BACKGROUND The aim of this study was to determine the involvement of S100A8/A9 in the development of arterial thrombosis. MATERIAL AND METHODS A total of 303 patients were enrolled in this study, with 110 having acute coronary syndrome (ACS) and 110 having coronary heart disease (CHD), and 83 subjects served as healthy blood donors. The concentrations of Toll-like receptor 4 (TLR-4), cyclooxygenase-2 (COX-2), and S100A8/A9 protein were determined in the sera of the participants and in peripheral blood mononuclear cells (PBMCs) derived from a rat carotid artery thrombosis model and in human aortic endothelial cells (HAECs). The mitogen-activated protein kinase (MAPK) inhibitor SB203580 and the TLR-4 blocker CLI-095 were used to investigate the role of the TLR-4-MAPK-COX2 signaling axis in thrombosis. RESULTS The levels of COX-2, TLR-4, and S100A8/A9 in the sera of patients with ACS and CHD were significantly higher than in healthy controls (P<0.05). S100A8/A9 expression was significantly correlated with TLR-4 and COX-2 in the ACS group and with TLR-4 in the CHD group. In the rat carotid thrombosis model, the expressions of TLR-4, COX-2, and p-p38 MAPK significantly increased until 14 days after thrombosis induction, whereas S100A8/A9 expression increased until day 7, but then decreased. Administration of SB203580 to rats reduced COX-2 expression in PBMCs after thrombosis induction, and incubation of HAECs with CLI-095 reduced their p-p38 MAPK and COX-2 response to S100A8/A9 stimulation. CONCLUSIONS S100A8/A9 is upregulated after blood vessel injury and is enhanced in combination with TLR-4 COX-2 induction via p38 MAPK activation.