TLR7 activation in platelets leads to Akt and p38-MAPK phosphorylation and translocation of P-selectin to the cell surface. (A-D) Platelets were isolated from humans and mice and treated with TLR7 agonist (Loxo) or pretreated (for 30 min) with the TRL7 antagonist, IRS661, and then stimulated with Loxo for the indicated time intervals. Protein was isolated and resolved by western-blot analysis. Phosphorylation (A) and quantitation (B) of kinases involved in α-granule release in human platelets. (C-D) Phosphorylation and quantitation, respectively, of kinases involved in α-granule release in murine platelets. (E) P-selectin surface expression and (F) quantitation in human platelets, post TLR7 agonist (Loxo) or thrombin (IIA) stimulation, resolved by flow cytometry. IC, isotype control. (G) Platelet P-selectin interacts with PSGL-1 on the granulocyte population measured by FlowSight image cytometer. (H-I) Five-week-old female WT and P-selectin (SELP) KO mice were injected with Loxo. (H) Heterotypic aggregates between CD41-platelets and Ly6G-PMNs and platelet count (I) 2 hours after Loxo injection. Data are average ± SD and were analyzed by Student t test, except the quantitation of the experiment with the inhibitors, analyzed by ANOVA (P < .0001). Analysis is based on n = 4 humans (A-B), n = 3 groups of mice (C-D), n = 4 for Loxo and n = 3 for thrombin (E), n = 4 (F), and n = 3 (G), n = 3-4 mice/group (H-I).