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Original Paper

Ecarin Chromogenic Assay – A New Method for Quantitative Determination of Direct Thrombin Inhibitors Like Hirudin

Lange U.a · Nowak G.b · Bucha E.a

Author affiliations

aHaemoSys GmbH and bResearch Unit ‘Pharmacological Hemostaseology’, Medical Faculty, Friedrich Schiller University, Jena, Germany

Corresponding Author

Dr. Ute Lange

HaemoSys GmbH

Winzerlaer Strasse 2

DE–07745 Jena (Germany)

Tel. +49 36 41 50 83 21, Fax +49 36 41 50 83 01, E-Mail u.lange@haemosys.de

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Pathophysiol Haemost Thromb 2003;33:202–205

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A new sensitive and precise method for quantitative determination of direct thrombin inhibitors is described, the ecarin chromogenic assay (ECA). Ecarin is used as the specific prothrombin-activating principle. The cleavage of a chromogenic substrate by meizothrombin is inhibited in a concentration-dependent fashion by direct thrombin inhibitors. For the ECA, the linear measuring range is about 0.1–3.0 µg hirudin/ml plasma. Coefficients of variations between 2.3 and 4% over the whole concentration range were achieved. The ECA has proved to be more sensitive than the compared tests (ecarin clotting time and a thrombin-based chromogenic assay); a detection limit of 0.011 µg hirudin/ml and a quantitation limit of 0.032 µg hirudin/ml were calculated. The ECA is independent of the variations of the coagulation variables fibrinogen and prothrombin. Neither heparin nor oral anticoagulants interfere with the ECA.

© 2004 S. Karger AG, Basel


  1. Tripodi A, Chantarangkul V, Arbini AA, Moia M, Mannucci PM: Effects of hirudin on activated partial thromboplastin time determined with ten different reagents. Thromb Haemost 1993;70:286–288.
  2. Nurmohamed MT, Berckmans RJ, Morrien-Salomons WM, Berends F, Hommes DW, Rijnierse JJ, Sturk A: Monitoring anticoagulant therapy by activated partial thromboplastin time: Hirudin assessment. An evaluation of native blood and plasma assays. Thromb Haemost 1994;72:685–692.
  3. Cullberg M, Eriksson UG, Larsson M, Karlsson MO: Population modelling of the effect of inogatran, a thrombin inhibitor, on ex vivo coagulation time (APTT) in healthy subjects and in patients with coronary artery disease. Br J Clin Pharmacol 2001;51:71–79.
  4. Despotis GJ, Hogue CW, Saleem R, Bigham M, Skubas N, Apostolidou I, Qayum A, Joist JH: The relationship between hirudin and activated clotting time: Implications for patients with heparin-induced thrombocytopenia undergoing cardiac surgery. Anesth Analg 2001;93:28–32.
  5. Reid TJ 3rd, Alving BM: A quantitative thrombin time for determining levels of hirudin and hirulog. Thromb Haemost 1993;70:608–616.
  6. Spinner S, Stoffler G, Fink E: Quantitative enzyme-linked immunosorbent assay (ELISA) for hirudin. J Immunol Methods 1986;87:79–83.
  7. Spinner S, Scheffauer F, Maschler R, Stoffler G: A hirudin catching ELISA for quantitating the anticoagulant in biological fluids. Thromb Res 1988;51:617–625.
  8. Iyer L, Adam M, Amiral J, Fareed J, Bermes E Jr: Development and validation of two enzyme-linked immunosorbent assay (ELISA) methods for recombinant hirudin. Semin Thromb Hemost 1995;21:184–192.
  9. Griessbach U, Stürzebecher J, Markwardt F: Assay of hirudin in plasma using a chromogenic thrombin substrate. Thromb Res 1985;37:347–350.
  10. Stürzebecher J: Methods for determination of hirudin. Semin Thromb Hemost 1991;17:99–102.
  11. Spannagl M, Bichler J, Birg A, Lill H, Schramm W: Development of a chromogenic substrate assay for the determination of hirudin in plasma. Blood Coagul Fibrinolysis 1991;2:121–127.
  12. Groetsch H, Damm D, Ben Youssef R, Haertel D: Comparison of two different methods for the determination of rDNA-hirudin in plasma samples: HPLC vs a chromogenic thrombin substrate. Thromb Res 1991;64:273–277.
  13. Hafner G, Fickenscher K, Friesen HJ, Rupprecht HJ, Konheiser U, Ehrenthal W, Lotz J, Prellwitz W: Evaluation of an automated chromogenic substrate assay for the rapid determination of hirudin in plasma. Thromb Res 1995;77:165–173.
  14. Calatzis A, Spannagl M, Gempeler-Messina P, Kolde HJ, Schramm W, Haas S: The prothrombinase-induced clotting test: A new technique for the monitoring of anticoagulants. Haemostasis 2000;30(suppl 2):172–174.
    External Resources
  15. Nowak G, Bucha E: A new method for the therapeutic monitoring of hirudin. Thromb Haemost 1993;69:306–310.
    External Resources
  16. Nowak G: Monitoring of the action of antithrombin agents by ecarin clotting time; in Pifarré R (ed): New Anticoagulants for the Cardiovascular Patient. Philadelphia, Hanley & Belfus, 1997, pp 539–550.
  17. Nowak G: Clinical monitoring of hirudin and direct thrombin inhibitors. Semin Thromb Hemost 2001;27:537–541.
  18. Hafner G, Roser M, Nauck M: Methods for the monitoring of direct thrombin inhibitors. Semin Thromb Hemost 2002;28:425–430.
  19. Ratnoff OD, Menzie C: A new method for the determination of fibrinogen in small samples of plasma. J Lab Clin Med 1954;37:316–320.
  20. Nowak G, Bucha E: Quantitative determination of hirudin in blood and body fluids. Semin Thromb Hemost 1996;22:197–202.
  21. Morita T, Iwanaga S, Suzuki T: The mechanism of action of bovine prothrombin by an activator isolated from Echis carinatus venom and characterization of the new active intermediates. J Biochem (Tokyo) 1976;79:1089–1108.
  22. Rhee MJ, Morris S, Kosow DP: Role of meizothrombin and meizothrombin-(des F1) in the conversion of prothrombin to thrombin by the Echis carinatus venom coagulant. Biochemistry 1982;21:3437–3443.
  23. Cote HC, Stevens WK, Bajzar L, Banfield DK, Nesheim ME, MacGillivray RT: Characterization of a stable form of human meizothrombin derived from recombinant prothrombin (R155A, R271A, and R284A). J Biol Chem 1994;269:11374–11380.
  24. Schoen P, Lindhout T: The in situ inhibition of prothrombinase-formed human alpha-thrombin and meizothrombin (des F1) by antithrombin III and heparin. J Biol Chem 1987;262:11268–11274.
  25. Lindhoff-Last E, Piechottka GP, Rabe F, Bauersachs R: Hirudin determination in plasma can be strongly influenced by the prothrombin level. Thromb Res 2000;100:55–60.
  26. Lange U, Wiesenburg A, Olschewski A, Nowak G, Bucha E: Quantitative determination of direct thrombin inhibitors using the ecarin chromogenic assay (ECA) – both POCT and automated method. Ann Hematol 2003;82 (suppl 1):S53.
  27. Kathiresan S, Shiomura J, Jang IK: Argatroban. J Thromb Thrombolysis 2002;13:41–47.
  28. Fenyvesi T, Jörg I, Harenberg J: Monitoring of anticoagulant effects of direct thrombin inhibitors. Semin Thromb Hemost 2002;28:361–368.

Article / Publication Details

First-Page Preview
Abstract of Original Paper

Received: July 26, 2004
Accepted: September 14, 2004
Published online: December 02, 2004
Issue release date: July – August

Number of Print Pages: 8
Number of Figures: 7
Number of Tables: 2

ISSN: 1424-8832 (Print)
eISSN: 1424-8840 (Online)

For additional information: https://www.karger.com/PHT

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