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

Summary

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 Villa 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.

• References

• 1 Rosenberg RD, Damus PS. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem 1973; 248: 6490-6505
• 2 Damus PS, Hicks M, Rosenberg RD. Anticoagulant action of heparin. Nature 1973; 246: 355-357
• 3 Holmer E, Soderstrom G, Andersson LO. Effect of heparin in antithrombin III depleted plasma. Thromb Haemost 1979; 42: 09
• 4 Egeberg o. Inherited antithrombin deficiency causing thrombophilia. Thromb Diath Haemorrh 1965; 13: 516-530
• 5 Spero JA, Lewis JH, Hasiba U. Disseminated intravascular coagulation. Findings in 346 patients. Thromb Haemost 1980; 43: 28-33
• 6 Kauffmann RH, Veltkamp JJ, van TilburgNH, van EsLA. Acquired antithrombin III deficiency and thrombosis in the nephrotic syndrome. Am J Med 1978; 65: 607-613
• 7 Marciniak E, Gockerman J. Heparin-induced decrease in circulating antithrombin III. Lancet 1977; 02: 581-584
• 8 Schipper HG, Jenkins CP, Kahle LH, ten CateJW. Antithrombin III transfusion in disseminated intravascular coagulation. Lancet 1978; 22: 854-856
• 9 Lechner K, Kyrle PA. Antithrombin III concentrates – are they clinical useful? Thromb Haemost. 1995; 73: 340-48
• 10 Hirsh J, Levine MN. Low molecular weight heparin. Blood 1992; 79: 01-17
• 11 Tollefsen DM, Perka CA, Monafo WJ. Activation of heparin cofactor II by dermatan sulfate. J Biol Chem 1983; 258: 6713-6716
• 12 Suzuki N, Kitazato K, Takamatsu J, Saito H. Antithrombotic and anticoagulant activity of depolymerized fragment of the glycosaminoglycan extracted from Stichopus japonicus Selenka. Thromb Haemost 1991; 65: 369-373
• 13 Nagase H, Enjyoji K, Minamiguchi K, Kitazato TK, Kitazato K, Saito H, Kato H. 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 II-dependent inhibition of thrombin. Blood 1995; 85: 1527-1534
• 14 Kitazato K, Kitazato TK, Nagase H, Minamiguchi K. DHG, a new depolymerized holothurian glycosaminoglycan, exerts an antithrombotic effect with less bleeding than unfractionated or low molecular weight heparin, in rats. Thromb Res 1996; 84: 111-120
• 15 Yoshida K, Minami Y, Nemoto H, Numata K, Yamanaka E. Structure of DHG, a depolymerized glycosaminoglycan from sea cucumber, Stichopus japonicus. Tetrahedron Lett 1992; 33: 4959-4962
• 16 Tran TH, Lämmle B, Zbinden B, Duckert F. Heparin cofactor II:Purification and antibody production. Thromb Haemost 1986; 55: 19-23
• 17 Curtis JE, Helgerson SL, Parker ET, Lollar P. Isolation and characterization of thrombin-activated human factor VIII. J Biol Chem 1994; 269: 6246-6251
• 18 Kumada T, Dittman WA, Majerus PW. A role for thrombomodulin in the pathogenesis of thrombin-induced thromboembolism in mice. Blood 1987; 71: 728-733
• 19 Koide T. Isolation and characterization of antithrombin III from human, porcine and rabbit plasma, and rat serum. J Biochem 1979; 86: 1841-1850
• 20 Kumada T, Abiko Y. Preparation and immunological determination of rat antithrombin III. Thromb Res 1980; 17: 789-799
• 21 Lamontagne L, Gauldie J. Ontogeny of mouse and rat antithrombin III. Thromb Res 1980; 20: 417-424
• 22 Odegard OR, Lie M, Abildgaard U. Antifactor Xa activity measured with amidolytic methods. Haemostas 1976; 05: 265-275
• 23 Kumada T, Abiko Y. Comparative study on heparin and a synthetic thrombin inhibitor No. 805 (MD-805) in experimental antithrombin Ill-deficient animals. Thromb Res 1981; 24: 285-298
• 24 Aoki Y, Ohishi R, Takai R, Matsuzaki o, Mohri M, Saitoh K, Gomi K, Sugihara T, Kiyota T, Yamamoto S, Ishida T, Maruyama I. Effects of recombinant human soluble thrombomodulin (rhs-TM) on a rat model of disseminated intravascular coagulation with decreased levels of plasma antithrombin III. Thromb Haemost 1994; 71: 452-455
• 25 Sandset PM, Andersson TR. Coagulation inhibitor levels in pneumonia and stroke:changes due to consumption and acute phase reaction. J Intern Med 1989; 225: 311-316