Pun at 130g, and the obtained platelet-rich plasma (PRP) was PubMed ID…

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 NaH 2 PO 4 , 1 mmol/L MgCl 2 ?6 H 1-(Cyclopropylsulfonyl)-1,4-diazepane 2 O, 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 interactionPlatelet aggregation was measured by using the turbidimetric method described by Born [25]. Human or murine PRP was obtained by centrifugation (130g) of whole citrated blood drawn from human cubital 3-Bromo-5,6-dihydro-1,6-naphthyridin-5-one veins or by cardiac puncture in mice. ADP-, collagen-, or thrombin receptor-activating peptide (TRAP)-induced platelet aggregation was measured photometrically by using a twochannel aggregometer (ChronoLog 490-2D; Chrono-log Corporation, Havertown, PA, USA) PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/10572343 under continuous stirring at 1,000 revolutions per minute at 37 . Written consent was obtained from platelet donors. To assess the effects of endothelial-derived cytokines, 100 of supernatant of stimulated human microvascular endothelial cells (HMECs) was added to 300 of PRP and incubated for 90 minutes before agonist-induced aggregation was measured.Cell cultureHuman umbilical vein endothelial cells (HUVECs) were isolated and cultured as described previously [26]. The procedure was approved by a university ethics review board. HMECs were provided by Ades and colleagues [27] 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 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 sixvessel 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 maximumFor superoxide measurements, the cytochrome c reduction method was used as previously described [28]. 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 expressionHMECs or HUVECs were grown as described and incubated with sham or TNFa (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 s.