DENV activates platelets through an NS1 autocrine loop. (A-B) Platelets from healthy donors were infected with DENV-2 (MOI = 1). At 1.5 hours after infection, unbound viruses were washed out, and after 3, 6, 12, and 24 hours postinfectious platelet pellets and supernatants were collected for quantification of viral RNA (A) and analysis of NS1 synthesis and secretion (B). β-Actin was used as the loading control. (C-G) Platelets were infected with DENV-2 (MOI = 1) in the presence of anti-TLR4 (C) or anti-NS1 (D-G) neutralizing antibodies or isotype-matched IgG. (C-D) The percentage of platelets expressing surface P-selectin (CD62P), and the concentrations of RANTES/CCL5 (E), MIF (F), and IL-1β (G) were quantified at 6 hours after infection. (H-I) The percentage of P-selectin surface expression on platelets pretreated with cycloheximide (10 µM, 30 minutes) and infected with DENV (MOI = 1) (H) or thrombin (0.5 U/mL) (I) for 6 hours. Dots or bars represent mean ± standard error of the mean of 3 to 4 independent experiments. *P < .05 compared with mock; #P < .05 between selected groups.