Platelet Phosphatidylcholine Research Articles

Gender-specific fatty acid profiles in platelet phosphatidyl-choline and -ethanolamine

Previous studies suggested that women synthesise docosahexaenoic acid (DHA) more efficiently from their precursors than men. This study investigated the relationship between diet, platelet phospholipids fatty acids and gender. Dietary intake and platelet phosphatidyl-choline (PC) and phosphatidylethanolamine (PE) fatty acids were determined in Caucasian 40 men and 34 women. Absolute and %energy intakes of arachidonic acid (AA), eicosapentaenoic acid (EPA), and DHA, and the ratios of total n-6/n-3 PUFA and linoleic/alpha-linolenic acids did not differ between the sexes. However, women had higher DHA in PC (1.19 vs 1.05 wt%, p<0.05) and PE (3.62 vs 3.21 wt%, p<0.05) than men. Also EPA (1.10 vs 0.93 wt%, p<0.05) was higher in women's PE. Conversely, men had elevated AA and total n-6 fatty acids in PC. The higher platelet DHA levels and lower platelet AA/EPA and AA/DHA ratios in women of child-bearing age compared with men, may lead to less platelet aggregation and vaso-occlusion.

Biochemical properties of platelet microparticle membranes formed during exocytosis resemble organelles more than plasma membrane

Studies of [ 3H]glycerol turnover in phosphatidylcholine (PC) in platelets revealed two metabolic pools, a ‘low turnover PC’ in collagen-induced microparticles with specific radioactivity only 10% of that found in the ‘high turnover PC’ of bulk platelet PC. Isolated organelle fractions of [ 3H]glycerol-labelled platelets contained [ 3H]PC with specific radioactivities about 20% of that in membrane fractions. These results together with studies on distribution of concanavalin A-FITC and GPlb, a plasma membrane receptor, indicate that microparticles formed during exocytosis are not simple vesiculations of plasma membrane, but they seem rather to originate from a relatively metabolically static membrane pool not accessible to extracellular reagents.

Effect of dietary olive and sunflower oils on the lipid composition of the aorta and platelets and on blood eicosanoids in rats.

The effects on aortic and platelet fatty acid compositions and on blood levels of prostacyclin and thromboxane A2 of low- and high-fat diets containing olive oil or sunflower oil were studied. For 4 weeks, four groups of weanling rats were fed a basal diet containing 5% or 25% olive oil or sunflower oil. Rats fed olive oil diets showed higher levels of 18:1(n-9) and polyunsaturated fatty acids of the n-3 series and lower percentages of 18:0 and 18:2(n-6) in aortic and platelet phosphatidylcholine and phosphatidylethanolamine than those fed the sunflower oil diets. Arachidonic acid increased in platelet phosphatidylethanolamine and aortic phosphatidylcholine of rats fed the diet containing 5% sunflower oil compared with those fed 5% olive oil. Plasma 6-ketoprostaglandin F1 alpha increased in both groups of animals fed olive oil while these rats also showed the lowest levels of serum thromboxane B2 and plasma cholesterol. Olive oil feeding leads to changes in lipid metabolism of the vascular compartment that could be favorable in the prevention of thrombosis and atherosclerosis.

Increased concentrations of phosphatidylinositol (PI) and decreased esterification of arachidonic acid into phospholipids in platelets from patients with schizoaffective disorders or atypic phasic psychoses

The concentration of various phospholipids (PLs) and sphingomyelin in platelets and the amount of [ 14C] arachidonic acid ([ 14C]-AA) esterified in phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were measured. The platelet-rich plasmas from unmedicated patients with psychiatric disorders and healthy controls were incubated for 30 min with 1 μM [ 14C]-AA. Platelets from patients with a schizoaffective disorder according to RDC criteria, a schizophreniform disorder (DSM III criteria) or an atypical phasic psychosis according to FC criteria contained twice as much PI and had significantly increased concentrations of PC as compared to controls (p < 0.05, t-test). A highly significant (40–70%) reduced rate of esterification of [ 14C]-AA into PI PS , PC and PE was found in platelets from patients with schizophreniform, schizoaffective and major depressive disorders but not in platelets from patients with chronic schizophrenia. The largest reduced esterification of [ 14C]-AA (about 70%) was found in PI PS of platelets from patients with schizoaffective disorders (1.9 ± 0.7 vs 6.3 ± 1.7 μmol [ 14C]-AA/mol PI PS ; p < 10 −4, t-test). The results indicate that changes in the metabolism of arachidonic acid and phosphatidylinositol and, to a lesser degree, of phosphatidylcholine in platelets are characteristic of patients with a likely favorable outcome of a psychotic episode.

Species variation in the atherogenic profile of monkeys: Relationship between dietary fats, lipoproteins, and platelet aggregation

Because lipoproteins and platelet aggregation have been implicated in atherogenesis, relative differences in the response of these variables to dietary fat saturation were compared in three species of monkeys differing in their susceptibility to atherosclerosis (cebus, rhesus, and squirrel monkeys). Both long-term (8-12 years) and short-term (8 weeks) responses to diets containing 31% fat calories were examined in the same monkeys. As expected, long-term feeding of coconut oil by comparison to corn oil produced significantly higher plasma concentrations of total cholesterol, LDL cholesterol, apoB, and triglycerides, as well as higher ratios of LDL/HDL cholesterol and apo B/apo A-I. These responses were characteristic of all species with cebus being most responsive and rhesus the least. The short-term plasma cholesterol response to animal fats (butter, lard, beef tallow) was significantly less than that to coconut oil. When fish oil was substituted for two-thirds of either corn oil or coconut oil, exceptional decreases occurred in plasma cholesterol and triglycerides, as well as in HDL cholesterol and apo A-I concentrations despite the fact that the fish oil diets contained more saturated fat and less polyenes than the corn oil diet. Platelet aggregation tended to increase with saturated fat consumption and greatly decreased with fish oil intake in all monkeys, although cebus monkeys were ten-fold more resistant to platelet aggregation than the other two species. The molecular species of platelet phosphatidylcholine (PC) varied with both the dietary fat fed and species of monkey. An inverse correlation (r = -0.60; p less than 0.001) was found between changes in one such PC molecular species (18:0-20:4) induced by diet and the platelet aggregation threshold. These results demonstrate that the lipemic and platelet responses to dietary saturated fat depend upon both the type of fat (i.e., the specific combination of dietary fatty acids, including the chain length of saturated fatty acids and the degree of polyunsaturation) and the species of monkey (genetic component) in which the response is elicited.

Platelet phosphatidylcholine turnover in experimental hypertension.

To gain insight into the membrane alteration that could account for the hyperresponsiveness of platelets in hypertension, we have investigated whether, in resting platelets of hypertensive rats, the metabolism of phospholipids was modified. Because preliminary results indicated a specific acceleration of phosphatidylcholine turnover in spontaneously hypertensive rats, the possible relation between such an abnormality and hypertension was investigated by studying phosphorus-32 labeling of phosphatidylcholine (taken as an index of its turnover) in various experimental models of hypertension. The data showed that phosphatidylcholine turnover 1) was considerably increased in platelets from spontaneously hypertensive (even at the prehypertensive stage) and stroke-prone rats compared with Wistar or Wistar-Kyoto control rats, 2) did not differ between deoxycorticosterone-salt-treated hypertensive and control rats, and 3) was increased in Dahl salt-sensitive rats fed a high NaCl diet (hence hypertensive rats), compared with either the rats fed a low NaCl diet or the salt-resistant rats. These results indicate that an increase in phosphatidylcholine turnover is a consequence of neither hypertension nor high salt intake and appears likely to be of genetic origin. These data allow us to suggest the existence, in platelets, of a relation between phosphatidylcholine turnover, free cytoplasmic Ca2+, and responsiveness to stimuli. Because phosphatidylcholine is assumed to participate in signal transduction, an increase in its turnover in platelets might be considered as a primary membrane abnormality that, in primary hypertension, results in platelet hyperresponsiveness.

Aggregation and/or oxygenated products of arachidonic acid are not required for collagen-induced deacylation of phosphatidylcholine in human platelets

In the present study the effects of collagen on platelet aggregation and arachidonic acid (AA) mobilization, specifically from phosphatidylcholine (PC), were investigated in the presence and absence of BW755C, a selective inhibitor of cyclo-oxygenase and lipoxygenases. The inhibition of cyclo-oxygenase and lipoxygenase(s) by BW755C (75 microM) resulted in severe impairment in collagen-induced platelet aggregation. In the presence of BW755C, the aggregation response amounted to 14, 26, 37 and 49% of the corresponding controls (without BW755C) at 10, 25, 50 and 100 micrograms of collagen respectively. On the contrary, the amount of AA released from PC, which ranged from 3.5 to 8.6 nmol/10(9) platelets, in response to the action of collagen (10-100 micrograms) remained unaffected by the presence of BW755C. Substantial amounts of non-esterified AA were detected in the free fatty acid fractions obtained from collagen-stimulated platelets in the presence as well as in the absence of BW755C. However, the presence of BW755C caused a greater accumulation of free AA (mass) and this ranged from 4 to 16 nmol, depending upon the amount of collagen. In addition, small increases in free stearic and oleic acids were observed in collagen-stimulated platelets as compared with unstimulated platelets. The amount of AA lost from PC represented 67, 80, 49 and 52% of the free AA obtained at 10, 25, 50 and 100 micrograms of collagen respectively. Our results adhesion of platelets to collagen fibres may be responsible for much of the AA release from PC Furthermore, these results demonstrate that aggregation and/or cyclo-oxygenase/lipoxygenase metabolites are not obligatory for the release of AA from PC in collagen-stimulated human platelets.

The effect of a dietary supplement of n-3 polyunsaturated fat on platelet lipid composition, platelet function and platelet plasma membrane fluidity in healthy volunteers.

1. Eight healthy male volunteers (aged 22-39 years) supplemented their normal daily diet with 15 g encapsulated fish oil (MaxEPA) for a 6 week period. Fasting blood samples were taken before, at the completion of and 3 months after the period of supplementation. 2. Evaluation of nutrient intakes showed that the intake of n-3 polyunsaturated fatty acids rose significantly (P less than 0.01) during supplementation. This was reflected in changes in the fatty acid composition of platelet phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) without any changes in phosphatidyl serine, phosphatidyl inositol or sphingomyelin. 3. In both PC and PE there were significant (P less than 0.05) increases in the levels of 18:1n-9 and 20:5n-3 fatty acids and a significant (P less than 0.05) decrease in 20:4n-6 during supplementation. 16:0 rose significantly in PC (P less than 0.05) while in PE, 18:0 fell and both 22:5n-3 and 22:6n-3 rose significantly (P less than 0.05). 4. There were no significant effects of fish-oil supplementation on serum lipids, platelet cholesterol: phospholipid, collagen-induced platelet aggregation or collagen-induced platelet thromboxane B2 production. However, there was a significant correlation (P less than 0.001; r + 0.63) between total phospholipid arachidonic acid and platelet thromboxane production. 5. The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was used to determine whether fish-oil supplementation altered fluorescence polarization of isolated platelet plasma membrane and, by inference, platelet plasma membrane fluidity. No significant effect of fish-oil supplementation on fluorescence polarization was seen.

Enhanced turnover of phosphatidylcholine in platelets of hypertensive rats. Possible involvement of a phosphatidylcholine-specific phospholipase C

In an attempt to determine the mechanism involved in the hyperreactivity of platelets in primary hypertension, the dynamic behavior of phospholipids was investigated in quiescent platelets of spontaneously hypertensive rats (SHR) compared to normotensive controls. By using 32P i, [methyl- 3H]choline or [ 3Higlycerol as the radioactive precursors, the labeling of phosphatidylcholine (PC) was shown to be markedly enhanced (10-20-times) in SHR compared to controls. This difference between SHR and controls could not be ascribed to differences either in the actual amount of PC or m the uptake of various labels, suggesting that PC turnover was markedly enhanced in platelets of SHR. The [methyl-H]choline labeling of phosphocholine and of CDP-choline was twice as high m SHR as in controls; chase experiments showed that when the label disappeared from phosphocholine, it was rapidly converted to PC. The results indicated that in rat platelets, PC synthesis occurred mainly via the CDP-choline pathway, and suggested that CTP: phosphocholine cytidylyltransferase was the rate-limiting step; they also indicated that the activity of this enzyme and that of choline kinase might be enhanced m SHR platelets compared to Wistar-Kyoto rat (WKY) platelets, and may thus be responsible for the enhanced PC synthesis. From these results, the existence of a PC-specific phospholipase C activity involved in PC turnover in SHR platelets can be envisaged.

Kinetic parameters of lysophospholipid acyltransferase systems in diet-induced modifications of platelet phospholipid acyl chains.

Competitive effectiveness and relative Km values of polyunsaturated fatty acids were determined in the acyl-CoA : lysophospholipid acyltransferase systems in platelet membranes. Some CoA esters of fatty acids such as 18 : 2n-6 (linoleic), 18 : 3n-3(α-linolenic), 18 : 3n-6, 20 : 3n-6 (dihomo-γ-linolenic) and 20 : 5n-3 (eicosapentaenoic) were found in vitro to be relatively good competitive inhibitors of arachidonate (20 : 4n-6) incorporation into phosphatidylcholine, while in the acylation of lysophosphatidylinositol, 18 : 2n-6, 20 : 2n-6, 20 : 3n-6, 20 : 5n-3 and 22 : 4n-6 were relatively good inhibitors. When vegetable oil-supplemented diets rich in 18 : 2n-6 or 18 : 3n-3 were fed to rats, these fatty acids were converted to highly unsaturated fatty acids such as 20 : 4n-6 and 20 : 5n-3, and were esterified to phospholipids in platelets. The kinetic parameters determined in vitro were useful in predicting grossly the relative abundance of eicosanoid precursors (20 : 4n-6 and 20 : 5n-3), but not that of 18-carbon polyunsaturated fatty acids. It was found that arachidonate was conserved but n-3 fatty acids were excluded relatively more strictly in phosphatidylinositol than in phosphatidylcholine in platelets of rats on diets containing different proportions of 18 : 2n-6 and 18 : 3n-3.

Effects of calmodulin antagonists on serine phospholipid base-exchange reaction in rabbit platelets.

Effects of the calmodulin antagonists chlorpromazine, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide on phospholipid metabolism were examined in rabbit platelets using [3H]serine, [3H]ethanolamine, [3H]choline, and [3H]glycerol. All these drugs markedly stimulated the incorporation of [3H]serine into phosphatidylserine. On the other hand, these drugs had only a slight effect on the rate of incorporation of [3H]ethanolamine and [3H]choline into the corresponding phospholipid. When [3H]glycerol was used as a precursor of the phospholipids, 3H-labeled phospholipids were mainly composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Although the phosphorus content of phosphatidylserine was about 40% of that of phosphatidylcholine in rabbit platelets, the amount of phosphatidylserine labeled with [3H]glycerol was less than 2% of that of the labeled phosphatidylcholine, and calmodulin antagonists slightly stimulated the incorporation of [3H]glycerol into phosphatidylserine. Treatment with calmodulin antagonists caused a marked decrease in the content of endogenous free serine with concomitant increase in the contents of endogenous free ethanolamine and choline. On the other hand, the contents of other free amino acids, including essential and non-essential amino acids, were unchanged. These results suggest that the calmodulin antagonists we used did not affect de novo synthesis of phosphatidylserine, but did stimulate the serine phospholipid base-exchange reaction in rabbit platelets.

Inhibition of transmembrane movement and metabolism of platelet activating factor (PAF-acether) by a specific antagonist, BN 52021

Incorporation of 1-[ 3H]-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine ([ 3H] PAF -acether) into rabbit platelet phosphatidylcholine (PC) was inhibited by a specific antagonist, BN 52021 (IC 50 5.6 × 10 −6 M, maximal effect, i.e 70 % inhibition, at 10 −4 M). Under the same conditions, [ 3H] lyso-PAF-acether incorporation remained 9 fold lower, compared to PAF-acether, without any effect of BN 52021. Upon cell lysis, both phospholipids attained the same rate of metabolic conversion, corresponding to a 1.15-fold and a 12-fold increase for PAF-acether and lyso-PAF-acether, respectively. In none of these cases was BN 52021 effective. It is concluded that transmembrane movement of the two phospholipids represents the limiting step of their metabolism. The higher rate of PAF-acether conversion by intact platelets could involve its binding to a membrane receptor, as suggested by the inhibitory effect of BN 52021, the significance of which is discussed.

Turnover of arachidonic acid in the major diacyl and ether phospholipids of human platelets.

In this work, the uptake and release of [3H]arachidonic acid by the diacyl and ether species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in human platelets were studied. Uptake of [3H]arachidonic acid into 1,2-diacyl-PC and 1,2-diacyl-PE was much greater than into the ether phospholipids of the same class. In [3H]arachidonoyl-labeled platelets stimulated by thrombin, there was a decrease in total [3H] arachidonoyl-PC. This was accounted for mostly by a decrease in 1-acyl-2-[3H]arachidonoyl-PC while the level of 1-O-alkyl-2-[3H]arachidonoyl-PC (a precursor for platelet-activating factor) increased slightly. However, in ionophore A23187-stimulated platelets, the reduction of total [3H]arachidonoyl-PC was due to a decrease in both 1-acyl-2-[3H]arachidonoyl-PC and 1-O-alkyl-2-[3H] arachidonoyl-PC, suggesting that ionophore should yield more platelet-activating factor than thrombin. In both thrombin- and ionophore-stimulated platelets, there was a net increase in total [3H]arachidonoyl-PE. This consisted of a decrease in 1,2-diacyl-PE, which was essentially complete by 1 min, followed by an increase in 1-O-alk-1'-enyl-2-[3H]arachidonoyl-PE, which was slower and not apparent until 3-5 min after thrombin. During reincubation of labeled platelets with saline, the 1-O-alkyl-2-[3H]arachidonoyl-PC increased by a factor of 2, between 0 and 4 h, with no significant change in the radioactivity of any other phospholipid. Thus, upon stimulation of human platelets, arachidonic is released from both 1,2-diacyl-PC and 1,2-diacyl-PE for metabolism by platelet cyclooxygenase and lipoxygenase, while certain ether pools of PC and PE also collect arachidonic acid.

Altered phospholipid composition of plasma membranes from thrombin-stimulated human platelets

The individual phospholipid concentrations, and their respective fatty acid distributions, in whole platelet lysates and plasma membranes derived from unstimulated and thrombin-stimulated intact human platelets were studied. This was of interest, since previous work had led to the suggestion that altered phospholipid concentrations in plasma membranes of intact stimulated cells may be of importance in mediating cellular responses. The concentrations (nmol/mg protein) of phosphatidylinositol in whole platelet lysates and plasma membranes derived from thrombin-activated platelets decreased by 37 and 45%, respectively, as compared to their corresponding controls. As well, concentrations of plasma membrane phosphatidylcholine and phosphatidylethanolamine in thrombin-stimulated platelets decreased by 20 and 9%, respectively, when compared with their control values. The amounts of phosphatidylserine and sphingomyelin in whole platelet lysates and plasma membranes were unchanged by exposure to thrombin. Fatty acid analyses revealed that thrombin stimulation of intact human platelets induced a decrease in the arachidonate content (from 37.7 to 33.1 wt.% of total fatty acid) of plasma membrane phosphatidylinositol. Similar shifts in the wt% of arachidonic acid in plasma membrane phosphatidylcholine were found. These results indicate that thrombin stimulation of intact human platelets produces a significant decrease in the mass of phosphatidylinositol in plasma membranes and raises the suggestion that the preferential depletion of the plasma membrane in arachidonoyl-containing phosphatidylinositol may be of importance in mediating cellular responses to external stimuli.

Changes of linoleic, arachidonic and eicosapentaenoic acids in rat platelet, aorta and plasma lipids after changing from a sardine oil diet to a corn oil diet.

When rats were fed on a diet containing sardine oil (SO) for 10 days, 20:5 (eicosapentaenoic acid) was incorporated into the platelet, aorta and plasma lipids. The 20:5/20:4 (arachidonic acid) ratios of phospholipids were higher in platelets than in aortas. When the diet was changed from SO to corn oil (CO), the levels of 20:5 in the plasma lipids and platelet phospholipids decreased with an increase of 20:4. Although the 20:5 levels of phospholipids in the aortas decreased, 20:4 did not increase during feeding with the CO diet for 4 days. The 20:5/20:4 ratios of the phosphatidylcholine in platelets and aortas rapidly changed from 5.0 and 1.1 to 2.4 and 0.6, respectively. The linoleic acid level of phosphatidylcholine in platelets and aortas increased rapidly by changing from the SO diet to the CO diet, but this fatty acid was not incorporated into phosphatidylinositol of the platelets and aortas.

Inhibition of platelet aggregation and thromboxane synthesis after intake of small amount of icosapentaenoic acid

Elderly people ingested 150 mg/day of icosapentaenoic acid (20:5n-3) or a placebo for one month. Platelet aggregation, platelet arachidonate metabolism and the fatty acid composition of both plasma and platelet lipids were investigated before and after the intake. Platelet aggregation induced by collagen, epinephrine or low concentrations of ADP was significantly reduced after 20:5n-3 intake. Besides, the main oxygenated product formation from endogenous platelet arachidonate under thrombin stimulation was markedly decreased after the 20:5n-3 supplementation. Such a decrease was absent after placebo. Moreover, no modification in the fatty acid composition of both plasma lipids and platelet phosphatidylcholine could be observed. We conclude that intake of low amounts of 20:5n-3 by elderly people, is able to lower their platelet sensitivity to aggregating agents, probably by decreasing the endogenous formation of platelet thromboxane A 2, although no modification in the fatty acid composition was detected.

The incorporation of [3H]glycerol and 1-[14C]acyl-sn-glycero-3-phosphocholine into different molecular species of phosphatidylcholine in human platelets.

The molecular species of phosphatidylcholine which are formed by de novo synthesis and by the acylation of 1-acyl-sn-glycero-3-phosphocholine were characterized and compared in human platelets. For this purpose, intact human platelets were incubated in the presence of [3H]glycerol or 1-[14C]palmitoyl-sn-glycero-3-phosphocholine and the newly formed radioactive phosphatidylcholine was isolated by thin-layer chromatography. The labelled phosphatidylcholines were converted to their 1,2-diacylglycerol acetate derivatives and fractionated into their various chemical classes (saturates, monoenes, dienes, trienes, tetraenes, and greater than tetraenes) by argentation thin-layer chromatography. Regardless of the precursor used, the radioactivity distributions among the various classes did not correspond closely to that of the mass. The highest percentage of the radioactivity incorporated from [3H]glycerol was found in the saturates (25% of total), followed closely by the tetraenes (21%) and monoenes (18%), with lesser amounts in the dienes (15%), pentaenes plus hexaenes (14%), and trienes (7%). These results indicate that the de novo pathway is capable of substantial synthesis of tetraenoic (1-acyl 2-arachidonoyl) phosphatidylcholine in human platelets in contrast to previous observations in rat liver. In close agreement with work in rat liver, 59% of the radioactivity in the [14C]phosphatidylcholine derived from 1-[14C]palmitoyl-sn-glycero-3-phosphocholine was found in the tetraenoic species. The present results, together with the existence of phospholipase A2 and acyltransferase activity in platelets, support the potential importance of a deacylation-acylation cycle enrichment of human platelet phosphatidylcholine in arachidonoyl species.

Arachidonate synthesis and uptake in isolated guinea-pig megakaryocytes and platelets

Arachidonic acid (20:4) synthesis and uptake were studied in guinea-pig megakaryocytes and platelets. Isolated megakaryocytes and platelets were incubated with ( 3H]20:4, 8,11,14-[ 14C]eicosatrienoic acid (γ-20:3) and [ 14C]linoleic acid (18:2) and their lipids were analyzed for radioactivity. The study showed that there was 0.153 μM of unesterified 20:3 and 0.237 μM of 20:4 in guinea-pig plasma in nonfasting animals. At these concentrations, 42.6 pmol of γ-20:3 and 53.3 pmol of 20:4 were taken up per h per 10 5 megakaryocytes in vitro. Megakaryocytes desaturated 27% of the γ-20:3 to 20:4 but could not desaturate 18:2. Platelets could not desaturate γ-20:3 or 18:2. In megakaryocytes, the distribution of 20:4 synthesized by the desaturation of γ-20:3 and 20:4 taken up reflected the endogenous distribution of 20:4 in megakaryocyte phospholipids and 20:4 was predominantly incorporated into phosphatidylethanolamine (PE). In contrast, the distribution of [ 3H]20:4 taken up into platelets did not reflect the endogenous distribution. 20:4 was primarily incorporated into platelet phosphatidylcholine and phosphatidylinositol. The study showed that megakaryocytes but not platelets possess a Δ 5 desaturase and can synthesize 20:4 from γ-20:3. Neither cell was shown to have a Δ 6 desaturase. Megakaryocytes appear to have the capacity to determine the composition of all pools of platelet 20:4 either by uptake or synthesis of 20:4. Platelets, most likely, have a limited capacity to alter structural pools of 20:4 contained primarily in PE and phosphatidylserine (PS).

Relative degradation of different molecular species of phosphatidylcholine in thrombin-stimulated human platelets.

The relative degradation of the various molecular species of [3H]phosphatidylcholine in response to thrombin was studied in human platelets following prelabeling with [3H]glycerol and compared to results obtained following labeling with [14C]oleic, [14C]linoleic, or [14C]arachidonic acids. This was of interest since previous work using radioactive fatty acids had led to the conclusion that the 1-acyl-2-arachidonoyl species of phosphatidylcholine is exclusively hydrolyzed in thrombin-stimulated platelets. Within 90 s, the thrombin-dependent release of [14C]arachidonic acid from phosphatidylcholine amounted to 25% but only 3 and 6% for oleic and linoleic acids, respectively, in general agreement with previous work. However, for [3H]glycerol-labeled phosphatidylcholine, all molecular species (saturates, monoenes, dienes, trienes, tetraenes, and greater than tetraenes) were subject to significant hydrolysis in the presence of thrombin within 90 s, ranging from 12-24% across the various classes. Furthermore, the degradation of the tetraenoic species (1-acyl-2-arachidonoyl) of [3H]phosphatidylcholine was found to be only 1.5 and 1.4 times that for the monoenoic (predominantly 1-acyl-2-oleoyl) and dienoic (predominantly 1-acyl-2-linoleoyl) species, respectively. A much heavier proportional labeling of plasma membrane relative to whole platelet phosphatidylcholine was observed with [3H]glycerol as compared to [14C] oleate or [14C]arachidonate. These results indicate that the 1-acyl-2-arachidonoyl species of phosphatidylcholine are not exclusively degraded by phospholipase A2 activity in thrombin-stimulated platelets and suggest that the differential compartmentation of molecular species of phosphatidylcholine according to their metabolic origins can influence their apparent susceptibility to hydrolysis.

The role of Ca2+ uptake in the response of human platelets to adrenaline and to 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor).

Thrombin induces Ca2+ uptake into both stirred and unstirred human platelets in the presence or absence of acetylsalicylate. This Ca2+ uptake is closely correlated with adenine nucleotide secretion in accord with previous observations [Massini, P. and Lüscher, E.F. (1974) Biochim. Biophys. Acta 372, 109-121] but a low level of secretion is observed in the absence of significant Ca2+ uptake. 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkylAcGEPC) also induces Ca2+ uptake into both stirred and unstirred human platelets in the presence and absence of acetylsalicylate. Aggregation and adenine nucleotide secretion induced by 1-O-alkylAcGEPC can be observed in the absence of added fibrinogen, but addition of fibrinogen causes a very marked shift to the left in the dose/response curves for aggregation and Ca2+ uptake induced by 1-O-alkylAcGEPC. In the absence of added fibrinogen a close correlation is observed between Ca2+ uptake and adenine nucleotide secretion induced by 1-O-alkylAcGEPC. In the presence of added fibrinogen significant aggregation can be observed in the absence of detectable Ca2+ uptake. Adrenaline induces Ca2+ uptake only into stirred human platelets in the presence of added fibrinogen. Blockade of secretion, e.g. by addition of acetylsalicylate, also prevents Ca2+ uptake. Addition of adrenaline fails to cause breakdown of phosphatidylinositol or phosphatidylcholine in acetylsalicylate-treated platelets under conditions where such a response is observed on addition of thrombin. We conclude that Ca2+ uptake into human platelets induced by thrombin, 1-O-alkylAcGEPC and adrenaline is closely associated with the secretory response and in some circumstances, e.g. stimulation by thrombin, is clearly a consequence of this latter response. Previous reports of Ca2+ uptake as an initiating event in the response of human platelets to adrenaline [Owen, N.E., Feinberg, H. and Le Breton, G.C. (1981) Am J. Physiol. 239, H483-488] have not been confirmed in this study.