Effects of antioxidants on cyclooxygenase and lipoxygenase activities in intact human platelets: Comparison with indomethacin and etya

Five antioxidative agents (BW755C, 1-naphtol, NDGA, propylgallate and quercetin) were compared with indomethacin and ETYA for their effects on (¹⁴C) arachidonic acid metabolism by cyclooxygenase (CO) and lipoxygenase (LPO) enzymes in intact human platelets. All tested compounds inhibited CO activity in a concentration-dependent manner. LPO activity was suppressed by NDGA, propylgallate, quercetin and ETYA but strongly enhanced by BW755C, 1-napthol and indomethacin. Whereas NDGA and ETYA showed almost equipotent inhibitory effects towards both fatty acid oxygenases, propylgallate and quercetin were found to be respectively 6.5 and 4 times better inhibitors of LPO than of CO activities.These data indicate that antioxidants affect arachidonic acid metabolism in intact human platelets in different ways: BW755C and 1-naphtol exerted the same activity as indomethacin, a selective CO blocker, whereas NDGA, propylgallate and quercetin behaved as ETYA, a dual CO-LPO inhibitor. Considering their inhibition selectivity, propylgallate and quercetin may serve as prototypes for more specific blockers of LPO activity.     

Arachidonic acid metabolim in rat pancreatic acinar cells: Calcium-mediated stimulation of the lipoxygenase system

Isolated rat pancreatic acini were employed to demonstrate that the exocrine pancreas can metabolize [¹⁴C]-arachidonic acid by way of the lipoxygenase pathway as well as the cyclooxygenase pathway. Analysis by high performance liquid chromtography delineated a monohydroxy acid, presumably 12-L-hydroxy-5,8–10,14-eicosatetraenoic acid (12-HETE) as the major lipoxygenase product. The formation of this hydroxy arachidonic derivative was stimulated by the calcium ionophore ionomycin. Stimulation of lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF_1α, PGF_2α, and PGE₂ were identified; and ionomycin, carbamylcholine, and caerulein enhanced the formation of these metabolites of the cyclooxygenase pathway. Ionomycin induced stimulation of HETE formation was inhibited by ETYA and nordihydroguaiaretic acid, but spontaneous and evoked enzyme secretion was unaffected. Thus, although ionomycin, a pancreatic secretagogue, stimulates the lipoxygenase pathway, the precise role of these arachidonate metabolites in the physiology of the exocrine pancreas is still obscure.     

Slow reacting substance (SRS) from ionophore A23187-stimulated peritoneal mast cells of the normal rat. II. Evidence for a precursor role of arachidonic acid and further purification.

The generation of slow reacting substance (SRS) from ionophore A23187-stimulated rat peritoneal mast cells was enhanced by arachidonic acid (AA). This SRS generation was inhibited by 5,8,11,14-eicosatetraynoic acid (ETYA), an acetylenic analogue of AA and an inhibitor of both fatty acid cyclooxygenase and lipoxygenase. Indomethacin, a fatty acid cyclooxgenase inhibitor, had an enhancing effect upon SRS generation. This suggests SRS generation occurred through an ETYA sensitive step--perhaps a lipoxygenase. Radiolabel from [14C]-AA was incorporated into SRS with comigration of radioactivity and bioreactivity in silicic acid and thin layer chromatographies. Upon silicic acid chromatography, the active principle was eluted in the methanol fraction. Two-dimensional thin layer chromatography revealed chromatographic separation from other known spasmogenic substances and phospholipids. Mast cell SRS was found to display physiochemical properties similar to those of rat basophilic leukemia cell SRS, namely: that mast cell SRS generation was 1) enhanced by arachidonic acid; 2) inhibited by ETYA but not by indomethacin; 3) incorporation of [14C]-AA into the active principle; and 4) similar behavior during purification in silicic acid and thin layer chromatographies.     

Regeneration of vitamin E in human platelets

Human platelets possess active lipoxygenase and cyclooxygenase which convert arachidonic acid to (12S)-12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) plus (12S)-12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and thromboxane B2 plus 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), respectively. When platelet homogenates were incubated with arachidonate, there was a rapid consumption of platelet tocopherol. Time course analysis revealed that within 0.5 min, over half of arachidonate and tocopherol were metabolized. Mass formation of 12-HPETE and 12-HETE or thromboxane B2 and HHT exceeded that of the mass of tocopherol oxidized. Preincubation with the lipoxygenase inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) completely abolished this arachidonate-induced tocopherol oxidation whereas cyclooxygenase inhibitors (indomethacin and aspirin) further potentiated tocopherol oxidation, indicating that this oxidation is closely linked with platelet 12-lipoxygenase activity. Incubation with lipoxygenase metabolites of arachidonic acid showed that only 12-HPETE caused a rapid tocopherol oxidation which was followed by a gradual tocopherol regeneration. By using nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor which is also a strong reductant, over 60% of the arachidonate-induced oxidized tocopherol was regenerated. Tocopherol regeneration declined with increasing oxidation time induced by arachidonate, and after 30-60 min virtually no regeneration could be observed, suggesting that the precursor molecule was unstable. We postulate that the precursor molecule is the tocopheroxyl radical. In the presence of ETYA, a lipoxygenase inhibitor without antioxidant properties, either ascorbate or GSH provided significant tocopherol regeneration. Kinetic studies showed that tocopherol regeneration after the addition of ascorbate was essentially completed by 1 min. By contrast, GSH addition caused a steady increase in tocopherol which peaked after 10 min of its addition. To determine whether this rapid regeneration is chemical or enzymic, regeneration was studied in the presence of chloroform and methanol. Comparison of various reductants in this denaturing condition for enzymes showed that ascorbate and NDGA afforded significant regeneration whereas GSH was ineffective, indicating that there are distinct enzymic and non-enzymic mechanisms for tocopherol regeneration. This study provides direct evidence from mass analysis that tocopherol can be regenerated in human cell homogenates. This finding implies that maintenance of membrane tocopherol status may be an essential function of ascorbate and GSH which operate in concert to ensure maximum membrane protection against oxidative damage.     

An imbalance of arachidonic acid metabolism in asthma

Metabolism of arachidonic acid via the cyclooxygenase and lipoxygenase pathways was studied in washed platelets from normal and asthmatic subjects. The platelets were incubated with [1-¹⁴C] arachidonic acid and the metabolites formed were separated by high pressure liquid chromatography (HPLC). The platelets from asthmatic patients had a 40% decrease in cyclooxygenase-derived metabolites and a 70% increase in lipoxygenase-derived product when compared with metabolites generated by platelets from normal subjects. The ratio of cyclooxygenase to lipoxygenase products was 3.24 ± 0.26 for platelets from normal subjects, and 1.14 ± 0.15 with platelets from the asthmatic patients. These results indicate an imbalance of arachidonic acid metabolism in platelets from asthmatic patients.     

Conversion of arachidonic acid to cyclooxygenase and lypoxygenase products,and incorporation into phospholipids in the mouse neuroblastoma clone,Neuro-2A

Arachidonic acid (AA) incorporation into phospholipids and cyclooxygenase and lipoxygenase mediated metabolism of arachidonic acid were studied in homogenized and intact Neuro-2A cells. When 3H8-AA was added to homogenized cells and incubated 20 minutes, 39% of the label was converted to prostaglandins (PGs), 10% to hydroxy-eicosatetraenoic acid (HETE) and 26% was incorporated into phospholipids. PGE2 and PGF2a were the major PGs produced. Synthesis of PGs was blocked by 10 microM indomethacin and synthesis of PGs and HETE was blocked by 10 microM eicosatetraynoic acid (ETYA). The cell homogenate produced the 13,14-dihydro-15-keto metabolites of PGE2 and PGF2a from 3H8-AA and also converted exogenous 3H7-PGE2 and 3H8-PGF2a to metabolites. When intact cells were labeled for 24 hours with 14C1-AA and the cells and media then analyzed, 75% of the radioactivity was incorporated into cellular phospholipids, 0.8% was converted to PGs and metabolites and 0.7% converted to HETE. Cells prelabeled for 24 hours were washed and incubated for 30 minutes in fatty acid free media. There was a 23% release of AA from phospholipids. One-fifth of the released AA was converted to HETE. PG synthesis in the intact resting cells was low. In summary, the Neuro-2A cell provides a good model system for studying arachidonic acid metabolism and incorporation into phospholipids in cells of neuronal origin.     

Arachidonic acid metabolism in rat pancreatic acinar cells: calcium-mediated stimulation of the lipoxygenase system

Isolated rat pancreatic acini were employed to demonstrate that the exocrine pancreas can metabolize [14C]-arachidonic acid by way of the lipoxygenase pathway as well as the cyclooxygenase pathway. Analysis by high performance liquid chromatography delineated a monohydroxy acid, presumably 12-L-hydroxy-5,8-10,14-eicosatetraenoic acid (12-HETE) as the major lipoxygenase product. The formation of this hydroxy arachidonate derivative was stimulated by the calcium ionophore ionomycin. Stimulation of the lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF1 alpha, PGF2 alpha, and PGE2 were identified; and ionomycin, carbamylcholine, and caerulein enhanced the formation of these metabolites of the cyclooxygenase pathway. Ionomycin induced stimulation of HETE formation was inhibited by ETYA and nordihydroguaiaretic acid, but spontaneous and evoked enzyme secretion was unaffected. Thus, although ionomycin, a pancreatic secretagogue, stimulates the lipoxygenase pathway, the precise role of these arachidonate metabolites in the physiology of the exocrine pancreas is still obscure.     

Epidermal arachidonate lipoxygenase

Guinea pig skin was found to display a high lipoxygenase activity, evidenced by the formation of a hydroxyeicosatetraenoic acid (HETE) from exogenous [14C]arachidonic acid. The lipoxygenase activity was localized to the epidermal layer of the skin, was completely inhibited by eicosatetraynoic acid (ETYA) and slightly enhanced by indomethacin. Susceptibility to inactivation by sulfhydryl-directed reagents indicated that an essential sulfhydryl is present in a hydrophobic region of the molecule. The enzyme exhibited a broad pH activity optimum and a Km of 2.48 . 10(-5) M. The cytosolic enzyme has been partly purified by ammonium sulfate precipitation and two steps of of column chromatography and exhibited an apparent high molecular weight. The lipoxygenase and hydroperoxidase activities were resolvable from one another. The physiological and pathophysiological roles of the enzyme remain to be elucidated.     

On the mechanism of the oxygenation of arachidonic acid by human platelet lipoxygenase

Formation of 12L-hydroxy-5,8,10,14-eicosatetraenoic acid from [10L-³H; 3-¹⁴C]arachidonic acid in suspensions of human platelets occurred with extensive loss of tritium and was accompanied by an isotope effect. These experiments showed that there is an antarafacial relation between the elimination of hydrogen from C-10 and insertion of oxygen at C-12 by human platelet lipoxygenase, and that the hydrogen elimination probably occurs as the initial step of the conversion. (Endo) peroxide intermediates formed by the fatty acid cyclo-oxygenase pathway activated platelet lipoxygenase.     

Stimulation of phosphatidic acid production in platelets precedes the formation of arachidonate and parallels the release of serotonin.

Thrombin rapidly induces the formation of labeled phosphatidic acid from platelets prelabeled with [17C]arachidonate or 32PO34- and specifically decreases by 50--75% the content of phosphatidylinositol. Ionophore A23187 also stimulates phosphatidate labeling, but less effectively than thrombin. This effect on phosphatidic acid is blocked by increasing the levels of cyclic AMP by preincubation with dibutyryl cyclic AMP, cyclic AMP-phosphodiesterase inhibitors or prostacyclin. Indomethacin and eicosatetraynoic acid do not alter the production of phosphatidate, indicating independence from cyclooxygenase or lipoxygenase products. Increased turnover of [14C]- or [32P]phosphatidate occurs within 2--5 s after platelet activation by thrombin and is observed before endogenous, 14C-labeled arachidonate can be detected. The rate of phosphatidate formation parallels the induced rate of serotonin release. Release of [3H]serotonin is not affected by eicosatetraynoic acid. Phosphatidate production reflects the generation of diacylglycerol by C-type phospholipase degradation of phosphatidylinositol. Diacylglycerol and phosphatidic acid may participate in the membrane modification related to the early changes in platelet shape, release reactions or aggregation which occur on stimulation.     

In vitro effect of trichosanic acid, a major component of Trichosanthes japonica on platelet aggregation and arachidonic acid metabolism in human platelets

The in vitro effect of trichosanic acid (TCA; C18:3, omega-5), a major component of Trichosanthes japonica, on platelet aggregation and arachidonic acid (AA) metabolism in human platelets was studied. TCA dose-dependently suppressed platelet aggregation of platelet rich plasma and washed platelets. TCA decreased collagen (50 micrograms/ml)-stimulated production of thromboxane B2 (TXB2) and 12-hydroxyhepta-decatrienoic acid (HHT) in a dose-dependent manner, while that of 12-hydroxyeicosatetraenoic acid (12-HETE) was rather enhanced. The conversion of exogenously added [14C]AA to [14C]TXB2 and [14C]HHT in washed platelets was dose-dependently reduced by the addition of TCA, while that to [14C]12-HETE was increased. Similar observations were obtained when linolenic acid (LNA; C18:3, omega-3) was used. These results suggest that TCA may decrease TXA2 formation in platelets, probably due to the inhibition of cyclooxygenase pathway, and thereby reduce platelet aggregation.     

The stimulation of arachidonic acid metabolism in human platelets by hydrodynamic stresses

Even though shear-induced platelet activation and aggregation have been studied for about 20 years, there remains some controversy concerning the arachidonic acid metabolites formed during stress activation and the role of thromboxane A2 in shear-induced platelet aggregation. In this study, platelets were labelled with [1-14C]arachidonic acid to follow the metabolism of arachidonic acid in stimulated platelets using HPLC and scintillation counting. Platelets activated by thrombin formed principally thromboxane A2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). In contrast, for platelets activated by shear--though arachidonic acid metabolism was stimulated--only 12-HETE was formed and essentially no cyclooxygenase metabolites were detected. This indicates that physical forces may initiate a different pathway for eicosanoid metabolism than most commonly used chemical stimuli and perhaps also implies that regulation of the cyclooxygenase activity may be a secondary level of regulation in eicosanoid metabolism.     

Measurement of arachidonic acid liberation in thrombin-stimulated human platelets. Use of agents that inhibit both the cyclooxygenase and lipoxygenase enzymes

The formation of radiolabelled oxygenated products of arachidonic acid in thrombin-stimulated, [3H]arachidonic acid-prelabelled human platelets is inhibited in a concentration-dependent manner by BW 755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline) or propyl gallate, both of which are combined inhibitors of lipoxygenase and cyclooxygenase. These compounds do not inhibit the thrombin-induced decrease in the radioactivity of platelet phospholipids but, instead, allow the accumulation of free radiolabelled arachidonic acid. Thrombin causes an increase in the levels of free, endogenous palmitic, stearic, oleic, linoleic and arachidonic acids of up to 10 nmol/10(9) platelets. In the presence of BW 755C or propyl gallate, further increases in the level of free arachidonic acid, of 20-50 nmol/10(9) platelets, occur. The enzyme inhibitors do not affect the accumulation of the other free fatty acids. The increase in arachidonic acid is optimal at 1 U/ml thrombin and 60% complete by 1 min at 37 degrees C. In the platelets from eight donors, the average increases in free fatty acids (in nmol/10(9) platelets) induced by 5 U/ml thrombin in 5 min at 37 degrees C in the presence of 100 microM BW 755C were 1 for linoleic acid, 3.6 for oleic acid, 4.5 for palmitic acid, 7.6 for stearic acid and 32.0 for arachidonic acid.     

Antiplatelet effects of conjugated linoleic acid isomers

Conjugated diene isomers of linoleic acid (CLA) are normal constituents of certain foods and exhibit anticarcinogenic and antiatherogenic properties. In the present study, the effects of several CLA isomers on human platelet aggregation and arachidonic acid metabolism were examined. It was found that 9c,11t-CLA, 10t, 12c-CLA and 13-hydroxy-9c,11t-octadecadienoic acid (13-HODE) inhibited arachidonic acid- and collagen-induced platelet aggregation with I50s in the 5-7 microM range. The nonconjugated 9c, 12c-LA was about 300% and 50%, respectively, less potent an inhibitor with these aggregating agents. Using either thrombin or the calcium ionophore A23187 as aggregating agents, a CLA isomer mix was also found to be more inhibitory than 9c,12c-LA. The 9c,11t- and 10t,12c-CLA isomers as well as the CLA isomer mix inhibited formation of the proaggregatory cyclooxygenase-catalyzed product TXA2, as measured by decreased production of its inactive metabolite [14C]TXB2 from exogenously added [14C]arachidonic acid (I50s=9-16 microM). None of the CLA isomers tested inhibited production of the platelet lipoxygenase metabolite [14C]12-HETE. The additional presence of a hydroxyl group gave opposite results: 13-HODE (I50=3 microM) was about 4-fold more potent a cyclooxygenase inhibitor than the 9c,11t-CLA isomer but 9-HODE was 2- to 3-fold less effective an inhibitor (I50=34 microM) of [14C]TXB2 formation than the corresponding 10t,12c-CLA. In both the aggregation and arachidonic acid metabolism experiments, the inhibitory effects of CLA on platelets were reversible and dependent on the time of addition of either the aggregating agent or the [14C]arachidonic acid substrate. These studies suggest that CLA isomers may also possess antithrombotic properties.     

Glucose-stimulated protein phosphorylation in the pancreatic islet

The effect of inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism on steroidogenesis in rat testis Leydig cells and rat tumour Leydig cells has been investigated. In the presence of nordihydroguaiaretic acid [NDGA; 4,4'-(2,3- dimethylbutan -1,4- diyl )bis[1,2- benzendiol ]], 5,8,11,14-eicosatetraynoic acid (ETYA), BW 755C [3-amino-1-[3-(trifluoromethyl)phenyl]-2-pyrazoline hydrochloride] and benoxaprofen [ Opren ; 2-(2-p-chlorophenyl- benzoxazol -5-yl)propionic acid)] (which inhibit lipoxygenase activity), but not indomethacin and aspirin (which inhibit cyclo-oxygenase activity), a dose-related inhibition of lutropin (LH)-stimulated testosterone and pregnenolone production was obtained (ID50 values of 2.5, 30, 25 and 30 microM for NDGA, ETYA, BW 755C and benoxaprofen were obtained, respectively). BW 755C and benoxaprofen had no significant effect on LH-stimulated cyclic AMP production except at the highest concentrations examined (330 and 380 microM, respectively), whereas NDGA and ETYA inhibited LH-stimulated cyclic AMP production in a dose-dependent manner (ID50 7.0 and 22 microM respectively). However, NDGA and ETYA also caused a dose-dependent inhibition of dibutyryl cyclic AMP-stimulated testosterone and pregnenolone production. The metabolism of exogenous ( 22R )-hydroxycholesterol or pregnenolone to testosterone by Leydig cells was not inhibited by either NDGA, ETYA or indomethacin. At low concentrations of NDGA and ETYA a significant increase in the conversion of both pregnenolone and ( 22R )-hydroxycholesterol to testosterone was obtained. Studies in which the metabolism of [14C]arachidonic acid by purified rat tumour Leydig cells was investigated indicate that products are formed by tumour Leydig cells that have similar mobilities in a thin layer chromatography system to 5-L-hydroxy-6,8,11,14-eicosatetraenoic acid, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and leukotriene B4. The formation of these products was inhibited to varying degrees by NDGA, BW 755C and benoxaprofen but not by aspirin and indomethacin. These studies demonstrate for the first time that inhibition of lipoxygenase activity but not cyclo-oxygenase activity causes an inhibition of LH- and dibutyryl cyclic AMP-stimulated steroid production and suggest a stimulatory role for products of the lipoxygenase pathway of arachidonic acid metabolism in steroidogenesis. The site of this stimulation is apparently distal to the production of cyclic AMP and before the side chain cleavage of cholesterol.     

Arachidonic acid metabolism of the murine eosinophil. II. Involvement of the lipoxygenase pathway in the response to the lymphokine eosinophil stimulation promoter

Eosinophil stimulation promoter (ESP) is a murine lymphokine that enhances the migration of eosinophils. Exogenous arachidonic acid between 0.5 and 2 micrograms/ml potentiated the activity of ESP on murine eosinophil migration, whereas such concentrations did not affect migration in the absence of ESP. Among the lipoxygenase products identified from an enriched population of murine eosinophils, leukotriene B4 (optimal activity at 100 ng/ml) and 12-HETE (optimal activity at 2 micrograms/ml) stimulated migration of these cells. Another lipoxygenase product from these cells 15-HETE inhibited ESP-induced migration; between 5 and 10 micrograms/ml 15-HETE decreased by one-half both stimulated migration and 12-HETE biosynthesis. Structurally diverse drugs at concentrations that inhibited HETE biosynthesis inhibited ESP-induced migration. The concentrations that decreased migration activity by one-half were 5 microM NDGA, 10 microM ETYA, and 150 microM BW755C. Aspirin and indomethacin at concentrations reported to inhibit prostaglandin biosynthesis did not substantially inhibit ESP activity, but concentrations of indomethacin above 20 microM caused concentration-dependent inhibition of migration. The selective lipoxygenases inhibitor 134,7,10,13-eicosatetraynoic acid was more potent than ETYA in inhibition of ESP-induced migration, and the selective cyclooxygenase inhibitor 6,9,12-octadecatriynoic acid did not effect inhibition. These results are consistent with the hypothesis that stimulation of eosinophils by the lymphokine ESP involves the generation of lipoxygenase products from arachidonic acid, which positively and negatively regulate the migratory activities of these cells.     

Interference of some flavonoids and non-steroidal anti-inflammatory drugs with oxidative metabolism of arachidonic acid by human platelets and neutrophils

The effect of ten flavonoids was studied on the stimulation of washed human platelets by either arachidonic acid or thrombin. The oxygenated metabolites released were analyzed by radioimmunoassay, glass-capillary-column gas chromatography and high-pressure liquid chromatography. No effect was evidenced for naringenin, rutinose and phloridzin up to 1000 microM. Thromboxane B2 and 12-hydroxyeicosatetraenoic acid production was depressed simultaneously by all other compounds at different IC50. When tested for their effect on reversibility, however, cyclooxygenase and lipoxygenase inhibition was found to be different depending upon the flavonoid used. All compounds, except morin and rutin, inhibited platelet aggregation and [14C]serotonin release with parallel inhibition of thromboxane synthesis when tested on arachidonic acid-induced platelet-rich plasma stimulation. Some flavonoids inhibited the metabolism of human neutrophils stimulated by ionophore A23187 as assessed by high-performance liquid chromatography. Our results show that flavonoids interfere with the different oxidative metabolisms of arachidonic acid. No clearcut specificity could be found between one compound and one metabolic pathway.     

Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets

The metabolism of linoleic acid by washed human platelets was investigated. [1.14C] linoleic acid was converted to [1.14C] hydroxy octadecadienoic acids (HODEs) at about the same rate with which [1.14C] 12-HETE was produced from [1.14C] arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by CG-MS. The production of HODEs by intact washed platelets was inhibited by indomethacin (IC50:5 x 10(-7) M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10(-3) M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10(-5) M) and baicalein (10(-5) M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.     

Enrichment of human platelet phospholipids with linoleic acid diminishes thromboxane release

We have investigated whether exposure of human platelets to elevated concentrations of linoleic acid, the principal dietary polyunsaturate, would influence platelet thromboxane A₂ release. Platelets were incubated with albumin-bound linoleic acid at 30°C for 24 h, with prostaglandin E₁ added to prevent aggregation. The linoleic acid supplemented platelets released, on averaged, 50% less thromboxane A₂ in response to stimulation with thrombin than corresponding control platelets. Other fatty acids were without appreciable effect. The inhibition of thrombin-stimulated thromboxane A₂ release was dependent on the time and temperature of incubation, as well as on the concentration of added linoleic acid. Supplementation increased the amount of linoleic acid in the platelet phospholipids, but the arachidonic acid content of the phospholipids was reduced. [1-¹⁴C]Linoleic acid was not converted to arachidonic acid by the platelets. Linoleic acid was released exclusively form the inositol phosphoglycerides when the enriched platelets were stimulated with thrombin. The linoleate-enriched platelets converted less [1-¹⁴C]arachidonic acid to all prostaglandin products, suggesting that the platelet cyclooxygenase was partially inhibited.