RSS-Feed abonnieren
DOI: 10.1055/s-0038-1661631
New Chromogenic Substrates for Thrombin with Increased Specificity
Publikationsverlauf
Received 06. Juni 1985
Accepted 02. Juli 1986
Publikationsdatum:
20. Juli 2018 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/198602/lookinside/thumbnails/10-1055-s-0038-1661631-1.jpg)
Summary
Chromogenic substrates for thrombin with high specificity are necessary for several functional assays, especially for the performance of photometric PT and APTT. A new approach to improve the specificity of chromogenic peptide substrates is made coupling tripeptide sequences selective for thrombin to derivatives of 5-amino-2-nitro benzoic acid (ANBA). Especially when the chromophore’s side chain is substituted by amines or amino acids hydrolysis rates by other enzymes like kallikrein, plasmin or factor Xa are decreased significantly compared to corresponding para-nitroanilides of the same amino acid sequence. On the other hand, most of these compounds are still sensitive thrombin substrates. KM-values for thrombin and other enzymes are in the same order of magnitude as corresponding pNA-peptides. ANBA peptide substrates may be useful to measure thrombin selectively in a mixture of other proteases like plasmin, factor Xa or kallikrein and for the colorimetric determination of PT and APTT.
-
References
- 1 Triplett DA. Chromogenic substrates: A revolution in the diagnostic coagulation laboratory. Clin Lab Ann 1982; 1: 243-287
- 2 Friberger P. Chromogenic substrates. Scand J Clin Lab Invest 1982; 42: 162
- 3 Lijnen HR, Collen D. Synthetic substrates in clinical blood coagulation assays. Verstraete M. (eds.) Martinus Nijhoff Publishers; The Hague - Boston - London: 1980
- 4 Fareed J, Messmore HL, Walenga JM, Bermes Jr EW. Diagnostic efficacy of newer synthetic substrates methods for assessing coagulation variables: A critical overview. Clin Chem 1983; 29: 225-236
- 5 Aiach M, Leon M, Michand A, Caparon L. Adaption of synthetic peptide substrate based assays on a discrete analyser. Semin Thromb Haemostas 1983; 9: 206-216
- 6 Bartl K, Becker U, Lill H. Application of several chromogenic substrate assays to automated instrumentation for coagulation analysis. Semin Thromb Haemostas 1983; 9: 301-320
- 7 Becker U, Jering H, Bartl K, Jilek F. Automated prothrombin time test with use of a chromogenic substrate and a centrifugal analyzer. Clin Chem 1984; 30: 524-528
- 8 Kolde H-J, Fuhge P. Global assays for coagulation function with a specific chromogenic substrate: Photometric determination of the prothrombin time (PT) and the activated partial thromboplastin time (aPTT). Blut 1984; 49: 145-146
- 9 Yamada K, Meguro T. A new APTT assay employing a chromogenic substrate and a centrifugal autoanalyzer. Thromb Res 1979; 15: 351-358
- 10 Heber H, Eberle R, Teetz V. Europaische Patentanmeldung 0110306. 1984
- 11 Fenton JW II, Fasco MJ, Stackrow AB, Aronson DC, Young AM, Finlayson JS. Human thrombins. Production, evaluation, and properties of a-thrombin. J Biol Chem 1977; 252: 3587-3598
- 12 Heber H, Geiger R, Heimburger N. Human plasma kallikrein: Purification, enzyme characterization and quantitative determination in plasma, Hoppe-Seyler’s. Z Physiol Chem 1978; 359: 659-669
- 13 Wiman B, Wallen P. Activation of human plasminogen by an insoluble derivative of urokinase. Eur J Biochem 1973; 36: 25-31
- 14 Farmer DA, Hagemann JH. Use of N-benzoyl-L-tyrosine-thio benzylester as a protease substrate. J Biol Chem 1975; 250: 7366-7371
- 15 Bang NU, Mattler LE. Sensitivity and specificity of plasma serine chromogenic substrates. Haemostasis 1978; 7: 98-104
- 16 Svendsen L, Blomback B, Blomback M, Olsson PJ. Synthetic chromogenic substrates for determination of trypsin, thrombin and thrombin-like enzymes. Thromb Res 1972; 1: 267-278
- 17 McRae BJ, Kurachi K, Heimark RL, Fujikawa K, Davie EW, Powers JC. Mapping the active site of bovine thrombin, factor IXa, factor Xa, factor XIa, factor XIIa, plasma kallikrein and trypsin with amino acid and peptide thioesters: Development of new sensitive substrates. Biochemistry 1981; 20: 7196-7206
- 18 Cho K, Tanaka T, Cook RC, Kisiel W, Fujikawa K, Kurachi K, Powers JC. Active-site mapping of bovine and human blood coagulation serine proteases using synthetic peptide 4-nitroanilide and thio ester substrates. Biochemistry 1984; 23: 644-650
- 19 Morita T, Kato H, Iwanaga S, Takada K, Kimura T, Sakakibara S. New fluorogenic substrates for a-thrombin, factor Xa, kallikreins and urokinase. J Biochem (Tokyo) 1977; 82: 1495-1498
- 20 Leytus SP, Patterson WL, Mangel WF. New class of sensitive and selective fluorogenic substrates for serine proteinases. Biochem J 1983; 215: 253-260
- 21 Castillo MJ, Kurachi K, Nishino N, Otikubo J, Power JC. Reactivity of bovine blood coagulation factor IXap, factor Xap and factor XIa toward fluorogenic peptides containing the activation site sequences of bovine factor IX and factor X. Biochemistry 1983; 22: 1021-1029
- 22 Lottenberg R, Hell JA, Bluider M, Binder EP, Jackson CM. The action of thrombin on peptide p-Nitroanilide substrates: Substrate selectivity and examination of hydrolysis under different reaction conditions. Biochim Biophys Acta 1983; 742: 539-557
- 23 Lottenberg R, Hall JA, Fenton JW II, Jackson CM. The action of thrombin on peptide p-nitroanilide substrates: Hydrolysis of Tos-Gly-Pro-Arg-pNA and D-Phe-Pip-Arg-pNA by human a- and y- and bovine a- and P-thrombin. Thromb Res 1982; 28: 313-312
- 24 Karges HE, Heber H, Heimburger N. Neue chromogene Substrate zur Bestimmung von Gerinnungsenzymen und anderen Serin-proteasen. Blut 1980; 40: 37