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 (F 1+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. F 1+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.

Fig. 1

Effect of dabigatran on thrombin generation. The effect of various range of dabigatran (0–566 ng/ml) on thrombin generation in normal, protein S-deficient or protein C-deficient plasma. Low concentrations of dabigatran (70 ng/ml) paradoxically enhanced the expression of thrombin generation (a). The phenomenon did not exist in protein S-deficient or protein C-deficient plasma (b and c). Levels of C_max and ETP initially increased at low concentrations of dabigatran and decreased at higher concentrations of dabigatran (d and e). ^∗P < 0.05 vs. respective controls. ETP, endogenous thrombin potential.

Fig. 2

Effect of dabigatran on prothrombin fragments 1+2 (F1+2) assay. Low concentrations of dabigatran dose-dependent increased F₁₊₂ in normal plasma (a). In protein S-deficient or protein C-deficient plasma, the paradoxical enhancement of thrombin generation was absent. F1+2 decreased in a dose-dependent manner as the concentrations of dabigatran increased (b and c). ^∗P < 0.05 vs. respective controls.

Fig. 3

Effect of dabigatran on activated protein C levels. In normal plasma, thrombin generation increased but free protein S and activated protein C decreased (a). In protein S-deficient plasma, we showed that thrombin generation and APC gradually decreased as the concentrations of dabigatran increased (b). ^∗P < 0.05 vs. respective controls. APC, activated protein C.

Fig. 4

Effect of dabigatran on factor Va generation and degradation. (a) The expression of FVa^HC and FVa^307–506 levels after activation of coagulation cascade in normal plasma. (d) Cleaved FVa increased after activation of coagulation cascade. In protein S-deficient plasma, degradation of FVa and generation of FVa^307–506 were inhibited (b and e). In normal plasma spiking with 70 ng/ml dabigatran, generation of FVa was delayed and degradation of FVa inhibited. (c and f) ^∗P < 0.05 vs. respective controls.