<p>S100A8/A9 heterodimer is an inflammatory mediator that promotes atherosclerotic thrombosis (AT) and can also be used as a biomarker of the disease. However, its origin and mechanism of action remain largely unknown. This study aimed to elucidate the role of S100A8/A9 heterodimer produced by endothelial cells stimulated by neutrophil extracellular traps (NETs) in AT. The effects of NETs on human umbilical vein endothelial cells (HUVECs) were studied in a co-culture system. We detected the reactive oxygen species (ROS) activity, pyroptosis and S100A8/A9 heterodimer production of HUVECs, as well as the NF-κB signaling pathway activation. The effects of S100A8/A9 heterodimer on platelet activation and underlying mechanisms were investigated in washed human platelets, and then validated in FeCl₃-injured carotid thrombosis model in rats. NETs stimulated HUVECs were found to produce ROS, then activating the NF-κB signaling pathway, inducing pyroptosis and the production of S100A8/A9 heterodimer. Additionally, S100A8/A9 heterodimer was found to induce the activation of platelets through activation of epidermal growth factor receptor (EGFR) signaling pathway. Subsequently, S100A8/A9 heterodimer mediated platelet activation thus inducing thrombus formation via the EGFR/PI3K/AKT and EGFR/p38 MAPK axes in platelets. Our results suggest that S100A8/A9 heterodimer produced by endothelial cells stimulated by NETs induces platelet activation by regulating the EGFR/PI3K/AKT and EGFR/p38 MAPK axes, thus promoting thrombosis. Our data provide new insights into the pathogenesis and therapeutic targets for AT.</p>