Murine TNFα was from Chemicon International (now part of Millipore Corporation, Billerica, MA, USA), human TNFα was from ReliaTech (Wolfenbüttel, Germany), collagen was from Nycomed (now part of Takeda Pharmaceuticals International, Zürich, Switzerland), and anti-human tissue factor-FITC, anti-human P-selectin-RPE, and respective negative controls were from AbD Serotec (Oxford, UK). RNeasy Mini Kit, small interfering RNA (siRNA) against TNFR1 and TNFR2, and Effectene transfection kit were from Qiagen (Hilden, Germany), and predesigned glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TNFR1, TNFR2, plasminogen activator inhibitor 1 (PAI-1), tissue factor, and thrombomudulin TaqMan primers were from Applied Biosystems (Foster City, CA, USA). All other chemicals were from Sigma-Aldrich (Munich, Germany).
Animal experiments were performed in wild-type (WT) or TNFα receptor-deficient C57BL/6 mice. WT mice were purchased from Charles River (Sulzfeld, Germany). TNFR1−/− and TNFR2−/− mice were originally obtained from the Jackson Laboratory (Bar Harbor, ME, USA), and TNFR1−/−R2−/− mice were subsequently generated by cross-breeding. Surgical procedures were performed under short-term anesthesia induced by a single intraperitoneal injection of midazolam 3 mg/kg (Ratiopharm, Ulm, Germany), fentanyl 0.03 mg/kg (CuraMED Pharma, Baden-Württemberg, Germany), and medetomidinhydrochloride 0.3 mg/kg (Pfizer, Berlin, Germany; produced by Orion Pharma, Espoo, Finland) diluted in 0.9% NaCl. After the experiments, the animals were killed by injection of an overdose (2 g/kg) of sodium pentobarbital (Merial, Hallbergmoos, Germany). All experiments were conducted in accordance with the German animal protection law and approved by the district government of Upper Bavaria (approval reference number AZ 55.2-1-54-2531-162-08). The investigation conforms to Directive 2010/63/EU of the European Parliament.
Intravital microscopy in the dorsal skinfold chamber microcirculatory model
The dorsal skinfold chamber microcirculatory model was used in mice as described previously . Animals with an intact microcirculation underwent carotid artery catheterization for application of drugs or injection of isolated platelets, respectively. Intravital fluorescence microscopy was performed by using a modified microscope (Zeiss Axiotech Vario; Carl Zeiss, Oberkochen, Germany). Images were recorded with a digital camera (AxioCam HSm; Carl Zeiss). For all in vivo experiments, TNFα was administered via carotid artery catheter at a dose of 0.4 µg/kg. This was calculated to match plasma levels of approximately 5 ng/mL, which caused effects in vitro.
Intravital assessment of arteriolar thrombosis
Intravital thrombotic vessel occlusion time was assessed in arterioles of WT or TNFα receptor-deficient C57BL/6 mice in the dorsal skinfold chamber model. For induction of intra-arteriolar thrombosis, the ferric chloride superfusion method was used as described previously [23, 24]. Before the experiments, blood vessel flow was digitally recorded and regular blood flow was confirmed for all analyzed arterioles. To visualize vessel lumina before vessel injury, 50 µL of a 5% fluorescein isothiocyanate-labeled dextran solution (FITC-dextran, molecular weight 150,000) was infused via the carotid catheter. Injury to the vascular wall was then performed by application of 30 µL of a ferric chloride solution (25 mmol/L) onto arterioles by using a standardized protocol, and movies were recorded until blood flow ceased.
Mouse platelet isolation and staining for in vivo studies
Whole blood was drawn from anesthetized mice by cardiac puncture. To prevent blood from clotting, syringes contained 10% of sodium citrate. The citrated whole blood was spun at 130g, and the obtained platelet-rich plasma (PRP) was incubated with carboxyfluorescin (carboxyfluorescein diacetate succinimidyl ester 17 µmol/L; Bachem, Bubendorf, Switzerland) in the dark for 30 minutes. Labeled platelets were then spun at 340g and resuspended in a buffered calcium-free physiologic solution (138 mmol/L NaCl, 2.7 mmol/L KCl, 12 mmol/L NaHCO3, 0.4 mmol/L NaH2PO4, 1 mmol/L MgCl2 × 6 H2O, 5 mmol/L D-glucose, 5 mmol/L Hepes; pH 7.35). For centrifugation, iloprost (10 ng/mL; Schering, Berlin-Wedding, Germany) was added to prevent platelet activation. The ability of the isolated and stained platelets to aggregate was tested by platelet aggregometry.
Intravital analysis of platelet-vessel wall interaction
For intravital studies of platelet interaction with the intact vessel wall, isolated and fluorescent-stained murine platelets from a donor animal were injected via a carotid artery catheter and observed in the dorsal skinfold chamber model. Movie sequences of 30 seconds in four- to six-vessel segments in each animal were recorded and analyzed by using AxioVision Software (Carl Zeiss). Vessels with abnormal flow were excluded from analysis. From the resulting length of the platelet trace in single images, velocities of single platelets were calculated by using the exposure time of each single picture. PVWI was expressed in frequency histograms consisting of all platelet velocities analyzed. Histograms were normalized to the maximum platelet speed within a vessel to exclude biasing influences of altered blood flow velocities between different arterioles. As a consequence, a rightward shift in platelet velocity distribution within a histogram expresses less PVWI, whereas a leftward shift signalizes increased PVWI at the arteriolar wall. Platelets with less than 5% of the velocity of the fastest platelets were defined as rolling platelets.
Platelet aggregation was measured by using the turbidimetric method described by Born . Human or murine PRP was obtained by centrifugation (130g) of whole citrated blood drawn from human cubital veins or by cardiac puncture in mice. ADP-, collagen-, or thrombin receptor-activating peptide (TRAP)-induced platelet aggregation was measured photometrically by using a two-channel aggregometer (ChronoLog 490-2D; Chrono-log Corporation, Havertown, PA, USA) under continuous stirring at 1,000 revolutions per minute at 37°C. Written consent was obtained from platelet donors. To assess the effects of endothelial-derived cytokines, 100 µL of supernatant of stimulated human microvascular endothelial cells (HMECs) was added to 300 µL of PRP and incubated for 90 minutes before agonist-induced aggregation was measured.
Human umbilical vein endothelial cells (HUVECs) were isolated and cultured as described previously . The procedure was approved by a university ethics review board. HMECs were provided by Ades and colleagues  and cultured in M199 media supplemented with 10% fetal calf serum, 10% endothelial growth media (PromoCell, Heidelberg, Germany), and 1% penicillin/streptomycin. The investigation conforms to the principles outlined in the Declaration of Helsinki.
Measurement of endothelial superoxide (O2 -)
For superoxide measurements, the cytochrome c reduction method was used as previously described . The O2--dependent part of cytochrome c reduction was calculated from the difference in absorbance (550 nm) between samples incubated with or without superoxide dismutase.
Endothelial surface molecule expression
HMECs or HUVECs were grown as described and incubated with sham or TNFα (5 ng/mL) as indicated. Cells were stained by using anti-p-selectin-RPE and anti-tissue factor-FITC or corresponding RPE- or FITC-labeled negative control. For measuring, a FACSCanto II flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was used. Data were analyzed by using FACSDiva software (Becton, Dickinson and Company).
Real-time polymerase chain reaction in endothelial cells
HMECs were incubated with sham or TNFα for indicated time points. RNA isolation and real-time polymerase chain reaction (PCR) were performed as described previously . Commercially available pre-developed TaqMan reagents were used for the human target genes thrombomodulin, PAI-1, tissue factor, TNFR1, and TNFR2, and GAPDH was used as a reference housekeeping gene. All measurements were performed in duplicates.
Endothelial-dependent blood clotting
Cells were stimulated with TNFα as indicated and then lysed with 15 mM n-octyl-D-glycopyranosidase in 0.1 M imidazol buffer; 20 μL of cell lysate and 20 μL of 200 mmol/L CaCl2 for re-calcification were added to 300 μL of citrated (3.13% sodium citrate) human whole blood from healthy volunteers, and clotting time was measured by thrombelastometry (Rotem; Tem Innovations, Munich, Germany). Stimulation with human recombinant tissue factor was used as a positive control.
Role of autocrine factors
To assess the role of TNFα-induced autocrine factors released by endothelial cells, cells were stimulated with TNFα 5 ng/mL for 3 hours. After removal of the TNFα-containing medium and a washing step, fresh medium without TNFα was added to the cells to allow secretion of prothrombotic factors. After 2 hours, this medium was used to stimulate untreated cells in which several parameters then were measured.
Small interfering RNA knockdown
Endothelial cells were transfected with 50 nM siRNA against human TNFR1 and TNFR2 by using the magnetofection method in combination with the Effectene kit from Qiagen as previously described . Transfected cells were left 48 hours to allow siRNA-mediated knockdown, which was confirmed by real-time PCR and Western blot, which was performed as described elsewhere .
SigmaStat Software (SYSTAT, Chicago, IL, USA) was used to calculate statistical differences. Data were analyzed by using Student t test for normally distributed variables or the Mann-Whitney rank sum test, when normal distribution was not given. Data are expressed as mean ± standard error of the mean. Differences were considered significant when the error probability level was a P value of less than 0.05.