Platelets are sensitive cells and are easily activated by different stimulants in the circulation system. It is known that tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine and plays a role in inflammation. The role of TNF-a in the apoptotic process in blood platelets is unknown. In order to study the formation of apoptosis in platelets after incubation with TNF-alpha and/or ADP, several biomarkers were chosen: phosphatidylserine (PS) exposure and P-selectin binding; cGMP, Cyt-c, and Ca2+ levels and NOS activation; and gene and protein expression of caspase-3 and iNOS. Platelets were incubated with 100 pg/mL TNF-a and/or 50 mM iNOS specific inhibitor, such as at 1400 W for 1 h at 37 degrees C in the presence of 5 mu M ADP. According to the results, the levels of PS exposure and P-selectin binding were significantly higher in TNF-alpha + ADP platelets, which decreased with the addition of 1400 W. A significant induction in cGMP, Cyt-c, and Ca2+ levels was observed in platelets treated with TNF-alpha + ADP. On the other hand, the upregulation of the apoptotic gene caspase-3 and iNOS expression levels in TNF-alpha-treated and ADP-activated platelets was significantly reversed with the addition of 1400 W. These data demonstrate that TNF-alpha promotes iNOS expression through caspase-3 stimulation in human platelets, and its concomitance leads to the triggering of apoptosis-mediated inflammation upon platelet activation.