DENV2 NS1 binds and activates TLR4 on platelets. (A-B) Platelets from healthy donors were labeled with FITC-conjugated LPS (LPS-FITC, 5 or 10 µg/mL) for 5 minutes in the presence of NS1 produced by HEK cells (0, 0.5, 5 and 50 μg/mL) (A), or unlabeled LPS (0, 0.1, 1, and 10 µg/mL) (B). Each dot represents the fluorescence (mean fluorescence intensity; [MFI]) of LPS-FITC–labeled platelets. Nonlinear regression was traced according to the distribution of the dots. (C-G) Platelets were incubated with neutralizing antibodies against TLR4 (C-D), TLR2 (E), TLR6 (F), or TLR6-binding peptide (G; TLR6-BP) for 30 minutes, and stimulated with NS1 produced in HEK cells (5 µg/mL) or LPS (10 µg/mL) for 3 hours. (H) Platelets from healthy volunteers were stimulated with NS1 produced in HEK cells (5 µg/mL) or LPS (10 µg/mL) for 3 hours in the presence or absence of 10% autologous human serum. (I) Platelets resuspended in medium containing 10% human serum were incubated with neutralizing antibodies against TLR4/MD2 complex for 30 minutes and stimulated with NS1 produced in HEK cells (5 µg/mL) or LPS (10 µg/mL) for 3 hours. The percentage of platelets expressing surface P-selectin (CD62P) was evaluated in each condition. Bars represent mean ± standard error of the mean of 4 to 6 independent experiments. *P < .05 compared with unstimulated platelets; #P < .05 compared with isotype-matched IgG.