Initially, prostaglandins (PGs) were considered to only exist as free acid mediators. Although, formation of PG glycerol esters and PG ethanolamides by cellular cyclooxygenase (COX)-2 has been reported, generation of complex oxidised lipids via COX-1 has not been considered. In this study, formation of sixteen unique PG-containing phospholipids generated by agonist-activated human platelets is demonstrated using lipidomic approaches. Precursor scanning-tandem mass spectrometry identified a group of specific lipids comprising PGE2, PGD2 and two previously undescribed PG-like molecules (named PGb and PGc), attached to four phosphatidylethanolamine (PE) phospholipids (16:0p/, 18:1p/, 18:0p/ and 18:0a/). PGb and PGc were also detected as free eicosanoids and their structures remain to be characterised. These novel lipids formed within 2-5 minutes of platelet activation by thrombin, collagen or ionophore and required activation of several intracellular signalling intermediates, including cytosolic phospholipase A2 (cPLA2), p38 mitogen-activated protein kinase (MAPK), src tyrosine kinases, phospholipase C (PLC) and cytosolic calcium. Unlike free PGs that are secreted, PG-PEs remain cell associated, suggesting an autocrine mode of action. Aspirin supplementation in vivo (75 mg/day) or in vitro (1 mM) blocked their generation, indicating that COX-1 is required. Pharmacological studies using inhibitors of fatty acyl re-esterification significantly reduced formation of PG-PEs. Furthermore, purified COX-1 was unable to directly oxidise PE in vitro. Collectively, these indicate that PG-PEs are initially formed as free PGs via COX-1, and then rapidly esterified into PEs. In summary, this is the first demonstration of acute generation of PG-PEs in agonist-activated human platelets from endogenous substrate via COX-1. These unique lipids may represent additional bioactive molecules from this key platelet enzyme.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:611008 |
| Date | January 2013 |
| Creators | Aldrovandi, MacEler |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/60146/ |
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