Acetaminophen (APAP) is one of the most widely used antipyretic and analgesic drugs in both children and adults. Although APAP is relatively safe when it is used within its recommended dosing, overdosing with APAP is the most common cause of acute liver failure in the U.S. Because treatment options for APAP hepatotoxicity are limited, many studies have been conducted to understand the mechanism of APAP hepatotoxicity. It is hoped that a greater understanding of mechanisms will lead to supplemental or alternative treatments. APAP-induced liver injury in humans is associated with robust coagulation cascade activation and thrombocytopenia. However, it is not known whether coagulation-driven platelet activation and thrombin generation participates causally in acetaminophen hepatotoxicity. Here, we hypothesized that platelets and thrombin contribute to APAP hepatotoxicity. Our results in a mouse model suggest that thrombin, the thrombin receptor, protease activated receptor-4 (PAR-4), and platelets contribute to the progression of APAP-induced liver injury. Also, our results suggest that platelets and thrombin contribute to the release of nitric oxide (NO) needed for generation of peroxynitrite. Results in isolated murine hepatocytes suggest that thrombin does not enhance hepatocellular injury directly but more likely plays a role by stimulating NPCs in the liver. Treatment of mice with lepirudin after APAP significantly decreased hepatic injury, however, potential hemorrhagic complications may limit use of thrombin inhibitors or other direct coagulation inhibitors in human patients. Further evaluation of the mechanisms of platelet and thrombin enhancement of APAP-induced liver injury is therefore warranted.Interestingly, during the development of the in vitro experiments using primary mouse hepatocytes (HPCs), an issue arose regarding the appropriate APAP concentration to use, since other investigators have used many different APAP concentrations (0.1-50 mM) in studies of APAP cytotoxicity in vitro. It is widely accepted that APAP toxicity requires bioactivation by cytochromes P450 (CYPs), yet our concentration-response study in murine HPCs suggested contribution of a CYP-independent mechanism. This led to another hypothesis that APAP toxicity in vitro includes CYP-independent mechanism(s). Our results suggest that at least two mechanisms contribute to APAP cytotoxicity. One is a CYP-dependent mechanism that operates at small, cytotoxic APAP concentrations and that occurs rapidly and is limited both in degree and duration. This mechanism is related to CYP-dependent NAPQI production. The second is a CYP-independent mechanism that predominates at larger APAP concentrations and that is slower to develop and highly lethal. Deacetylation of APAP to PAP appears to be a contributor to the CYP-independent mechanism and might enhance cell death by its own mechanism or by acting on cells already stressed by NAPQI. Whether the CYP-dependent and CYP-independent initiating mechanisms activate similar or different cell death signaling pathways remains to be determined.
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