Thrombin-activated human platelets acutely generate oxidized docosahexaenoic-acid-containing phospholipids via 12-lipoxygenase.
Arachidonate-containing oxidized phospholipids are acutely generated by 12-LOX (12-lipoxygenase) in agonist-activated platelets. In the present study, formation of structurally related lipids by oxidation of DHA (docosahexaenoic acid)-containing phospholipids is demonstrated using lipidomic approaches. Precursor scanning reverse-phase LC (liquid chromatography)-MS/MS (tandem MS) identified a new family of lipids that comprise phospholipid-esterified HDOHE (hydroxydocosahexaenoic acid). Two diacyl and two plasmalogen PEs (phosphatidylethanolamines) containing predominantly the 14-HDOHE positional isomer (18:0p/14-HDOHE-PE, 18:0a/14-HDOHE-PE, 16:0a/14-HDOHE-PE and 16:0p/14-HDOHE-PE) were structurally characterized using MS/MS and by comparison with biogenic standards. An involvement of 12-LOX was indicated as purified recombinant human 12-LOX also generated the 14-HDOHE isomer from DHA. Pharmacological studies using inhibitors and recombinant platelet 12-LOX indicate that they form via esterification of newly formed non-esterified HDOHE. HDOHE-PEs formed at significant rates (2-4 ng/4×10(7) cells) within 2-180 min of thrombin stimulation, and their formation was blocked by calcium chelation. In summary, a new family of oxidized phospholipid was identified in thrombin-activated human platelets.
Morgan
Lloyd T
LT
Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK.
Thomas
Christopher P
CP
Kühn
Hartmut
H
O'Donnell
Valerie B
VB
D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D001792:Blood Platelets / Q000201:enzymology* / Q000378:metabolism; D004281:Docosahexaenoic Acids / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*
D010714:Phosphatidylethanolamines; D010743:Phospholipids; D011994:Recombinant Proteins; D004281:Docosahexaenoic Acids; D001092:Arachidonate 12-Lipoxygenase; D013917:Thrombin