DENV NS1 binds to platelets and induces activation through TLR4 signal transduction.

(A) The protein expression levels of TLR4 and β actin, an internal control, in human-isolated platelets from 3 different donors were detected using Western blotting (50 μg protein/lane). (B) Human-isolated platelets were preincubated with αTLR4 or a control Rabbit IgG for 1 h, and the binding of NS1 on platelet surfaces was determined by flow cytometry using FITC-conjugated anti-NS1 monoclonal antibodies (33D2-FITC) (n = 3). (C) The binding of NS1 on platelets isolated from wild-type or TLR4 knockout mice was determined by flow cytometry (n = 4). Platelets were preincubated with or without different concentrations of (D) αTLR4 (E) the TLR4 antagonist LPS-Rs, (F) the TLR4 signaling inhibitor TAK242, or the LPS inhibitor polymyxin B (10 μg/ml) for 30 min, followed by BSA or NS1 (10 μg/ml) stimulation for 1 h (n = 4). The percent fluorescence of NS1 binding and the P-selectin surface expression on platelets were analyzed by FACSCalibur flow cytometry. *P<0.05, **P<0.01; Kruskal-Wallis ANOVA (panels B to panel F).