Dornase alfa

Kaolin was obtained from Sigma Aldrich. The activated partial thromboplastin time (aPTT) reagent Dade^® Actin^® FS containing ellagic acid and soybean phospholipids and Pathromtin SL^® were purchased from Siemens Healthcare Diagnostics. FXII contact activators STA^® - C.K. Prest^® and STA^® - Cephascreen^® were obtained from Diagnostica Stago (Asnières, France), while SynthAFax^®, SynthASil^® and APTT SP reagent were purchased from IL Werfen (München, Germany). The following synthetic polyphosphates with different lengths were obtained from ICL Pharmaceuticals: ammonium polyphosphate (P42, #2 2846), ammonium polyphosphate (P30, #2 2840), sodium polyphosphate (P75, #7 9990), sodium polyphosphate (P70, #7 1480), sodium polyphosphate (P68, #7 1480). Dornase alfa, the recombinant human DNaseI (Pulmozyme^®), was purchased from Roche. FXII purified from human plasma was obtained from Haematologic Technologies. The FXIIa-specific inhibitor rHA-Infestin-4 was supplied by CSL Behring. Thrombin generation assays were performed with the fluorogenic substrate Z-Gly-Gly-Arg-AMC, purchased from Bachem Bubendorf, Switzerland. The thrombin calibrator (α2-macroglobulin-thrombin complex, α2M-T) and MP reagent (containing phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine at a ratio of 20:60:20) were obtained from Diagnostica Stago.

Total DNase activity was measured using single radial enzyme diffusion technique as previously described [22 (link)], with modifications. Salmon testes DNA (Sigma-Aldrich, D1626-1G) was dissolved at a concentration of 100 µg/ml in assay buffer containing divalent cations and a DNA-binding fluorescent dye (35 mM Tris–HCl, pH 7.8, 20 mM MgCl₂, 2 mM CaCl₂, 2.5 × SYBR Safe [Invitrogen, S33102]) as substrate for DNases. After heating the solution to 50 °C for 10 min, an equal volume of 2% ultra-pure agarose (Invitrogen, 16500–500) was added. The mixture was poured into a plastic tray to allow solidification. Then, 2 µl sample or standard were loaded into wells with a diameter of 1 mm and incubated at 37 °C for 20 h. Remaining fluorescence of gels was recorded using Biorad ChemiDoc XRS + fluorescence scanner. DNase activity was calculated according to a standard curve (Dornase alfa, Roche). DNase activity was measured by an investigator blinded to genotypes.

Total DNase activity was measured using a single radial enzyme diffusion (SRED) assay as previously published [7 (link)]. An example is shown in Appendix A, Figure A1. Salmon testes DNA serving as substrate for DNases (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in assay buffer [35 mM Tris–HCl, 20 mM MgCl2, 2 mM CaCl2, 2.5 × SYBR Safe (Invitrogen), pH 7.8] at a concentration of 100 µg/mL. The solution was kept at 50 °C for ten minutes, then mixed 1:2 with 2% ultra-pure agarose (Invitrogen) and poured into plastic trays. After solidification, 2 µL sample or standard (Dornase alfa; Roche, Basel, Switzerland) were loaded into wells of 1 mm diameter. Gels were incubated for ten hours at 37 °C. At six and ten hours, remaining fluorescence was recorded with a Fusion FX imaging system (Vilber, Marne-la-Vallée, France). DNase activity of samples was compared to a six-point standard curve with a detection limit of 0.78 mU/mL.