Phe174-Mutated Human Factor X As Bypassing Agent to the Direct FXa Inhibitors

The direct thrombin and factor Xa (FXa) inhibiting oral anticoagulants have emerged as the preferred anticoagulant therapy, having major benefits for the patient's quality of life due to similar or lower risks of bleeding compared to the vitamin K antagonists. Unfortunately, in the case of bleeding-related adverse effects, no reversal agent is available to counteract the FXa-targeted anticoagulant therapy and restore hemostasis.

The direct FXa inhibitors effectively block its catalytic activity by reversible high affinity occupation of the FXa active site. X-ray crystal structures of human FXa in complex with the FXa inhibitors apixaban or rivaroxaban revealed their specific binding mode and demonstrated occupation of the FXa substrate binding S1 and S4 subsites. To better understand the molecular requirements for direct inhibitor binding in FXa, we performed 750 ns Molecular Dynamics (MD) simulations of the FXa-apixaban complex. During MD, the narrow distribution of close range contacts (≤ 0.5 nm) between the side chain atoms of the S4 pocket residues Tyr99 and Phe174 (chymotrypsinogen numbering) and apixaban confirmed stabilization of apixaban in the S4 subsite. To directly assess the contribution of S4 pocket residues Tyr99 and Phe174 to apixaban binding, we generated human FX(a) variants comprising either Tyr99Ala, Phe174Ala, or both. While substitution of either one of these residues reduced the sensitivity for apixaban by ~8-fold, replacement of both increased the IC50 >100-fold (5 nM wt-FXa; 34 nM Y99A-FXa; 45 nM F174A-FXa; 616 nM Y99A/F174A-FXa). The FX clotting activity, on the other hand, was drastically reduced by substituting Tyr99 (6% residual activity) and essentially lost with both replacements. In contrast, following Phe174 replacement full FX clotting activity was maintained. Since this points to Phe174 substitution as a promising target for abrogated inhibitor binding while maintaining FX activity, we continued with assessing the efficacy of Phe174-mutated FX variants as bypassing agent for the direct FXa inhibitors.

To do so, the human variants FX-F174A and FX-F174S were stably expressed in HEK293 cells and purified to homogeneity using ion-exchange chromatography. Evaluation of the specific FX clotting activity revealed that the Phe174-substituted FX variants exhibited a similar clotting activity (84 mU/µg FX-F174A; 122 mU/µg FX-F174S) relative to human wild-type (wt-)FX (105 mU/µg). This indicates that substitution of Phe174 does not interfere with the binding and subsequent conversion of the FXa substrate. Inhibition of the RVV-X-activated FX variants employing peptidyl substrate conversion revealed a reduced sensitivity to apixaban, demonstrated by a 10-fold increase in IC50 (2 nM wt-FX; 19 nM FX-F174A; 20 nM FX-F174S). Consequently, a decrease in sensitivity was also observed for inhibition by rivaroxaban (3-8-fold) and edoxaban (4-7-fold). To determine whether the FX variants supported tissue factor-initiated (6 pM) thrombin formation in normal human plasma, we performed FXa inhibitor titrations using the calibrated automated thrombin generation assay. While thrombin formation reached half-maximum inhibition at 55 nM apixaban in wt-FX-spiked plasma, plasma spiked with the FX variants displayed a ~10-fold reduced sensitivity to apixaban inhibition (IC50: 640 nM FX-F174S; 405 nM FX-F174A). A similar loss of inhibitor-sensitivity was observed for inhibition by rivaroxaban (6-10-fold) and edoxaban (17-fold), demonstrating that the Phe174-mutated FX variants are able to support thrombin generation in the presence of physiological plasma concentrations of inhibitor. Furthermore, at supra-physiological inhibitor concentrations (1 µM), little if any thrombin was formed upon adding up to 60 µg/ml wt-FX. In contrast, supplementation with 30-45 µg/ml Phe174-mutated FX restored the thrombin peak up to 80% in edoxaban-treated plasma, while the thrombin peak was fully restored in the presence of apixaban or rivaroxaban.

Collectively, our findings indicate that human FX variants comprising a single point mutation at position Phe174 in the S4 subsite are able to restore hemostasis in plasma inhibited by the direct FXa inhibitors. As such, these variants have the potential to serve as rescue therapeutic agents to overcome the effect of the direct FXa inhibitors in case of potential life-threatening bleeding events or emergency surgical interventions.