Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

Summary

We examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

• References

• 1 Esmon CT, Owen WG. Identification of an endothelial cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci USA 1981; 78: 2249-2252
• 2 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
• 3 Marlar R, Kleiss R, Griffin J. Mechanism of action of human activated protein C, a thrombin dependent anticoagulant enzyme. Blood 1982; 59: 1067-1072
• 4 Vehar GA, Davie EW. Preparation and properties of bovine factor VIII (antihemophilic factor). Biochemistry 1980; 19: 401-410
• 5 Van Hinsbergh VW M, Bertina RM, van WijingaardenA, van TiburgN, Emeis JJ, Havekate F. Activated protein C decreases plasminogen activator-inhibitor activity in endothelial cell-conditioned medium. Blood 1985; 65: 444-451
• 6 Maruyama I, Bell CE, Majerus PW. Thrombomodulin is found on endothelium of arteries, veins, capillaries and lymphatics and on syncytiotropho- blast of human placenta. J Cell Biol 1985; 101: 363-371
• 7 Ishii H, Majerus PW. Thrombomodulin is present in human plasma and urine. J Clin Invest 1985; 76: 2178-2181
• 8 Ishii H, Nakano M, Tubouti J, Ishikawa T, Uchiyama H, Hiraishi S, Tahara C, Miyajima Y, Kazama M. Establishment of enzyme immunoassay of human thrombomodulin in plasma and urine using monoclonal antibodies. Thromb Haemostas 1990; 63: 157-162
• 9 Yamamoto S, Mizoguchi T, Tamaki T, Ohkuchi M, Kimura S, Aoki N. Urinary thrombomodulin, its isolation and characterization. J Biochem 1993; 113: 433-440
• 10 Jackson D E, Tetaz T J, Salem HH, Mitchell CA. Purification and characterization of two forms of soluble thrombomodulin from human urine. Eur J Biochem 1994; 221: 1079-1087
• 11 Kurosawa S, Aoki N. Preparation of thrombomodulin from human placenta. Thromb Res 1985; 37: 353-364
• 12 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 13 Ohno H, Kambayashi J, Chang S W, Kosaki G. FOY:[Ethyl p-(6-guanidi-nohexanoyloxy) benzoate] methanesulfonate as a serine protease inhibitor. In vivo effect of coagulofibrinolytic system in comparison with heparin or aprotinin. Thromb Res 1981; 24: 445-452
• 14 Hahn IF, Pickenhahn P, Lenz W, Brandis H. An avidin-biotin ELISA for the detection of staphylococcal enterotoxins A and B. J Immunol Methods 1986; 92: 25-29
• 15 Gomi K, Zushi M, Honda G, Kawahara S, Matsuzaki O, Kanabayashi T, Yamamoto S, Maruyama I, Suzuki K. Antithrombotic effect of recombinant human thrombomodulin on thrombin-induced thromboembolism in mice. Blood 1990; 75: 1396-1399
• 16 Weinstein RE, Walker FJ. Species specificity of the fibrinolytic effects of activated protein C. Thromb Res 1991; 63: 123-131
• 17 Kumada T, Dittman WA, Majerus PW. A role for thrombomodulin in the pathogenesis of thrombin-induced thromboembolism in mice. Blood 1988; 71: 728-733
• 18 Nawa K, Itani T, Ono M, Sakano K, Marumoto Y, Iwamoto M. The glycosaminoglycan 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
• 19 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
• 20 Mohri M, Oka M, Aoki Y, Gonda Y, Hirata S, Gomi K, Kiyota T, Sugihara T, Yamamoto S, Ishida T, Maruyama I. Intravenous extended infusion of recombinant human soluble thrombomodulin prevented tissue factor-induced disseminated intravascular coagulation in rats. Am J Hematol 1994; 45: 298-303
• 21 Solis MM, Cook J, Glaser C, Light D, Morser J, Yu S, Fink L, Eidt JF. Intravenous recombinant soluble thrombomodulin prevents venous thrombosis in a rat model. J Vascular Surg 1991; 14: 599-604
• 22 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
• 23 Giles AR. Disseminated intravascular coagulation. In: Haemostasis and Thrombosis. vol 2 Bloom AR, Forbes CD, Thomas DP, Tuddenham EG D. eds Churchill Livingstone; Edinburgh, U K: 1994. pp 969-986
• 24 Takano S, Kimura S, Ohdama S, Aoki N. Plasma thrombomodulin in health and diseases. Blood 1990; 76: 2024-2029
• 25 Takahashi H, Ito S, Hanano M, Niwano H, Seki Y, Shibata A. Circulating thrombomodulin as a novel endothelial cell marker: comparison of its behavior with von Willebrand factor and tissue-type plasminogen activator. Am J Hematol 1992; 41: 32-39
• 26 Amano K, Tateyama M, Inaba H, Fukutake K, Fujimaki M. Fluctuations in plasma levels of thrombomodulin in patients with DIC. Thromb Haemost 1992; 68: 404-406
• 27 Shimano H, Takahashi K, Kawakami M, Gotoda T, Harada K, Shimada M, Yazaki Y, Yamada N. Elevated serum and urinary thrombomodulin levels in patient with non-insulin-dependent diabetes mellitus. Clin Chim Acta 1994; 225: 89-96
• 28 Dittman WA, Majerus PW. Structure and function of thrombomodulin: A natural anticoagulant. Blood 1990; 75: 329-336