Murine S100A8/A9 activates mouse platelets through GPIbα and CD36. Washed murine platelets (10⁶ platelets/condition) were incubated with different doses of recombinant mouse S100A8/A9 for 30 minutes at 37°C. Platelet activation was determined by flow cytometry using (A) anti–P-selectin antibody and (B) annexin-V binding. (C-K) Washed platelets isolated from WT, GPVI (GPVI^−/−), CLEC-2 (CLEC-2^−/−), GPVI/CLEC-2 knock-out (GPVI^−/−/CLEC-2^−/−), or GPIbα^−/− mice (IL4R/GPIbα−Tg mice) were incubated with S100A8/A9 (10 μg/mL) for 30 minutes at 37°C. The percentage of platelets positive for P-selectin, GPIIb/IIIa activation (JON/A), and annexin-V binding were measured by flow cytometry. Data are shown as percentage platelets (CD41⁺) positive for different markers. (K) Platelets isolated from WT or IL4R/GPIbα−Tg mice were pretreated with SSO (25 μM) before addition of S100A8/A9 (10 μg/mL) for 30 minutes at 37°C, and annexin-V binding was assessed by flow cytometry. The statistical significance was analyzed using nonparametric test (Kruskal-Wallis test) (A-B), ordinary two-way ANOVA with Sidak’s multiple comparison test (C,D,F,H,J), and two-way ANOVA with Tukey’s multiple comparison test (K). Data are shown as mean ± SD. ∗P < .05, ∗∗P < .005, ∗∗∗P < .001.