Aim: To investigate the effect of factor Xa (FXa), thrombin and platelets, under activation or resting conditions, on neutrophil extracellular traps (NETs) formation (NETosis). Materials and Methods: Neutrophils, isolated from apparently healthy volunteers, were challenged with 25-50 nM FXa, 0.2-8 U/mL thrombin, 5-100 μM adenosine diphosphate (ADP), 0.1-0.5 mM arachidonic acid (AA), 5-15 μg/mL collagen, 100 μM protease-activated receptor (PAR)-4 activating peptide, 10-100 μM PAR-2 activating peptide or 25-100 nM phorbol 12-myristate 13-acetate (PMA). Moreover, neutrophils were challenged with platelet-rich plasma (PRP), activated with ADP (at a concentration of either 20 μM, in an aggregometer, or of 50 μM, in situ). Neutrophils were incubated for 3.5 h at 37οC and 5% CO2 and an immunofluorescence protocol followed, in order to stain neutrophils and NETs for myeloperoxidase and DNA. Finally, NETs and neutrophils were microscopically visualized and the percentage of NET-releasing neutrophils of each of the above experimental conditions was evaluated. Results and Conclusion: Among the agonists studied, only PMA and AA, induced robust NETosis, at a threshold concentration of 25 nM and 0.25 mM, respectively. Furthermore, platelets pre-activated with ADP, but not resting platelets induce the formation of NETs, a phenomenon that is also observed when platelets were activated with ADP in situ, in the presence of neutrophils. The present results provide an additional mechanism through which platelets contribute to various pathophysiological conditions in which NETs are implicated, such as inflammation and thrombosis. The whole spectrum of platelet mediators that are implicated in NETosis needs to be further investigated.