TFPIα inhibition by andexanet alfa is partially restored by protein S and factor V-short

Andexanet alfa (AndA) is a modified, inactive form of coagulation factor Xa (FXa) and is the only selective reversal agent for direct oral anti-FXa inhibitors. Due to its similarity to FXa, AndA binds the endogenous anticoagulant, tissue factor pathway inhibitor α (TFPIα). We determined how AndA affects TFPIα function and enhancement by its cofactors, protein S and FV-short. AndA reversed rivaroxaban-mediated suppression of thrombin generation in plasma. In the absence of rivaroxaban, AndA (0.25μM-4μM) increased peak thrombin ∼22-fold, but had no impact on the presence of anti-TFPIα antibodies, suggesting AndA inhibited TFPIα anticoagulant function. However, AndA did not fully block TFPIα, with ∼20% to 30% function remaining at 2μM-4μM AndA. This preserved TFPIα function was fully inhibited by anti-protein S antibodies, suggesting partial protection of TFPIα function by its cofactors. In pure-component FXa inhibition assays, AndA fully blocked TFPIα-mediated FXa inhibition, both in the presence and absence of protein S and FV-short. Similarly, AndA-bound TFPIα was unable to inhibit FIXa and FXa generation by tissue factor (TF)-FVIIa in the absence of protein S and FV-short. However, in their presence, AndA-TFPIα inhibited FIXa generation, with a 50% inhibitory concentration of 0.14nM compared with that of 0.05nM for FXa-TFPIα. Similarly, the assays of FXa generation showed inhibition of TF-FVIIa-mediated FX activation at saturating concentrations of AndA in the presence of protein S and FV-short. Although AndA reduces TFPIα function by competing with FXa for TFPIα interactions, it does not fully block TFPIα. Protein S and FV-short enable AndA-bound TFPIα to locate at the membrane surface to inhibit TF-FVIIa.