12-Oxo-10-glutathionyl-5,8,14-eicosatrienoic acid (TOG10), a novel glutathione-containing eicosanoid generated via the 12-lipoxygenase pathway in human platelets

The 12-lipoxygenase pathway of arachidonic acid metabolism in platelets and other cells is bifurcated into a reduction route yielding 12-hydroxyeicosatetraenoic acid (12-HETE) and an isomerization route forming hepoxilins. Here we show for the first time the presence of phospholipid hydroperoxide glutathione peroxidase (PHGPx) protein and its activity in platelets. The ratio of the activity of PHGPx to that of cytosolic glutathione peroxidase (GPx-1) was consistently found to be approx. 1:60 in platelets and UT7 megakaryoblasts. Moreover, short-lived PHGPx mRNA was detected in megakaryocytes but not in platelets. Carboxymethylation of selenium-containing glutathione peroxidases by iodoacetate, which results in the inactivation of PHGPx and GPx-1 without inhibition of 12-lipoxygenase, markedly altered the pattern of arachidonic acid metabolism in human platelets. Whereas the formation of 12-HETE was inhibited by 80%, a concomitant accumulation of 12-hydroperoxyeicosatetraenoic acid (12-HpETE) by two orders of magnitude as well as the formation of hepoxilins A3 and B3 were observed. The formation of hepoxilins also occurred when 12-HpETE was added to untreated platelets. In selenium-deficient UT7 cells, which were devoid of GPx-1 but not of PHGPx, the reduction of 12-HPETE was retained, albeit with a lower rate than in control cells containing GPx-1. We therefore believe that both GPx-1 and PHGPx are involved in the regulatory network of the 12-lipoxygenase pathway in platelets and other mammalian cells. Moreover, the diminution of hydroperoxide tone in platelets incubated with arachidonic acid leads primarily to the formation of 12-HETE, whereas the increase in hydroperoxide tone (a situation found under oxidative stress or selenium deficiency or on incubation with 12-HPETE) partly diverts the 12-lipoxygenase pathway from the reduction route to the isomerization route, thus resulting in the formation of hepoxilins.

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Potentiating Effect Of 5, 8, 11-Eicosatrienoic Acid (20:3ω9) On Platelet Aggregation

10.1055/s-0038-1652608 ◽

1981 ◽

Author(s):

M Lagarde ◽

M Burtin ◽

H Sprecher ◽

M Dechavanne ◽

S Renaud

Keyword(s):

Saturated Fat ◽

Eicosatrienoic Acid ◽

Human Platelets ◽

Ionophore A23187 ◽

Lipoxygenase Pathway ◽

Fatty Acid Deficiency ◽

Vitro Effect ◽

Induced Aggregation ◽

Saturated Fat Intake

The in vitro effect of 20:3ω9 (5×10-6M) was tested on human platelet suspensions, with aggregation induced by thrombin, ionophore A23187, ADP, collagen and arachidonic acid. When 20:3ω9 was added simultaneously with the aggregating agent, the response was increased by 100 to 300 % with all agents. It was solely to thrombin and ionophore that the aggregation was increased (by 200 to 300 %) when 20:3ω9 was preesterified in the platelet phospholipids by a 2 hour incubation period and subsequent platelet washing. Aspirin at a concentration inhibiting cyclooxygenase activity, did not block the potentiating effect of 20:3ω9. However, that effect was inhibited by a higher aspirin concentration, also inhibiting the peroxydase from the lipoxygenase pathway. Monohydroperoxy (HOO) - and monohydroxy (HO) - 20:3ω9 have been prepared from human platelets and purified by TLC. When added to platelets in vitro, solely the HO-20:3ω9 (0.2×10-6M) increased by several folds the response to thrombin and ionophore. Finally, both thrombin and ionophore induced the formation of HO-20:3ω9 (detected by a radiochemical technique) from platelets enriched with 20:3ω9. Thus, an increase level of 20:3ω9 in the platelet phospholipids seems to markedly enhance the susceptibility of platelets to aggregation by thrombin (and ionophore), probably through the lipoxygenase end product. Consequently the increase in the susceptibility of platelets to thrombin induced aggregation reported in essential fatty acid deficiency as well as with high saturated fat intake, both in man and in animals, might be due to the higher level of 20:3ω9 in the platelet phospholipids, which has been observed under those conditions.

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Evidence for the presence of phospholipid hydroperoxide glutathione peroxidase in human platelets: implications for its involvement in the regulatory network of the 12-lipoxygenase pathway of arachidonic acid metabolism

Biochemical Journal ◽

10.1042/0264-6021:3530091 ◽

2000 ◽

Vol 353 (1) ◽

pp. 91 ◽

Cited By ~ 12

Author(s):

Mark SUTHERLAND ◽

Pattabhiraman SHANKARANARAYANAN ◽

Tankred SCHEWE ◽

Santosh NIGAM

Keyword(s):

Arachidonic Acid ◽

Glutathione Peroxidase ◽

Regulatory Network ◽

Acid Metabolism ◽

Human Platelets ◽

Arachidonic Acid Metabolism ◽

Phospholipid Hydroperoxide Glutathione Peroxidase ◽

Lipoxygenase Pathway ◽

Phospholipid Hydroperoxide ◽

12 Lipoxygenase

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Lipoxin synthase activity of human platelet 12-lipoxygenase

Biochemical Journal ◽

10.1042/bj2960127 ◽

1993 ◽

Vol 296 (1) ◽

pp. 127-133 ◽

Cited By ~ 64

Author(s):

M Romano ◽

X S Chen ◽

Y Takahashi ◽

S Yamamoto ◽

C D Funk ◽

...

Keyword(s):

Human Platelet ◽

Fold Increase ◽

Eicosatrienoic Acid ◽

Human Platelets ◽

Dual Function ◽

Lipoxin A4 ◽

Suicide Inactivation ◽

Leukotriene A4 ◽

12 Lipoxygenase

Human platelets and megacaryocytes generate lipoxins from exogenous leukotriene A4 (LTA4). We examined the role of human 12-lipoxygenase (12-LO) in lipoxin generation with recombinant histidine-tagged human platelet enzyme (6His-12-LO), partially purified 12-LO from human platelets (HPL 12-LO) and, for the purposes of direct comparison, permeabilized platelets. Recombinant and HPL 12-LO catalysed the conversion of intact LTA4 into both lipoxin A4 (LXA4) and lipoxin B4 (LXB4). In contrast, only negligible quantities of LXA4 were generated when recombinant 12-LO was incubated with the non-enzymic hydrolysis products of LTA4.6His-12-LO also converted a non-allylic epoxide, 5(6)-epoxy-(8Z,11Z,14Z)-eicosatrienoic acid. The apparent Km and Vmax. for lipoxin synthase activity of 6His-12-LO were estimated to be 7.9 +/- 0.8 microM and 24.5 +/- 2.5 nmol/min per mg respectively, and the LXB4 synthase activity of this enzyme was selectively regulated by suicide inactivation. Aspirin gave a 2-fold increase in lipoxin formation by platelets but did not enhance the conversion of LTA4 by the recombinant 12-LO. These results provide direct evidence for LXA4 and LXB4 synthase activity of human platelet 12-LO. Moreover, they suggest that 12-LO is a dual-function enzyme that carries both oxygenase and lipoxin synthase activity.

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Albumin modifies the metabolism of hydroxyeicosatetraenoic acids via 12-lipoxygenase in human platelets

The Journal of Lipid Research ◽

10.1016/s0022-2275(20)32129-5 ◽

1999 ◽

Vol 40 (5) ◽

pp. 940-947 ◽

Cited By ~ 2

Author(s):

Marina Dadaian ◽

Par Westlund

Keyword(s):

Human Platelets ◽

12 Lipoxygenase ◽

Hydroxyeicosatetraenoic Acids

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Metabolism of arachidonic acid in nervous system of marine mollusk Aplysia californica

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1991.260.5.r844 ◽

1991 ◽

Vol 260 (5) ◽

pp. R844-R848 ◽

Cited By ~ 1

Author(s):

D. Piomelli

Keyword(s):

Arachidonic Acid ◽

Intracellular Signaling ◽

High Performance ◽

Aplysia Californica ◽

Gas Chromatography Mass Spectrometry ◽

Neural Cells ◽

Lipoxygenase Pathway ◽

Marine Mollusk ◽

Fmrf Amide ◽

12 Lipoxygenase

Studies of the marine mollusk Aplysia californica indicate that products of the 12-lipoxygenase pathway may be involved in neuronal intracellular signaling. The nervous tissue of Aplysia has a 12-lipoxygenase activity that converts both exogenous and endogenous arachidonic acid to an array of products, which include 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and its metabolites hepoxilin A3, hepoxilin B3, 12-ketoeicosatetraenoic acid, and 12-oxododecatrienoic acid. These eicosanoids were identified using a combination of high-performance liquid chromatography, ultraviolet spectrometry and gas chromatography-mass spectrometry. Generation of 12-lipoxygenase products was stimulated by application of the neurotransmitters, histamine and FMRF-amide, or by stimulation of identified neural cells. In electrophysiological studies of identified L14 and sensory neurons it was found that 12-HPETE and its metabolic products exert physiological actions that resemble those of histamine and FMRF-amide. These results suggest that products of 12-HPETE metabolism may act as second messengers in Aplysia neurons.

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Inhibition Of Platelet Aggregation, Malonyldialdehyde And Thromboxane Formation By Hydroperoxides Of Arachidonic Acid

10.1055/s-0038-1652786 ◽

1981 ◽

Author(s):

D Aharonv ◽

J B Smith ◽

M J Silver

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Lipoxygenase Pathway ◽

Dose Dependent Fashion ◽

Induced Aggregation ◽

Layer Chromatography ◽

Dose Dependent ◽

Thromboxane Formation

The arachidonate hydroperoxides 12-HPETE and 15-HPETE were biosynthesized from arachidonic acid using partially purified human platelet lipoxygenase or soybean lipoxidase respectively, and isolated by thin layer chromatography. Both compounds inhibited the arachidonic acid- induced aggregation of washed human platelets, suspended in calcium-free Krebs Henseleit solution, in a dose dependent fashion at concentrations between 1 and 50 uM. No inhibition was seen with up to 100 uM of these hydroperoxides when platelet -rich plasma was used. 12-HPETE (in micromolar concentrations) inhibited the formation of both thromboxane B2 (radioimmunoassay) and malonyldialdehyde (spectrophotometrie assay) when washed platelets were incubated with arachidonic acid. The 12-hydroxide, 12-HETE also inhibited platelet aggregation and thromboxane formation, but was less potent than 12-HPETE. We suggest that arachidonate hydroperoxide generated in platelets via the lipoxygenase pathway modulates platelet aggregation induced by arachidonic acid by inhibiting thromboxane formation.

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