MONITORING OF RECOMBINANT HIRUDIN: ASSESSMENT OF A PLASMA-BASED ECARIN CLOTTING TIME ASSAY

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• 作者：Pö ; tzsch ; Bernd ; Hund ; Simone ; Madlener ; Katharina ; Unkrig ; Christoph ; Mü ; ller-Berghaus ; Gert
• 关键词：r-hirudin ; ecarin clotting time ; systemic anticoagulation
• 刊名：Thrombosis Research
• 出版年：1997
• 出版时间：June 1, 1997
• 年：1997
• 卷：86
• 期：5
• 页码：373-383
• 全文大小：650 K

文摘

Assay conditions of a plasma-based ecarin clotting time (ECT) were evaluated and the precision of the ECT in monitoring plasma levels of r-hirudin assessed. The snake venom enzyme ecarin converts prothrombin to meizothrombin possessing only weak coagulant but strong esterase activity. In r-hirudin containing plasma samples, meizo thrombin is rapidly neutralized by r-hirudin resulting in a dose-dependent prolongation of the clotting times. Among the different assay conditions tested, addition of 20 μl of ecarin (4 U/ml) to 100 μl of undiluted citrate-anticoagulated plasma gave optimal results with respect to precision and reproducibility. The measuring range of the ECT performed in this way is about 0.02 – 5.0 μg/ml r-hirudin. In vitro studies performed on r-hirudin-spiked plasma samples of 50 healthy individuals demonstrate remarkably low interindividual differences in r-hirudin responsiveness as indicated by CV-values below 5 % and 7 % at r-hirudin concentrations between 0 – 3 μg/ml and 4 – 5 μg/ml, respectively. The specificity for r-hirudin of the ECT is further demonstrated by the strong correlation (r=0.94) between the results of a chromogenic assay and the ECT-measured r-hirudin concentrations obtained on 67 ex vivo blood samples. Depending on the concentration of r-hirudin the ECT is sensitive to plasma levels of prothrombin. In the absence of r-hirudin the critical prothrombin concentration was found to be 20 % but increasing to 60 % in the presence of 2.0 μg/ml r-hirudin. A fibrinogen concentration of 50 mg/dl was found to be the minimal concentration independent of the r-hirudin concentration. The precision of the ECT in measuring plasma levels of r-hirudin is not influenced by treatment with heparin, aprotinin or oral anticoagulants. The data demonstrate that the ECT is a rapid and easily performable clotting assay which allows accurate measurement of r-hirudin plasma levels. ECT-monitored hirudin treatment will help to establish optimal dose regimens that are more efficacious but still as safe as heparin. © 1997 Elsevier Science Ltd