Monitoring of recombinant hirudin: assessment of a plasma-based ecarin clotting time assay

Abstract

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 50 microliters of ecarin (4 U/ml) to 100 microliters 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 micrograms/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 micrograms/ml and 4-5 micrograms/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 micrograms/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 perfor mable 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.