Reverting Rivaroxaban Effect By Activated Factor X: Assessment Using Thrombin Generation Assay
Authors: Grenier Dominique , Samama Meyer Michel , Chtourou Sami , Plantier Jean-Luc 
Published: November 2019

Abstract
Specific anti-activated factor X molecules are currently used for the prevention and the treatment of various thromboembolic disorders. However, despite a growing use of these molecules, they are still devoid of a reliable antidote.
Rivaroxaban is a specific anticoagulant targeting activated factor X (FXa). Its potential in inhibiting FXa in vitro and in vivo was demonstrated during the characterization of the molecule. However, the use of FXa to revert the effect of Rivaroxaban in plasma was never studied. To do so the measurement of thrombin generation (TG) using the calibrated automatic thrombinoscope was performed. The ability of purified human FXa (Haematologic Technologies at 10, 50, 100, 500 and 1000 ng/ml) to induce TG in a platelet-poor plasma (PPP) without the induction of the coagulation was first evaluated. There was a FXa dose-dependent TG. The TG profile at concentrations up to 50 ng/ml of FXa was similar than the control profile obtained by a PPP activated by tissue-factor (0.5 pM) and phospholipids. Above 50 ng/ml FXa, the lag time decreased and the endogeneous thrombin potential (ETP) increased with the dose. This pattern revealed the thrombogenic potential of FXa and demonstrated that a dose of 50 ng/ml (or ≈1 nM) FXa was the maximum safer dose identified by this assay. A similar experiment was performed following the activation of plasma with 0.5 pM Tissue-Factor (TF) and 4 µM phospholipids (PL) and adding FXa at 31, 62, 125, 250 and 500 ng/ml. The kinetics of TG in the presence of the different amounts of FXa differed less than when coagulation was not induced. The lag times varies from 3 to 1.83 min with the increasing concentrations of FXa and the peak heights from 120 to 212 nM, being the two most affected parameters. Following the addition of 62 ng/ml (or ≈1.25 nM) FXa, the TG was more effective than a control plasma identically stimulated.
Rivaroxaban was then spiked in the PPP at the therapeutic dose of 0.35 µg/ml (or 0.8 µM). Following 0.5 pM TF/4 µM PL stimulation, this dosage completely inhibits the TG. Increasing doses of FXa (31, 62, 125, 250 and 500 ng/ml) were then added and dose-dependently restores the TG. All the parameters of the TG profile were affected by the presence of FXa. The normalization was attained at the dose of 250 ng/ml (or 5 nM) FXa. A similar set of experiment was repeated by activating the plasma with cephalin, used as a model to mimic the initiation of the contact phase coagulation. The pattern of TG was different than following FT/PL activation. With cephalin and for all FXa concentrations identical peak aspects (velocity, ETP and peak height) were obtained differing only by their lag times and times-to-peak. Lag times and times to peak were shortened by the addition of FXa from 10.7 to 3.7 min and 13.2 to 6 min respectively. Plasma were then spiked by Rivaroxaban (0.35 µg/ml) and activated by cephalin in the presence of various concentrations of FXa (31, 62, 125, 250 and 500 ng/ml). A dose-dependent TG was demonstrated with the ETP, the peak height and the velocity increasing with the amount of FXa spiked whereas the lag time and time to peak were shortened. Following the induction by cephalin, the presence of FXa systematically shortened the TG when Rivaroxaban was present or not, when compared to the TG from control plasma.
This work aimed to establish the antidote potential of the natural substrate of the anti-Xa molecules and limiting the risk in promoting a thrombotic response. The calibrated thrombin generation assay was used to determine the in vitro efficiency of FXa to induce a normal thrombin generation without primary induction or following an induction by TF/PL or cephalin. The doses of FXa required to normalize coagulation in the presence of Rivaroxaban and following induction were identified. These conditions will now be assessed in vivo in Rivaroxaban treated-mice. In addition of establishing the antidote properties of FXa, this data paved the way to compare its capacities, which are optimal to inhibit such inhibitor, to further antidote in development.

Disclosures
Grenier: LFB BIotechnologies: Employment. Chtourou:LFB Biotechnologies: Employment. Plantier:LFB Biotechnologies: Employment.