Horm collagen

Anandamide (Arachidonylethanolamide; Sigma-Aldrich, St Louis, MO) was dissolved in dimethylsulfoxide (DMSO; Merck Chemicals Internationals, Darmstadt, Germany) and stored at −20°C. Thrombin receptor activating peptide (TRAP; SFLLRN-trifluoroacetate salt) specific for proteinase-activated receptor (PAR-1) was purchased from Bachem (Bubendorf, Switzerland), cross-linked collagen related peptide (CRP-XL) was a generous gift from Prof. dr. R. Farndale (Cambridge, UK). Horm Collagen (Kollagenreagens Horm suspension) was purchased by Nycomed (Linz, Austria); human α-thrombin and human fibrinogen from Kordia (Leiden, The Netherlands); H-D-Phe-Pro-Arg-chloromethylketone (PPACK) from Santa Cruz Biotechnology (Santa Cruz, CA); pentasaccharide (Arixtra) from GlaxoSmithKline (Greenford, UK); ADP from Sigma (Saint Louis, MO). U46619 was purchased from Cayman Chemical (Ann Arbor, MI). URB597 was purchased from Enzo Life Science GMbH (Lörrach, Germany) and indomethacin from Sigma (Saint Louis, MO). Glass coverslips for perfusion studies were from Menzel Glaser (Braunschweig, Germany). Human serum albumin (Immuno fraction V) was purchased from MP Biomedicals, Amsterdam, The Netherlands.

Horm collagen and collagen diluent were purchased from Nycomed (Munich, Germany). CRP (ten glycine-proline-hydroxproline [GPO] repeats) was crosslinked as described [26]. Rhodocytin was purified in the Eble lab (University of Münster, Germany) from the crude venom of Calloselasma rhodostoma. The mouse monoclonal antibodies (mAbs) anti-phosphotyrosine clone 4G10 (05–321) and rabbit polyclonal anti-FcR γ-chain (06–727) were purchased from Merck Millipore (Watford, UK). The rabbit polyclonal antibody anti-Syk (sc-1077), the mouse mAbs anti-Syk 4D10 (sc-1240) and anti-FcR γ-chain (sc-390222) were purchased from Santa Cruz (Wembley, UK). All other reagents including losartan, honokiol and the anti-mouse IgG (Fc specific) F(ab′)₂ fragment antibody were purchased from Sigma-Aldrich (Poole, UK), or came from described sources [3]. losartan was dissolved in water and honokiol in DMSO. The mouse monoclonal mAb IV.3 against the low affinity immune receptor FcγRIIA was purified from the hybridoma obtained from the American Type Culture Collection. 1G5-Fab against Pan-GPVI was gift from Elizabeth Gardiner (Australian National University, Canberra, Australia).

Heparin-anticoagulated whole blood was incubated with vehicle (PBS), Horm collagen
(Nycomed, St. Peter, Austria), thrombin receptor-activating peptide (TRAP)-6 amide
(Bachem, Heidelberg, Germany), Ca²⁺ ionophore, A23187 (Sigma-Aldrich,
Poole, United Kingdom), for 30 min, or with LPS (Sigma-Aldrich), triacylated
lipoprotein CSK4 (Pam3CSK4; InvivoGen, Toulouse, France), bisacylated lipoprotein
CGDPKHPKSF (FSL-1; InvivoGen), polyinosinic:polycytidylic acid [poly(I:C);
Sigma-Aldrich], or IL-1β (Invitrogen, Life Technologies, Paisley, United
Kingdom) for 18 h in the presence or absence of diclofenac (10 μM;
Sigma-Aldrich). Levels of (C-X-C motif) ligand-8 (CXCL8; R&D Systems,
Abingdon, United Kingdom), PGE₂ (Cisbio, Saclay, France), or
TXB₂ (Cayman Chemical, Cambridge Bioscience, Cambridge, United Kingdom)
were measured by immunoassay or total eicosanoids by gas chromatography–tandem
mass spectrometry (see below) in the conditioned plasma.

The characteristics of the flow chamber used in this study are described in our previous paper [4 (link)]. The flow chamber used was a transparent, polycarbonate-made, parallel-plate type (height, 50 μm; width, 3 mm; length, 30 mm) and was equipped with a metal (medical steel) inlet and an outlet installed at an angle of 15° in relation to the flow axis. Disposable coverslips (0.2 mm thick, degreased in a mixture of 2 M HCl and 50% ethanol) were used to create a detachable bottom for the chamber. To facilitate the formation of the thrombus under flow conditions, the coverslips were coated with a microspot of type I collagen suspension (diluted with 1.67 mM acetic acid to a concentration of 50 μg/mL, Horm collagen; Nycomed, Zurich, Switzerland). The spot had a diameter of 1 mm. After overnight incubation at 4 °C in a humid chamber, the coverslips were washed with saline (to remove unbound collagen) and blocked for 30 min with 1% (w/v) bovine serum albumin (BSA) in Hepes buffer (138 mM NaCl, 2.8 mM KCl, 8.9 mM NaHCO₃, 0.8 mM KH₂PO₄, 0.8 mM MgCl₂, 5.5 mM glucose, 3.5 mg/mL albumin, and 10 mM HEPES, pH 7.4) in order to minimize nonspecific interactions of blood constituents with glass. After blocking, the coverslips were rinsed again with saline before the experiment. Then, the chamber was assembled and filled with Hepes buffer containing 0.1% BSA and glucose (5.5 mM).

FluoZin3/AM (F24195, Invitrogen), N,N,N′,N′-tetrakis 2-pyridylmethyl ethylene diamine (TPEN) (ALX-450-011-M100, Enzo). Horm collagen (11207690, Nycomed), tissue factor (TF) was from Innovin (B4212-40, Dade-Behring, Marburg, Germany). Membrane dye DiOC₆ was from AnaSpec. AF568-annexin A5 was from Life Technology (A13202, Eugene, USA) and AF647-labeled fibrinogen from Molecular Probes (F35200, Life Technology). Human fibrinogen (F4883) was from Sigma-Aldrich and thrombin from (10602400001) was from Roche Diagnostics. Phalloidin-AF647 (65906) was from Sigma-Aldrich. NucleoSpin®RNA II Kit was from Macherey-Nagel. High Capacity cDNA RT Kit was from Applied Biosystems and FastStart Universal SYBR Green Master Mix was from Roche. All reagents were bought from German suppliers unless otherwise stated.