Quantitative Eicosanoid Profiling in Platelets

Blood was incubated with vehicle, aspirin (100 µM), the P2Y₁₂ receptor antagonist prasugrel active metabolite (PAM) (3 µM; a gift from AstraZeneca), or aspirin + PAM. Part of the blood was centrifuged (175 g; 15 min) to obtain platelet-rich plasma (PRP). Platelets in both whole blood and PRP were then activated under static conditions (in initial experiments) or under stirring conditions in a light transmission aggregometer by the addition of collagen (30 µg/ml) (Takeda Pharmaceuticals, Deerfield, IL, USA), TRAP-6 (30 µM) (Sigma-Aldrich, St. Louis, MO, USA), or A23187 (50 µM) and incubation for 5 min. Plasma was then quickly separated from the samples by centrifugation (2000 g, 5 min, 4°C) and stored at −80°C for eicosanomic analysis, as previously described (14 (link), 15 (link)). In brief, HyperSep Retain PEP SPE cartridges (Thermo Fisher Scientific, Waltham, MA, USA) were preconditioned with a solution of 0.1% acetic acid/5% methanol and spiked with 30 ng each of internal standards. Plasma (0.25 ml) was diluted in 0.1% acetic acid/5% methanol containing 0.009 mM butylated hydroxytoluene and added to the column. Samples were then washed with two volumes of 0.1% acetic acid/5% methanol, eluted in 1 ml of ethyl acetate and 1 ml methanol, dried by vacuum centrifugation at 37°C, and reconstituted in 30% ethanol. AA-derived metabolites were then separated by reverse-phase HPLC on a 1 × 150 mm, 5 μm Luna C18 (2 (link)) column (Phenomenex, Torrance, CA, USA) and quantified using a MDS Sciex API 3000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, USA) with negative-mode electrospray ionization and multiple reaction monitoring. Data were captured and analyzed using Analyst 1.5.1 software. Relative response ratios of each analyte were used to calculate concentrations, and extraction efficiency for each sample was calculated based on recovery of the internal standards.