Acquired coagulant factor VIII deficiency induced by Bacillus anthracis lethal toxin in mice

Abstract

Mice treated with anthrax lethal toxin (LT) exhibit hemorrhage caused by unknown mechanisms. Moreover, LT treatment in mice induced liver damage. In this study, we hypothesized that a suppressed coagulation function may be associated with liver damage, because the liver is the major producing source of coagulation factors. The hepatic expression of coagulant factors and the survival rates were analyzed after cultured cells or mice were exposed to LT. In agreement with our hypothesis, LT induces cytotoxicity against hepatic cells in vitro. In addition, suppressed expression of coagulation factor VIII (FVIII) in the liver is associated with a prolonged plasma clotting time in LT-treated mice, suggesting a suppressive role of LT in coagulation. Accordingly, we further hypothesized that a loss-of-function approach involving treatments of an anticoagulant should exacerbate LT-induced abnormalities, whereas a gain-of-function approach involving injections of recombinant FVIII to complement the coagulation deficiency should ameliorate the pathogenesis. As expected, a sublethal dose of LT caused mortality in the mice that were non-lethally pretreated with an anticoagulant (warfarin). By contrast, treatments of recombinant FVIII reduced the mortality from a lethal dose of LT in mice. Our results indicated that LT-induced deficiency of FVIII is involved in LT-mediated pathogenesis. Using recombinant FVIII to correct the coagulant defect may enable developing a new strategy to treat anthrax.

Keywords: Anthrax; coagulation factor VIII; hemorrhage; lethal toxin.

Figure 1.

LT-induced liver damage. Mortality of mice treated with PA (4.4 mg/kg) and LT (4.4 mg/kg, lethal dose; or 3 mg/kg, sublethal dose) (A) (n = 8) was plotted as Kaplan–Meier curves (PA vs. LT, **P < 0.01). Analyses data were obtained from animals with the same type of treatments as indicated in Panel A (B–E). Hematoxylin and eosin staining of liver sections from PA (B) and lethal dose LT (C) treated mice (96-h treatment). Arrows indicate the blood cell leaked into liver sinusoid (C). Liver retained Evans blue levels, which indicates the plasma leakage (D) (96-h treatment). Serum aspartate aminotransferase (ASL) and alanine aminotransferase (ALT) levels of mice treated with PA and LT for 0, 72, and 96 h, respectively; *P < 0.05, compared with respective PA groups (E). n = 6 (D, E).

Figure 2.

D-dimer, TAT and clotting time analysis. D-dimer analysis of plasma from untreated mice or mice treated with PA (4.4 mg/kg), LT (4.4 mg/kg), and LPS (5 ng/kg) for 0, 6, 24 and 72 h, respectively (A). TAT analysis of plasma from untreated, PA, LT, and LPS treated mice (72 h) (B). Recalcification plasma clotting time analysis of plasma samples collected from untreated, PA, LT, and LPS treated mice (72 h) (C), or normal mice plasma treated with or without PA, LT or anti-PA Ig preincubated LT (LT + anti-PA) (2 h in vitro) (D). Activated partial thromboplastin time (APTT) and prothrombin time (PT) analysis of plasma from untreated, PA, and LT treated mice (E). *P < 0.05, **P < 0.01, compared to respective PA groups. n = 6 (A–E).

Figure 3.

Cytotoxicity analysis. Relative cellular surviving rates of human hepatoma HepG2 (A) or Huh-7 (B) cells treated with or without PA (4 μg/mL), or LT (0.2, 2, and 4 μg/mL). *P < 0.05, **P < 0.01, compared to respective PA groups. n = 6 (A, B).

Figure 4.

mRNA expression of coagulant factors in mice liver. The RT-PCR analysis for coagulant-factor-mRNA was measured according to gel intensities. The mRNA levels of coagulant factors FI, FII, FV, FVII, FVIII, FIX, FX, FXI, and FXIII were detected using RT-PCR and liver samples from mice treated with or without PA or LT for 72 h. Data were first normalized with internal control actin; untreated groups were subsequently adjusted to 100% during each time course. *P < 0.05, **P < 0.01, compared to respective PA-treated groups (n = 6).

Figure 5.

Analysis of FVIII deficiency. The FVIII defects were analyzed using FVIII-deficient plasma (A) and an FVIII ELISA kit (B), respectively. After treatment with PA or LT for 72 h, APTT analysis was performed using various ratios of combined FVIII-deficient plasma and mouse sample plasma (A). The mice plasma + normal plasma groups (mice plasma/normal plasma = 1/5 [v/v]) served as a basal level, in which the deficiency of coagulation factor in mice plasma was complemented by normal plasma and thus exhibited the fastest clotting response (n = 6). For ELISA analyses, plasma levels of factor VIII (FVIII) and factor VII (FVII) were measured (B) (n = 4). Respective untreated groups were normalized to 100%. *P < 0.05, **P < 0.01, compared to respective PA-treated groups.

Figure 6.

Mortality analysis. Sublethal doses PA (3 mg/kg) and LT (1, 2, and 3 mg/kg) were used to challenge mice with (warfarin) or without (vehicle) 10-d pretreatment of sublethal warfarin (100 mg/kg/day) (A, B) (n = 8), plotted as Kaplan–Meier curves (PA vs. LT, **P < 0.01). No mortality occurred when sublethal doses of LT (1, 2, and 3 mg/kg) were used to challenge mice without warfarin preconditioning (A). Recombinant FVIII (rFVIII; 40 IU/kg/4hr) was used to rescue lethal-dose LT (4.4 mg/kg) challenged mice (C) (n = 12). **P < 0.01, LT vs. LT + rFVIII.