Thrombin-activated human platelets acutely generate oxidized docosahexaenoic-acid-containing phospholipids via 12-lipoxygenase

Abstract

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.

Esterified eicosanoids are acutely generated by 5-lipoxygenase in primary human neutrophils and in human and murine infection

“…This is similar to previous observations for 12-LOX-derived esterified lipids generated by human platelets. 9,29 In addition, a previous report showed that, although 15-HETE is avidly esterified into neutrophil PLs, little 5-HETE is incorporated. 13 However, we note that these results were obtained with in vitro approaches, and in the future, if it becomes possible to determine the fate of endogenous 5-HETE in vivo, different results may be obtained.…”

Directing eicosanoid esterification into phospholipids

“…In platelets, 12-HETE or 14-HDOHE attached to PE or PC are found, whereas EET-PLs in liver predominate as positional isomers reflecting cytochrome P-450 activities (5,12,13). More recently, eoxPLs generated by COX-1 have been found in human platelets.…”

“…To characterize the molecular species of complex lipids, precursor scanning LC-MS/MS was used, thus "fishing" for molecular ions that incorporated a HETE functional group. Between 2007 and 2012, several families of eoxPLs generated by LOXs were uncovered using this approach, not only in monocytes but also in human platelets, neutrophils, and airway epithelial cells (9)(10)(11)(12)(13)(14). The most abundant were phosphatidylethanolamines (PEs) but PC-derived forms were detected with many being plasmalogens.…”

Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation

“…Although AA is widely studied in platelets, 12-LOX may oxidize a number of other fatty acids on the platelet membrane following stimulation ( 22,44 ). Because thrombin is the most potent activator of platelet function, we investigated the ability of these metabolites to modulate platelet activation following stimulation of the thrombin receptor proteaseactivated receptor-1 (PAR1).…”