Dabigatran increases thrombin generation by inhibiting protein S and FVa degradation

Background: Low dose of dabigatran paradoxically increased thrombin generation (TG) through inhibition of protein C (PC) activation. Protein S (PS) is a co-factor in the activation of PC. However, the role of PS in the enhancement of TG has not been addressed. Methods: Firstly, we measured TG by calibrated automated thrombinography (CAT) and prothrombin fragments 1+2 (F 1+2 ) assays. Secondly, we assessed the coagulation and anticoagulation factors activity in normal plasma spiking with dabigatran. Then, free PS and PC activation were measured. Finally, heavy chain of FVa and its degradation products were detected by WB. Results: CAT assay showed that 70-141ng/mL dabigatran paradoxically increased TG in normal plasma. However, dabigatran suppressed TG in a concentration-dependent manner. F 1+2 assay showed the similar results. ELISA assay was not affected by clot methods. Interestingly, results from ELISA showed that dabigatran (2-566ng/mL) suppressed free PS level in normal plasma. Combined with WB results, dabigatran inhibited PS and subsequently suppressed FVa degradation. Conclusions: PS participated in the paradoxical enhancement of TG in normal plasma spiking with low concentrations of dabigatran.