Protein S contributes to the paradoxical increase in thrombin generation by low-dose dabigatran in the presence of thrombomodulin

Abstract

Low dose of dabigatran paradoxically increased thrombin generation through inhibition of protein C activation. Protein S is a co-factor in the activation of protein C. However, the role of protein S in the enhancement of thrombin generation has not been addressed. Firstly, we measured thrombin generation by calibrated automated thrombinography (CAT) and prothrombin fragments 1+2 (F1+2) assays. Secondly, we assessed activated protein C (APC) formation in normal or protein S-deficient plasma spiking with dabigatran. Then, protein C activation was measured. Finally, heavy chain of factor Va (FVa) and its degradation products were detected by western blot. CAT assay showed that 70–141 ng/ml dabigatran paradoxically increased thrombin generation in normal plasma. However, higher concentrations of dabigatran (283 ng/ml) suppressed the level of ETP. F1+2 assay showed the similar results. In protein S-deficient or protein C-deficient plasma, the paradoxical increase in thrombin generation was absent. Level of generated APC was to a similar extent inhibited by dabigatran in normal and protein S-deficient plasma. Low-dose dabigatran inhibited the protein S-dependent inactivation of factor Va. Protein S participated in the paradoxical enhancement of thrombin generation in normal plasma spiking with low concentrations of dabigatran. Increased thrombin generation at low dabigatran can be explained by reduced thrombin–thrombomodulin mediated APC formation and subsequent reduced FVa inactivation that is protein S-dependent.