Blood-derived HMGB1 causes venous thrombosis. (A) Thrombus weight in Balb/c mice treated with PBS (n = 8) compared with BoxA (n = 6). (B) Left, Immunofluorescence staining of cross-sections of the IVC for propidium iodide (PI) (red) and DAPI (blue) 6 hours after flow reduction. Dotted line indicates endothelium; bar, 200 µm. Right, Higher magnification of intraluminal propidium iodide–positive cells (arrowheads); bar, 70 µm. (C) Immunofluorescence staining of cross-sections of the IVC 1, 6, and 12 hours after flow reduction for CD41 (red) and HMGB1 (green). Nuclei are counterstained with DAPI (blue). Dotted line indicates endothelium; bar, 100 µm. Images representative of n = 3 experiments. (D) Immunofluorescence staining of cross-sections of the IVC 48 hours after flow reduction. Top, Presence of HMGB1 on NETs (arrowheads) as shown by staining for HMGB1 (green), Ly6G (red), and DAPI (blue); bar, 20 µm. Bottom, HMGB1 (green) colocalizes with platelets (red). Dotted line indicates endothelium; bar, 100 µm. Nuclei are counterstained with DAPI (blue). Images representative of n = 3 experiments. (E) Thrombus weight in Hmgb1^+/+ fetal liver cell chimeras (n = 7) compared with Hmgb1^−/− chimeras (n = 6). (F) Thrombus weight in Hmgb1^−/− bone marrow chimeras (n = 6) compared with Hmgb1^−/− chimeras receiving wild-type platelets (n = 10), Hmgb1^−/− platelets (n = 8), or wild-type neutrophils (n = 9). (A,E,F) Line indicates mean. The Student t test was used to compare results between 2 groups, 1-way ANOVA followed by LSD–post hoc test for 3 groups.