Ecarin Clotting Time: a Predictive Coagulation Assay for the Antithrombotic Activity of Argatroban in the Rat

Summary

We studied the use of the Ecarin Clotting Time (ECT) as a predictive assay of the antithrombotic effects of argatroban in a new tissue factor-dependent model of venous thrombosis and a model of arterial thrombosis in the rat. Heparin was used as a reference anticoagulant.

Infusions of argatroban dose-dependently increased the ECT across the range of doses required for antithrombotic activity in models of venous and arterial thrombosis (1.25-40 μg/kg/min). The TT was only useful as a marker in the case of venous thrombosis, since, in the arterial thrombosis model, the clotting times were >200 s in the majority of animals receiving antithrombotic doses. The aPTT is not sufficiently sensitive to be predictive of an antithrombotic effect in the venous model, and shows only modest increases in the arterial thrombosis model. Heparin did not significantly increase the ECT at antithrombotic doses in the venous thrombosis model, and only increased the ECT by 53% at 40 μg/kg/min in the arterial model, despite a marked antithrombotic effect. Both the TT and aPTT were dose-dependently increased by heparin at doses active in the venous model, whereas both parameters were >200 s at doses active in the arterial thrombosis model.

Thus, the ECT provides a predictive marker for the antithrombotic activity of argatroban in both venous and arterial thrombosis, at least in the rat.

• References

• 1 Berry CN, Girard D, Lochot S, Lecoffre C. Antithrombotic actions of argatroban in rat models of venous, ‘mixed’ and arterial thrombosis, and its effects on the tail transection bleeding time.. Br J Pharmacol 1994; 113: 1209-14.
• 2 Carteaux J-P, Gast A, Tschopp T, Roux S. Activated clotting time as an appropriate test to compare heparin and direct thrombin inhibitors such as hirudin or Ro46-6240 in experimental arterial thrombosis.. Circulation 1995; 91: 1568-74.
• 3 Peters RF, Lees CM, Mitchell KA, Tweed MF, Talbot MD, Wallis RB. The characterisation of thrombus development in an improved model of arteriovenous shunt thrombosis in the rat and the effects of recombinant desulphatohirudin (CGP 39393), heparin, and iloprost.. Thromb Haemost 1991; 65: 268-74.
• 4 Bucha E, Nowak G. The Ecarin Coagulation Time (ECT) the first specific method suited for quick determination of direct thrombin inhibitors in whole blood, plasma and body fluids.. Int Angiology 1995; 14 (Suppl. 01) 174.
• 5 Kornalik F, Blombäck B. Prothrombin activation induced by ecarin – a pro thrombin converting enzyme from Echis carinatus venom.. Thromb Res 1975; 6: 53-63.
• 6 Callas DD, Fareed J. Comparative anticoagulant effects of various thrombin inhibitors, as determined in the ecarin clotting time method.. Thromb Res 1996; 83: 463-8.
• 7 Kikumoto R, Tamao Y, Tezuka T, Tonomura S, Hara H, Ninomiya K, Hijikata A, Okamoto S. Selective inhibition of thrombin by (2R4R)-4-methyl-1-[N²-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)sulfonyl]-L-Arginyl)]-2-piperidinecarboxylic acid.. Biochemistry 1984; 23: 85-90.
• 8 Okamoto S, Hijikata A, Kikumoto R, Tonomara S, Hara H, Ninomiya K, Maruyama A, Sugano M, Tamao Y. Potent Inhibition of Thrombin by the Newly Synthesized Arginine Derivative No. 805. The Importance of Stereo-Structure of its Hydrophobic Carboxamide Portion.. Biochem Biophys Res Commun 1981; 101: 440-6.
• 9 Tamao Y, Yamamoto T, Kikumoto R, Hara H, Itoh J, Hirata T, Mineo K, Okamoto S. Effect of a selective thrombin inhibitor MCI-9038 on fibrinolysis in vitro and in vivo.. Thromb Haemost 1986; 56: 28-34.
• 10 Imura Y, Stassen J-M, Collen D. Comparative Antithrombotic Effects of Heparin, Recombinant Hirudin and Argatroban in a Hamster Femoral Vein Platelet-Rich Mural Thrombosis Model.. J Pharm Exp Ther 1992; 261: 895-8.
• 11 Jang IK, Gold HK, Ziskind AA, Leinbach RC, Fallon JT, Collen D. Prevention of Platelet-Rich Arterial Thrombosis by Selective Thrombin Inhibition.. Circulation 1990; 81: 219-25.
• 12 Fujioka K. The Effect of MD-805 on Experimental Acute Arterial Thrombosis in Relation to Vasoactive Prostanoids (TXA₂ and PGI₂).. J Jap Coll Angiol 1989; 29: 57-64.
• 13 Eidt JF, Allison P, Noble S, Ashton J, Golino P, Mcnatt J, Buja LM, Willerson JT. Thrombin is an Important Mediator of Platelet Aggregation in Stenosed Canine Coronary Arteries with Endothelial Injury.. J Clin Invest 1989; 84: 18-27.
• 14 Duval N, Lunven C, O’Brien DP, Grosset A, O’Connor SE, Berry CN. Antithrombotic actions of the thrombin inhibitor, argatroban, in a canine model of coronary cyclic flow: comparison with heparin.. Br J Pharmacol 1996; 118: 727-33.
• 15 Schumacher WA, Heran CL, Steinbacher TE. Low molecular weight heparin (Fragmin) and thrombin active site inhibitor (Argatroban) compared in experimental arterial thrombosis and bleeding time.. J Cardiovascular Pharmacol 1996; 28: 19-25.
• 16 Hladovec J. Experimental thrombosis in rats with continuous registration.. Thromb Haemost 1971; 26: 407-10.
• 17 Berry CN, Lunven C, O’Brien DP, Girardot C, Girard D, Charles MC, Ferrari P. Ecarin clotting time: a predictive marker for the antithrombotic effects of argatroban in rat venous and arterial thrombosis.. Thromb Haemost. 1998 77. (Suppl) 92.
• 18 Béguin S, Lindhout T, Hemker C. The mode of action of heparin in plasma.. Thromb Haemost 1988; 60: 457-62.
• 19 GUSTO IIa investigators.. Randomized trial of intravenous heparin versus recombinant hirudin for acute coronary syndromes.. Circulation 1994; 90: 1631-7.
• 20 Verstraete M, Nurmohamed M, Kienast J, Siebeck M, Silling-Engel-hardt G, Büller H, Hoet B, Bichler J, Close P. Biological effects of recombinant hirudin (CGP 39393) in human volunteers.. JACC 1993; 22: 1080-8.
• 21 Marbert GA, Verstraete M, Kienast J, Graf P, Hoet B, Tsakiris DA, Silling-Engel-hardt G, Close P. Clinical pharmacology of intravenously administered recombinant desulfatohirudin (CGP 39393) in healthy volunteers.. J Cardiovasc Pharm 1993; 22: 364-72.
• 22 Nurmohamed MT, Berckmans RJ, Morrien-Salomons WM, Berends F, Hammes WD, Rijnierse JJMM, Sturk A. Monitoring anticoagulant therapy by activated partial thromboplastin time: Hirudin assessment.. Thromb Haemost 1994; 72: 685-92.
• 23 Schwarz RP, Becker JCP, Brooks RL, Joffrion JL, Knappenberger GD, Kogan TP, Kogan PW, McKinney AA. The preclinical and clinical pharmacology of Novastan (argatroban): a small-molecule, direct thrombin inhibitor.. J Clin Appl Thromb Hemost. 1998 (in press).
• 24 Pötzsch B, Madlener K, Seelig C, Riess CF, Greinacher A, Müller-Berghaus G. Monitoring of r-hirudin anticoagulation during cardiopulmo-nary bypass – Assessment of the whole blood ecarin clotting time.. Thromb Haemost 1998; 77: 920-5.
• 25 Mize PD, Ruebsaman K. Dry-chemistry ecarin clotting time for ex vivo monitoring of thrombin inhibitors.. Thromb Haemost. 1998 77. (Suppl.) 436.
• 26 Lewis BE, Iqbal O, Hoppensteadt D, Ahsan A, Ahmad S, Messmore HL, Schwarz RP, Walenga JM. Clinical pharmacokinetic and pharmacodynamic studies on argatroban to optimize the anticoagulant dosage in interventional cardiovascular procedures.. Thromb Haemost 1998; 77 (Suppl.) 492.
• 27 Morita T, Iwanaga S, Suzuki T. The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venom and characterization of the new active intermediates.. J Biochem 1976; 79: 1089-108.
• 28 Verstraete M. Pharmacotherapeutic aspects of unfractionated and low molecular weight heparins.. Drugs 1990; 40: 498-530.
• 29 Stocker K, Fischer H, Brogli M. Chromogenic assay for the prothrombin activator ecarin from the venom of the saw-scaled viper (Echis carinatus).. Toxicon 1986; 24: 81-9.