Figure 7.
Activation of FSAP by exposed histones during the removal of the first wave of NETs contributes to the second wave of the traps. Endotoxemia (LPS IP; 1 mg/kg b.w.) was induced to follow the events occurring between days 13 and 14 after its initiation. (A) Impact of neutralization of histone activity by administration of polyanions (suramin or heparin) on day 13 on the influx of neutrophils into the liver and NET formation on day 14 (regime as in supplemental Figure 5). NETs adhering to the liver endothelium were estimated by IVM: extDNA, NE, and histones H2A.X. Total concentration (B) and the enzymatic activity (C) of FSAP protease present in the blood plasma were measured by ELISA and the activity assay, respectively. The standard imaging time was 10 AM, but on day 13, data were also collected 9 and 17 hours later (day 13 + 9 hours and day 13 + 17 hours, respectively). (D) To block FSAP activity, carminic acid or a C1 esterase inhibitor were applied on day 13 (regime as in supplemental Figure 5), and neutrophil numbers and NET components (extDNA, NE, and H2A.X histones) were followed on day 14 with IVM. (E) Representative images of NET formation on day 14 in animals treated with carminic acid or a C1 esterase inhibitor on day 13. Histones were stained with AF568–anti-H2A.X Abs (red), NE with AF647–anti-NE Abs (violet), and extDNA with SYTOX Green (bright green). The colocalization of NET components was visualized by overlaying the images from individual channels. The scale bar indicates 50 μm. (F) FSAP activity at day 14 after induction of endotoxemia measured in the blood plasma of control mice and animals treated with inhibitors and neutralizing compounds used in panels A (heparin and suramin) and panel D (carminic acid and C1 esterase inhibitor), along with ADAMTS13, DNase I, or their combination. The data in the graphs are expressed as the mean ± SD of at least 3 fields of view. Statistically significant differences according to the 1-way ANOVA are designated by letters, in which the same letter indicates no differences between groups, and different letters indicate statistical differences (Bonferroni post hoc; n = 3-5). OD, optical density; sFSAP, soluble Factor VII-activating protease.

Activation of FSAP by exposed histones during the removal of the first wave of NETs contributes to the second wave of the traps. Endotoxemia (LPS IP; 1 mg/kg b.w.) was induced to follow the events occurring between days 13 and 14 after its initiation. (A) Impact of neutralization of histone activity by administration of polyanions (suramin or heparin) on day 13 on the influx of neutrophils into the liver and NET formation on day 14 (regime as in supplemental Figure 5). NETs adhering to the liver endothelium were estimated by IVM: extDNA, NE, and histones H2A.X. Total concentration (B) and the enzymatic activity (C) of FSAP protease present in the blood plasma were measured by ELISA and the activity assay, respectively. The standard imaging time was 10 AM, but on day 13, data were also collected 9 and 17 hours later (day 13 + 9 hours and day 13 + 17 hours, respectively). (D) To block FSAP activity, carminic acid or a C1 esterase inhibitor were applied on day 13 (regime as in supplemental Figure 5), and neutrophil numbers and NET components (extDNA, NE, and H2A.X histones) were followed on day 14 with IVM. (E) Representative images of NET formation on day 14 in animals treated with carminic acid or a C1 esterase inhibitor on day 13. Histones were stained with AF568–anti-H2A.X Abs (red), NE with AF647–anti-NE Abs (violet), and extDNA with SYTOX Green (bright green). The colocalization of NET components was visualized by overlaying the images from individual channels. The scale bar indicates 50 μm. (F) FSAP activity at day 14 after induction of endotoxemia measured in the blood plasma of control mice and animals treated with inhibitors and neutralizing compounds used in panels A (heparin and suramin) and panel D (carminic acid and C1 esterase inhibitor), along with ADAMTS13, DNase I, or their combination. The data in the graphs are expressed as the mean ± SD of at least 3 fields of view. Statistically significant differences according to the 1-way ANOVA are designated by letters, in which the same letter indicates no differences between groups, and different letters indicate statistical differences (Bonferroni post hoc; n = 3-5). OD, optical density; sFSAP, soluble Factor VII-activating protease.

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