Human Urinary Soluble Thrombomodulin (MR-33) Improves Disseminated Intravascular Coagulation without Affecting Bleeding Time in Rats: Comparison with Low Molecular Weight Heparin

Summary

We compared the antithrombotic and hemorrhagic effects of naturally existing human urinary soluble thrombomodulin (MR-33) with those of low molecular weight heparin (LMW-heparin) in rats. In in vitro experiments, MR-33 prolonged APTT in a dose-dependent fashion; its effect in this respect was as potent as that of LMW-heparin, but it was less potent than unfractionated heparin (UF-heparin). MR-33 was effective on endotoxin- or thromboplastin-induced disseminated intravascular coagulation (DIC) in rats. In both DIC models, infusion of MR-33 improved hematological abnormalities compatible with DIC in a dose-dependent fashion without excessive prolongation effect on APTT. Although LMW-heparin and UF-heparin also improved both DIC models, excessive prolongation of APTT was observed at high doses. It is well-known that the excessive prolongation of APTT with antithrombotic drugs like heparins is an index for hemorrhage, which is a major side effect in the treatment of DIC. We therefore further compared the antithrombotic (Benefit: dose required for 50% inhibition of fibrinogen decrease: ED50) and hemorrhagic (Risk: minimum dose required for significant prolongation of bleeding time) effects of MR-33 and LMW-heparin in the thromboplastin-induced DIC model. As a result, Benefit-Risk ratio was 1:27 for MR-33 and 1:3 for LMW heparin. These results indicate that MR-33 may be a clinically useful antithrombotic agent with reduced risk for hemorrhage compared with LMW-heparin.

• References

• 1 Maruyama I, Bell CE, Majerus PW. Thrombomodulin is found on endothelium of arteries, veins, capillaries and lymphatics and on syncytiotrophoblast of human placenta. J Cell Biol 1985; 101: 363-371
• 2 Ishii H, Majerus PW. Thrombomodulin is present in human plasma and urine. J Clin Invest 1985; 76: 2178-2181
• 3 Yamamoto S, Mizoguchi T, Tamaki T, Ohkuchi M, Kimura S, Aoki N. Urinary thrombomodulin, its isolation and characterization. J Biochem 1993; 113: 433-440
• 4 Jackson DE, Tetaz TJ, Salem HH, Mitchell CA. Purification and characterization of two forms of soluble thrombomodulin from human urine. Eur J Biochem 1994; 221: 1079-1087
• 5 Takahashi Y, Hosaka Y, Niina H, Nagasawa K, Naotsuka M, Sakai K, Uemura A. Soluble thrombomodulin purified from human urine exhibits a potent anticoagulant effect in vitro and in vivo. Thromb Haemost 1995; 73: 805-811
• 6 Esmon CT, Owen WG. Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci USA 1981; 78: 2249-2252
• 7 Esmon NL, Owen WG, Esmon CT. Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C. J Biol Chem 1982; 257: 859-864
• 8 Marlar RA, Kleiss AJ, Griffin JH. Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Blood 1982; 59: 1067-1072
• 9 Vehar GA, Davie EW. Preparation and properties of bovine factor VIII (antihemophilic factor). Biochemistry 1980; 19: 401-410
• 10 Van Hinsbergh VWM, Bertina RM, van WijngaardenA, van TilburgNH, Emeis JJ, Haverkate F. Activated protein C decreases plasminogen activator-inhibitor activity in endothelial cell-conditioned medium. Blood 1985; 65: 444-451
• 11 Rubin RN, Colman RW. Disseminated intravascular coagulation. Drugs 1992; 44: 963-971
• 12 Giles AR. Disseminated intravascular coagulation. In: Haemostasis and Thrombosis. vol 2 Bloom AR, Forbes CD, Thomas DP, Tuddenham EGD. eds. Churchill Livingstone, Edinburgh, UK: 1994. pp 969-986
• 13 Holmer E, Mattson C, Nilsson S. Anticoagulant and antithrombotic effects of heparin and low molecular weight heparin fragments in rabbits. Thromb Res 1982; 25: 475-485
• 14 Carter CJ, Kelton JG, Hirsh J, Cerskus A, Santos AV, Gent M. The relationship between the hemorrhagic and antithrombotic properties of low molecular weight heparin in rabbits. Blood 1982; 59: 1239-1245
• 15 Hamano S, Kinukawa M, Komatsu H, Ikeda S, Sakuragawa N. Effects of low molecular weight heparin (FR-860) on coagulative and fibrinolytic activities. Folia pharmacol Japon 1989; 94: 243-249
• 16 Hamano S, Kinukawa M, Komatsu H, Miyata H, Sakuragawa N. Effects of low molecular weight heparin (FR-860) on the experimental disseminated intravascular coagulation models. Folia pharmacol Japon 1991; 98: 53-62
• 17 Hirsh J. Low molecular weight heparin. Thromb Haemost 1993; 70: 204-207
• 18 Barrowcliffe TW, Thomas DP. Heparin and low molecular weight heparin. In: Haemostasis and Thrombosis, vol 2. Bloom AR, Forbes CD, Thomas DP, Tuddenham EGD. eds. Churchill Livingstone, Edinburgh, UK: 1994. pp 1417-1438
• 19 Nawa K, Itani T, Ono M, Sakano K, Marumoto Y, Iwamoto M. The glycos-aminoglycan of recombinant human soluble thrombomodulin affects antithrombotic activity in a rat model of tissue factor-induced disseminated intravascular coagulation. Thromb Haemost 1992; 67: 366-370
• 20 Aoki Y, Takei R, Mohri M, Gonda Y, Gomi K, Sigihara T, Kiyota T, Yamamoto S, Ishida T, Maruyama I. Antithrombotic effects of recombinant human soluble thrombomodulin (rhs-TM) on arteriovenous shunt thrombosis in rats. AmJHematol 1994; 47: 162-166
• 21 Yoshikawa T, Furukawa Y, Murakami M, Takemura S, Kondo M. Protection of endotoxin-induced disseminated intravascular coagulation in rats by gabexate mesilate. Acta Haematol JPN 1982; 45: 633-640
• 22 Stella L, Donati MB, Gaetano G. Bleeding time in laboratory animals. I. Aspirin does not prolong bleeding time in rats. Thromb Haemost 1975; 7: 709-716
• 23 Pixley RA, Cadena RDL, Page JD, Kaufman N, Wyshock EG, Chang A, Taylor Jr FB, Colman RW. The contact system contributes to hypotension but not disseminated intravascular coagulation in lethal bacteremia. J Clin Invest 1993; 91: 61-68
• 24 Gonda Y, Hirata S, Saitoh K, Aoki Y, Mohri M, Gomi K, Sugihara T, Kiyota T, Yamamoto S, Ishida T, Maruyama I. Antithrombotic effect of recombinant human soluble thrombomodulin on endotoxin-induced disseminated intravascular coagulation in rats. Thromb Res 1993; 71: 325-335
• 25 Ohno H, Kambayashi J, Chang SW, Kosaki G. FOY: [Ethyl-p-(6-guanidi-nohexanoyloxy) benzoate] methanesulfonate as a serine protease inhibitor. II. In vivo effect of coagulofibrinolytic system in comparison with heparin or aproti-nin. Thromb Res 1981; 24: 445-452
• 26 Schwarz RS, Bauer KA, Rosenberg RD. et al Clinical experience with antithrombin III concentrate in treatment of congenital and acquired deficiency of antithrombin. Am J Med 1989; 87 (Suppl. 03) B 53-60
• 27 Menache D, O’Malley JP, Schorr JB, Wagner B, Williams C. Evaluation of the safety, recovery, half-life and clinical efficacy of antithrombin III (human) in patients with hereditary antithrombin III deficiency. Blood 1990; 75: 33-39
• 28 Aoki N, Matsuki K, Sakata Y. The use of antithrombin III concentrates in disseminated intravascular coagulation. Jpn J Clin Hematol 1982; 23: 804-811
• 29 Tsuji H. Antithrombin III. Jpn J Clin Hematol 1990; 31: 750-755
• 30 Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolysis cascades through the thrombin-throm-bomodulin complex. J Biol Chem 1996; 271: 16603-16608
• 31 Dosne AM, Bendetowicz AV, Kher A, Samama M. Marked potentiation of the plasminogenolytic activity of pro-urokinase by unfractionated heparin and a low molecular-weight heparin. Thromb Res 1988; 51: 627-630
• 32 Bacher P, Welzel D, Iqbal O, Hoppensteadt D, Callas D, Walenga JM, Fareed J. The thrombolytic potency of LMW-heparin compared to urokinase in a rabbit juglar vein clot lysis model. Thromb Res 1992; 66: 151-158
• 33 Gruber A, Griffin JH, Harker LA, Hanson SR. Inhibition of platelet-dependent thrombus formation by human activated protein C in a primate model. Blood 1989; 73: 639-642
• 34 Hara T, Yokoyama A, Tanabe K, Ishihara H, Iwamoto M. DX-9065a, an orally active, specific inhibitor of factor Xa, inhibits thrombosis without affecting bleeding time in rats. Thromb Haemost 1995; 74: 635-639
• 35 Ono M, Nawa K, Marumoto Y. Antithrombotic effects of recombinant human soluble thrombomodulin in a rat model of vascular shunt thrombosis. Thromb Haemost 1994; 72: 421-425
• 36 Taylor Jr FB, Chang A, Esmon CT, D’Angelo A, Vigano-D’Angelo S, Blick KE. Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon. J Clin Invest 1987; 79: 918-925
• 37 Gruber A, Hanson SR, Kelly AB, Yan BS, Bang N, Griffin JH, Harker LA. Inhibition of thrombus formation by activated protein C in a primate model of arterial thrombosis. Circulation 1990; 82: 578-585
• 38 Araki H, Nishi K, Ishihara N, Okajima K. Inhibitory effects of activated protein C and heparin on thrombotic arterial occlusion in rat mesenteric arteries. Thromb Res 1991; 62: 209-216
• 39 Katsuura Y, Aoki K, Tanabe H, Kiyoki M, Funatsu A. Characteristic effects of activated human protein C on tissue thromboplastin-induced disseminated intravascular coagulation in rabbits. Thromb Res 1994; 76: 353-362