Identification of novel oxidized phospholipids that activate platelet-activating factor receptor using HPLC fractionation and comprehensive LC-MS/MS analysis

Platelet-activating factor receptor (PAFR) is involved in various physiological processes, including the immune system and inflammatory responses. In addition to PAF, several oxidized phospholipids have been shown to act as ligands for PAFR. We have previously developed a comprehensive analysis method for oxidized phospholipids, and in this study, we employed this method to test whether additional oxidized phospholipids can activate PAFR. From an oxidized phosphatidylcholine mixture, we identified that 1-palmitoyl-2-(4'-oxo-butanoyl)-sn-glycero-3-phosphocholine (POBPC) functions as a novel PAFR activator, using preparative HPLC and comprehensive LC-MS/MS analysis of fractionated oxidized phospholipids. Next, multiple assays confirmed that POBPC acts as a bona fide PAFR agonist. The H248W mutation of PAFR attenuated the response to POBPC. Finally, POBPC induced phosphorylation of extracellular signal-regulated kinase in mouse peritoneal macrophages, which endogenously express PAFR. Our findings provide valuable insight into the biological functions of oxidized phospholipids, advancing our understanding of their roles in cellular processes.