Evaluation of procoagulant enhancement of HB-causing FIX variants by emicizumab. (A) The FIX activity of conditioned media from HEK293 cells that transiently expressed recombinant select HB-causing FIX variants with 0 (gray) or 300 nM (red) emicizumab. Only variants with a greater than or equal to twofold enhancement with emicizumab are shown; nonrescuable variants are listed in supplemental Table 1. For clarity, variants with FIX activity with emicizumab >5% of normal are shown on the left, whereas variants with FIX activity with emicizumab 1% to 5% of normal are shown on the right. FIX activity was determined using an activated partial thromboplastin time (aPTT)–based assay with standard curves for 0 or 300 nM emicizumab (supplemental Figure 2). Values are normalized to recombinant FIX-WT activity expressed in the same experiment. Bars represent the mean ± standard error of mean of ≥9 independent transfections. (B) FIX activity of conditioned media with recombinant FIX protein controls with 0 (gray) or 300 nM emicizumab (blue). (C) Enhancement with emicizumab as a function of recombinant FIX antigen. (D) Enhancement with 300 nM emicizumab (red) or 1000% normal FVIII (light blue) of select HB-causing FIX variants. (E) Thrombograms of FIX variants with 0 nM (gray) or 300 nM emicizumab (red). Thrombograms of controls FIX-WT and UC with 0 nM (gray) or 300 nM emicizumab (blue). Coagulation was triggered with FXIa. Results are representative of ≥2 thrombograms from ≥2 independent transfections. Additional thrombograms of rescuable HB-causing variants are provided in supplemental Figure 3. (F) An aPTT-based clotting time of plasma from patients with moderate HB with FIX-I397T variant (patient 1) with increasing amounts of emicizumab in comparison with commercial severe HB plasma with undetectable FIX antigen (cross reactive material [CRM] negative). Points indicate the mean ± standard deviation of 4 measurements. The horizontal gray line represents the clotting time of normal human plasma. (G) Rotational thromboelastometry (ROTEM) thromboelastograms of whole blood from a patient with moderate HB with an FIX-I397T variant (patient 2) with 300 nM emicizumab or 100% recombinant FIX added ex vivo. Results are representative of 2 measurements. UC, unconditioned media.