Summary
A number of direct thrombin inhibitors are successfully used clinically and experimentally
as novel antithrombotics and specific anticoagulants. They are also used as anticoagulants
in certain blood collection tubes for the analysis of platelet function. A series
of platelet function tests have emerged to measure adequate responses to antiplatelet
therapy. For comparative and practical reasons, it would be of advantage to use the
same anticoagulant in blood collection tubes for different methods, e.g. thrombin
inhibitors. However, there are little data on the effects of thrombin inhibitors on
platelet signalling pathways that could influence results. We examined the applicability
of thrombin inhibitor containing blood for platelet reactivity index (PRI) measurements
of the VASP assay and investigated the effects of two thrombin inhibitors (hirudin
and lepirudin) on cAMP- and cGMP-mediated signalling pathways in washed human platelets.
We show that induction of VASP phosphorylation by PGE1 is markedly reduced in lepirudin
containing blood samples. In consequence, PRI levels were highly variable compared
to routinely used citrated blood. Surprisingly, in vitro incubation of platelets with thrombin inhibitors increases platelet cGMP levels and
induces NOS independent sGC/PKG-mediated VASP phosphorylation. We conclude that thrombin
inhibitors activate sGC/PKG-dependent pathways resulting in an increase of VASP phosphorylation
which contributes to deviations in PRI measurements. These effects of thrombin inhibitors
on sGC- and cGMP-mediated pathways including increased VASP phosphorylation may indicate
the presence of an important additional platelet-based mechanism for the reduction
of thrombus formation and thromboembolism by thrombin inhibitors.
Keywords
VASP - PRI - lepirudin - hirudin - sGC/ PKG/ cGMP pathway
