Direct Thrombin Inhibitors, but Not Factor Xa Inhibitors, Enhance Thrombin Formation in Human Plasma by Interfering with the Thrombin–Thrombomodulin–Protein C System

Activation of protein C is dependent on thrombin complexed with thrombomodulin (TM). Activated protein C (APC), together with its cofactor protein S, degrades coagulation Factors Va and VIIIa, thereby limiting further thrombin formation. Thus, in addition to suppressing the procoagulant effects of thrombin, direct thrombin inhibitors (DTIs) may also downregulate anticoagulant effects of thrombin-mediated feedback mechanisms. By contrast, direct Factor Xa (FXa) inhibitors block the formation of thrombin, but not its actions. The objective of this study was to investigate whether the direct FXa inhibitor, rivaroxaban, and the DTIs, dabigatran and melagatran, inhibit the negative-feedback reaction of the thrombin–TM complex/APC (thrombin–TM/APC) system and thereby increase thrombin formation. Experiments were conducted in plateletpoor plasma from healthy donors (normal plasma) and in pooled protein C-deficient plasma, both substituted with 1.33 μM phospholipids, in the presence or absence of 10 nM TM with increasing concentrations of rivaroxaban, dabigatran, melagatran, or the appropriate vehicles. Thrombin formation was initiated by adding 1.67 pM tissue factor (TF) and assessed by measuring the cleavage of the fluorogenic substrate Z-Gly-Gly-Arg-AMC (Bachem) using the Calibrated Automated Thrombogram (CAT, Thrombinoscope® BV) method. The parameters assessed were lag time, time to peak thrombin generation (tmax), peak thrombin generation (Cmax), and endogenous thrombin potential (ETP). In addition, formation of prothrombin fragments 1+2 (F1+2) was determined by ELISA (Enzygnost® F1+2 monoclonal [Dade Behring]). Rivaroxaban potently inhibited thrombin formation in the absence and presence of TM across all parameters in a concentration-dependent manner in both normal plasma and protein C-deficient plasma (see Table). In the absence of TM, melagatran and dabigatran also inhibited thrombin formation in a concentration-dependent manner, both in normal plasma and protein C-deficient plasma. In the presence of TM, DTIs prolonged lag time and tmax in a concentration-dependent manner. However, only high concentrations of the DTIs reduced ETP, Cmax, and F1+2, In normal plasma,lower concentrations even increased ETP, Cmax, and F1+2. Increased thrombin formation was observed with melagatran 119–474 nM or dabigatran 68–545 nM. DTIs did not increase thrombin formation in protein C-deficient plasma, suggesting that both protein C and TM are needed for the DTI-mediated increase in thrombin formation. The results suggest that low concentrations of DTIs suppress the anticoagulant effects of the thrombin–TM/APC system by inhibiting activation of protein C by the thrombin–TM complex, and thereby enhance thrombin formation. Conversely, rivaroxaban does not increase thrombin formation, suggesting that it does not suppress the negative-feedback reaction by inhibition of protein C activation. This hypothesis is supported by the absence of enhanced thrombin formation in protein C-deficient plasma. Enhanced thrombin formation might explain the hypercoagulation observed with DTIs in a rat model of TF-induced intravascular coagulation (Furugohri, et al. 2005; Morishima, et al. 2005; Perzborn, et al. 2008) and suggests that DTIs could cause activation of coagulation at lower plasma concentrations. Table. Effect of rivaroxaban, dabigatran, and melagatran on peak thrombin formation (Cmax [nM thrombin]) in the absence or presence of thrombomodulin (TM) in normal plasma (NP) from healthy volunteers (n=8–12), and in pooled protein C-deficient plasma (PPC) in the presence of TM (n=3). Results were obtained by the CAT method and are a mean of n plasma samples. Prothrombin fragments F1+2 (nM F1+2) results were obtained in the presence of TM (mean results; n=3 [NP]). Thrombin (nM) in normal and in protein C-deficient plasma n.d:, no data. Rivaroxaban (nM) 0 18 91 182 363 1,090 Cmax: − TM in NP 273 222 113 71 43 15 Cmax: + TM in NP 100 67 32 18 10 3 Cmax: + TM in PPC 290 249 168 117 80 38 F1+2: + TM (in NP) 184 89 43 18 n.d. 2 Dabigatran (nM) 0 68 136 273 545 1,090 Cmax: − TM in NP 261 287 289 282 239 102 Cmax: + TM in NP 81 156 203 240 218 96 Cmax: + TM in PPC 298 301 300 292 253 116 F1+2: + TM in NP 252 n.d. 362 422 351 98 Melagatran(nM) 0 24 119 237 474 948 Cmax: − TM in NP 276 290 297 289 251 127 Cmax: + TM in NP 101 133 215 251 237 113 Cmax: + TM in PPC 294 296 299 292 256 132 F1+2: + TM in NP 213 n.d. 389 427 393 123