Thrombotic challenge of WT EPCR^WT/R84A and EPCR^R84A/R84A mice. (A) Anesthetized mice were given an IV injection (via tail vein) of bovine thrombin (1 nmol/kg) or 0.9% NaCl. At 8 minutes postinjection, blood samples were collected via the inferior vena cava (IVC) in the presence of 0.01 M benzamidine. APC levels were measured by enzyme capture assay. The solid line indicates the lower limit detection for the assay. (B-C) Anesthetized mice were given an IV injection (via tail vain) of 50 pmol/kg FXa and 75 nmol/kg phospholipid vesicles (PCPS) in the presence of 0.1% BSA and 2 mM CaCl₂. At 10 minutes postinjection, blood samples were collected via the IVC. TAT (B) and APC (C) levels were measured by ELISA. (D) Immunohistochemical detection of fibrin deposition in the lungs after FXa/PCPS injection. Red alkaline phosphatase reaction product identifies fibrin under brightfield (top) and fluorescent (bottom) microscopy. (E) Fluorescence quantification shows that the MFI of fibrin staining is greater in the lungs of EPCR^R84A/R84A mice when compared with WT mice. (F) Immunohistochemical detection of fibrin deposition in the ventricles of the heart after FXa/PCPS injection. Images are representative of 3 mice per group. Data represents the mean ± SE; *P < .05, **P < .01, ***P < .001; n > 3 per group. ELISA, enzyme-linked immunosorbent assay.