Sequences and secondary structures of DNA- and RNA-oligonucleotides.

<p>Described are the secondary structures of (A) DNA- and (B) RNA-oligonucleotides as predicted by the mFold DNA or RNA database. Delta G (ΔG) values represent changes in free enthalpy representative for the stability of the compounds with negative (exergonic) or positive values (endergonic).</p

Stability of DNA- and RNA-oligonucleotides in human plasma.

<p>(A) Integrity of 21mer-L1 and (B) 21mer-H1 DNA- and RNA-oligonucleotides was confirmed by polyacrylamide gel electrophoresis without or after preincubation in pooled human plasma for 1 or 5 min, respectively. Each panel represents one representative experiment out of three independent ones.</p

Binding of biotinylated DNA-oligonucleotides to different coagulation factors of the intrinsic coagulation pathway.

<p>Microtiter plate wells were coated with 10 µg/mL each of (A) kininogen or (B) prekallikrein and binding of increasing concentrations of the biotinylated DNA-oligonucleotides 21mer-H1 (closed circles), 21mer-H3 (open triangles), 21mer-L1 (closed squares) was assessed. All data represent mean ± SD (n = 3; *p<0.05; 21mer-L1 and 21mer-H3 vs. 21mer-H1) of one representative experiment out of three independent ones. (C) Microtiter plate wells were coated with 10 µg/mL kininogen, factor XI (FXI) or factor XII (FXII) each and incubated with 25 µg/mL each of different biotinylated DNA-oligonucleotides: 21mer-H1 (black bars), 21mer-H3 (white bars) or 21mer-L (hatched bars). All data represent mean ± SD (n = 3) of one representative experiment out of three independent ones. (D) Increasing concentrations of the biotinylated DNA-oligonucleotides 21mer-H1 (closed circles), 21mer-H3 (open triangles) or 21mer-L1 (closed squares) were analyzed for prekallikrein auto-activation. All data represent mean ± SEM (n≥3; *p<0.05; 21mer-H1 vs. 21mer-H3).</p

Influences of DNA-aptamers on the intrinsic coagulation pathway.

<p>(A) The activation of prekallikrein was followed in the presence of increasing doses of the DNA-aptamers 15mer-thrombin (open circles, interrupted line), 44mer-APC (closed squares), 26mer-AS1411 (closed circles) or 25mer-VEGF (closed triangles, dotted line). (B) Turbidity clot-lysis assays were performed in the absence (black bars) or presence of 1.25 µg/mL (white bars) and 10 µg/mL (striped bars) of the DNA-aptamers 15mer-thrombin, 44mer-APC, 26mer-AS1411 or 25mer-VEGF, respectively. Coagulation was initiated by recalcification, clotting times were defined as respective time points of maximal absorbance. The clotting time of untreated plasma was defined as 100%. All data represent mean ± SEM (n = 3; *p<0.05; 1.25 µg/mL or 10 µg/mL vs. control). (C) The activation of prekallikrein was followed in the presence of increasing doses of the oligonucleotide 21mer-H1 (closed circles) and 21mer-H1-HEG (closed squares). All data represent mean ± SEM (n = 6).</p

Procoagulant activity of snRNAs.

<p>(A) Increasing concentrations of U6snRNA (closed triangles, black line) or poly (I:C) (closed diamonds, dotted line) were analyzed for prekallikrein auto-activation. All data represent mean ± SEM (n = 3). (B) Integrity of U6snRNA was confirmed by agarose gelelectrophoresis.</p

Procoagulant activity of different linear and hairpin-forming DNA-oligonucleotides.

<p>Increasing concentrations of the linear DNA-oligonucleotides 21mer-L1 (closed squares), 21mer-L2 (closed triangels) or 21mer-L3 (closed circles) were analyzed for (A) prekallikrein auto-activation or (B) procoagulant activity in a turbidity clot-lysis assay. The clotting time of untreated plasma was defined as 100%. All data represent mean ± SEM (n = 3). Increasing concentrations of the hairpin-forming DNA-oligonucleotides 21mer-H1 (closed circles), 21mer-H2 (open squares) or 21mer-H3 (open triangles) were analyzed for (C) prekallikrein auto-activation or (D) procoagulant activity in a turbidity clot-lysis assay. The clotting time of untreated plasma was defined as 100%. All data represent mean ± SEM (n≥3; *p<0.05; 21mer-H1 vs. 21mer-H3; #p<0.05; 21mer-H2 vs. 21mer-H3). (E) The sizes of DNA-oligonucleotides were analyzed by polyacrylamide gel electrophoresis. Shown is one representative experiment out of three independent ones.</p

APC decreases epithelial cytotoxicity induced by histones but not by NET.

<p>(A) Histones (200 or 100 µg/ml), pre-incubated for 1 h at 37°C in the absence or presence of 100 nM human APC, were incubated with A549 cells for 16 h, followed by analysis of cytotoxicity. (B) NET were incubated with APC (mass ratio APC: NET proteins, 1∶5, 1∶2 and 1∶1) or without APC for 1 h at 37°C, followed by incubation with A549 cells for 16 h and measurement of cytotoxicty. APC alone or active-site blocked APC (APC+PPACK) were incubated with A549 cells for control. (C) DNase-digested and (D) undigested forms of NET were pre-incubated with 100 nM APC for 20 to 80 min before incubation with A549 cells for 16 h, followed by determination of cytotoxicty. Shown are representative data of four independent experiments (mean SD), ***<i>p</i><0.001 and ns = non-significant.</p

NET induce cytotoxicity in epithelial and endothelial cells independent of digestion.

<p>(A) The extent of cytotoxicity was measured after treatment of A549 cells for 16 h with undigested NET (−), completely (DNase), partially digested (MNase) or boiled forms of NET. The same concentration of DNA alone as DNA-NET (3.4 µg/ml) as well as DNase or MNase alone were used as controls. Shown are representative data of five independent experiments (mean SD), ***<i>p</i><0.001, and ns = non-significant. (B) The degree of cytotoxicity was measured after treatment of HUVEC, HPAEC, AT-II or MLE-12 cells for 16 h with undigested NET (−) or completely (DNase) forms of NET as well as DNA alone. Shown are representative data of three (except for AT-II, n = 2) independent experiments (mean SD), ns = non-significant.</p

Time-dependent cytokine expression from macrophages treated with eRNA/Pam2CSK4.

<p>Following preincubation of eRNA (10 μg/ml) and Pam2CSK4 (0.1 ng/ml) for 1 h at 37°C, macrophages were treated with these combined agonists (eRNA+Pam2CSK4), eRNA, Pam, or buffer for different time periods as indicated. Supernatants of cells were analyzed for the release of TNF-α (A) and IL-6 (B) by ELISA. mRNA expression of TNF-α (C), IL-6 (D), IL-1β (E), and MCP-1 (F) was assessed from cell lysates by qRT-PCR. Values are expressed as mean ± SEM; N = 3; *P < 0.05 versus buffer-treated group.</p

Histones induce epithelial and endothelial cell death.

<p>(A) A549 cells were treated for 16 h with different concentrations of histone type II-A, and the cell morphology was evaluated. (B) A549 cell numbers were counted after treatment with various concentrations of histones for 16 h. (C) HUVEC or A549 cells were treated with 200 µg/ml histones for 16 h or left untreated (control). (D) HUVEC were treated for 16 h with different concentrations of histones, and the extent of cytotoxity was measured. B and D are representative data of three independent experiments, and in A and C pictures are representative pictures from three independent experiments at 20× magnification.</p