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/4×107 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.
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Research Article|
September 14 2010
Thrombin-activated human platelets acutely generate oxidized docosahexaenoic-acid-containing phospholipids via 12-lipoxygenase
Lloyd T. Morgan;
Lloyd T. Morgan
*Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff CF14 4XN, U.K.
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Christopher P. Thomas;
Christopher P. Thomas
*Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff CF14 4XN, U.K.
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Hartmut Kühn;
Hartmut Kühn
†Institute of Biochemistry, Humboldt University, 10115 Berlin, Germany
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Valerie B. O'Donnell
Valerie B. O'Donnell
1
*Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff CF14 4XN, U.K.
1To whom correspondence should be addressed (email o-donnellvb@cardiff.ac.uk).
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Publisher: Portland Press Ltd
Received:
March 22 2010
Revision Received:
July 22 2010
Accepted:
July 23 2010
Accepted Manuscript online:
July 23 2010
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem J (2010) 431 (1): 141–148.
Article history
Received:
March 22 2010
Revision Received:
July 22 2010
Accepted:
July 23 2010
Accepted Manuscript online:
July 23 2010
Citation
Lloyd T. Morgan, Christopher P. Thomas, Hartmut Kühn, Valerie B. O'Donnell; Thrombin-activated human platelets acutely generate oxidized docosahexaenoic-acid-containing phospholipids via 12-lipoxygenase. Biochem J 1 October 2010; 431 (1): 141–148. doi: https://doi.org/10.1042/BJ20100415
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