Monitoring of r-Hirudin Anticoagulation during Cardiopulmonary Bypass – Assessment of the Whole Blood Ecarin Clotting Time

Summary

The use of recombinant ® hirudin as an anticoagulant in performing extracorporeal circulation systems including cardiopulmonary bypass (CPB) devices requires a specific and easy to handle monitoring system. The usefulness of the celite-induced activated clotting time (ACT) and the activated partial thromboplastin time (APTT) for r-hirudin monitoring has been tested on ex vivo blood samples obtained from eight patients treated with r-hirudin during open heart surgery. The very poor relationship between the prolongation of the ACT and APTT values and the concentration of r-hirudin as measured using a chromogenic factor Ila assay indicates that both assays are not suitable to monitor r-hirudin anticoagulation. As an alternative approach a whole blood clotting assay based on the prothrombin-activating snake venom ecarin has been tested. In vitro experiments using r-hirudin- spiked whole blood samples showed a linear relationship between the concentration of hirudin added and the prolongation of the clotting times up to a concentration of r-hirudin of 4.0 µg/ml. Interassay coefficients (CV) of variation between 2.1% and 5.4% demonstrate the accuracy of the ecarin clotting time (ECT) assay. Differences in the interindividual responsiveness to r-hirudin were analyzed on r-hirudin- spiked blood samples obtained from 50 healthy blood donors. CV- values between 1.8% and 6% measured at r-hirudin concentrations between 0.5 and 4 µg/ml indicate remarkably slight differences in r-hirudin responsiveness. ECT assay results of the ex vivo blood samples linearily correlate (r = 0.79) to the concentration of r-hirudin. Moreover, assay results were not influenced by treatment with aprotinin or heparin. These findings together with the short measuring time with less than 120 seconds warrant the whole blood ECT to be a suitable assay for monitoring of r-hirudin anticoagulation in cardiac surgery.

• References

• 1 Markwardt F. The development of hirudin as an antithrombotic drug. Thromb Res 1994; 74: 01-23
• 2 Glusa E, Markwardt F. Platelet functions in recombinant hirudin-anticoagulated blood. Hemostasis 1990; 20: 112-118
• 3 van den Bos AA, Deckers JW, Heyndrickx GR, Laarman G-J, Surya-pranata H, Zijlstra F, Close P, Rijnierse JJ MM, Buller HR, Serruys PW. Safety and efficacy of recombinant hirudin (CGP 39 393) versus heparin in patients with stable angina undergoing coronary angioplasty. Circulation 1993; 88: 2058-2066
• 4 Zolhelyi P, Webster MWI, Fuster V, Grill DE, Gaspar D, Edwards SJ, Cabot CF, Chesebro JH. Recombinant hirudin in patients with chronic, stable coronary artery disease – Safety, half-life, and effect on coagulation parameters. Circulation 1993; 88: 2015-2022
• 5 Flather M. Recombinant hirudin in the treatment of patients with unstable angina pectoris: Preliminary results of the oasis pilot study. [Abstract] Annals Hematol 1996; 72: A92
• 6 Greinacher A, Völpel H, Pötzsch B. Recombinant hirudin in the treatment of patients with heparin-associated thrombocytopenia type II (HAT). [Abstract] Annals Hematol 1996; 72: A92
• 7 Pötzsch B, Greinacher A, Riess F-C, Madlener K, Völpel H, MüllerBerghaus G. Recombinant hirudin as anticoagulant in cardiac surgery: Experiencies with eleven patients. [Abstract] Annals Hematol 1996; 72: A4
• 8 Riess F-C, Löwer C, Seelig C, Bleese N, Kormann J, Müller-Berghaus G, Pötzsch B. Recombinant hirudin as a new anticoagulant during cardiac operations instead of heparin: Successful for aortic valve replacement in man. J Thorac Cardiovasc Surg 1995; 110: 265-267
• 9 Riess F-C, Pötzsch B, Bader R, Bleese N, Greinacher A, Löwer C, Madlener K, Müller-Berghaus G. A case report on the use of recombinant hirudin as an anticoagulant for cardiopulmonary bypass in open heart surgery. Eur J Cardio-thorac Surg 1996; 10: 386-388
• 10 Koza MJ, Walenga JM, Fareed J, Pifarre R. A new approach in monitoring recombinant hirudin during cardiopulmonary bypass. Semin Thromb Hemostas 1993; 19: 90-96
• 11 Verstraete M, Nurmohamed M, Kienast J, Siebeck M, Silling-Engelhardt G, Büller H, Hoet B, Bichler J, Close P. Biological effects of recombinant hirudin (CGP 39393) in human volunteers. JACC 1993; 22: 1080-1180
• 12 Marbet GA, Verstraete M, Kienast J, Graf P, Hoet B, Tsakiris DA, Silling-Engelhardt G, Close P. Clinical pharmacology of intravenously administered recombinant desulfatohirudin (CGP 39393) in healthy volunteers. J Cardiovasc Pharm 1993; 22: 364-372
• 13 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
• 14 Walenga JM, Bakhos M, Messmore HL, Koza M, Wallock M, Orfei E, Fareed J, Pifarre R. Comparison of recombinant hirudin and heparin as an anticoagulant in a cardiopulmonary bypass model. Blood Coagulation and Fibrinolysis 1991; 2: 105-111
• 15 Terrell MR, Walenga JM, Koza MJ, Pifarré R. Efficacy of aprotinin with various anticoagulant agents in cardiopulmonary bypass. Ann Thorac Surg 1996; 62: 506-511
• 16 Nowak G, Bucha E. A new method for the therapeutical monitoring of hirudin. [Abstract] Thromb Haemost 1993; 69: 1306
• 17 Nishida S, Fujita T, Kohno N, Atoda H, Morita T, Takeya H, Kido I, Paine MJI, Kawabata S-I, Iwanaga S. cDNA cloning and deduced amino acid sequence of prothrombin activator (ecarin) from kenyan echis carinatus venom. Biochemistry 1995; 34: 1771-8
• 18 Boskovic DS, Giles AR, Nesheim ME. Studies of the role of factor Va in the factor Xa-catalyzed activation of prothrombin, fragment 1.2-prethrombin-2, and dansyl-L-glutamyl-glycyl-L-arginine-meizothrombin in the absence of phospholipid. JBC 1990; 265: 10497-10505
• 19 Novoa E, Seegers WH. Mechanisms of alpha-thrombin and beta-thrombin- E formation: use of ecarin for isolation of meizothrombin 1. Thromb Res 1980; 18: 657-668
• 20 Hafner G, Fickenscher K, Friesen H-J, Rupprecht H-J, Konheiser U, Ehrental W, Lotz J. Prellwitz. Evaluation of an automated chromogenic substrate assay for the rapid determination of hirudin in plasma. Thrombosis Research 1995; 77: 165-173
• 21 Solano C, Cobcroft G, Scott DC. Prediction of vitamin K response using the echis time and echis-prothrombin time ratio. Thromb Haemost 1990; 64: 353-367