Acetaminophen (N-acetyl-p-aminophenol [APAP]) is one of the leading causes of acute liver failure, and APAP hepatotoxicity is associated with coagulopathy in humans. We tested the hypothesis that activation of the coagulation system and downstream protease-activated receptor (PAR)-1 signaling contribute to APAP-induced liver injury. Fasted C57BL/J6 mice were treated with either saline or APAP (400 mg/kg intraperitoneally) and were euthanized 0.5-24 hours later. Hepatotoxicity and coagulation system activation occurred by 2 hours after administration of APAP. Treatment with APAP also caused a rapid and transient increase in liver procoagulant activity. In addition, significant deposition of fibrin was observed in the liver by 2 hours, and the concentration of plasminogen activator inhibitor-1 in plasma increased between 2 and 6 hours. Pretreatment with heparin attenuated the APAP-induced activation of the coagulation system and hepatocellular injury and diminished hepatic fibrin deposition at 6 hours. Loss of hepatocellular glutathione was similar in APAP-treated mice pretreated with saline or heparin, suggesting that heparin did not diminish bioactivation of APAP. In mice deficient in tissue factor, the principal cellular activator of coagulation, APAP-induced liver injury, activation of coagulation, and hepatic fibrin deposition were reduced at 6 hours. A cetaminophen (N-acetyl-p-aminophenol [APAP]) is the leading cause of drug-induced hepatic failure in humans. Early results in animal models revealed that APAP is bioactivated to a reactive metabolite that is responsible for the hepatotoxicity. 1,2 Many subsequent studies have identified numerous factors that appear to contribute to APAP-induced liver injury, including mitochondrial alterations, reactive oxygen and nitrogen species, and cytokines such as tumor necrosis factor-␣. [3][4][5][6][7][8] Despite extensive study, the factors and mechanisms involved in the initiation and progression of hepatocellular lesions during APAP hepatotoxicity are not fully understood.Disturbances in the hemostatic system are well documented in human patients with APAP hepatotoxicity. During hemostasis, formation and lysis of clots is regulated by the balance among coagulant, anticoagulant and fibrinolytic pathways. 9 The coagulation system is activated by tissue factor (TF), and this culminates in the generation of thrombin and formation of insoluble fibrin clots. Dissolution of fibrin clots is mediated by plasmin and is inhibited by plasminogen activator inhibitor-1 (PAI-1). In people who develop APAP-induced liver injury, prothrombin time increases, and this change correlates with the severity of toxicity. [10][11][12] Moreover, the concentrations of several coagulation factors are decreased in APAP-poisoned patients, 13 an effect that could be interpreted to be a consequence of decreased production of coagulation factors by the injured liver. An alternative interpretation, however, is that the decrease in coagula-

Acetaminophen (APAP)-induced hepatotoxicity accounts for nearly half of acute liver failure cases in the United States. The doses that produce hepatotoxicity vary considerably and many risk factors have been proposed, including liver inflammation from viral hepatitis. Interestingly, inflammatory stress from another stimulus, bacterial endotoxin (lipopolysaccharide, LPS), renders the liver more sensitive to hepatotoxicity from numerous xenobiotic agents. The purpose of these studies was to test the hypothesis that inflammation induced by LPS or infection with reovirus increases sensitivity to APAP-induced liver injury. For LPS-induced inflammation, C57BL/6J mice were treated with either saline or LPS (44 x 10(6) EU/kg, ip) 2 h before treatment with APAP (100-400 mg/kg, ip) or saline. No elevation in serum alanine aminotransferase (ALT) activity was observed in mice that received vehicle or LPS alone. LPS co-treatment produced a leftward shift of the dose-response curve for APAP-induced hepatotoxicity and led to significantly greater tumor necrosis factor-alpha (TNF) production than APAP alone. Reovirus serotype 1 (10(8) PFU, iv) induced inflammation in Balb/c mice as evidenced by increases in hepatic mRNAs for macrophage inhibitory protein-2, interleukin-6, and TNF. Co-administration of reovirus and APAP at doses of 450 and 700 mg/kg (2 h after reovirus) led to increases in serum ALT activity, whereas neither reovirus nor APAP alone produced liver injury. Consistent with the increases in serum ALT activity, histopathologic examination revealed centrilobular necrosis with marked neutrophilic accumulation only in livers of mice treated with LPS/APAP or with reovirus/APAP. The results suggest that normally noninjurious doses of APAP are rendered hepatotoxic by modest inflammation, whether bacterial or viral in origin.