Recombinant S100A8/A9 induces human platelet activation in vitro. Human washed platelets (10⁶ platelets/condition) were incubated with different concentrations of recombinant heterodimeric S100A8/A9 (10, 20, and 40 μg/mL) for 30 minutes at 37°C. (A-B) Platelet activation was determined by flow cytometry using anti–P-selectin antibody. (A) Representative plots from a healthy donor; histograms normalized to mode (each peak normalized to its mode for each condition). (B) Percentage of CD41⁺P-selectin⁺ platelets. (C) The percentage of platelet-neutrophil aggregates (CD66b⁺/CD41⁺) in whole blood after addition of S100A8/A9 for 30 minutes at 37°C (n = 7). Blood was diluted 1:5. (D) Anti-CD41/CD61 PAC-1 antibody (against activated GPIIb/IIIa) binding to platelets was assessed by flow cytometry and presented as percentage of CD41⁺ platelets positive for PAC-1 (n = 17). (E-F) Washed platelets (2×10⁸/mL) were incubated with S100A8/A9 (40 μg/mL) or CRP (10 μg/mL), and platelet aggregation was assessed for 20 minutes by light transmission aggregometry (n = 3). (G-H) Washed platelets (2 × 10⁸/mL) were incubated with S100A8/A9 (40 μg/mL) or CRP (10 μg/mL), and ATP generation was assessed for 6 minutes with the CHRONO-LUME luciferin:luciferase assay kit from Chronolog (n = 3). (E,G) Representative traces. (I-J) Washed platelets (2 × 10⁸/mL) were incubated with S100A8/A9 (20 μg/mL or 40 μg/mL) for 6 minutes under stirring condition followed by the addition of exogenous fibrinogen (200 μg/mL). Platelet aggregation was assessed for 6 minutes by light transmission aggregometry (n = 3). (K-L) Alexa-Fluor 488-labeled fibrinogen binding to platelets. (K) Representative plot for fibrinogen binding. (L) Percentage of platelets positive for fibrinogen-Alexa Fluor 488, CRP (10 μg/mL) and TRAP-6 (100 μM) were used as positive control. Data are shown as mean ± SD. The statistical significance was analyzed using ordinary one-way ANOVA. ∗P < .05, ∗∗P < .005, ∗∗∗P < .001, ∗∗∗∗P < .0001.