Effect of MPC1609 and MAPC1591 on the anticoagulant and cytoprotective activities of APC. (A) Varying concentrations of MPC1609 and MAPC1591 were incubated with murine APC (20 pM) and phospholipids (25 µM) for 1 hour. Then, FVa (20 pM) was added to the APC and phospholipid mixture and incubated for 30 minutes. APC inactivation of FVa was measured in prothrombinase (IIase) assay by adding factor Xa (0.4 nM) and prothrombin (1 µM) and determining thrombin generation in a chromogenic assay. IIase activity measured in the absence of APC or the antibodies and not subjected to any preincubation was taken as 100%. Green filled circle indicates relative the IIase activity in a reaction mixture containing APC but no antibodies. The blue triangle indicates relative IIase activity in the absence of both APC and antibodies. (B) Confluent monolayers of bEND3 endothelial cells were treated with control vehicle (CV), MAPC (50 nM), or MAPC (50 nM) plus control IgG, MPC1609, or MAPC1591 (200 µg/mL) for 1 hour. After 1 hour, the endothelial cells were stimulated with IL-1β (10 ng/mL) for 16 hours. At the end of 16 hours, supernatant media were collected and IL-6 levels in the media were determined by ELISA. UN, untreated; first CV (red bar) indicates cells treated with control vehicle alone; the second CV (orange bar) indicates cells treated with a CV, followed by APC. (C-D) FVIII^−/− mice were administered with control IgG, MAPC1591, or MPC1609 via the tail vein (1 mg/kg). After 1 hour, the mice were subjected to the saphenous vein incision, and the average time to achieve hemostasis (C) and the blood loss (D) was determined as described in “supplemental Methods” (8 to 9 mice per group). The data shown for WT mice for comparative purposes were from the earlier study of the authors’ laboratory.³⁴ ns, not a statistically significant difference. **P < .01; ***P <.001; ****P < .0001. conc., concentration.