Journal: Blood

Article Title: In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps

doi: 10.1182/blood-2016-06-722298

Figure Lengend Snippet: Fluorescence and immunofluorescence microscopy of stimulated human neutrophils for NETs formation. Stimulated normal human neutrophils were seeded on poly-l-lysine–coated slides and incubated at 37°C + 5% CO2 for 3 hours. After incubation, the preparations were fixed using 4% paraformaldehyde and stained using 2.5 μM Sytox Green, a DNA staining dye. Unstimulated neutrophils did not form NETs (A) and were intact after treatment with DNAase (20 μM) (B). Robust NET release after stimulation with 600 nM PMA (C) or 5 μM A23187 (E) as shown by the bright and scattered Sytox Green signal pattern. Digestion of PMA-stimulated (D) and A23187-stimulated (F) NETs after treatment with DNAase (20 μg/mL), indicating the extracellular location of NETs. (G) Dual immunofluorescence staining of NETs by Sytox Green (2.5 μM) for DNA and citrillunated histone H3 (red). A23187, calcium ionophore.

Article Snippet: Recombinant human histones H3 and H4 were from New England Biolabs (Ipswich, MA); TRAP6 was from R&D Systems (Minneapolis, MN).

Techniques: Fluorescence, Immunofluorescence, Microscopy, Incubation, Staining

Journal: Blood

Article Title: In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps

doi: 10.1182/blood-2016-06-722298

Figure Lengend Snippet: hnDNA and individual human histone H3 and H4 trigger coagulation in plasma. TG in recalcified normal PFP (A) and normal PRP (B) containing hnDNA. TG in recalcified FXII-deficient (FXII-Def), FXI-deficient (FXI-Def), or FVII-deficient (FVII-Def) PFP in the presence or absence of 30 µg/mL of hnDNA (C). No TG was observed in any individual deficient plasma after recalcification in the absence of DNA, represented by a single flat curve (PFP no DNA, panel C). Quantification of FXIa-AT after activation of the contact system by hnDNA in the synthetic contact system activation assay as described in “Methods” (D). TG in recalcified PRP containing recombinant human histone H3 (rH3) (E) or recombinant human histone H4 (rH4) (F). Effect of citrullination of histones H3 (G) and H4 (H) on TG in recalcified PRP. All the figures are representative of at least 3 independent experiments.

Article Snippet: Recombinant human histones H3 and H4 were from New England Biolabs (Ipswich, MA); TRAP6 was from R&D Systems (Minneapolis, MN).

Techniques: Coagulation, Activation Assay, Recombinant

Journal: Blood

Article Title: In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps

doi: 10.1182/blood-2016-06-722298

Figure Lengend Snippet: Individual human histones H3 and H4, but not octameric core histones, trigger TG in PRP. TG performed in recalcified PRP containing individual human histone proteins was compared with that of recombinant core histone octamers (A) or purified calf thymus histones (B). CThist, mixture of purified calf thymus histones; rOctamer, octameric core histone reconstituted with recombinant human histone proteins. Panels A and B are representative of 3 independent experiments.

Article Snippet: Recombinant human histones H3 and H4 were from New England Biolabs (Ipswich, MA); TRAP6 was from R&D Systems (Minneapolis, MN).

Techniques: Recombinant, Purification