The prothrombotic effects of HMGB1 depend on the redox state. (A) Thrombus weight in Hmgb1^−/− chimeras (n = 10) treated with buffer compared with Hmgb1^−/− chimeras (n = 5 each) receiving 3S- or disulfide HMGB1. Lines indicate mean. (B) Quantification of NETs, (C), monocytes, (D), and platelet covered area from immunofluorescence stainings of Hmgb1^−/− chimeras receiving buffer, 3S-, or disulfide HMGB1 (n = 3 each). (E) Extem (left) and Fibtem (right) 6 hours after IV injection of 3S- (n = 3), disulfide HMGB1 (n = 3), or sulfonyl (n = 3) compared with control (n = 5). (F) Fold increase in whole-blood aggregation after incubation with 3S-, disulfide, or sulfonyl HMGB1 compared with control stimulated with buffer. This was followed by stimulation by ADP in the groups indicated (n = 4-6). (G) Results from RT-PCR for TF (left), IL-1β (middle), and IL-6 (right) of peripheral blood human monocytes incubated with different redox forms of HMGB1 and LPS for 3 hours shown as fold increase compared with control stimulated with the respective buffer (n = 3 each). (H) Left, Quantification of NET formation in vitro after stimulation of isolated human neutrophils with buffer, 3S-, disulfide, or sulfonyl HMGB1 (n = 3 each). Right, Representative images of immunofluorescence stainings for MPO (red) and Hoechst (blue); bar, 50 µm. Results are mean ± SEM. *P < .05. The Student t test was used to compare results between 2 groups, 1-way ANOVA followed by LSD–post hoc test for 3 groups.