In vitro heparin characterization. (A) In vitro effects of varying amounts of UFH and AADH on thrombin generation in normal pooled mouse plasma as measured by CAT.12 Variable amounts of UFH (B) or AADH (C) and histone H3 (H3) were added to EA.hy926 cells and cell viability was quantitated with flow cytometry. Each bar represents the average ± SD of at least 5 independent experiments. (D) Surface plasmon resonance–analysis of AADH binding to immobilized histone H3 with collected data points in blue and data fit in black. Saturation of binding was verified by analysis of the binding data (insert). (E-F) Fluorimaging analysis of phorbol myristate acetate–stimulated human neutrophils stained with propidium iodide (red), FITC-labeled AADH (green), or anti-histone H2B antibody (blue). (E) A detail showing colocalization (yellow, arrow) of histones, DNA, and AADH on extracellular DNA fibers. (F) Representative presentation of a larger field with NETs, where colocalization (yellow) was observed for histones, DNA, and AADH. Arrows indicate several of the colocalization sites. Bar represents 20 µm for histology. Individual images for the composites (E-F) have been added as supplemental figures. Statistical significance (P < .05) was tested using one-way analysis of variance with Dunnett post hoc test. *Indicates significant difference compared with control samples.
