Introduction <p>Acetaminophen (APAP) overdose remains a common cause of liver injury, primarily due to its toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). This study sought to investigate APAP-induced platelet aggregation in vitro, and the implication of CYP2E1 in the metabolism of APAP and hepatic cell toxicity.</p> Methods <p>Co-cultures of platelets and hepatic cells that do not (HepG2) and do express CYP2E1 (HepG2^E47) were exposed to APAP (0-20&#xa0;mM), NAPQI (0-250&#xa0;µM), APAP in the absence/presence of inhibitors of glutathione (50&#xa0;μM buthionine sulphoximine (BSO)), or APAP in the absence/presence of inhibitors CYP2E1 (chlormethiazole (CMZ, 100&#xa0;µM), or 4-methylpyrazole (4-MP, 5&#xa0;mM)). Platelet aggregation, cell viability and reactive oxygen species (ROS) were analyzed. Changes in platelet aggregation was determined in platelets directly exposed to APAP/NAPQI.</p> Results <p>Exposure to APAP decreased platelet aggregation under co-culture conditions but not in platelet-only cultures. Conversely, NAPQI exposure decreased platelet aggregation in both co-culture and platelet-only conditions. Both APAP and NAPQI reduced cell viability in HepG2 and HepG2^E47 cells, with BSO enhancing APAP toxicity, while 4-MP mitigated it. Acetaminophen exposure led to ROS production in HepG2^E47 cells, with no effect of CMZ and 4-MP.</p> Conclusions <p>Acetaminophen exposure impacts platelet aggregation in co-cultures of platelets and HepG2/HepG2^E47 cells with increased ROS production in HepG2^E47 cells and 4-MP preventing APAP-induced cytotoxicity in HepG2^E47 cells. While APAP had no direct effect on platelets, NAPQI exposure acted to decrease platelet aggregation. These findings enhance our understanding of the mechanisms of APAP-induced hepatotoxicity and the potential role of APAP-induced hepatocellular toxicity in platelet aggregation.</p>