Tissue factor pathway inhibitor-reduced and antithrombin-reduced hemophilia state exhibit different coagulation responses to bypassing agents

Abstract Background : Non-factor therapies have been developed to overcome the limitations of conventional treatments in people with hemophilia (PwH), regardless of inhibitor status. Fitusiran is an investigational siRNA which reduces antithrombin (AT) to rebalance hemostasis in PwH. Moreover, monoclonal antibodies (mAbs) against tissue factor pathway inhibitor (TFPI) have been developed as TFPI-reduced hemostatic rebalance therapy for PwH. In PwH receiving these non-factor therapies, bypassing agents (BPAs) such as recombinant factor (rF)VIIa and activated prothrombin complex concentrates (aPCC) are typically used during bleeding episodes. A plasma-derived (pd-)FVIIa/FX product demonstrates high efficacy against bleeding episodes in PwH and inhibitors (PwH-inh) in Japan (Shirahata A. Haemophilia. 2013). In addition, previous basic studies have demonstrated that even FX supplementation augments emicizumab-driven hemostasis (Shimizu K. Thromb Haemost. 2024). However, the influence of supplementation with pd-FVIIa/FX or FX alone on coagulation potentials in TFPI-reduced or AT-reduced hemophilia state remains unknown. Aim : To assess the coagulation potentials of pd-FVIIa/FX and FX in TFPI-reduced or AT-reduced PwH model plasmas. Methods : TFPI-deficient plasma supplemented with full-length TFPI at 10% level was pre-incubated with FVIII-depleted or FIX-depleted plasmas to generate TFPI-reduced PwH state. The pd-FVIIa/FX (0.187, 0.375, and 1.5 µg/mL as FVIIa; corresponding to 7.5, 15, and 60 μg/kg), recombinant (r)FVIIa (2.2 µg/mL; corresponding to 90 μg/kg), or FX (65, 130, and 520 nM; corresponding to 0.5, 1, and 4 IU/mL) were added to the TFPI-reduced PwH model plasmas. Pd-FVIIa/FX (0.75 and 1.5 µg/mL as FVIIa), rFVIIa (1.1 and 2.2 µg/mL), or FX (260 and 520 nM) were added to FVIII-depleted or FIX-depleted AT-deficient plasmas at AT level of 10% and 30% (AT-reduced model). FX (0-1,040 nM) was incubated with FVIII-depleted or FIX-depleted FⅩ-deficient plasmas to assess FVIIa/TF-mediated FX activation. Coagulation potential was assessed by thrombin generation (TG) assay. Results : We firstly examined the coagulation function following the addition of rFVIIa (2.2 μg/mL) in AT-reduced PwH model plasmas and TFPI-reduced PwH model plasmas. Addition of rFVIIa improved the TG potential to normal levels in TFPI-reduced PwH state. The addition of rFVIIa, however, had little effect on TG in AT-reduced PwH model. We previously demonstrated poor responsiveness to rFVIIa in PwHA-inh with reduced FX levels due to FX consumption (Yada K. Thromb Res. 2024). These results supported that FX level in AT-reduced PwH model may be lower than that in TFPI-reduced PwH model. We next assessed the coagulation function after the addition of pd-FVIIa/FX or FX alone in AT-reduced PwH plasmas and TFPI-reduced PwH plasmas. The TG potential in TFPI-reduced PwH plasmas was augmented beyond normal levels by the addition of pd-FVIIa/FX (1.5 µg/mL). Similarly, supplementation with the addition of FX (520 nM) increased TG potential above normal levels. In addition, even low concentrations of pd-FVIIa/FX (0.187 and 0.375 µg/mL) or FX (65 and 130 nM) restored TG potentials to normal levels and the obtained parameters were comparable to those of rFVIIa (2.2 μg/mL). By contrast, the addition of pd-FVIIa/FX (1.5 µg/mL) or FX (520 nM) to AT-reduced PwH model plasmas improved TG potentials within the normal range. The results indicated that the coagulation potential by the addition of pd-FVIIa/FX (1.5 µg/mL) or FX (520 nM) alone in TFPI-reduced PwH model plasmas appeared to be greater than that in AT-reduced PwH model plasma. Further experiment showed that the addition of FX(up to 1,040 nM) to FVIII-depleted or FIX-depleted FX-deficient plasma increased TG potentials, indicating that the increased coagulation potential could be due to FVIIa/TF-triggered FXa generation in a FX dose-dependent manner. Taken together, it seemed likely that FVIIa/TF-mediated FXa activation following the addition of FX in TFPI-reduced PwH plasma may be greater than that in AT-reduced PwH plasma. Conclusion : FX or pd-FVIIa/FX supplementation increased coagulation potentials in TFPI-reduced PwH model plasmas and AT-reduced PwH model plasmas due to FVIIa/TF-induced activation of FX. However, caution is warranted because the effects of rFVIIa, pd-FVIIa/FX and FX were different between TFPI-reduced PwH state and AT-reduced PwH state. この文面が相手に理解してもらえるかどうかですね？