Figure - PMC

Skip to main content

View full-text article in PMC

. Author manuscript; available in PMC: 2020 Aug 17.

Published in final edited form as: Circ Res. 2019 Jun 28;125(5):507–519. doi: 10.1161/CIRCRESAHA.118.314571

Figure 2. — A, B, r-huADAMTS13 was incubated with r-huPAD4. A, ADAMTS13 was immunopurified using an anti-ADAMTS13 antibody or B, citrullinated proteins were isolated using an anti-pan-citrulline antibody. Membranes were blotted with the indicated antibody (left side of blots) and stripped and re-probed with an anti-ADAMTS13 antibody (A; lower panel). The band at approximately 190 kDa indicates ADAMTS13. C, Plasma from Adamts13^−/− mice injected with r-huADAMTS13 was collected and incubated with r-huPAD4. ADAMTS13 was immunopurified with an anti-ADAMTS13 antibody and detected by Western blot using an anti-pan-citrulline antibody (upper panel; lower molecular weight plasma proteins shown to non-specifically bind to Sepharose beads were excluded from figure). Membranes were stripped and re-probed using an anti-ADAMTS13 antibody (lower panel). C, D, E, Citrullination of ADAMTS13 was performed by incubating r-huADAMTS13 with r-huPAD4 for 15, 90, and 180 minutes. C, Citrullination of r-huADAMTS13 was detected by Western blot with an anti-pan-citrulline antibody as a band of 190 kDa (upper arrow). The band at 74 kDa (lower arrow) corresponds to auto-citrullinated r-huPAD4. D, E, Activity of r-huADAMTS13 was determined by FRETS-VWF73 assay (60 minutes). D, Fluorescent counts changes in FRETS-VWF73 as a function of time. E, ADAMTS13 activity of the different samples expressed as percentage of that observed for r-huADAMTS13 in the absence of PAD4 (One-way ANOVA, Tukey’s multiple comparison test; ** p=0.0054, *** p=0.0001). F, G, H, Citrullination of ADAMTS13 was performed by incubating r-huADAMTS13 with r-huPAD4 for 180 minutes with or without Cl-amidine. F, Citrullination of r-huADAMTS13 observed by Western blot. G, H, Activity of r-huADAMTS13 determined by FRETS-VWF73 assay (One-way ANOVA, Tukey’s multiple comparison test; *** p=0.0001). Data are representative of 3 independent experiments and expressed as mean ± SEM. I, Western blot of citrullinated-ADAMTS13 and ADAMTS13 from plasma of young healthy donors, septic patients, and co-morbidity patients. Citrullinated ADAMTS13 was labeled with biotin-PG and immunopurified using streptavidin beads. ADAMTS13 was then detected by western blot with anti-ADAMTS13 antibody. As control, healthy donor samples 8 and 5, and co-morbidity donor sample 1, were immunopurified without modification by biotin-PG. Graph represents the relative amount of citrullinated ADAMTS13 for each sample. Septic patient number 7* was excluded from analysis because ADAMTS13 levels could not be detected. (One-way ANOVA, Tukey’s multiple comparison test, p value 0.01; n=7–8 donors).

Figure 2. — A, B, r-huADAMTS13 was incubated with r-huPAD4. A, ADAMTS13 was immunopurified using an anti-ADAMTS13 antibody or B, citrullinated proteins were isolated using an anti-pan-citrulline antibody. Membranes were blotted with the indicated antibody (left side of blots) and stripped and re-probed with an anti-ADAMTS13 antibody (A; lower panel). The band at approximately 190 kDa indicates ADAMTS13. C, Plasma from Adamts13^−/− mice injected with r-huADAMTS13 was collected and incubated with r-huPAD4. ADAMTS13 was immunopurified with an anti-ADAMTS13 antibody and detected by Western blot using an anti-pan-citrulline antibody (upper panel; lower molecular weight plasma proteins shown to non-specifically bind to Sepharose beads were excluded from figure). Membranes were stripped and re-probed using an anti-ADAMTS13 antibody (lower panel). C, D, E, Citrullination of ADAMTS13 was performed by incubating r-huADAMTS13 with r-huPAD4 for 15, 90, and 180 minutes. C, Citrullination of r-huADAMTS13 was detected by Western blot with an anti-pan-citrulline antibody as a band of 190 kDa (upper arrow). The band at 74 kDa (lower arrow) corresponds to auto-citrullinated r-huPAD4. D, E, Activity of r-huADAMTS13 was determined by FRETS-VWF73 assay (60 minutes). D, Fluorescent counts changes in FRETS-VWF73 as a function of time. E, ADAMTS13 activity of the different samples expressed as percentage of that observed for r-huADAMTS13 in the absence of PAD4 (One-way ANOVA, Tukey’s multiple comparison test; ** p=0.0054, *** p=0.0001). F, G, H, Citrullination of ADAMTS13 was performed by incubating r-huADAMTS13 with r-huPAD4 for 180 minutes with or without Cl-amidine. F, Citrullination of r-huADAMTS13 observed by Western blot. G, H, Activity of r-huADAMTS13 determined by FRETS-VWF73 assay (One-way ANOVA, Tukey’s multiple comparison test; *** p=0.0001). Data are representative of 3 independent experiments and expressed as mean ± SEM. I, Western blot of citrullinated-ADAMTS13 and ADAMTS13 from plasma of young healthy donors, septic patients, and co-morbidity patients. Citrullinated ADAMTS13 was labeled with biotin-PG and immunopurified using streptavidin beads. ADAMTS13 was then detected by western blot with anti-ADAMTS13 antibody. As control, healthy donor samples 8 and 5, and co-morbidity donor sample 1, were immunopurified without modification by biotin-PG. Graph represents the relative amount of citrullinated ADAMTS13 for each sample. Septic patient number 7* was excluded from analysis because ADAMTS13 levels could not be detected. (One-way ANOVA, Tukey’s multiple comparison test, p value 0.01; n=7–8 donors).