Stimulation with NS1 partially reproduces the inflammatory pathways of DENV-infected platelets. (A-D) Platelets from healthy volunteers were kept unstimulated (Unst) or stimulated with NS1 (5 µg/mL) produced by transfected E coli (NS1_EC), mammalian HEK cells (NS1_HEK), or insect SF9 cells (NS1_SF9) or were infected with DENV (MOI = 1) for 3 hours. (A) The percentage of P-selectin (CD62P) surface expression on platelets; and the concentrations of RANTES/CCL5 (B), MIF (C), and IL-1β (D) in platelet supernatants were evaluated. (E-I) Platelets were stimulated with NS1 produced in SF9 cells (5 µg/mL) and/or ATP (5 mM) or infected with DENV (MOI = 1) for 3 hours. The expression of pro-IL-1β and β-actin in platelet lysates (E); the expression of IL-1β in platelet supernatants (F); the concentration of IL-1β in platelet supernatants (G); labeling of active caspase-1 in intact platelets (H); and the percent of platelet CD63 surface expression (I) were evaluated in each condition. Platelets stimulated with thrombin (Thr, 0.5 U/mL) were used as the positive control for CD63 expression. (J) Platelets were infected with DENV or stimulated with NS1+ATP in the presence of dimethyl sulfoxide (DMSO; vehicle) or BBG. Caspase-1 activity was evaluated in each condition. Bars represent mean ± standard error of the mean of 4 to 10 independent experiments. *P < .05 compared with unstimulated; #P < .05 between platelets treated with vehicle or BBG. ns, nonsignificant.