Figure 1.
Figure 1. Procoagulant activities of DNA, polyP, and silica particles. (A-D) Shown are plasma clot times versus concentration, with horizontal dashed lines showing the mean clot time without activator (± standard error of the mean as horizontal dotted lines). (A) Clot times with: HEK 293 cell DNA isolated with DNeasy Blood & Tissue (open diamond); HEK 293 cell DNA isolated with phenol/chloroform (solid diamond); NET-derived DNA isolated with DNeasy Blood & Tissue kit (open inverted triangle); or NET-derived DNA isolated with phenol/chloroform (solid inverted triangle). Each data set represents the mean clot time for 3 separate purifications as detailed in supplemental Figure 2A (HEK293 cell DNA) and supplemental Figure 2B (NET DNA). (B) Clot times with silica particles: glass milk (solid triangle) or homogenized DNeasy column matrix (open triangle). (C) Clot times with water elutions from 3 different lots of DNeasy columns (open triangle) or 2 different lots of Econospin columns (open circle). On the x-axis, fold concentration refers to the concentration relative to the volume eluted from the Qiagen column, with 1 equaling the original elution volume. (D) Clot times of λ phage DNA before (blue square) or after (open square) repurification on DNeasy columns; or of short-chain polyP before (blue circle) or after (open circle) repurification on DNeasy columns. The purification data sets represent mean clot times for 2 different purifications as detailed in supplemental Figure 4. (E) Clot times of various samples before (red bars) or after (blue bars) digestion with a combination of Benzonase and calf intestine alkaline phosphatase. Samples include buffer control (no activator), 20 µg/mL HEK 293 cell DNA purified using DNeasy, 20 µg/mL NET DNA purified using DNeasy, water applied to DNeasy column as described in panel C (water elution), or 2 µg/mL long-chain polyP. Digestion of DNA by Benzonase and polyP by phosphatase was confirmed by gel electrophoresis (supplemental Figure 1). (F) Clot times of various samples before (red bars) or after (blue bars) acid hydrolysis. Samples include buffer control (no activator), 20 µg/mL HEK 293 cell DNA purified using DNeasy, 20 µg/mL NET DNA purified using DNeasy, water applied to DNeasy column (water elution), 2 µg/mL long-chain polyP, or 5 µg/mL polyguanylate (polyG). (G) Clot times in the presence of varying concentrations of histones with (open triangle) or without (solid triangle) a 10-fold concentrated water elution (prepared as described for panel C). (H) Tissue factor–triggered clot times in the presence of varying concentrations of histones.

Procoagulant activities of DNA, polyP, and silica particles. (A-D) Shown are plasma clot times versus concentration, with horizontal dashed lines showing the mean clot time without activator (± standard error of the mean as horizontal dotted lines). (A) Clot times with: HEK 293 cell DNA isolated with DNeasy Blood & Tissue (open diamond); HEK 293 cell DNA isolated with phenol/chloroform (solid diamond); NET-derived DNA isolated with DNeasy Blood & Tissue kit (open inverted triangle); or NET-derived DNA isolated with phenol/chloroform (solid inverted triangle). Each data set represents the mean clot time for 3 separate purifications as detailed in supplemental Figure 2A (HEK293 cell DNA) and supplemental Figure 2B (NET DNA). (B) Clot times with silica particles: glass milk (solid triangle) or homogenized DNeasy column matrix (open triangle). (C) Clot times with water elutions from 3 different lots of DNeasy columns (open triangle) or 2 different lots of Econospin columns (open circle). On the x-axis, fold concentration refers to the concentration relative to the volume eluted from the Qiagen column, with 1 equaling the original elution volume. (D) Clot times of λ phage DNA before (blue square) or after (open square) repurification on DNeasy columns; or of short-chain polyP before (blue circle) or after (open circle) repurification on DNeasy columns. The purification data sets represent mean clot times for 2 different purifications as detailed in supplemental Figure 4. (E) Clot times of various samples before (red bars) or after (blue bars) digestion with a combination of Benzonase and calf intestine alkaline phosphatase. Samples include buffer control (no activator), 20 µg/mL HEK 293 cell DNA purified using DNeasy, 20 µg/mL NET DNA purified using DNeasy, water applied to DNeasy column as described in panel C (water elution), or 2 µg/mL long-chain polyP. Digestion of DNA by Benzonase and polyP by phosphatase was confirmed by gel electrophoresis (supplemental Figure 1). (F) Clot times of various samples before (red bars) or after (blue bars) acid hydrolysis. Samples include buffer control (no activator), 20 µg/mL HEK 293 cell DNA purified using DNeasy, 20 µg/mL NET DNA purified using DNeasy, water applied to DNeasy column (water elution), 2 µg/mL long-chain polyP, or 5 µg/mL polyguanylate (polyG). (G) Clot times in the presence of varying concentrations of histones with (open triangle) or without (solid triangle) a 10-fold concentrated water elution (prepared as described for panel C). (H) Tissue factor–triggered clot times in the presence of varying concentrations of histones.

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