S100A8/A9 induces the formation of procoagulant platelets. Human washed platelets (10⁶ platelets/condition) were incubated with different concentrations of S100A8/A9 (10, 20 and 40 μg/mL) for 30 minutes at 37°C. (A-C) PS exposure was determined by flow cytometry using PE-Cy7–labeled annexin-V (n = 17). CRP (10 μg/mL) and TRAP-6 (100 μM) were used as positive control. (A) Representative histogram for annexin-V staining. Representative histograms normalized to mode (each peak normalized to its mode for each condition). (B) Percentage of CD41⁺annexin-V⁺ platelets. (C) Fold change in the mean fluorescent intensity (MFI) of agonist-activated platelets over control. (D) The percentage of CD41⁺MVs positive for annexin-V (n = 6). (E-F) Human washed platelets spreading on collagen or S100A8/A9. Representative differential interference contrast (DIC) images of spread platelets. (F) Representative annexin-V (magenta) and P-selectin (green) staining for adherent platelets. (G) Quantification of annexin-V–positive, P-selectin–positive, or annexin-V and P-selectin–double positive platelets adherent on collagen and S100A8/A9 (positive platelets/total platelets). The statistical significance was analyzed using two-way ANOVA with Tukey’s multiple comparison test between all groups. (H-I) Assessment of intracellular Ca²⁺ release from platelets spread over surfaces coated with S100A8/A9 (20 and 40 μg/mL), fibrinogen, or bovine serum albumin assessed using BAPTA-1-AM Ca²⁺ sensitive dye. (H) Representative Ca²⁺ traces assessed using Oregon Green-488 BAPTA-1-AM. (I) Peak fluorescence at time point zero (F0/Fmax). Data are shown as mean ± SD. The statistical significance was analyzed using a nonparametric test (Kruskal-Wallis test). ∗P < .05, ∗∗P < .005, ∗∗∗P < .001, ∗∗∗∗P < .0001.