Effect of a Selective Thrombin Inhibitor MCI-9038 on Fibrinolysis In Vitro and In Vivo

Summary

The effect of a selective thrombin inhibitor, (2R, 4R)-4-methyl-1- [N²- [(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)sulfo-nyl]-L-arginyl]-2-piperidinecarboxylic acid (MCI-9038), on the fibrinolysis induced by t-PA and u-PA was studied in vitro and in vivo. MCI-9038 remarkably reduced the lysis time of the plasma clot generated by the addition of calcium chloride to the plasma at the concentration ranging from 0.01 to 0.3 μM. Heparin also reduced the plasma clot lysis time with a lower effect than MCI-9038. The fibrin crosslinkage in the plasma clot was inhibited by MCI-9038 or heparin. MCI-9038 potently inhibited the factor XIIIa generation from factor XIII by thrombin.

The effect on the in vivo thrombolysis was studied on the arterial thrombosis generated by the endothelial cell injury of the rabbit carotid artery by acetic acid. t-PA dissolved the thrombi with the infusion at 0.96 mg/kg over 2 h without a significant activation of a systemic fibrinolysis. u-PA dissolved the thrombi with the infusion at 180,000 and 360,000 IU/kg over 2 h. At a dose of 0.48 mg/kg t-PA or 90,000 IU/kg u-PA, the thrombi were not dissolved, but the combined use of MCI-9038 at 1.2 mg/kg over 2 h effectively dissolved the thrombi. Thus, combination of MCI-9038 with plasminogen activators accelerated thrombolysis of an experimental thrombosis in rabbits.

Keywords

Tissue-type plasminogen activator - Urokinase-type plasminogen activator - Thrombin inhibitor MD-805 - Plasma clot lysis - Experimental thrombosis

• References

• 1 Okamoto S, Hijikata A, Kikumoto R, Tonomura S, Hara H, Ninomiya K, Maruyama A, Sugano M, Tamao Y. Potent inhibition of thrombin by the newly synthesized arginine derivatives No. 805. The importance of stereostructure of its hydrophobic carboxamide portion. Biochem Biophys Res Commun 1981; 101: 440-446
• 2 Kikumoto R, Tamao Y, Tezuka T, Tonomura S, Hara H, Ninomiya K, Hijikata A, Okamoto S. Selective inhibition of thrombin by (2R, 4R)-4-methyl-l-[N²-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)sul-fonyl]-L-arginyl]-2-piperidinecarboxylic acid. Biochemistry 1984; 23: 85-90
• 3 Ikoma H, Ohtsu K, Tamao Y, Kikumoto R, Okamoto S. Effect of a potent thrombin inhibitor, MCI-9038, on novel experimental arterial thrombosis. Blood & Vessel 1982; 13: 72-77
• 4 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
• 5 Verheijen JH, Mullaart E, Chang GT G, Kluft C, Wijngaards G. A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thromb Haemostas 1982; 48: 266-269
• 6 Weber K, Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 1969; 244: 4406-4412
• 7 McKee PA, Mattock O, Hill RL. Subunit structure of human fibrinogen, soluble fibrin, and cross-linked insoluble fibrin. Proc Nat Acad Sci USA 1970; 66: 738-744
• 8 Lorand L, Urayama T, DeKiewiet JW C, Nossel HL. Diagnostic and genetic studies on fibrin-stabilizing factor with a new assay based on amine incorporation. J Clin Invest 1969; 48: 1054-1064
• 9 Cleland WW. Statistical analysis of enzyme kinetic data. Methods Enzymol 1979; 63: 103-138
• 10 Ratnoff OD, Menzie C. A new method for the determination of fibrinogen in small samples of plasma. J Lab Clin Med 1951; 37: 316-320
• 11 Matsuo O, Rijken DC, Collen D. Comparison of the relative fibrinogenolytic, fibrinolytic and thrombolytic properties of tissue plasminogen activator and urokinase in vitro. Thromb Haemostas 1981; 45: 225-229
• 12 Blomback B, Hessel B, Hogg D, Therkildsen L. A two-step fibrinogen-fibrin transition in blood coagulation. Nature 1978; 275: 501-505
• 13 Gaffney PJ, Whitaker AN. Fibrin crosslinks and lysis rates. Thromb Res 1979; 14: 85-94
• 14 Sakata Y, Mimuro J, Aoki N. Differential binding of plasminogen to crosslinked and noncrosslinked fibrin: its significance in hemostatic defect in factor XIII deficiency. Blood 1984; 63: 1393-1401
• 15 Sakata Y, Aoki N. Crosslinking of α₂-plasmin inhibitor to fibrin by fibrin-stabilizing factor. J Clin Invest 1980; 65: 290-297
• 16 Francis CW, Markham Jr RE, Marder VJ. Demonstration of in situ fibrin degradation in pathologic thrombi. Blood 1984; 63: 1216-1224
• 17 Collen D, Stassen JM, Marafino Jr BJ, Builder S, De Cock F, Ogez J, Tajiri D, Pennica D, Bennett WF, Salwa J, Hoyng CF. Biological properties of human tissue-typ plasminogen activator obtained by expression of recombinant DNA in mammalian cells. J Pharm Exp Therap 1984; 231: 146-152
• 18 Korninger C, Matsuo O, Suy R, Stassen JM, Collen D. Thrombolysis with human extrinsic (tissue-type) plasminogen activator in dogs with femoral vein thrombosis. J Clin Invest 1982; 69: 573-580
• 19 Gold HK, Fallon JT, Yasuda T, Leinbach RC, Khaw BA, Newell JB, Guerrero JL, Vislosky FM, Hoyng CF, Grossbard E, Collen D. Coronary thrombolysis with recombinant human tissue-type plasminogen activator. Circulation 1984; 70: 700-707