Lipid composition of blood platelets and erythrocytes of southern elephant seal (Mirounga leonina) and antarctic fur seal (Arctocephalus gazella)

Erythrocyte and blood platelet phospholipid compositions were studied in three elephant seals and two fur seals, two species of marine mammals living in the Subantarctic region feeding on preys rich in (n-3) polyunsaturated fatty acids. Results were compared with those reported for related species and humans. In erythrocytes, the phospholipid (PL) and cholesterol (CHOL) contents were lower in pinnipeds than in humans. Phosphatidylcholine (PC) levels were higher in elephant seals than in fur seals, with a reverse trend for phosphatidylethanolamine (PE) and phosphatidylserine (PS). Both species had lower SM/PC ratios and PE plasmalogen concentrations than human. Erythrocytes were richer in (n-3) fatty acids (FA) in pinnipeds than in humans. In platelets, the PL content was lower and the CHOL content higher in elephant seals than in humans or in other phocid seal species studied to date. The SM/PC ratio was much higher than in other seal species or in man. In both species, the proportion of PE plasmalogens was higher in platelets than in erythrocytes. PL were more saturated in elephant seals than in fur seals. These results suggest that the erythrocytes and platelets of wild marine mammals may prove useful models to study the influence of dietary lipids on the structure and hemostatic function of these cells.     

Arachidonate cannot be released directly from diacyl-sn-glycero-3-phosphocholine in thrombin-stimulated platelets.

The origin of the arachidonate released from platelets on stimulation with thrombin was investigated by comparing the specific activities of released arachidonate and of arachidonoyl-containing phospholipids using rat platelets prelabelled with arachidonate. Quantification of the released arachidonate was determined in the presence of BW 755 C, a dual cyclo-oxygenase/lipoxygenase inhibitor, which was found not to modify the arachidonate mobilization between the platelet phospholipids. The phospholipid molecular species were analysed by h.p.l.c. of diradylglycerol benzoate derivatives of diacyl, alkylacyl and alkenylacyl classes. The labelled/unlabelled arachidonate ratio varied greatly in the phospholipids depending on whether an ether or acyl bond was present in sn-1 position of the glycerol, on the length and degree of unsaturation of this fatty chain and on the polar head group. Between 15 s and 5 min of stimulation by thrombin, the released arachidonate kept a constant specific activity which was considerably lower than the specific activity of diacyl-GPC. The specific activity of the released arachidonate was intermediate between the specific activities of the 16:0-20:4 and 18:0-20:4 species of diacyl-GPI and diacyl-GPE, and corresponded to the mean specific activity of alkylacyl-GPC. The data indicate that the released arachidonate cannot come directly from diacyl-GPC, and that two phospholipids in particular can act as direct precursors of the released arachidonate. These are (1) the alkylacyl-GPC and (2) the diacyl-GPE whose hydrolysis would induce an arachidonate transfer from diacyl-GPC.     

Studies on the acyl-chain selectivity of cellular phospholipases A2

The selective release of arachidonate, as opposed to monoenoic and dienoic fatty acids, after stimulation of cells has suggested the involvement of arachidonate-selective phospholipases A2. Supportive evidence for the existence of such enzymes has also come from in vitro experiments. We have studied the acyl-chain selectivity of phospholipase A2 preparations obtained from human polymorphonuclear leukocytes, human platelets and rat platelets using sn-2-[14C]oleoylphosphatidylcholine and sn-2-[3H]arachidonoylphosphatidylcholine either as single substrates or in doubly labeled mixtures. In either case, no evidence for acyl-chain selectivity was observed for human PMN and rat platelet phospholipase A2. Additional experiments with human PMN homogenates and derived extracts yielded no indication for the selective loss of an arachidonate-selective phospholipase A2. Results with human platelet cytosol were highly suggestive for the presence of an arachidonoyl-selective phospholipase A2 when separate phosphatidylcholine species were assayed. This apparent selectivity was progressively lost when the substrates were mixed or embedded in a membrane of 1-palmitoyl-2-linoleoylphosphatidylcholine. The implications for occurrence of arachidonate-selective phospholipase A2 are discussed.     

Rapid return of cyclo-oxygenase active platelets in dogs after a single oral dose of aspirin

A single dose of oral aspirin in human subjects inhibits the aggregation response of platelets to arachidonate and other agents for approximately one week after ingestion. In the present study we have evaluated the rate at which cyclo-oxygenase active platelets return to the circulation in humans and dogs and compared the response curves obtained to improvements in cyclo-oxygenase activity produced by the aspirin platelets. After a single dose of aspirin, dog platelet function was compromised for several days. Normal responses to arachidonate and other aggregating agents were restored six days after aspirin, and the pattern of recovery was the same for dogs and human subjects. However, cyclo-oxygenase active platelets returned to the circulation in dogs more rapidly than in humans and chemical competence was restored in both species well before correction of the defective response to aggregating agents. The delay of 1-3 days before return of significant numbers of cyclo-oxygenase active platelets most likely reflects acetylation of bone marrow megakaryocytes by the drug. More rapid return of chemically competent cells in dogs than humans probably relates to the more rapid turnover and shorter life span of canine platelets. The basis for the discrepancy in return of chemical integrity compared to functional activity after aspirin in vivo compared to simultaneous correction of chemistry and function when 10% normal platelets are added to aspirin platelets in vitro remains unresolved.     

Very high proportion of disaturated molecular species in rat platelet diacyl-glycerophosphocholine: involvement of CoA-dependent transacylation reactions

The molecular species composition of rat platelet diacyl-glycerophosphocholine (GPC) was investigated by reverse-phase HPLC and by mass spectrometry. The two methods gave the same very high proportion of fully saturated phospholipids, the 16:0-16:0 and 16:0-18:0 species representing together about 40% of the overall molecular species. [14C]Palmitoyllyso-GPC was found to be acylated by resting platelets in equal amounts into 16:0-16:0 and into 16:0-20:4 species. The acylation rate of this lysophospholipid was increased by 3-fold and 14-fold when platelets were stimulated for 10 min with thrombin and the ionophore A23187, respectively. Essentially the same two molecular species were synthesized upon stimulation but with a higher preference for arachidonate than for palmitate. We investigated the mechanisms responsible for the incorporation of palmitate and arachidonate by examining the enzymatic acylation of [14C]palmitoyllyso-GPC by platelet homogenates. The percentage of the various molecular species formed when CoA, ATP, and Mg2+ were added excludes the CoA, ATP-dependent pathway as being involved in the acylation reactions previously observed. In the absence of ATP, CoA-independent transacylations appear to play a crucial role in the synthesis of the 16:0-20:4 species whereas the addition of CoA greatly favored dipalmitoyl-GPC synthesis. The involvement of CoA-dependent mechanisms in the synthesis of dipalmitoyl-GPC was demonstrated as follows: (i) the labeling in the sn-2 position of the dipalmitoyl-GPC synthesized in the presence of CoA was not modified when free unlabeled palmitic acid was added to the incubation medium and (ii) platelet homogenates were unable to esterify lysolecithin with added labeled palmitic acid in the presence of CoA only.     

Mepacrine-induced inhibition of human platelet cyclic-GMP phosphodiesterase

The effect of mepacrine (DL-quinacrine-HCI), a specific inhibitor of phospholipase C, on cyclic-GMP levels in human platelets was investigated. The concentrations of mepacrine producing 50% inhibition of human platelet aggregation induced by 5 microM ADP and 3 micrograms/ml of collagen were 50 +/- 8 and 70 +/- 15 microM, respectively. Addition of mepacrine to human platelet suspension resulted in increases in cyclic GMP. In contrast to cyclic-GMP levels, cyclic-AMP content was not affected by mepacrine. Mepacrine did not stimulate guanylate cyclase, but did specifically inhibit human platelet cyclic-GMP phosphodiesterase, separated from cyclic-AMP phosphodiesterase or other forms of phosphodiesterase on DEAE-cellulose columns. Stimulation by cyclic GMP of human platelet cyclic-GMP-stimulated cyclic-AMP phosphodiesterase activity was not inhibited by mepacrine. The IC50 value of the drug for cyclic-GMP phosphodiesterase was 40 microM, and IC50 for cyclic-AMP phosphodiesterase was 1.2 mM. Mepacrine was 30-times more potent as an inhibitor of human platelet cyclic GMP than of cyclic-AMP phosphodiesterase. Mepacrine blocks arachidonate release from human platelets by inhibiting phosphatidylinositol-specific phospholipase C. The increase in cyclic-GMP levels produced by addition of mepacrine will explain part of the pharmacological action of this drug.     

High concentrations of exogenous arachidonate inhibit calcium mobilization in platelets by stimulation of adenylate cyclase.

1. Exposure of platelets to exogenous arachidonic acid results in aggregation and secretion, which are inhibited at high arachidonate concentrations. The mechanisms for this have not been elucidated fully. In our studies in platelet suspensions, peak aggregation and secretion occurred at 2-5 microM-sodium arachidonate, with complete inhibition around 25 microM. 2. In platelets loaded with quin2 or fura-2, the cytoplasmic Ca2+ concentration, [Ca2+]i, rose in the presence of 1 mM-CaCl2 from 60-80 nM to 300-500 nM at 2-5 microM-arachidonate, followed by inhibition to basal values at 25-50 microM. Thromboxane production was not inhibited at 25 microM-arachidonate. Cyclic AMP increased in the presence of theophylline, from 3.5 pmol/10(8) platelets in unexposed platelets to 8 pmol/10(8) platelets at 50 microM-arachidonate; all platelet responses were inhibited with doubling of cyclic AMP contents. 3. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine attenuated the inhibitory effect of arachidonate, suggesting that it is mediated by increased platelet cyclic AMP and that it is unlikely to be due to irreversible damage to platelets. 4. Aspirin or the combined lipoxygenase/cyclo-oxygenase inhibitor BW 755C did not prevent the inhibition by arachidonate of either [Ca2+]i signals or aggregation induced by U46619. 5. Thus high arachidonate concentrations inhibit Ca2+ mobilization in platelets, and this is mediated by stimulation of adenylate cyclase. High arachidonate concentrations influence platelet responses by modulating intracellular concentrations of two key messenger molecules, cyclic AMP and Ca2+.     

Effect of vitamin E enrichment on arachidonic acid release and cellular phospholipids in cultured human endothelial cells

The effect of (R,R,R)-alpha-tocopherol on agonist-stimulated arachidonate release and cellular lipids was investigated in cultured human umbilical cord endothelial cells. Endothelial cells in culture incorporate added tocopherol in a dose-dependent manner at both physiological (23.2 microM) or pharmacological (92.8 microM) concentrations which were well tolerated by the cells, as judged by unaltered cell number and viability. Two experiments were conducted in which cells were either incubated with (R,R,R)-alpha-tocopherol followed by labelling with [1-14C]arachidonic acid or they were labelled with arachidonate followed by incubation with tocopherol. Irrespective of the sequence of incubation with arachidonate and tocopherol, (R,R,R)-alpha-tocopherol-enriched cells released significantly more labelled arachidonate when stimulated with thrombin (2.5 U/ml) or ionophore A23187 (1 microM) for 10 min. The magnitude of [1-14C]arachidonate release was higher from ionophore A23187 stimulation than from thrombin stimulation, but the trend of increased arachidonate release in tocopherol-enriched cells was the same. Results from these studies demonstrate that (R,R,R)-alpha-tocopherol can stimulate arachidonate release in human endothelial cells. This observation is in direct contrast to the role of tocopherol, which has been shown to inhibit platelet and cardiac phospholipase A2 activity in rats, and to reduce thrombin-stimulated thromboxane release in rat platelets.     

Asymmetry of arachidonic acid metabolism in the phospholipids of the human platelet membrane as studied with purified phospholipases

Human platelets were incubated with high density lipoproteins (HDL) doubly labelled with either free [14C]arachidonate/[3H]arachidonoylphosphatidylcholine or free [14C]oleate/[3H]oleoylphosphatidylcholine. Whereas [14C]arachidonate was incorporated at a 10-15-times higher rate than [14C]oleic acid, the exchange of both species of phosphatidylcholine occurred to the same extent. In both cases, free 3H-labelled fatty acids were generated during the labelling procedure, indicating phospholipase A2 hydrolysis. A redistribution of radioactivity to other phospholipids was noted after exchange of [3H]arachidonoylphosphatidylcholine only. (2) The exchange of phosphatidylcholine to platelets was confirmed using [14C]choline-labelled dipalmitoyl-and 1-palmitoyl-2-arachidonoylphosphatidylcholines. (3) Non-lytic degradation of platelet phospholipids by phospholipases revealed that free fatty acids were incorporated at the inside of the cells, whereas exchange was taking place on the platelet outer surface. However, 2-arachidonoylphosphatidylcholine displayed a more rapid movement towards the cell inside. The above findings suggest a topological asymmetry for the two pathways (acylation and exchange) of fatty acid renewal in platelets. The possible mechanisms and physiological relevance of the translocation of the external arachidonic acid pool across the membrane are discussed.     

Global analysis of the rat and human platelet proteome – the molecular blueprint for illustrating multi‐functional platelets and cross‐species function evolution

Emerging evidences indicate that blood platelets function in multiple biological processes including immune response, bone metastasis and liver regeneration in addition to their known roles in hemostasis and thrombosis. Global elucidation of platelet proteome will provide the molecular base of these platelet functions. Here, we set up a high‐throughput platform for maximum exploration of the rat/human platelet proteome using integrated proteomic technologies, and then applied to identify the largest number of the proteins expressed in both rat and human platelets. After stringent statistical filtration, a total of 837 unique proteins matched with at least two unique peptides were precisely identified, making it the first comprehensive protein database so far for rat platelets. Meanwhile, quantitative analyses of the thrombin‐stimulated platelets offered great insights into the biological functions of platelet proteins and therefore confirmed our global profiling data. A comparative proteomic analysis between rat and human platelets was also conducted, which revealed not only a significant similarity, but also an across‐species evolutionary link that the orthologous proteins representing “core proteome”, and the “evolutionary proteome” is actually a relatively static proteome.     

Arachidonyl transfer from diacyl phosphatidylcholine to ether phospholipids in rat platelets.

High levels of ether phospholipids were found in rat platelets. Alkylacyl compounds constituted 18 and 29% of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE). Alkenylacyl compounds, not detected in GPC, represented 40% of GPE. Arachidonate comprised 60%, 42% and 26% of the acyl residues in the sn-2 position of alkenylacyl-GPE, alkylacyl-GPE and alkylacyl-GPC respectively. Based on all arachidonate being linked to the sn-2 position of the diacyl species, the arachidonate level was 47% in diacyl-GPE and 30% in diacyl-GPC. The incorporation and metabolic fate of arachidonate in various phospholipid classes of resting platelets was examined. Arachidonate was essentially recovered in the diacyl phospholipids and very poorly in alkylacyl- and alkenylacyl-GPE and GPC after 30 min incubation in the presence of [14C]arachidonic acid. Upon reincubation of the platelets after removal of free arachidonate, the radioactivity was gradually lost from diacyl-GPC. Concomitantly, the radioactivities in alkylacyl-GPC, alkylacyl-GPE, alkenylacyl-GPE and to a lower extent in diacyl-GPE were increased. Labelling of glycerophosphoinositol was not changed. This labelling transfer was linear up to 5-6 h, except for alkylacyl-GPC; then labelling remained constant. These data strongly suggest that free arachidonate incorporation through the Lands pathway occurs only for diacyl species and that arachidonate incorporation into the ether phospholipids is achieved by exchange from diacyl-GPC. Based on specific activities related to phosphorus content, the arachidonate incorporation rates into diacyl-GPE and diacyl-GPC were approximately equivalent. The very large differences between specific radioactivities related to arachidonate observed at the starting reincubation time were strongly attenuated when labelling equilibrium was reached. The turnover rate by this exchange pathway was higher in alkylacyl-GPC than in alkyl- and alkenylacyl-GPE. This finding agrees with the selectivity for arachidonate observed in the acylation of PAF-acether in human neutrophils [Chilton, O'Flaherty, Ellis, Swendsen & Wykle (1983) J. Biol. Chem. 258, 7268-7271].     

Compound 48/80 is a potent inhibitor of phospholipase C and a dual modulator of phospholipase A2 from human platelet

Compound 48/80 inhibited phosphatidylinositol-specific phospholipase C activity from human platelets. Whereas 1 microgram/ml of compound 48/80 slightly stimulated Ca2+-dependent phospholipase A2, higher concentrations led to dose-dependent inhibition of this platelet enzyme. This biphasic effect was confirmed with phospholipases A2 purified from rat liver and human synovial fluid. The aggregation of human platelets induced by ADP and PAF-acether was inhibited by compound 48/80, whereas the aggregation induced by ionophore A23187 was not modified by this compound. These results demonstrate that the inhibition of platelet aggregation by compound 48/80 is not due solely to effects on calmodulin as previously reported, but that inhibition of phospholipases and probably arachidonate mobilization may also be involved.     

Calcium, collagen dose, gender and fasting affect the response of rat platelet thromboxane formation to extremes in dietary linoleate

Platelet thromboxane synthesis in response to supplemental linoleate in the diet has been very inconsistent. The objective of this study was to investigate potential confounding factors known to affect platelet thromboxane synthesis. Citrated whole blood was recalcified with varying Ca2+ concentrations and challenged with low or high dose collagen preparations to induce extreme ranges of thromboxane synthesis from endogenous arachidonate pools by rat platelets. Male and female weanling rats were fed 0.0, 1.0 or 23 energy percent linoleate for 11 to 13 weeks. Fasting tended to enhance thromboxane synthesis. Both fasted and fed females showed slightly faster rates of thromboxane synthesis than males. Essential fatty acid deficiency depressed (P less than 0.01) thromboxane synthesis; the degree of this depression was inversely related to the level of recalcification (68% for 0.0 mM Ca2+, 36% for 2.5 mM Ca2+ and 20% for 5.0 mM Ca2+) when challenged with the high dose collagen. Essential fatty acid deficiency depressed platelet phospholipid arachidonate concentration 26%. Only blood from fed females stimulated with a mild challenge responded to excess dietary linoleate, and a 62% (not statistically significant) depression in TX synthesis was observed and this was associated with a decrease in platelet phospholipid arachidonate concentration.     

Evidence for the release of arachidonic acid through the selective action of phospholipase A2 in thrombin-stimulated human platelets

The release of arachidonic acid from thrombin-stimulated platelets can be attributed to the action of phospholipase A2 on membrane phospholipid. Previously, analysis of individual subclasses of phospholipid demonstrated that 1-acyl-2-[3H]arachidonoyl-sn-glycerophosphocholine and to a lesser degree 1-acyl-2-[3H]arachidonoyl-sn-glycerophosphoethanolamine were the main source of [3H]arachidonic acid in thrombin-stimulated cells. In the present work, 1,2-diacyl phospholipid subclasses were analyzed as 1,2-diacylglycerobenzoates by high-pressure liquid chromatography in order to analyze arachidonate release as mass changes in individual molecular species of phospholipid. Following thrombin stimulation (5 U/ml, 5 min, 37 degrees C) all arachidonoyl-containing molecular species of 1,2-diacyl-sn-glycerophosphocholine decreased in mass and [3H]arachidonate content by almost 50%, while those of 1,2-diacyl-sn-glycerophosphoethanolamine decreased by 20%. The mass change was substantial and indicated that these phospholipids are a major source of arachidonate in stimulated cells. No variation was seen in the other non-arachidonate-containing molecular species of either subclass. Thus, deacylation of membrane 1,2-diacylglycerophosphocholine and 1,2-diacylglycerophosphoethanolamine by phospholipase A2 is selective for those molecular species of phospholipid containing arachidonic acid, suggesting that a certain proportion of arachidonoyl-containing molecular species of phospholipid are compartmentalized with the platelet membrane proximal to the site of action of this enzyme. These studies demonstrate that the human platelet is a cell poised and specialized to release rapidly substantial amounts of arachidonic acid upon stimulation.     

Ca2+ effect on ATP-independent lysophospholipid transacylases is indissociable from Ca2+ effect on phospholipase A2 activity in rat platelet sonicates

CoA-dependent transacylation and phospholipid hydrolysis were studied in parallel experiments using rat platelet sonicates. The decrease observed in palmitoyllyso-sn-glycero-3-phosphocholine (palmitoyllyso-GPC) transcylation as a function of Ca2+ concentration was found to be correlated with appearance of endogenous lysoderivatives. We also demonstrated that endogenously produced acyllyso-sn-glycero-3-phosphoethanolamine (acyllyso-GPE) induced CoA-dependent arachidonate transfer from diacyl-GPC. These results further argue for a two-step arachidonate release from diacyl-GPC when platelets are stimulated with thrombin.     

Human platelet activation by an alkylacetyl analogue of phosphatidylcholine

The present study has evaluated the influence of semi-synthetic platelet-aggregating factor, (PAF) i.e., alkylacetylglycerophosphocholine, on human platelet morphology, biochemistry and function in order to determine if PAF serves as the corrective factor restoring sensitivity to refractory platelets after treatment with epinephrine. Threshold concentrations of PAF caused irreversible platelet aggregation which could be blocked by agents elevating endogenous levels or cyclic AMP or inhibited by antagonists of platelet prostaglandin synthesis and secretion. PAF did not stimulate platelets through α-adrenergic receptors or receptors for arachidonate, endoperoxides or thromboxanes. 24 h after aspirin ingestion, platelets could be aggregated irreversibly by high concentrations, but not by threshold amounts of PAF, even though they were still insensitive to arachidonate. Another less potent PAF derivative, alkenylacetylglycerophosphocholine, blocked aggregation of 24-h aspirin platelets by PAF, but did not inhibit restoration of arachidonate sensitivity and irreversible aggregation when the samples were treated first with epinephrine. Our findings indicate that threshold amounts of PAF activate human platelets in a physiologic manner and cause irreversible aggregation which is dependent on prostaglandin synthesis and the release reaction. The results do not support the concept that PAF is the mediator of the mechanism of membrane modulation through which epinephrine induces correction of the refractory state in prostaglandin I₂-treated or dissociated platelets, or cells obtained from individuals following aspirin ingestion. Thus, the mechanism of platelet membrane modulation is capable of securing irreversible aggregation of secretion, prostaglandin synthesis or PAF formation.     

Indomethacin prevents the long-lasting inhibitory effect of aspirin on human platelet cyclo-oxygenase activity

Aspirin and indomethacin do interact with the same site on cyclo-oxygenase. This suggestion is based on in vitro studies on ram seminal vesicles and in vivo drug interaction studies on rat platelets. The purpose of the present study was to ascertain whether the same interaction also occurred after administration of both drugs to human volunteers. Platelet aggregation induced by sodium arachidonate or by collagen, and formation of platelet MDA and TxB2 were measured before, two and 48 hours after ingestion of either indomethacin (50 mg) or aspirin (500 mg) or of both drugs (30 minutes apart). While the inhibitory effect of indomethacin on these parameters was short lasting, that of aspirin persisted for at least 48 hours. However, when both drugs were given concurrently, the long-lasting effect of aspirin was no longer detectable. Since competition at levels other than platelets was unlikely, this study indicates that indomethacin and aspirin inhibit human platelet cyclo-oxygenase by the same basic mechanism. Acetylation of the enzyme appears to be a secondary mechanism which makes the inhibitory effect of aspirin persistent.     

Detection in human platelets of phospholipase A2 activity which preferentially hydrolyzes an arachidonoyl residue

It has been generally considered that highly specific liberation of arachidonic acid is induced upon the stimulation of the platelets, although the molecular mechanism of the regulation of its action has not been well understood. An aim of the present study is to clarify the role of phospholipase A2 in the arachidonic acid metabolism within human platelets. Phosphatidylcholine or phosphatidylethanolamine with arachidonate at the sn-2 position of glycerol was cleaved efficiently by phospholipase A2 activity in homogenates as well as in the cytoplasmic fraction of human platelets, leading to the selective liberation of free arachidonate, whereas phospholipids with linoleate were hardly hydrolyzed under the same conditions. Double-reciprocal plots of kinetic data further strengthened the conclusion that human platelet phospholipase A2 showed high selectivity for arachidonoyl residue. This enzyme may play a crucial role in the intracellular metabolism of the arachidonate of phospholipids.     

1-ether-linked phosphoglycerides. Major endogenous sources of arachidonate in the human neutrophil

This study has quantitated changes in the content of labeled and unlabeled arachidonate of neutrophil phosphoglyceride classes and subclasses during cell activation with ionophore A23187. The predominant pools of endogenous arachidonate in the resting neutrophil were found in ethanolamine (68%)-, choline (19%)-, and inositol (12.0%)-containing glycerolipids. Upon stimulation, endogenous arachidonate was lost from primarily ethanolamine (PE) greater than choline (PC) greater than inositol (PI)-linked phosphoglycerides. Released leukotriene B4 and 20-hydroxyleukotriene B4 accounted for 10-35% of the total arachidonate lost from all phosphoglyceride classes. In contrast to the mass loss, ionophore induced a decrease of labeled arachidonate from primarily PC and PI. In the resting neutrophil, 66% of the total arachidonate in PC was found in the 1-alkyl-linked fraction. Furthermore, loss of endogenous arachidonate from 1-alkyl-2-arachidonoyl sn-glycero-3-phosphocholine accounted for 62% of the decrease of arachidonate from choline-linked phosphoglycerides. In contrast, 60% of the release of labeled arachidonate from PC subclasses originated from 1-acyl molecular species. 1-Alk-1'-enyl-2-acyl-sn-glycero-3-PE contained 71% of the arachidonate in ethanolamine-linked phosphoglycerides and was the major PE subclass which was degraded during neutrophil activation with ionophore A23187. These findings demonstrate that human neutrophils contain large ether-linked stores of arachidonate and the capacity to mobilize these stores. In addition, this study points out major discrepancies between using mass or label to determine sources of arachidonate for eicosanoids.     

Phorbol ester, 1,2-diacylglycerol, and collagen induce inhibition of arachidonic acid incorporation into phospholipids in human platelets

We have shown that phorbol myristate acetate (PMA) enhanced A-23187-induced arachidonate release and thromboxane synthesis in human platelets (Mobley, A., and Tai, H. H. (1985) Biochem. Biophys. Res. Commun. 130, 717-723). The mechanism of enhancement by PMA was not elucidated. In the present study, we have shown that PMA-treated platelets exhibited significantly less [1-14C]arachidonate incorporation than did control platelets. However, no significant change in uptake of labeled linoleate or oleate was observed by PMA treatment. Examination of the two enzyme activities involved in arachidonate incorporation into phospholipids indicated that both arachidonoyl-coenzyme A (CoA) synthase and arachidonoyl-CoA lysophosphatide acyltransferase were inactivated following treatment with PMA or 1-oleoyl-2-acetyl glycerol. When platelets were stimulated with A-23187 plus PMA which produced a significant synergism in thromboxane synthesis, both enzyme activities were substantially less than those in platelets treated with A-23187 alone. In addition to PMA and 1-oleoyl-2-acetyl glycerol induced decreases in both enzyme activities, collagen, a platelet agonist which can activate protein kinase C (Ca2+/phospholipid-dependent enzyme), was also found to cause a concentration-dependent attenuation of both enzyme activities. These results suggest that protein kinase C activation induced by PMA or collagen may cause inactivation of both arachidonoyl-CoA synthase and arachidonoyl-CoA lysophosphatide acyltransferase resulting in inhibition of the reincorporation of arachidonate released by A-23187 and, consequently, greater availability of arachidonate for thromboxane synthesis.