Fig. 4.

DXA₃-PE formation requires the activity of PLC, calcium, cPLA₂ and COX-1, and occurs via esterification of free DXA_3.Panels A-C Effects of signaling inhibitors on DXA₃formation. Platelets were incubated with inhibitors 10 min prior to thrombin activation (0.2 U/ml for 30 min at 37 °C). Lipids were extracted and analyzed using LC-MS/MS monitoring precursor [M-H]^-m/z 351.2 → 165.1, as described in Methods. Data are representative of experiments repeated at least three times on different donors (n=3, mean±SEM). *** P<0.001 versus thrombin, using ANOVA and Bonferroni Post Hoc Test. Panel A. U-73112, 10 µM (PLC), or its negative control U-73343. Panel B. wortmannin, 100 nM (PI3 kinase), Gö 6850 (protein kinase C), 100 nM (PKC) or vehicle (DMSO, 0.5%), Panel C. EGTA 1 mM (extracellular Ca²⁺) or BAPTA-AM 10 mM (intracellular Ca²⁺). Panels D,E. Generation of DXA₃-PE is sensitive to COX inhibition in vitro. Platelets were incubated with 1 mM aspirin, 1 μM SC-560 or 10 μM indomethacin prior to thrombin activation (0.2 U/ml for 30 min at 37 °C). Lipids were extracted and analyzed as described in Methods. Levels are expressed as ratio analyte to internal standard. Data are representative of experiments repeated at least three times on different donors (n=3, mean±SEM). Panel F. In vivo aspirin supplementation blocks generation of DXA₃-PE. Lipids were analyzed following thrombin activation of washed platelets, before or after supplementation with 75 mg/day aspirin for 7 days. Data are representative of five independent donors (n=5, mean±SEM); ***p<0.001 versus thrombin alone, using ANOVA and Bonferroni Post Hoc Test. Levels of DXA₃-PEs are expressed as ratio analyte to internal standard. A/IS: analyte:internal standard.