The Glycosaminoglycan of Recombinant Human Soluble Thrombomodulin Affects Antithrombotic Activity in a Rat Model of Tissue Factor-Induced Disseminated Intravascular Coagulation

Summary

Previous studies on recombinant human soluble thrombomodulin (rsTM) from Chinese hamster ovary cells revealed that rsTM was expressed as two proteins that differed functionally in vitro due to the presence (rsTMp) or absence (rsTMa) of chondroitin-4-sulfate. The current study evaluates the in vivo behavior of rsTM in rats and in a rat model of tissue factor-induced disseminated intravascular coagulation (DIC). rsTMp was more potent than rsTMa for prolongation of the activated partial thromboplastin time (APTT) and their in vivo half-lives determined by ELISA were 20 min for rsTMp and 5.0 h for rsTMa. Injection of a tissue factor suspension (5 mg/kg) resulted in DIC as judged by decreased platelet counts and fibrinogen concentrations, prolonged APTT, and increased fibrin and fibrinogen degradation products (FDP) levels. A bolus injection of either rsTM (0.2 mg/kg) 1 min before induction of DIC essentially neutralized effects on platelets, fibrinogen, and FDP levels, and had only a moderate effect on APTT prolongation. The dose of anticoagulant to inhibit the drop in platelet counts by 50% (ED₅₀) was 0.2 mg/kg rsTMa, 0.07 mg/kg rsTMp, and 7 U/ kg heparin. The effect of increasing concentrations of rsTM and heparin on bleeding times were compared in experiments involving incision of the rat tail. Doubling of the bleeding times occurred at 5 mg/kg rsTMa, 3 mg/kg rsTMp or 90 U/kg heparin. These values represent a 25-fold increase over the ED₅₀ for rsTMa, 43-fold for rsTMp and 13-fold for heparin. These results suggest that rsTMp is a potent anticoagulant to inhibit the platelet reduction when injected prior to the induction of DIC in rats.

• References

• 1 Esmon CT. The roles of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem 1989; 264: 4743-4746
• 2 Dittman WA, Majerus PW. Structure and function of thrombomodulin. Blood 1990; 75: 329-336
• 3 Kisiel W, Canfield WM, Ericsson LH, Davie EW. Anticoagulant properties of bovine plasma protein C following activation by thrombin. Biochemistry 1977; 16: 5824-5831
• 4 Vehar GA, Davie EW. Preparation and properties of bovine factor VIII. Biochemistry 1980; 19: 401-410
• 5 Marlar RA, Kleis AJ, Griffin JH. Mechanism of activated protein C, a thrombin-dependent anticoagulant enzyme. Blood 1982; 59: 1067-1072
• 6 Esmon CT, Esmon NL, Harris KW. Complex formation between thrombin and thrombomodulin inhibits both thrombin catalyzed fibrin formation and factor V activation. J Biol Chem 1982; 257: 7944-7947
• 7 Bourin MC, Ohlin AK, Lane DA, Stenflo J, Lindahl U. Relationship between anticoagulant activities and polyanionic properties of rabbit thrombomodulin. J Biol Chem 1988; 263: 8044-8052
• 8 Preissner KT, Koyama T, Muller D, Tschopp J, Müller-Berghaus G. Domain structure of the endothelial cell receptor thrombomodulin as deduced from modulation of its anticoagulant function. J Biol Chem 1990; 265: 4915-4922
• 9 Bourin MC, Lindahl U. Functional role of the polysaccharide component of rabbit thrombomodulin proteoglycan. Biochem J 1990; 270: 419-425
• 10 Bourin MC, Lundgren-Akerlund E, Lindahl U. Isolation and characterization of the glycosaminoglycan component of rabbit thrombomodulin proteoglycan. J Biol Chem 1990; 265: 15424-15431
• 11 Parkinson JF, Grinnel BW, Moore RE, Hoskins J, Vlahos CJ, Bang NU. Stable expression of a secretable deletion mutant of recombinant human thrombomodulin in mammalian cells. J Biol Chem 1990; 265: 12602-12610
• 12 Nawa K, Sakano K, Fujiwara H, Sato Y, Sugiyama N, Teruuchi T, Iwamoto M, Marumoto Y. Presence and function of chondroitin-4-sulfate on recombinant human soluble thrombomodulin. Biochem Biophys Res Commun 1990; 171: 729-737
• 13 Kumada T, Dittman WA, Majerus PW. A role for thrombomodulin in the pathogenesis of thrombin-induced thromboembolism in mice. Blood 1988; 71: 728-733
• 14 Gomi K, Zushi M, Honda G, Kawahara S, Matsuzaki O, Kanabayasi T, Yamamoto S, Maruyama I, Suzuki K. Antithrombotic effect of recombinant human thrombomodulin on thrombin-induced thromboembolism in mice. Blood 1990; 75: 1396-1399
• 15 Freund M, Cazenave J-P, Courtney M, Degryse E, Roitsch C, Bernat A, Delebasseé D, Defreyn G, Maffrand J-P. Inhibition by recombinant hirudins of experimental venous thrombosis and disseminated intravascular coagulation induced by tissue factor in rats. Thromb Haemostas 1990; 63: 187-192
• 16 Hara H, Tamao Y, Kikumoto R, Okamoto S. Effect of a synthetic thrombin inhibitor MCI-9038 on experimental models of disseminated intravascular coagulation in rabbits. Thromb Haemostas 1987; 57: 165-170
• 17 Marbet GA, Griffith MJ. Tissue thromboplastin induced reversible DIC and heparin-enhanced inhibitors in dogs. Thromb Haemostas 1986; 55: 78-85
• 18 Salem HH, Maruyama I, Ishii H, Majerus PW. Isolation and characterization of thrombomodulin from human placenta. J Biol Chem 1984; 259: 12246-12251
• 19 Kurosawa S, Glavin JB, Esmon NL, Esmon CT. Proteolytic formation and properties of functional domains of thrombomodulin. J Biol Chem 1987; 262: 2206-2212
• 20 Nakane PK. Preparation and standardization of enzyme-labeled conjugates. In: Immunoassays in the Clinical Laboratory. Nakamura RM, Dito WR, Tucker ES. (eds). Alan R Liss Inc; New York: 1975. pp 81-87
• 21 Lowry OH, Rosebrough NJ, Farr AL, Rindall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-275
• 22 Wood KH, Curtis CG, Powell GM, Wusteman FS. The metabolic fate of intravenously injected peptide-bound chondroitin sulfate in the rat. Biochem J 1976; 158: 39-46