Effect of platelet activators on HIV-platelet interactions. (A) RNAscope probing for the HIV gag gene (green), followed by immunostaining for CD61 as a platelet marker (red) was also performed on these platelets. Images were taken using a confocal microscope (60× objective) and are representative of 2 independent trials. (B) Isolated platelets were treated with ADP (5 μM), sCD40L (1 μg/mL), thrombin (0.1 U/mL), or TRAP6 (10 μM) prior to the addition of HIV-1 BaL. Quantitative RT-PCR for HIV gag was performed and normalized to RNA extracted from the same viral stock. Data were analyzed by 1-way ANOVA. (C) Isolated platelets were treated with aspirin (10 μM), Syk inhibitor R406 (0.5 μM), ticagrelor (10 μΜ), or vehicle control prior to the addition of TRAP6 and HIV BaL-1; data were analyzed using 1-way ANOVA. (D) Platelets were treated with an IgG control or anti–DC-SIGN antibody prior to incubation with HIV-1 BaL. A ratio-paired Student t test was used to determine significance. (E) DC-SIGN and CLEC-2 expression was analyzed by flow cytometry. Mean fluorescence intensity (MFI) of DC-SIGN/CLEC-2 was measured on CD61-gated cells and normalized to the average MFI of the control.