Characterization of mFIX_gla-egf1FVIIa and hFIX_gla-egf1FVIIa. (A) Relative coagulant activity of mFIXgla-egf1FVIIa compared with recombinant mFVIIa in an APTT assay; 25 µL of mFVIIa or mFIXgla-egf1FVIIa (5 µg/mL) was added to 25 µL FIX -deficient human plasma and 25 µL APTT reagent, and the assay initiated with 25 µL CaCl. (B) Relative activity of mFIXgla-egf1FVIIa compared with recombinant mFVIIa in a PT assay; 25 µL of either mFVIIa or mFIXgla-egf1FVIIa (0.5 µg/mL) was added to 25 µL FVII-deficient plasma, and 100 µL Innovin was added to initiate the reaction. The Innovin concentration was determined by titration with human FVIIa. Data were derived from at least 3 experimental points. Data are shown as average ± 1 standard deviation (SD). (C) Functional binding of 5000 ng/mL mFVIIa and mFIXgla-egf1FVIIa to Innovin (human TF), measured by FXa generation and detected with an FXa chromogenic substrate. Absorbance was measured after 15 minutes. Backgrounds were subtracted. (D) Relative functional binding of 5000 ng/mL mFIXgla-egf1FVIIa compared with mFVIIa to mouse TF–positive microparticles. Substrate (S-2288, 2 mM final concentration) was added to the reaction and the absorbance at 405 nm was determined after 15 minutes. Backgrounds were subtracted. The Mann-Whitney U test was used for all statistical analysis. ND, not detectable; OD, optical density.