Platelet-HMGB1 promotes platelet-neutrophil interactions and neutrophil activations. (A) Lung immunofluorescence in mice at 18 hours after CLP. Green, CD41; Red, Ly6G; blue, nucleus. Scale bar, 50 µm (images in the right column were magnified ×5). Colocalization of CD41 and Ly6G is shown as yellow. Quantification was performed using NIS Elements. Percentage of Ly6G⁺ nuclei and percentage of CD41⁺ neutrophils were calculated. (B-C) Platelets were isolated from HMGB1 Flox and HMGB1 PF4 mice. Twenty million platelets were treated with (B) collagen (0.5 mM) or (C) thrombin (0.2 U/mL) for 2 minutes and incubated with 1 million isolated WT neutrophils for 5 minutes. Formation of platelet-neutrophil aggregation was assessed using flow cytometry. These experiments have been repeated 4 times. Data represent mean ± SD. Statistical difference was tested using Student t test; *P < .05.