Phenotypic appearance of mice following silencing of hepatic antithrombin and protein C production. (A) Effectiveness of silencing Serpinc1 and Proc in mouse liver. siRNAs targeting Serpinc1 and Proc were complexed, mixed, and intravenously injected into C57Black/6J mice at a dose of 3.5 mg/siRNA/kg (hatched bars, n = 13) or 7 mg/siRNA/kg (black bars, n = 6). At 2 days post siRNA injection, mice were removed from the experiment and euthanized; livers were subjected to Serpinc1 (left) or Proc (right) transcript analysis by quantitative polymerase chain reaction. β-actin was used as the internal control for quantification and normalization. The ΔCt values of the individual samples were related to the mean ΔCt of the reference group (siNEG-treated, 7 mg/kg, open bars, n = 11). (B) Plasma antithrombin (left) and protein C levels (right) in siNEG-injected mice (open circles), siSerpinc1/siProc-injected mice at 3.5 mg/siRNA/kg (open squares), and 7 mg/siRNA/kg (black squares). (C) Right eye of an siSerpinc1/siProc-injected animal. Unilateral severe exophthalmos and periocular hemorrhages are shown. (D) Multifocal hemorrhages in the mandibular and submandibular area and masseter muscle. (E) Severe multifocal hemorrhages within the eye (Hematoxylin and Eosin [HE]-stained 5-μm section, magnification 40×). (F) Eye region: presence of harderian glands with multifocal hemorrhages (h) and thrombus (t) (HE-stained 5-μm section, magnification 100×). (G) Cyanosis of the right hind leg. (H) Hemorrhages (arrow) and thrombi (arrowheads) were present in the subcutis but also among muscular fibers of the tibial and femoral areas (HE-stained 5-μm section, magnification 200×). (I) Liver (formalin-fixed specimen) presenting focally extensive areas of necrosis (asterisk). (J) Liver section presenting severe multifocal to coalescing coagulative necrosis (cn) and thrombosis (t) in hepatic vein (HE-stained 5-μm section, magnification 100×). Liver fibrin (K) and plasma fibrinogen (L) in siNEG-treated mice (open circles), siSerpinc1/siProc-injected animals at 3.5 mg/siRNA/kg (open squares), and 7 mg/siRNA/kg (black squares). (M) Treatment of siSerpinc1/siProc-injected mice (3.5-mg/kg dose) with DE. DE was administered by oral gavage of 3 mg per mouse at 7 am, 3 pm, and 11 pm for 5 days starting on the day before siRNA injection. This resulted in an aPTT of 66.9 ± 6.8 seconds to 36.2 ± 4.4 seconds, as determined in a parallel treated control group at 2 hours after dosing and 1 hour before the next dose (vehicle treated animals aPTT of 26.7 ± 5.1 seconds and 24.5 ± 0.8 seconds, respectively). Presence of periocular contusion (arrow heads) for vehicle-treated animals (left) and animals not treated with DE (right). (N) Liver fibrin deposition in siSerpinc1/siProc-treated mice (3.5 mg/siRNA/kg) treated with DE (open squares) or vehicle (filled squares). Data were analyzed using the Instat software (GraphPad, San Diego, CA). Statistical differences between control siNEG and siSerpinc1, siProc, siSerpinc1/siProc were evaluated using a Mann-Whitney rank sum test. P values < .05 were regarded as statistically significant. *P < .05, †P < .01, ‡P < .001.