Kinetic Studies on the Selectivity of a Synthetic Thrombin-Inhibitor Using Synthetic Peptide Substrates

Summary

The synthetic thrombin-inhibitor termed No. 205 (N-α-dansyl-L-arginine-4-ethyl-piperidine amide) found in our laboratories was studied kinetically using synthetic peptide substrates. The following results were obtained. 1. No. 205 inhibited thrombin competitively with bz-Phe-Val-Arg-pNA and the K_i value obtained was extremely small, 3.7 × 10^-8 M. 2. No. 205 also inhibited trypsin competitively with bz-Phe-Val-Arg-pNA but the K_i value obtained was far larger than that for thrombin, 1.0 × 10^-5 M. 3. No. 205 inhibited F. Xa, plasmin and urokinase only to a small extent when estimated using 2 × 10^-4 M D-Val-Leu- Lys-pNA, bz-Ile-Glu-Gly-Arg-pNA and Glu-Gly-Arg-pNA, respectively. 4. No. 205 differed from APPA in its specific inhibitory spectrum for thrombin as compared to trypsin, plasmin and F. Xa. The above results indicate that No. 205 is an extremely potent and highly selective reversible thrombin-inhibitor.

• References

• 1 Aurell L. 1977; A new sensitive and highly specific chromogenic peptide substrate for factor Xa. Thromb. Res. 11: 595
• 2 Bando M, Matsushima A, Hirano J, Inada Y. 1972; Thrombin-catalyzed conversion of fibrinogen to fibrin. J. Biochem. 71: 897
• 3 Blombäck B. 1967. Fibrinogen to fibrin transformation. In Seegers WH. (ed.) Blood Clotting Enzymology. Academic Press; New York: p 143
• 4 Blombäck B, Blombäck M, Olsson P, Svendsen L, Aberg G. 1969; Synthetic peptides with anticoagulant and vasodilating activity. J. Clin. Lab. Invest. Suppl. 107: 59
• 5 Geratz JD. 1970; Inhibition of thrombin, plasmin and plasminogen activation by amidinocom- pounds. Thrombos. Diathes. haemorrh. (Stuttg.) 23: 486
• 6 Geratz JD, Tidwell RR. 1977. The development of competitive reversible thrombin inhibitors. In Lundblad RL, Fenton II JW, Mann KG. (eds.) Chemistry and Biology of Thrombin. Ann Arbor Science; Michigan: p 179
• 7 Hijikata A, Okamoto S, Mori E, Kinjo K, Kikumoto R, Tonomura S, Tamao Y, Hara H. 1976; In vitro and in vivo studies of a new series of synthetic thrombin-inhibitors (OM-inhibitors). Thromb. Res. 8 Suppl (Suppl. 02) 83
• 8 Kettner C, Shaw E. 1977. The selective inactivation of thrombin by peptides of arginine chloromethyl ketone. In Lundblad RL, Fenton II JW, Mann KG. (eds.) Chemistry and Biology of Thrombin. Ann Arbor Science; Michigan: p 129
• 9 Lineweaver H, Burk D. 1934; The determination of enzyme dissociation constants. J. Amer Chem. Soc. 56: 658
• 10 Markwardt F, Landmann H, Walsmann P. 1968; Comparative studies on the inhibition of trypsin, plasmin and thrombin by derivatives of benzamidine. Eur. J. Biochem. 6: 502
• 11 Nesheim ME, Downing MR, Mann KG. 1977. Approaches to fluorescence studies with thrombin. In Lundblad RL, Fenton II JW, Mann KG. (eds.) Chemistry and Biology of Thrombin. Ann Arbor Science; Michigan: p 233
• 12 Okamoto S, Huikata A, Kinjo K, Kikumoto R, Ohkubo K, Tonomura S, Tamao Y. 1975; A novel series of synthetic thrombin-inhibitors having extremely potent and highly selective action. Kobe J. Med. Sci. 21: 43
• 13 Okamoto S, Huikata A, Ikezawa K, Kinjo K, Kikumoto R, Tonomura S, Tamao Y. 1976; A new series of synthetic thrombin-inhibitors (OM-inhibitors) having extremely potent and selective action. Thromb. Res. 8 Suppl (Suppl. 02) 77
• 14 Sherry S, Troll W. 1954; The action of thrombin on synthetic substrates. J. Biol. Chem. 208
• 15 Sherry S, Alkjaersig N, Fletcher AP. 1965; Comparative activity of thrombin on substituted arginine and lysine esters. Amer. J. Physiol. 209: 571
• 16 Stürzebecher J, Markwardt F, Walsmann P. 1976; Synthetic inhibitors of serine proteinases XIV. Inhibition of factor Xa by derivatives of benzamidine. Thromb. Res. 9: 637
• 17 Svendsen L, Blombäck B, Blombäck M, Olsson PI. 1972; Synthetic chromogenic substrates for determination of trypsin, thrombin and thrombin-like enzymes. Thromb. Res. 1: 267
• 18 Weinstein MJ, Doolittle RF. 1972; The effect of some synthetic arginyl and lysyl compounds on clotting and fibrinolysis. Thrombos. Diathes. haemorrh. (Stuttg.) 28: 289