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.
Keywords:
Bedside monitoring of hirudin - ecarin clotting time - meizothrombin-induced coagulation
- whole blood determination of hirudin
