Infused mFVIIa-FMR shows enhanced hemostatic potential in hemophilic mice compared with mFVIIa. (A) Hemophilia B mice (n = 3-5 per group) were subjected to a 7.5% FeCl₃ injury, and blood flow was monitored for 10 minutes. Subsequently, procoagulant (mFVIIa or mFVIIa-FMR) was administered at the indicated dose via the jugular vein, and time to vessel occlusion was determined. Wild-type (WT) mice (n = 4) are shown for reference (white bar). Data are shown as mean ± SD. *P < .05 vs mFVIIa for each procoagulant dose; ^#P < .05 vs WT; NS, nonsignificant difference. In all experiments, blood flow was monitored for 30 minutes following protein infusion, and the time to carotid artery occlusion was defined as the time following application of FeCl₃ (or protease infusion in the treated mice) until the blood flow has decreased by >90%. Hemophilic mice did not show vessel occlusion over the time of observation (data not shown). (B) Hemophilia B mice (n = 3-4 per group) were injected with 50 µg of EPCR-blocking antibody (RCR-252) or control isotype IgG. Thirty minutes later, they were subjected to a 7.5% FeCl₃ injury, and blood flow was monitored for 10 minutes. Subsequently, mFVIIa-FMR was administered at 3 mg/kg via the jugular vein, and time to vessel occlusion was determined. Data are shown as mean ± SD. *P < .05 vs IgG. In all experiments, blood flow was monitored for 30 minutes following protease infusion, and the time to carotid artery occlusion was defined as the time following protease infusion until the blood flow has decreased by >90%. (C) Hemophilia A mice (n = 4-5 per group) were subjected to the same experimental setup as in A. Data are shown as mean ± SD. *P < .05 vs mFVIIa. (D) Hemophilia B mice (black bar, n = 4-5 mice per group) or WT (white bar, n = 5 mice) were subjected to a tail transection, and blood loss (in µL) was measured for a period of 2 minutes (PRE period). Immediately after, 3 mg/kg of mFVIIa or mFVIIa-FMR was infused, and blood loss (in µL) was measured for an additional 8 minutes (AFTER period). Quantitative assessment of blood loss was determined by measuring total hemoglobin by absorbance at 575 nm following red cell lysis and converted to total blood loss (μL) using a standard curve. WT mice infused with PBS are shown as a reference. Data are shown as mean ± SD. *P < .05 vs WT mice; ^#P < .05 vs mFVIIa.