CD36 blockade partially decreases GPIIb/IIIa activation and PS exposure, whereas immunoreceptor tyrosine-based activation motif receptor inhibition reduces P-selectin expression. Human washed platelets (106 platelets/condition) were incubated with S100A8/A9 (20 μg/mL) with or without different inhibitors for 30 minutes at 37°C. Inhibitors were preincubated with platelets for 10 minutes before addition of S100A8/A9: Paquinimod (blocks S100A9 binding to TLR4) (10 μM), RAGE inhibitor Azeliragon (1 μM), CD36 inhibitor SSO (25 μM), Syk inhibitor PRT-060318 (10 μM), Src inhibitor PP2 (20 μM), and BTK inhibitor Ibrutinib (500 nM) were used. Platelet activation was determined by flow cytometry using (A,D,G) anti-CD41/CD61 PAC-1 antibody, (B,E,H) anti–P-selectin antibody, and (C,F,I) annexin-V binding. Data are shown as the percentage of platelets positive (CD41+) for these markers. Data are shown as mean ± SD. Statistical significance was analyzed using ordinary one-way ANOVA. ∗P < .05, ∗∗P < .005, ∗∗∗P < .001, ∗∗∗∗P < .0001.
