Phospholipid-esterified Eicosanoids Are Generated in Agonist-activated Human Platelets and Enhance Tissue Factor-dependent Thrombin Generation

Abstract

Here, a group of specific lipids, comprising phosphatidylethanolamine (PE)- or phosphatidylcholine (PC)-esterified 12S-hydroxyeicosatetraenoic acid (12S-HETE), generated by 12-lipoxygenase was identified and characterized. 12S-HETE-PE/PCs were formed within 5 min of activation by thrombin, ionophore, or collagen. Esterified HETE levels generated in response to thrombin were 5.85 ± 1.42 (PE) or 18.35 ± 4.61 (PC), whereas free was 65.5 ± 17.6 ng/4 × 107 cells (n = 5 separate donors, mean ± S.E.). Their generation was stimulated by triggering protease-activated receptors-1 and -4 and signaling via Ca2+ mobilization secretory phospholipase A2, platelet-activating factor-acetylhydrolase, src tyrosine kinases, and protein kinase C. Stable isotope labeling showed that they form predominantly by esterification that occurs on the same time scale as free acid generation. Unlike free 12S-HETE that is secreted, esterified HETEs remain cell-associated, with HETE-PEs migrating to the outside of the plasma membrane. 12-Lipoxygenase inhibition attenuated externalization of native PE and phosphatidylserine and HETE-PEs. Platelets from a patient with the bleeding disorder, Scott syndrome, did not externalize HETE-PEs, and liposomes supplemented with HETE-PC dose-dependently enhanced tissue factor-dependent thrombin generation in vitro. This suggests a role for these novel lipids in promoting coagulation. Thus, oxidized phospholipids form by receptor/agonist mechanisms, not merely as an undesirable consequence of vascular and inflammatory disease.

Platelet 12-LOX is essential for FcγRIIa-mediated platelet activation

“…Our study demonstrates that a significant increase in 12-HETE formation is reproducibly observed within 15 seconds following FcgRIIa crosslinking-mediated 12-LOX oxidation of AA (P 5 .0143) ( Figure 1C). Our data also suggests that 12-LOX may partially regulate FcgRIIa-induced platelet activation either through oxylipin formation, and subsequent metabolite signaling, 49,50 or directly through a protein complex formation within the cell. [51][52][53] Although both of these regulatory mechanisms are plausible, the kinetics by which 12-LOX inhibition attenuates FcgRIIa signaling may favor a direct role for 12-LOX in regulating the FcgRIIa pathway through nonenzymatic regulation of the FcgRIIa signaling cascade.…”

A simplified procedure for semi-targeted lipidomic analysis of oxidized phosphatidylcholines induced by UVA irradiation

“…Because of the tremendous excess of unoxidized lipids and the presence of isobaric peaks, analysis of individual OxPLs by mass spectrometry is only feasible after chromatographic separation of lipids, typically by reversed-phase HPLC. Previous publications described mass spectrometry-based methods for analysis of oxidized cardiolipin (15)(16)(17), phosphatidylserine ( 15,16,18 ), phosphatidylethanolamine ( 6,(19)(20)(21)(22), and phosphatidylcholine ( 23-25 and references below). The results show that different PL classes have variable propensity for oxidation.…”

Chronic Alcohol Exposure Increases Circulating Bioactive Oxidized Phospholipids

“…Because polyunsaturated fatty acyl residues predominate at the sn-2 position of the glycerol backbone of phospholipids, common products of oxidative attack are phospholipids containing sn-2 hydroperoxyoctadecadienoyl ester (HpODE) 2 from linoleoyl oxidation and hydroperoxyeicosatetraenoyl ester (HpETE) * This work was supported, in whole or in part, by National Institutes of derived from arachidonoyl peroxidation. Phospholipid hydroperoxides themselves have no established signaling role, although this may be changing (9), but are relevant because they reflect endogenous oxidative stress and so act as circulating biomarkers of oxidative stress (3). Phospholipid hydroperoxides may also be relevant because they are precursors of a host of oxidatively truncated phospholipids, which are formed by fragmentation of the fatty acyl chain at the site of the (hydro)peroxy function (10), that can be potent inflammatory and apoptotic agonists.…”