PB0215 - Emicizumab Impairs Antithrombin Regulation of Factor IXa

Background: Emicizumab, a bispecific monoclonal antibody, partially mimics activated factor VIII, binding to factor IXa (FIXa) and factor X (FX). Used for bleeding prophylaxis in hemophilia A (HA) patients, including those with inhibitors, its impact on FIXa regulation by antithrombin (AT) remains unevaluated. If emicizumab affects FIXa regulation by AT, it may explain the cases of thrombotic microangiopathy (TMA) reported in emicizumab-treated patients.

Aims: Our goal is to examine if emicizumab affects the kinetics of AT inhibition of FIXa and to understand the mechanism involved, with the hypothesis that it impairs AT inhibition of FIXa in a FX-dependent process.

Methods: We assessed AT inhibition kinetics of FIXa in a purified system by incubating AT with FIXa at 37°C. Residual FIXa activity was measured in FIX-deficient plasma using a thrombin generation assay after incubation. Emicizumab, at therapeutic concentrations, was added to evaluate its effects. The unactivatable variant FXR15Q was used to determine FX's impact on AT inhibition of FIXa.

Results: AT inhibited FIXa (half-life 38.6 min, Fig. 1a red) consistent with prior reports, while the addition of FXR15Q impaired FIXa inhibition by AT (half-life 75.2 min, Fig. 1a blue). Addition of emicizumab alone had minimal effect on AT inhibition of FIXa (half-life 41.5 min, Fig. 1b red), but in the presence of FXR15Q, impairment was substantial at 4 hours (Fig. 1b blue). Varying FXR15Q concentration in the presence of emicizumab demonstrated a loss of protection at low FXR15Q concentration with an apparent KD of 5 nM, consistent with the reported KM of FX for FIXa in the presence of emicizumab.

Conclusion(s): Our results support the hypothesis that FX binding to FIXa precludes AT inhibition. Emicizumab enhances this by increasing FX's affinity for FIXa. This implies that emicizumab-treated patients may experience prolonged FIXa activity, explaining the clinical etiology of TMA.