Stimulation of lipoxin synthesis from leukotriene A4 by endogenously formed 12-hydroperoxyeicosatetraenoic acid in activated human platelets

Human platelets are devoid of 5-lipoxygenase activity but convert exogenous leukotriene A₄ (LTA₄) either by a specific LTC₄ synthase to leukotriene C₄ or via a 12-lipoxygenase mediated reaction to lipoxins. Unstimulated platelets mainly produced LTC₄, whereas only minor amounts of lipoxins were formed. Platelet activation with thrombin, collagen or ionophore A23187 increased the conversion of LTA₄ to lipoxins and decreased the leukotriene production. Maximal effects were observed after incubation with ionophore A23187, which induced synthesis of comparable amounts of lipoxins and cysteinyl leukotrienes (LTC₄, LTD₄ and LTE₄). Chelation of intra- and extracellular calcium with quin-2 and EDTA reversed the ionophore A23187-induced stimulation of lipoxin synthesis from LTA₄ and inhibited the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) from endogenous substrate. However, calcium did not affect the 12-lipoxygenase activity in the 100 000 × g supernatant of sonicated platelet suspensions. Furthermore, the stimulatory effect on lipoxin formation induced by platelet agonists could be mimicked in intact platelets by the addition of low concentrations of arachidonic acid, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) or 13-hydroperoxyoctadecadienoic acid (13-HPODE). The results indicate that the elevated lipoxin synthesis during platelet activation is due to stimulated 12-lipoxygenase activity induced by endogenously formed 12-HPETE.     

Dihomogammalinolenic acid, but not eicosapentaenoic acid, activates washed human platelets

Dihomogammalinolenic acid (2.5-20 microM) added to suspensions of washed human platelets induces platelet shape change and the formation of 1,2-diacylglycerol and phosphatidic acid, indicating the activation of phospholipase C. It also stimulates the phosphorylation of a 40 kDa protein, indicating the activation of protein kinase C. Dihomogammalinolenic acid is converted mainly to 12-hydroxyheptadecadienoic acid and to a smaller extent to prostaglandin E1 and thromboxane B1. Small quantities of the lipoxygenase product 12-hydroxyeicosatrienoic acid are also observed. Indomethacin, by blocking platelet cyclooxygenase, prevents the activation of phospholipase C, protein kinase C, and platelet shape change induced by dihomogammalinolenic acid. Compound UK 38485, a specific thromboxane synthetase inhibitor, does not block platelet activation induced by dihomogammalinolenic acid. The results indicate that endoperoxides derived from dihomogammalinolenic acid, such as prostaglandin G1 or prostaglandin H1, may be responsible for the stimulation of phospholipase C and protein kinase C, and for the induction of platelet shape change. Eicosapentaenoic acid does not activate platelets and is poorly metabolized by platelet cyclooxygenase and lipoxygenase. Eicosapentaenoic acid is a better inhibitor of platelet activation induced by various agonists in washed platelets than dihomogammalinolenic acid. Eicosapentaenoic acid and dihomogammalinolenic acid are, however, equally effective in inhibiting aggregation induced by collagen in platelet-rich plasma. We suggest that eicosapentaenoic acid might be a better antithrombotic agent than dihomogammalinolenic acid.     

A comparative study of eicosapentaenoic acid metabolism by human platelets in vivo and in vitro

During long-term dietary n-3 fatty acid supplementation, eicosapentaenoic acid (EPA) is not incorporated into phosphatidylinositol or -serine of human platelets in vivo and is not detectable in phosphatidic acid upon stimulation with thrombin. However, EPA is released from platelet phospholipids and metabolized to thromboxane B3 (TXB3). In contrast, in vitro, platelets incorporate [14C]EPA into phosphatidylinositol, whether they contain endogenous EPA in their cellular lipids or not. Following platelet stimulation, [14C]EPA appears in phosphatidic acid, as free fatty acid, and is transformed to TXB3. We conclude that the fatty acid compositions of platelet phospholipid subclasses are regulated with a high degree of specificity in vivo. Qualitative differences exist between in vivo and in vitro uptake of EPA into platelet phospholipid subclasses. After in vivo incorporation, EPA is released by action of a phospholipase A2.     

The effect of superoxide dismutase and catalase on metabolism of 3H-arachidonic acid by washed platelets

Previous experiments have suggested that superoxide dismutase (SOD) and catalase (CAT) may inhibit prostaglandin synthesis. The purpose of this study was to determine if these free radical scavengers can alter the metabolism of free arachidonic acid (AA) by the cyclooxygenasse and lipoxygenase enzyme systems in platelets. In control experiments washed platelets were incubated with ³H-AA for 5 minutes, extracted and the products separated by reverse phase high pressure liquid chromatography (HPLC). In normal intact platelets 13.5 ± 0.6% of the radioactivity was found in TxB₂, 16.3 ± 1.4% in HHT, 61.3 ± 1.1% in 12-HETE and 9.0 ± 1.0% was unconverted AA. Pre-incubating the platelets for 1 minute with 10 μg/ml SOD or CAT or 10 μg/ml SOD plus 10 μg/ml CAT did not inhibit AA conversion or alter the percent product distribution. Similarly, SOD and CAT had no effect on AA metabolism in broken cells. However, as expected, pretreating platelets with indomethaoin blocked TxB₂,and HHT formation (P <.0001). We conclude that SOD and CAT do not inhibit cyclooxygenase or lipoxygenase metabolism of free AA in platelets.     

Prostacyclin prolongs viability of washed human platelets

The functional viability of stored human platelets, washed in the presence and absence of prostacyclin, was examined over a 96 h period. Platelet counts, aggregation responses and cyclic AMP levels were monitored as well as the spontaneous generation of thromboxane B2 and the liberation of labelled oleate from cellular phosphatides. In suspensions prepared without prostacyclin in the medium, platelet counts declined rapidly as did the sensitivity to aggregating agents. In addition, substantial amounts of thromboxane B2 were generated during preparation and storage and oleate liberation occurred at a rapid rate. In contrast, in prostacyclin-washed platelets, aggregation was maintained throughout the study period and there was little generation of thromboxane B2. Moreover, only a gradual decrease in platelet count and a slow increase in the rate of oleate liberation was observed when compared with controls. However, cyclic AMP levels rapidly declined when platelets were resuspended in prostacyclin-free medium.     

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.     

Regulation of arachidonate-induced platelet aggregation by the lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid

Two lines of genetically involved and control chickens were compared with regard to the onset of muscle dystrophy during the early stages of growth ex ovo. Definite structural and functional involvement of pectoralis muscle developed within the first 4-5 weeks. In parallel experiments, microsomal membranes were obtained weekly from pectoralis muscle during the first 14 weeks ex ovo. The microsomes were studied with respect to ultrastructural features, protein composition, Ca2+ uptake and ATPase activity. Microsomal preparations obtained from all newborn chickens contain two types of vesicles: one type reveals an asymmetric distribution and 'high density' of particles on freeze-fracture faces which is characteristic of sarcoplasmic reticulum (SR) membrane; the other type reveals a symmetric distribution and 'low density' of particles. The yield of 'low density' microsomes from muscle of normal birds is very much reduced as the chicks grow from 1 to 4-5 weeks ex ovo. On the contrary, it remains high in chicks developing muscle dystrophy. Ca2+ uptake and coupled ATPase activity are found to be of nearly identical specific activity in control and genetically involved newborn chicks. The specific activity of the control birds, however, increases as the chicks grow from 1 to 4-5 weeks of age, while the specific activity of the dystrophic birds remains low. Such a difference appears to be related to the relative representation of sarcoplasmic reticulum and 'low density' vesicles in the microsomal preparations. It is concluded that failure to obtain a normal differentiation of muscle cell membranes is a basic defect noted in the early growth of genetically involved chickens. This defect appears along with the earliest signs of the dystrophic process.     

Metabolism of 12-hydroperoxyeicosatetraenoic acid to vasodilatory trioxilin C3 by rabbit aorta

Arachidonic acid is metabolized by both the cyclooxygenase and lipoxygenase pathways by rabbit aorta. We investigated the metabolism of 12-hydroperoxyeicosatetraenoic acid by aortic homogenates and microsomes. Rabbit aortic homogenates were incubated in the presence of (14)C-arachidonic acid plus 12-lipoxygenase and analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Under these experimental conditions, there was a (14)C-metabolite that migrated at 17.6 min. This (14)C-metabolite was not observed when aortic homogenates were incubated in the absence of 12-lipoxygenase. Similar results were obtained with aortic microsomes. Further analysis using a different HPLC solvent system resolved the (14)C-metabolite into a number of products. Gas chromatography/mass spectrometric (GC-MS) analysis of the major product (labeled peak 3) after conversion to the methyl ester-trimethylsilyl derivative showed two major compounds (compounds A and B) eluting at 13.99 and 14.14 min. The two compounds differed in the intensities of the 213 and 243 m/z ions with 243 being greater than 213 in compound A and the opposite in compound B (relative abundance 213 vs. 243; 100% vs. 43% for compound A and 5% vs. 100% for compound B). Based on the mass spectra, peak 3 contained two metabolites identified as the methyl ester-trimethylsilyl ether derivatives of 8,11,12-trihydroxyeicosatrienoic acid (trioxilin A(3)) and 8,9,12-trihydroxyeicosatrienoic acid (trioxilin C(3)). Biological activity of the mixture of two trioxilins isolated from aortic homogenates was tested in phenylephrine-precontracted aortas and found to produce concentration-dependent relaxations (maximal relaxation: 20.1+/-7.6%). Further testing with authentic trioxilin A(3) and C(3) revealed that trioxilin C(3) was the active metabolite (maximal relaxation: 16.6+/-1.3%). In conclusion, trioxilin C(3) acid was isolated and identified as a novel biologically active arachidonic acid metabolite formed by rabbit aorta when 12-lipoxygenase is supplied exogenously.     

Optimization of an assay for studying the effects of agents on cyclooxygenase and lipoxygenase metabolism of arachidonic acid in washed human platelets

Before one can examine the effects of substances on the metabolism of arachidonic acid (AA) by the cyclooxygenase and lipoxygenase pathways, an assay system which allows one to detect increases or decreases in both pathways in needed. In order to develop such a system, we have examined nonaggregating washed human platelets (10(8) platelets/0.5 ml) incubated for various times with 2 microCi 3H-AA and increasing concentrations of AA. T/B2, HHT, 12-HETE, and AA were extracted and separated using reverse phase-HPLC. We first calculated the mass of AA products formed with 10(-7) to 10(-4) M AA and found that the cyclooxygenase was saturated with 10(-5) M AA whereas the lipoxygenase was not saturated with 10(-4) M AA. Cyclooxygenase products were more prevalent than 12-HETE below 10(-5) M AA, while lipoxygenase products predominated at 3 x 10(-5)-10(-4) M AA. Using 3 microM AA, which does not saturate the cyclooxygenase, we examined the effect of 0.25-10 minute incubation durations on the distribution of AA metabolites and found AA product formation to increase throughout this period without completely depleting the substrate. Since substrate depletion does not occur and further metabolism could be detected for both pathways with a 5 minute incubation with 3 microM AA, these incubation parameters were chosen in order to further test the assay system. Using these parameters, we found that 10(-4) M 5-hydroxytryptamine enhanced platelet 12-HETE formation and decreased T/B2 and HHT formation, thus demonstrating the capacity of this system to simultaneously detect changes in cyclooxygenase and lipoxygenase enzyme metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetyltransferase activity in human platelet microsomes and washed platelets

It has been demonstrated that human platelets form platelet-activating factor (PAF) when stimulated by thrombin, collagen and ionophore A-23187, but the mechanism of its formation has not been elucidated. In this study we demonstrated increased acetyltransferase activity (i.e., transfer of the acetyl moiety of [3H]acetyl-CoA to lyso-PAF (1-alkyl-sn-glycero-3-phosphocholine) to form PAF) occurring in human platelet microsomes made from platelets stimulated by thrombin or ionophore A-23187. This stimulation resulted in a 2-4-fold increase in acetyltransferase activity over unstimulated platelets. Acetyltransferase activity was also demonstrated by incubating [3H]acetate with whole platelets and stimulating with thrombin or ionophore A-23187. Radioactive PAF was detected when the platelets were stimulated. None was formed without stimulation. These findings indicate that acetyltransferase may play a role in the biosynthesis of PAF by human platelets.     

Aspirin inhibits Ca2t+-stimulated fatty acid release from human washed platelets

Ca²⁺ at 2mM concentration stimulates the release of saturated and unsaturated fatty acids from intact washed platelets incubated at 37°C with stirring. Aspirin at a concentration of 0.4mM inhibits both cyclo-oxygenase activity and fatty acid efflux induced by Cat+. Thus, in intact washed platelets, aspirin reduces formation of cyclo-oxygenase products by direct inhibition of the enzyme and by reducing the availability of precursor arachidonate.     

Binding of coagulation factor XI to washed human platelets

The binding of human coagulation factor XI to washed human platelets was studied in the presence of zinc ions, calcium ions, and high molecular weight kininogen. Significant factor XI binding occurred at physiological levels of these metal ions when high molecular weight kininogen was present. Binding required platelet stimulation and was specific, reversible, and saturable. Scatchard analysis of the binding yielded approximately 1500 binding sites per platelet with an apparent dissociation constant of approximately 10 nM. Since the concentration of factor XI in plasma is about 25 nM, this suggests that in plasma factor XI binding sites on stimulated platelets might be saturated. Calcium ions and high molecular weight kininogen acted synergistically to enhance the ability of low concentrations of zinc ions to promote factor XI binding. The similarity between the concentrations of metal ions optimal for factor XI binding and those optimal for high molecular weight kininogen binding, as well as the ability of high molecular weight kininogen to modulate these metal ion effects, implies that factor XI and high molecular weight kininogen may form a complex on the platelet surface as they do in solution and on artificial negatively charged surfaces.     

Production of platelet-activating factor by washed rabbit platelets

Production of platelet-activating factor by washed rabbit platelets under stimulation with the ionophore A23187 was investigated utilizing two groups of platelet preparations. The first platelet preparation contained 0.03 +/- 0.02% contaminating white cells, while the second preparation contained 0.48 +/- 0.27% white cells. The latter preparation produced platelet-activating factor, mainly 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, 8.3 +/- 6.3 pmol (mean +/- standard deviation) with a range of 2.6 to 21.4 pmol (n = 9), followed by small quantities of 1-octadecenyl- and 1-octadecyl-2-acetyl-sn-glycero-3-phosphocholine. In contrast, there was no production of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by the former platelet preparation having 0.03% leukocytes. These quantitative analyses were carried out by the selected ion monitoring technique and it was concluded that it is necessary to consider the presence of contaminating white cells in studies on the production of platelet-activating factor by platelets.     

Trans n-3 eicosapentaenoic and docosahexaenoic acid isomers exhibit different inhibitory effects on arachidonic acid metabolism in human platelets compared to the respective cis fatty acids

N-3 trans geometrical isomers of 20:5 n-3 and 22:6 n-3 were isolated from rats fed heated linseed oil. The ability of these acids to inhibit 20:4 n-6 metabolism by human platelets was examined. The concentrations required to inhibit 50% of platelet aggregation (IC50) induced by 2.5 microM 20:4 n-6 were higher for the 20:5 delta 17t isomer compared to all cis 20:5 n-3; means 29.2 and 7.6 microM, respectively (P less than 0.05). There were no significant differences in IC50 between 22:6 delta 19t and all cis 22:6 n-3; means 4.3 and 5.6 microM, respectively (P greater than 0.05). Inhibition of action of cyclooxygenase on 20:4 n-6 was similar for 20:5 delta 17t and 20:5 n-3 when examined at their IC50s, but comparison at equal concentrations indicated that 20:5 n-3 was a significantly better inhibitor (P less than 0.05). The ability to inhibit platelet aggregation was paralleled by cyclooxygenase inhibition as determined by thromboxane B2 and 12-hydroxyheptadecatrienoic acid formation. 22:6 delta 19t appeared to inhibit cyclooxygenase more completely than 22:6 n-3, examined at their IC50s or at similar concentrations (P less than 0.05). Isomers of 20:5 n-3 and 22:6 n-3 having an n-3 cis or trans bond appear to have similar modes of action, although levels required for effectiveness are different for the C20 acids.     

Requirement of free arachidonic acid for leukotriene B4 biosynthesis by 12-hydroperoxyeicosatetraenoic acid-stimulated neutrophils

Stimulation of human neutrophils with 12-hydroperoxyeicosatetraenoic acid (12-HPETE) led to formation of 5S, 12S-dihydroxyeicosatetraenoic acid (DiHETE), but leukotriene B4 (LTB4) or 5-hydroxyeicosatetraenoic acid (5-HETE) was not detectable by reversed-phase high-performance liquid chromatography analysis. N-formylmethionylleucylphenylalanine (FMLP) induced the additional synthesis of small amounts of LTB4 in 12-HPETE-stimulated neutrophils. The addition of arachidonic acid greatly increased the synthesis of LTB4 and 5-HETE by neutrophils incubated with 12-HPETE. In experiments using [1-14C]arachidonate-labeled neutrophils, little radioactivity was released by 12-HPETE alone or by 12-HPETE plus FMLP, while several radiolabeled compounds, including LTB4 and 5-HETE, were released by A23187. These findings demonstrate that LTB4 biosynthesis by 12-HPETE-stimulated neutrophils requires free arachidonic acid which may be endogenous or exogenous.     

12(S)-hydroperoxyeicosatetraenoic acid (12-HETE) increases mitochondrial nitric oxide by increasing intramitochondrial calcium

12(S)-Hydroxyeicosatetraenoic acid (12-HETE) is one of the metabolites of arachidonic acid involved in pathological conditions associated with mitochondria and oxidative stress. The present study tested effects of 12-HETE on mitochondrial functions. In isolated rat heart mitochondria, 12-HETE increases intramitochondrial ionized calcium concentration that stimulates mitochondrial nitric oxide (NO) synthase (mtNOS) activity. mtNOS-derived NO causes mitochondrial dysfunctions by decreasing mitochondrial respiration and transmembrane potential. mtNOS-derived NO also produces peroxynitrite that induces release of cytochrome c and stimulates aggregation of mitochondria. Similarly, in HL-1 cardiac myocytes, 12-HETE increases intramitochondrial calcium and mitochondrial NO, and induces apoptosis. The present study suggests a novel mechanism for 12-HETE toxicity.