Blood was drawn from WT mice before and 6, 24 and/or 48 h after IVC stenosis application (DVT) or sham surgery. Plasma was prepared and DNA levels determined by SytoxGreen dye; n = 3 – 8 per time point. *, P
Figure 2. Neutrophils expressing citrullinated histone H3…
Figure 2. Neutrophils expressing citrullinated histone H3 are present in deep vein thrombi
Figure 2. Neutrophils expressing citrullinated histone H3 are present in deep vein thrombi Frozen sections of a thrombus formed in the mouse IVC after 48 h IVC stenosis were stained for Gr-1 (brown), CitH3 (grey-black), and counterstained for DNA (pink). (A) Composite of photographs of a stained longitudinal section of the entire thrombus with white (remote from the suture towards the tail) platelet-rich and red (adjacent to the suture) RBC-rich parts designated (dotted green line approximately delineates the white part of the thrombus). (B) High magnification of the white thrombus part. Note abundant Gr-1-positive neutrophil staining and absent CitH3 staining. (C, D) High magnification of the red part of the thrombus. (C) Orange arrowhead indicates single Gr-1 staining; red arrowhead designates double Gr-1/CitH3 staining. (D) Red arrowheads indicate NETs-like CitH3-positive structures. Inset represents high magnification of the circled area with NETs-like structures. (E) Negative control without first antibody from the red (upper panel) and white (lower panel) parts of the thrombus. (A) bar 500 μm; (B – E), bar 50 μm.
Figure 3. Extracellular citrullinated histone H3 is…
Figure 3. Extracellular citrullinated histone H3 is partially associated with VWF in murine deep vein…
Figure 3. Extracellular citrullinated histone H3 is partially associated with VWF in murine deep vein thrombi (A, B) Widefield fluorescence microscopy analysis of longitudinal sections of a thrombus developed in the IVC after 48 h stenosis. Sections were immunostained for VWF (red) and for CitH3 (green) (upper panels). Incubation with only secondary antibodies served as a negative control (lower panels). (A) The “white part” of the thrombus (*) was positive for VWF but negative for citrullinated histones H3 whereas the “red part” of the thrombus was positive for both VWF and CitH3. The red part-associated CitH3 could be observed as a punctiform (intracellular) or diffuse (extracellular) staining. (A, C) Scale bars, 500 μm. (B) Higher magnification images show frequent co-distribution of extracellular CitH3 with VWF within the red part of thrombus as indicated by the arrowheads. (B, D) Scale bars, 50 μm. (E, F, H) Widefield fluorescence microscopy analysis of cross-sections of the red part of a thrombus developed in the IVC after 48 h stenosis and including vessel wall. (E, F, G) Sections were immunostained for VWF (red), CitH3 (green) and counterstained for DNA (Hoechst 33342, blue); (H) control staining without first antibody. (E, F) Prominent CitH3 staining could be found within the thrombus and was often in proximity to VWF (F, arrowheads). No extracellular CitH3 could be detected in a sham-operated vessel containing only blood (G). (E – H) Scale bars, 100 μm.
Figure 3. Extracellular citrullinated histone H3 is…
Figure 3. Extracellular citrullinated histone H3 is partially associated with VWF in murine deep vein…
Figure 3. Extracellular citrullinated histone H3 is partially associated with VWF in murine deep vein thrombi (A, B) Widefield fluorescence microscopy analysis of longitudinal sections of a thrombus developed in the IVC after 48 h stenosis. Sections were immunostained for VWF (red) and for CitH3 (green) (upper panels). Incubation with only secondary antibodies served as a negative control (lower panels). (A) The “white part” of the thrombus (*) was positive for VWF but negative for citrullinated histones H3 whereas the “red part” of the thrombus was positive for both VWF and CitH3. The red part-associated CitH3 could be observed as a punctiform (intracellular) or diffuse (extracellular) staining. (A, C) Scale bars, 500 μm. (B) Higher magnification images show frequent co-distribution of extracellular CitH3 with VWF within the red part of thrombus as indicated by the arrowheads. (B, D) Scale bars, 50 μm. (E, F, H) Widefield fluorescence microscopy analysis of cross-sections of the red part of a thrombus developed in the IVC after 48 h stenosis and including vessel wall. (E, F, G) Sections were immunostained for VWF (red), CitH3 (green) and counterstained for DNA (Hoechst 33342, blue); (H) control staining without first antibody. (E, F) Prominent CitH3 staining could be found within the thrombus and was often in proximity to VWF (F, arrowheads). No extracellular CitH3 could be detected in a sham-operated vessel containing only blood (G). (E – H) Scale bars, 100 μm.
Figure 4. Histone infusion promotes flow restriction-induced…
Figure 4. Histone infusion promotes flow restriction-induced thrombosis in mice
Histone mix (10 mg/kg) was…
Figure 4. Histone infusion promotes flow restriction-induced thrombosis in mice Histone mix (10 mg/kg) was infused into WT mice immediately before DVT surgery. Mice were sacrificed after 1 h stenosis and thrombi developed in the IVC were examined and harvested. Values for weight (A) and length (B) of the thrombi are shown with medians (horizontal bars). (C) Percentage of mice that developed a thrombus. Vehicle-treated mice, n = 14; Histone-treated mice, n = 9.
Figure 5. DNase 1 infusion protects mice…
Figure 5. DNase 1 infusion protects mice from flow restriction-induced thrombosis
Wild-type mice underwent IVC…
Figure 5. DNase 1 infusion protects mice from flow restriction-induced thrombosis Wild-type mice underwent IVC stenosis for 6 h (A – C) or 48 h (D – F). Mice received infusion of either vehicle or DNase 1 (50 μg i.p. and 10 μg i.v.) before the surgery (A – F) and every 12 h thereafter (D – F). (A, D) Thrombus weight and (B, E) thrombus length are presented; horizontal lines represent median. (C, F) Percentage of mice with a thrombus. 6 h vehicle, n = 14; 6 h DNase 1, n = 10; 48 h vehicle. n = 8; 48 h DNase 1, n = 12.