The interaction between DHG and factors VIII and V and thrombin was examined with a DHG-cellulofine column. DHG had strong affinity for factor V and thrombin, but slight affinity for factor VIII. The interaction of DHG with thrombin was analyzed, using fluorescein isothiocyanate-labeled DHG. One mole of DHG bound 2 mol of thrombin, with a dissociation constant (Kd) of 3.04 x 10(-6) M. These results suggest that DHG interferes with the interaction between thrombin and factor VIII, probably by making a binary complex through the anionic binding exosite II of thrombin.

Depolymerized Holothurian Glycosaminoglycan With Novel Anticoagulant Actions: Antithrombin III- and Heparin Cofactor II-Independent Inhibition of Factor X Activation by Factor IXa-Factor Villa Complex and Heparin Cofactor H-Dependent Inhibition of Thrombin

The inhibition mechanism of a polysaccharide anticoagulant, depolymerized holothurian glycosaminoglycan (DHG), was examined by analyzing its effects on the clotting time of human plasma depleted of antithrombin III (ATIII), of heparin cofactor II (HCII), or of both heparin cofactors. The effect exerted by this agent on the activation of prothrombin and factor X in purified human components were also examined and all effects were compared with those of other glycosaminoglycans (GAGs). The capacity of DHG to prolong activated partial thromboplastin time was not reduced in ATIII-depleted, HCII-depleted, HCII-depleted, or ATIII- and HCII-depleted plasma, whereas its capacity to prolong prothrombin time and thrombin clotting time was reduced in HCII-depleted plasma. DHG inhibited the amidolytic activity of thrombin in the presence of HCII with a second order rate constant of 1.2 x 10(8) (mol/L)-1 min-1. These results indicated that DHG has two different inhibitory activities, one being an HCII-dependent thrombin inhibition and the other an ATIII- and HCII-independent inhibition of the coagulation cascade. The heparin cofactors-independent inhibitory activity of DHG was investigated in the activation of prothrombin by factor Xa and in the activation of factor X by tissue factor-factor VIIa complex or by factor IXa. DHG significantly inhibited the activation of factor X by factor IXa in the presence of factor VIIIa, but not in the absence of factor VIIIa. The interaction between DHG and factors IXa, VIIIa, and X was investigated with a DHG-cellulofine column, on which DHG had strong affinity for factors IXa and VIIIa. These findings show that the heparin cofactors-independent inhibition exhibited by DHG was caused by inhibition of the interaction of factor X with the intrinsic factor Xase complex, probably by binding to the factor IXa-factor VIIIa complex.

Determination of a depolymerized holothurian glycosaminoglycan in plasma after intravenous administration by postcolumn HPLC.

Depolymerized holothurian glycosaminoglycan (DHG) produced artificially from sea cucumber has an anticoagulant and antithrombotic activity. In the present study, we attempted to determine the plasma level of DHG using a chemical procedure. A general method for determination of chondroitin sulfates by forming unsaturated disaccharides with chondroitinase digestion was difficult to apply to DHG, because it was resistant to any chondroitinase digestion. We therefore developed a highly sensitive postcolumn HPLC method for determination of intact DHG. DHG was applied to Asahipak NH2P-50, an amino-bonded column, eluted by alkaline solution and then detected with arginine under a strong alkaline condition as a postcolumn reagent. The limit of detection for DHG was 10 ng. The present method was applicable to the determination of DHG in plasma after intravenous administration.