This study showed that PS participated in the paradoxical enhancement of TG in plasma spiking with low concentrations of dabigatran. Dabigatran increased thrombin generation by inhibiting protein S and FVa degradation. Previous study demonstrated that low concentrations of dabigatran paradoxically increased TG in PC-dependent manner . Dabigatran inhibited thrombomodulin-bound thrombin more aggressively than thrombin at low concentrations resulting in the decrease of PC activation and FVa degradation. Our previous study speculated that higher concentrations of dabigatran may sufficiently suppress thrombin and coagulation cascade . PS is a cofactor to activated protein C. PS could cleave FVa and FVIIIa in PC dependent and independent manners . The effect of PS on the paradoxical enhancement of TG has not been well addressed.
It is known that a transient hypercoagulable state occurs within the first 12-60h of warfarin treatment because of inhibition of PC and PS . Previous study showed that low concentrations of dabigatran paradoxically increased TG. We aimed to disclosure whether the paradoxical enhancement of TG induced by low concentrations of dabigatran depends on PS. PS is a vitamin K-dependent glycoprotein that plays vital role in blood coagulation. PS circulates in plasma at a concentration of around 350nM which exists both in a free form (≈ 140nM) and in an inactive form (~ 60%) combined with the complement regulatory factor C4b-binding protein (C4BP) . Only the free PS exerts co-factor activity. Hence, the detection of free PS but not total PS was applied in the study .
Firstly, TG was assessed by CAT assay in normal, PS- or PC-deficient plasma containing 10nM rhs-TM. It has been previously shown that 10nM rhs-TM added to the plasma was appropriate to mimic the condition in vivo . Blood coagulation cascade includes two pathways, one of which is contact factor-dependent intrinsic pathway and the other is TF-induced extrinsic pathway. In most cases, TG was assessed in coagulation reaction induced by extrinsic pathway. In our study, final concentration of tissue factor, phospholipids, fluorogenic substrate, and CaCl2 were 5pM, 4µM, 417µM, and 16.7mM. Under these circumstances, coagulation cascade process could be induced completely. Wagenvoord et al.  reported that the paradoxical enhancement of TG induced by low concentrations of dabigatran may be caused by the presence of α2-macroglobulin-thrombin (α2MT) complex. CAT assay uses an algorithm to subtract α2MT activity from the total amidolytic activity. The transient enhancement of α2MT induced by dabigatran could not be subtracted which leads to a false increase of TG. However, there is study showing different opinion. Study reported α2M may not participate in the enhancement of TG . CAT assay showed that low concentrations of dabigatran paradoxically induced TG in normal plasma. Higher concentrations of dabigatran may sufficiently suppress thrombin and coagulation cascade.
Secondly, TG was assessed by F1 + 2 assay under the same circumstance. F1 + 2 is also a marker of TG. Our results showed that F1 + 2 increased in normal plasma spiking with 70ng/mL dabigatran. The paradoxical enhancement of F1 + 2 was absent in PS deficient plasma. The results are inconsistent between assays. However, the trend of CAT and F1 + 2 assays is same. Previous study showed that TG increased at 136-545ng/ml dabigatran. But only 273ng/ml dabigatran increased F1 + 2 . Our results are similar to other study.
Furthermore, FV, FVIII, PS and PC activity was measured to assess the haemostatic situation in clotting blood using the same batches of reagents. Previous study showed different batches of reagents and different coagulometers could lead to highly variable results . Dabigatran dose-dependent decreased FV and FVIII activity and increased PS and PC activity. It has been reported dabigatran would affect APTT more than PT. FV activity is measured by normalization of PT. Whereas FVIII and PC activity is associated with APTT. Hence, FVIII and PC activity would be more affected than FV. The activity of coagulation and anticoagulation factors using clot methods may be adversely affected by dabigatran . To avoid the influence by dabigatran, free PS and aPC-PCI ELISA were used. It is well known that dabigatran is a direct thrombin inhibitor. Interestingly, dabigatran could inhibit free PS levels. In normal plasma, the concentrations of dabigatran for inhibition of free PS and PC activation were almost the same as for enhancement of TG. Except for PC, participates in the paradoxical enhancement of TG in plasma spiking with low concentrations of dabigatran.
Last but not least, we used WB assay to evaluate the generation and degradation of FVa. In normal plasma, FVa307–506 increased in line with the decrease of FVaHC. In PS-deficient plasma, the degradation of FVa was absolutely suppressed. PS plays an important role in the degradation of FVa. In normal plasma spiking with 70ng/mL dabigatran, the generation of FVa was not affected. However, the degradation of FVa was inhibited. We speculated low concentrations of dabigatran suppressed PS and APC more than coagulation cascade leading the paradoxical enhancement of TG.
This study still has some limitations. In one hand, most parts of our results are based on CAT assay. It has been reported α2MT would affect the results. What is more, there may be some confounding factors affect the results. In the other hand, this study performed in vitro. Although the concentrations of dabigatran used in vitro are close to the condition in vivo, the results may not reflect the actually situation in vivo. It would be more interesting to perform the experiments in patients.