Quantitative Determination of Hirudin in Blood and Body Fluids

 

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Abstract

Recent clinical studies using hirudin as anticoagulant have demonstrated that an efficient method to determine the current blood level of hirudin is imperative for exact dose finding and adjustment. Only the exact determination of the hirudin content in blood, performed within a few minutes, prevents overdosage involving side effects or, otherwise, a subtherapeutic dose regimen. Therefore, a method for rapid, sensitive, and reproducible measurement of hirudin in blood, plasma, and other body fluids has been developed. The method, which is based on coagulation measurement, is called ecarin clotting time (ECT). In this test, ecarin, a purified enzyme of the Echis carinatus snake venom, acts as a prothrombin activator. In contrast to the “solid phase” prothrombin activation by prothrombinase, the ecarin-induced prothrombin activation proceeds in an alternative way, i.e., without the need of cofactors, resulting in intermediates such as meizothrombin. Compared to thrombin, meizothrombin has a lower procoagulant activity, but it still binds hirudin, which leads to the inhibition of meizothrombin. Depending on the sample's concentration of hirudin, ecarin forms a residual, nonhirudin-bound amount of intermediates of the prothrombin-thrombin conversion that are able to concentration-dependently convert fibrinogen to fibrin. There is an excellent linear correlation between ECT prolongation and the hirudin content of the sample in a range from 50 to 5,000 ng/mL blood or plasma. This allows immediate measurement not only of the therapeutic blood level of hirudin, but also of its concentration in blood following under- or overdosage. The ECT method is nearly independent of variations in the sample's content of fibrinogen (from 60% to 100%) and prothrombin (from 20% to 100%). Heparin is not able to catalyze the very low antithrombin inhibition of meizothrombin. Therefore, it is also possible to determine hirudin in blood containing varying amounts of heparin. Another advantage of the method is that it can be applied to different mechanical measuring systems used in coagulation diagnostics.