Points Nongenomic role for IκB kinase in platelet secretion: IKK phosphorylates

Points Nongenomic role for IκB kinase in platelet secretion: IKK phosphorylates SNAP-23 which affects granule-plasma membrane Scriptaid fusion. Given this central role understanding platelet secretion should identify potential therapeutic targets for modulating spurious thrombosis and ameliorating platelet-based bleeding disorders. Exocytosis is mediated by membrane proteins called Soluble for 10 minutes at room temperature TSPAN2 (RT). Platelet-rich plasma (PRP) was recovered and platelets were pelleted at 483 × for 10 minutes at RT. The pellets were resuspended in HEPES/Tyrode buffer (HT; 20 mM HEPES/KOH pH 6.5 128 mM NaCl 2.8 mM KCl 1 mM MgCl2 0.4 mM NaH2PO4 12 mM NaHCO3 5 mM d-glucose) supplemented with 1 mM EGTA 0.37 U/mL apyrase and 10 ng/mL PGI2. Platelets were washed and resuspended in HT (pH 7.4) without EGTA apyrase or PGI2. Platelets were counted with a Z2 Coulter Particle Analyzer (Beckman/Coulter Fullerton CA) and adjusted to the indicated concentrations. Washed human platelets were prepared as described in Karim et al.31 PRP was isolated in the presence of apyrase (0.37 U/mL) and PGI2 (10 ng/mL) Scriptaid by centrifugation at 150 × for 10 minutes at RT. PRP was centrifuged at 900 × for 10 minutes and platelets were resuspended in HT containing 1 mM EGTA apyrase and PGI2. Platelets were washed and resuspended in HT (pH 7.4) without EGTA apyrase or PGI2. Measurement of platelet granule cargo release Platelets were labeled with 0.4 μ Ci/mL [3H]5-HT (serotonin; Perkin-Elmer Waltham MA) for 1 hour at RT. After washing the platelets were resuspended in HT (pH 7.4) and CaCl2 (0.7 mM final) prior to stimulation with thrombin (0.05 U/mL; Chrono-log) for the indicated times. Hirudin (0.1 U/mL; Sigma-Aldrich) was added to stop the reaction. Platelets were incubated with BMS-345541 (5 μM) or TPCA-1 (0.5 μM) prior to stimulation. The samples were separated by centrifugation at 13 800 × for 1 Scriptaid minute the supernatants were recovered and the pellets were lysed with 1% Triton X-100 in phosphate-buffered saline. Equal volumes of both Scriptaid fractions were assayed for [3H]5-HT (serotonin) for dense granules PF4 for α-granules and β-hexosaminidase for lysosomes as described in Schraw et al.28 32 Preparation of SNARE-containing proteoliposomes All lipids were from Avanti Polar Lipids (Alabaster AL). Reconstitution of v-SNARE and t-SNARE vesicles was as described in Tucker et al.33 v-SNAREs were reconstituted using a mix of 27% 1-palmitoyl-2-oleoyl-phosphatidylethanolamine 55 1 2 phosphatidylcholine 15 1 2 phosphatidylserine 1.5% N-(7-Nitro-2-1 3 (NBD)-1 2 phosphatidylethanolamine (NBD-PE donor) and 1.5% N-(lissamine rhodamine B sulfonyl)-1 2 phosphatidylethanolamine (Rhodamine-PE acceptor). t-SNAREs were reconstituted in 30% palmitoyl-2-oleoyl-phosphatidylethanolamine 55 phosphatidylcholine and 15% phosphatidylserine v-SNARE (VAMP-8) and t-SNARE (SNAP-23 + syntaxin-2) vesicles were reconstituted to give ~60 copies and ~95 copies per vesicle respectively. Syntaxin-2 was a surrogate for syntaxin-11 as we cannot produce recombinant syntaxin-11 with the appropriate acylation. For fusion assays 10 μL t-SNARE vesicles were incubated with 0.5 mM ATP 10 mM MgCl2 and increasing IKK (0-2.0 μg/reaction) at RT. v-SNARE vesicles were incubated separately at RT. After 60 minutes v-SNARE and fifty percent from the t-SNARE vesicles had been combined in 25 mM HEPES pH 7.4 100 mM KCl 1 mM fusion and dithiothreitol was monitored at 37°C. Calcium mineral (1 mM last) was added at t = 20 mins. The upsurge in NBD fluorescence was assessed utilizing a Bio-TEK FLx800 Microplate Fluorescence Audience (Bio-Tek U.S. Winooski VT) and KC4 software program with data acquisition every 1.five minutes. After 60 mins 15 μL of 5% n-dodecyl-β-d-octylglucoside was put into obtain the optimum fluorescence. Fusion was plotted as the percent of optimum fluorescence as time passes. Some aliquot of t-SNAREs had been examined by immunoblotting using anti-phospho-Ser95 and anti-SNAP-23 antibodies. For inhibitor research 5 μM BMS-345541 or 0.5 μM TPCA-1 had been put into the t-SNARE mixture including 1.0 μg fusion and IKK was supervised after 60 minutes. Immunoblotting Platelet proteins had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and used in.