EPCR blocking antibody augments hemostatic activity of a low dose of FVIIa in hemophilia A mice. (A) Average hemostatic times of hemophilia A and wild-type mice following saphenous vein injury and the effect of varying doses of FVIIa in restoring hemostasis in hemophilia A mice. Saline or varying doses of FVIIa (1, 4, and 10 mg/kg body weight) in 100 µL saline were given to hemophilia A mice via the tail vein. Five minutes after FVIIa administration, mice were subjected to saphenous vein incision following the procedure described by Buyue et al8 with a few modifications. Briefly, mice were anesthetized with ketamine (100 mg/kg ketamine and 8.5 mg/kg xylazine, ∼100 µL volume, intraperitoneally) and placed in the supine position on a heating mat. Saphenous vein from the ventral hind limb of the right leg of the mouse was exposed by dissecting the skin lengthwise over the saphenous neurovascular bundle. The exposed vein was overlaid with warm saline. Then, at the midway point of the exposed vein, an entry hole in the vein was made by inserting the tip of a 23-gauge needle into the vein. Blood was absorbed on a Kimwipe by gently touching the blood drop away from the puncture site. Immediately following the initial hemostasis, an ∼1-mm longitudinal distal cut was made using a Student Vannas spring scissors by inserting 1 blade into the vessel using the needle hole as the entry. Bleeding was observed for 30 minutes from this cut. After each hemostasis incident, the clot was disrupted gently by stroking the clot with a blunted 30-gauge needle in the direction of the blood flow to reinitiate a new bleeding episode. Duration of each bleeding episode in the 30-minute time period was noted, and average time to ATH was calculated. (B) Same as in (A), but volume of blood loss in 30-minute experimental time frame was determined. Throughout the 30-minute experimental time period, blood was adsorbed on a Kimwipe at every 20 seconds. Hemoglobin was extracted by soaking the wipes in 20 mL of solution of ABX Lysebio for 2 hours or more. Hemoglobin was also extracted from known volumes of freshly collected mouse blood to generate a standard curve for calculating the volume of blood loss (n = 4-6 mice per group). *Average clot time to hemostasis in these experimental groups was significantly shorter than that which was noted in hemophilia A mice not subjected to FVIIa treatment or given 1 mg/kg of FVIIa (P < .05). ns, not statistically significant; **P < .001 compared with values noted for hemophilia A mice not subjected to FVIIa treatment. (C-D) Effect of EPCR antibodies on hemostasis in hemophilia A mice. Hemophilia A mice were injected with EPCR nonblocking or blocking antibodies intraperitoneally (4 mg/kg body weight) 35 minutes prior to saphenous vein incision. Five minutes before saphenous vein incision, either saline (none) or a low dose of FVIIa (1 mg/kg) was administered via the tail vein. The indicated EPCR mAbs (4 mg/kg) were included with the saline and FVIIa injections. (C) Average time to hemostasis and (D) blood loss in the 30-minute experimental time frame were determined as described above. ns, not statistically significant; **P < .001; and ***P < .0001 (n = 8-9 mice per group). (E) Plasma levels of APC. EPCR nonblocking and EPCR blocking antibodies were given to hemophilia A mice as described in (C) and (D). Blood (100 µL) was collected from the mice via mandibular vein prior to giving FVIIa (1 mg/kg body weight) and the saphenous vein incision. At the end of 30-minute bleeding, blood was collected from the heart by cardiac puncture. In both cases, blood was collected in 0.38% sodium citrate and 0.01 M benzamidine hydrochloride (final concentrations). APC levels in plasma were assayed as described earlier.9 (n = 6 mice per group). ***P < .0001.
