Thrombin receptor activator peptide 6 trap 6

The conjugation of highly purified human fibrinogen with fluorescein isothiocyanate (FITC) via FITC-celite was performed as previously described^{64 (link)}. Sytox-Green was from Molecular Probes and Hoechst33342 from Life Technologies. Anti-CD42a-PE, mouse IgG clone Beb1, anti-CD62P-FITC, PAC-1-FITC, and CD16b were from BD Pharmingen. Anti Mac-1 activation-dependent epitope antibody (CBRM1/5) was from eBioscience. Anti-human defensin antibody was purchased from Hycult Biotech and anti-FDP-lysine antibody was from Abcam. Anti-mouse IgG-FITC (Sigma-Aldrich) was used as secondary antibody. Resveratrol (≥98% trans-3,4′,5-Trihydroxystilben) was purchased from Carl Roth, human antithrombin III from Enzyme Research Laboratories, human serum, taurine, phorbol 12-myristate 13-acetate (PMA), bovine thrombin, ADP, Thrombin Receptor Activator Peptide 6 (TRAP-6) and α1-antitrypsin were from Sigma-Aldrich and reduced glutathione for i.v. injection (Tationil®) was from Roche (Italy). IgG depleted human serum was from Sunnylab. RPMI 1640 medium was from Biochrom. Melagatran (for injection) was from Astrazeneca (Wedel, Germany). Recombinant staphylokinase (SAK) was kindly provided by Dr. Bernhard Schlott (Leibnitz Institute for Age Research, Jena, Germany) and was prepared as previously described^{65 (link)}.

Myricetin, gallic acid, ADP, thrombin, phorbol-12-myristate-13-acetate (PMA), thrombin Receptor Activator Peptide 6 (TRAP-6), human fibrinogen, and 1,4-Dithiothreitol (DTT) and 3,3′-Dihexyloxacarbocyanine iodide (DIOC₆) were purchased from Sigma-aldrich (Dorset, UK). PAPA-NONOate was purchased from Tocris (Abingdon, UK). PE/Cy5 anti human CD62P and PAC-1 FITC antibodies were purchased from BD Biosciences (Wokingham, UK). FITC-conjugated fibrinogen was purchased from Agilent (Stockport, UK). Collagen was purchased from Nycomed (Munich, Germany) whereas Collagen-Related Peptide (CRP) was obtained from Prof Richard Farndale (University of Cambridge, Cambridge, UK). Anti-phospho-vasodilator-stimulated phospho-protein (VASP) (Ser239) was purchased from Cell Signalling (Hitchin, UK), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Proteintech (Manchester, UK), and Alexa-488 conjugated phalloidin secondary antibody was bought from Life Technologies (Paisley, UK)

PMNs (1x10⁶ cells/mL) were seeded in flat-bottomed 96-well plates with DMEM/F-12 medium for 1 hour at 37°C until attached. After 1h, cells were washed with pre-warmed PBS and co-cultured with or without washed platelets (ratio 1 PMN vs. 50 platelets) in the presence of the following stimuli: 10µg/mL Dengue virus-2 non-structural protein-1 (DENV2 NS1, strain Thailand/16681/84; Native Antigen, Oxford, UK) or 156 µM Thrombin Receptor Activator Peptide 6 (TRAP-6) (Sigma-Aldrich, Zwijndrecht, The Netherlands) for 3 hours at 37°C. For induction of NET formation by patient plasma, 10% plasma was added, together with 20μM D-Phe-Pro-Arg-CMK (PPACK, Santa Cruz Biotechnology, Dallas, TX, USA) to inhibit clot formation. Heat-inactivated DENV2 (0.5x10⁷ median tissue culture infectious dose (TCID)/ml) or mock (medium control) were incubated with PMNs in the presence or absence of autologous washed platelets. Next, adherent NETs were digested with 5U/mL micrococcal nuclease (Worthington Biochemical Corporation, Lakewood, NJ, USA) in fresh DMEM/F-12 medium for 20 minutes at 37°C. NETs in the culture supernatant were measured using the assays as outlined below.