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, a 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.     

Involvement of the Na+/H+ exchanger in membrane phosphatidylserine exposure during human platelet activation

Platelet membrane phosphatidylserine (PS) exposure that regulates the production of thrombin represents an important link between platelet activation and the coagulation cascade. Here, we have evaluated the involvement of the Na+/H+ exchanger (NHE) in this process in human platelets. PS exposure induced in human platelets by thrombin, TRAP, collagen or TRAP+ collagen was abolished in a Na+ -free medium. Inhibition of the Na+/H+ exchanger (NHE) by 5-(N-Ethyl-N-Isopropyl) Amiloride (EIPA) reduced significantly PS exposure, whereas monensin or nigericin, which mimic or cause activation of NHE, respectively, reproduced the agonist effect. These data suggest a role for Na+ influx through NHE activation in the mechanism of PS exposure. This newly identified pathway does not discount a role for Ca2+, whose cytosolic concentration varies together with that of Na+ after agonist stimulation. Ca2+ deprivation from the incubation medium only attenuated PS exposure induced by thrombin, measured from the uptake of FM1-43 (a marker of phospholipid scrambling independent of external Ca2+). Surprisingly, removal of external Ca2+ partially reduced FM1-43 uptake induced by A23187, known as a Ca2+ ionophore. The residual effect can be attributed to an increase in [Na+]i mediated by the ionophore due to a lack of its specificity. Finally, phosphatidylinositol 4,5-bisphosphate (PIP2), previously reported as a target for Ca2+ in the induction of phospholipid scrambling, was involved in PS exposure through a regulation of NHE activity. All these results would indicate that the mechanism that results in PS exposure uses redundant pathways inextricably linked to the physio-pathological requirements of this process.     

Quantitative loss of individual eicosapentaenoyl-relative to arachidonoyl-containing phospholipids in thrombin-stimulated human platelets

The thrombin-dependent losses of eicosapentaenoate (EPA) from the various phospholipids of platelets derived from human subjects ingesting a fish lipid concentrate (MaxEPA) were quantitatively assessed and studied in relation to arachidonate (AA). The net loss of AA and EPA from the total phospholipid, phosphatidylcholine (PC) + phosphatidylethanolamine (PE) + phosphatidylserine (PS) + phosphatidylinositol (PI) (loss from phosphatidylinositol minus accumulated phosphatidate), amounted to 44.4 and 7.3 nmol/2 x 10(9) platelets (mean values, n = 4 subjects), respectively, in response to thrombin (2 units/ml). The phosphatidylcholine, phosphatidylethanolamine (including alkenylacyl), phosphatidylserine, and phosphatidylinositol contributed 46, 17, less than 5, and 33%, respectively, of the AA loss; in contrast to these distributions, the corresponding phospholipid contributions to the net loss of EPA were 71, 27, less than 1, and less than 2%, respectively. Furthermore, the inhibition of AA- and EPA-phospholipid degradation by trifluoperazine indicated that almost all of the release of EPA occurs from PC and PE (greater than 95% of total EPA loss) upon thrombin stimulation and is mediated predominantly via phospholipase A2 activity with almost no contribution from PI. Similarities in the molar ratios of AA/EPA in the PC (3.9) or PE (3.7) which were degraded with those in the corresponding phospholipids from resting platelets suggested no marked selectivity by the phospholipase A2 in intact thrombin-stimulated human platelets in the hydrolysis of AA-PC (or AA-PE) versus EPA-PC (or EPA-PE). Quantitation of the newly released free AA and EPA was determined in the presence of BW755C, a dual cyclooxygenase/lipoxygenase inhibitor which was found not to influence the degradation of individual AA- and EPA-containing phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of the skeletal sarcoplasmic reticulum Ca2(+)-pump: influence of negatively charged phospholipids.

The Ca2(+)-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in the presence of phosphatidyl choline using the freeze-thaw sonication technique. The effect of incorporation of negatively charged phospholipids, phosphatidylserine and phosphatidylinositol phosphate, into the phosphatidylcholine proteoliposomes was investigated. Various ratios of phosphatidylserine or phosphatidylinositol phosphate to phosphatidylcholine were used, while the total amount of phospholipid in the reconstituted vesicles was kept constant. Enrichment of phosphatidylcholine proteoliposomes by phosphatidylserine or phosphatidylinositol phosphate was associated with activation of Ca2(+)-uptake and Ca2(+)-ATPase activities. The highest activation was obtained at a 50:50 molar ratio of phosphatidylserine:phosphatidylcholine and at a 10:90 molar ratio of phosphatidylinositol phosphate:phosphatidylcholine. The initial rates of Ca2(+)-uptake obtained at 1 microM Ca2+ were 2.6 +/- 0.1 mumol/min per mg of phosphatidylserine:phosphatidylcholine proteoliposomes and 1.5 +/- 0.1 mumol/min per mg of phosphatidylinositol phosphate:phosphatidylcholine proteoliposomes, compared to 0.9 +/- 0.05 mumol/min per mg of phosphatidylcholine proteoliposomes. These findings suggest that negatively charged phospholipids may be involved in the activation of the reconstituted skeletal muscle sarcoplasmic reticulum Ca2(+)-pump.     

Prothrombinase activity of human platelets is inhibited by beta 2-glycoprotein-I

In the present paper the influence of beta 2-glycoprotein-I, also known as apolipoprotein H, upon the prothrombinase activity of platelets and phospholipid vesicles was investigated. The results can be summarized as follows. 1. The prothrombinase activity of resting, non-activated platelets, lysed platelets and vesicles composed of phosphatidylserine and phosphatidylcholine at different molar ratios is inhibited by beta 2-glycoprotein-I in a dose-dependent manner. The concentration of glycoprotein which produces marked inhibition is within the physiological plasma concentration range of beta 2-glycoprotein-I. 2. The time dependence of this inhibition is a relatively slow process, which is not fully expressed before 1 h of incubation. 3. The effect of the glycoprotein is not due to a direct interaction with the components of the prothrombinase complex, i.e. factors Xa, Va, Ca2+ or prothrombin, nor is the inhibitory action abolished by increasing concentrations of coagulation factors Xa and Va. This suggests that beta 2-glycoprotein-I causes a reduction of the prothrombinase binding sites of these coagulation factors to platelets or phospholipid vesicles. 4. The prothrombinase activity of platelets stimulated with ionophore A23187 or with collagen plus thrombin is also inhibited by beta 2-glycoprotein-I in a manner similar to that observed for phospholipid vesicles or for lysed platelets. These findings suggest a regulatory role for beta 2-glycoprotein-I in the pathway of blood coagulation.     

Elevated glucose alters A23187-induced release of arachidonic acid from porcine aortic endothelial cells by enhancing reacylation.

Cultured porcine aortic endothelial cells were conditioned in normal (5.2 mM) and elevated (15.6 mM) glucose, prelabeled with [14C]arachidonic acid and stimulated with ionophore A23187. Elevated glucose cultures released less radiolabeled products and less [14C]arachidonic acid. Analysis of cellular lipids revealed that elevated glucose reduced net loss of radiolabel from diacylphosphatidylethanolamine, did not affect early phosphatidylinositol hydrolysis, and increased net loss from diacylphosphatidylcholine and alkenylacylphosphatidylethanolamine. Uptake of radiolabel upon stimulation was examined to measure the role of reacylation on the diminished net release of radiolabel in elevated glucose cultures. Enhanced acylation of [3H]arachidonic acid into cellular lipids, especially PI, was observed in stimulated and resting cultures with elevated glucose. Further, pretreatment of the cultures with an acyltransferase inhibitor, thimerosal, prior to A23187 stimulation in radiolabeled cultures, abolished the effects of glucose on eicosanoid and arachidonic acid release. Differences in the ionophore-induced net loss of radiolabel from diacylphosphatidylethanolamine and phosphatidylinositol of the two glucose treatments were diminished by thimerosal exposure, while net loss of radiolabel from diacylphosphatidylcholine and alkenylacylphosphatidylethanolamine were unaffected. The data indicate that elevated glucose alters deacylation and enhances reacylation of arachidonic acid into endothelial cells and particularly into phosphatidylinositol. Enhanced reacylation may explain some of the altered lipid pathways that have been observed in experiments that elevate glucose concentrations or involve diabetes.     

Correlation between calpain-mediated cytoskeletal degradation and expression of platelet procoagulant activity. A role for the platelet membrane-skeleton in the regulation of membrane lipid asymmetry?

The relationship between platelet calpain-activity and platelet procoagulant-activity was investigated by comparison of the time course of their generation after platelet stimulation by calcium ionophore A23187, or by the combined action of collagen and thrombin, or during exposure of platelets to the local anesthetics dibucaine or tetracaine. In addition, the Ca2+ dose-response curves of both activities in intact platelets, obtained by stimulation with A23187 in the presence of Ca2+/HEDTA-buffers, were compared. Platelet procoagulant activity was determined by assaying for prothrombinase activity in the presence of saturating concentrations of factors Xa, Va, and prothrombin. Platelet calpain activity was monitored by the degradation of its major substrates (filamin, talin, myosin) and the formation of their fragments as judged from protein patterns after gel electrophoresis. Platelet stimulation by A23187 resulted in a fast increase in prothrombinase activity, reaching its maximum level after about 20 seconds. Filamin and talin were completely hydrolysed within 15 s, and myosin was partly degraded between 15 and 30 s after platelet activation. When platelets were activated by collagen plus thrombin, prothrombinase activity was generated with a sigmoid time course, the steepest increase being observed between 1 and 2 min after platelet activation. Proteolysis of filamin and talin occurred between 0.5 and 1.5 min after platelet activation, while degradation of myosin became visible after 2 to 2.5 min. Dibucaine and tetracaine were both found to be potent stimulators of prothrombinase activity, with half-maximal activities obtained at 0.7 and 2.8 mM, respectively. Using suboptimal concentrations of both local anesthetics, it was found that the generation of prothrombinase activity closely paralleled that of calpain activity over a time course of 1 hour. Ca2+ titration of intact platelets using A23187 and Ca2+/HEDTA buffers, revealed half-maximal response at about 15 microM free Ca2+ for both calpain and prothrombinase activity. These findings strongly suggest a causal relationship between generation of a procoagulant platelet surface and calpain-mediated degradation of filamin, talin, and myosin. Since an increased procoagulant activity reflects an increased exposure of phosphatidylserine at the platelet outer surface, the present findings suggest that platelet cytoskeletal proteins are involved in the regulation of membrane lipid asymmetry.     

Physical properties of phosphatidylcholine-phosphatidylinositol liposomes in relation to a calcium effect

Physical properties of binary mixtures of dipalmitoylphosphatidylcholine and yeast phosphatidylinositol were studied by ESR analysis using TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) and lipid spin probes, freeze-fracture electronmicroscopy and particle microelectrophoresis, and they were compared with those of phosphatidylcholine/bovine brain phosphatidylserine mixtures. The phase diagram of the binary mixtures of dipalmitoylphosphatidylcholine and phosphatidylinositol was obtained from the thermal features of TEMPO spectral parameter in the lipid mixtures. The phase diagram provided evidence that these two phospholipids in various combinations were miscible in the crystalline state. The addition of 10 mM Ca2+ slightly shifted the phase diagram upward. TEMPO titration of the binary mixture of dipalmitoylphosphatidylcholine and bovine brain phosphatidylserine revealed that 10 mM Ca2+ caused the complete phase separation of this lipid mixture. Studies of phase separations using phosphatidylcholine spin probe manifested that 10 mM Ca2+ induced almost complete phase separation in egg yolk phosphatidylcholine/bovine brain phosphatidylserine mixtures but only slight phase separation in egg yolk phosphatidylcholine/yeast phosphatidylinositol mixtures. However, some phase changes around the fluidus and the solidus curves were visualized by the freeze-fracture electronmicroscopy. The molecular motion of lipid spin probe was decreased by the addition of Ca2+ in the liposomes containing phosphatidylinositol. The temperature dependence of electrophoretic mobility was also examined in the absence and presence of 1 mM Ca2+. Liposomes of dipalmitoylphosphatidylcholine-phosphatidylinositol (90 : 10, mol/mol) exhibited a clear transition in the thermal features of electrophoretic mobilities. Raising the phosphatidylinositol content up to 25 mol% rendered the transition broad and unclear. The addition of 1 mM Ca2+ decreased the electrophoretic mobility but did not change its general profile of the thermal dependence. These results suggest that the addition of calcium ions induced a small phase change in the binary mixture of phosphatidylcholine and phosphatidylinositol while Ca2+ causes a remarkable phase separation in phosphatidylcholine/phosphatidylserine mixture. The physical role of phosphatidylinositol is discussed related to the formation of diacylglycerol.     

Polyphosphoinositide inclusion in artificial lipid bilayer vesicles promotes divalent cation-dependent membrane fusion.

Recent studies suggest that phosphoinositide kinases may participate in intracellular trafficking or exocytotic events. Because both of these events ultimately require fusion of biological membranes, the susceptibility of membranes containing polyphosphoinositides (PPIs) to divalent cation-induced fusion was investigated. Results of these investigations indicated that artificial liposomes containing PPI or phosphatidic acid required lower Ca2+ concentrations for induction of membrane fusion than similar vesicles containing phosphatidylserine, phosphatidylinositol, or phosphatidylcholine. This trend was first observed in liposomes composed solely of one type of phospholipid. In addition, however, liposomes designed to mimic the phospholipid composition of the endofacial leaflet of plasma membranes (i.e., liposomes composed of combinations of PPI, phosphatidylethanolamine, and phosphatidylcholine) also required lower Ca2+ concentrations for induction of aggregation and fusion. Liposomes containing PPI and phosphatidic acid also had increased sensitivity to Mg(2+)-induced fusion, an observation that is particularly intriguing given the intracellular concentration of Mg2+ ions. Moreover, the fusogenic effects of Ca2+ and Mg2+ were additive in vesicles containing phosphatidylinositol bisphosphate. These data suggest that enzymatic modification of the PPI content of intracellular membranes could be an important mechanism of fusion regulation.     

Phospholipid and detergent effects on (Ca2+ + Mg2+)ATPase purified from human erythrocytes

(Ca2+ + Mg2+)ATPase (EC 3.6.1.3) was solubilized from human erythrocyte membranes by detergent extraction with Triton N-101 (0.5 mg/mg membrane protein) and purified by calmodulin affinity chromatography. ATPase activity was assayed in mixtures of Triton N-101 and phospholipid, without reconstitution into bilayer vesicles. At low levels of phospholipid (5 micrograms/ml), the ATPase activity was highly sensitive to the detergent concentration, with maximal activity occurring at or near the critical micelle concentration of the detergent. With increased amounts of phospholipid (50 micrograms/ml), detergent concentrations greater than the critical micelle concentration were required for maximal activity. Detergent alone did not support ATPase activity. Sonicated phospholipid in the form of vesicles was equally ineffective. Activity seemed to be dependent on the presence of detergent/phospholipid mixed micelles. The acidic phospholipids, phosphatidylserine and phosphatidylinositol, as well as the commercial phospholipid preparation, Asolectin, gave activities five to eight times greater than the same amount of phosphatidylcholine. Mixtures of phosphatidylserine and phosphatidylcholine produced intermediate ATPase activities, with the maximal value dependent on the phosphatidylserine concentration. Addition of phosphatidylcholine to fixed concentrations of phosphatidylserine caused a rise in activity that was independent of the ratio of the two phospholipids or the total phospholipid concentration. Phosphatidylcholine may therefore be irreplaceable for some aspect of ATPase function. The number of phospholipid molecules present in mixed micelles at maximal ATPase activity was calculated to be near 50. This value implied that the hydrophobic surface of the ATPase molecule must be completely coated by a single layer of phospholipid molecules for maximum activity to occur.     

Liberation and oxygenation of polyenoic acids in stimulated platelets

Radiolabeled polyenoic acids were incorporated into human platelet lipids using albumin as vector. Platelets were then triggered with 0.1 or 1 U/ml thrombin, and 0.5 or 2 x 10(-6) M calcium ionophore A23187. Lipid extracts were analyzed for neutral lipids, free fatty acids, monohydroxylated acids, prostanoids and glycocerophospholipid subclasses. During platelet activation induced by thrombin or by ionophore, arachidonic and eicosapentaenoic acids were liberated from phospholipids in large amounts and were subsequently oxygenated via platelet oxygenases. Substantial amounts of lipoxygenase products and thromboxanes were produced from these acids. Liberation and oxygenation of linoleic, alpha-linolenic, and docosahexaenoic acids were much less pronounced. Polyenoic acid liberation from phospholipid subclasses also behaved quite differently. Apart from alpha-linolenic and adrenic acids, which were poorly liberated, all the others were freed from phosphatidylinositol. In addition, arachidonic, eicosapentaenoic, and 5, 8, 11-eicosatrienoic acids were liberated from phosphatidylcholine at high concentrations of agonists and partially reincorporated into phosphatidylethanolamine. Finally, linoleic acid was deacylated from phosphatidylinositol and phosphatidylserine and almost entirely reacylated into phosphatidylcholine, whereas docosahexaenoic acid was deacylated from phosphatidylcholine and phosphatidylinositol reacylated into phosphatidylethanolamine, respectively. It is concluded that these polyenoic acids, all for which modulate platelet functions, exhibit very different metabolisms. They may act via their oxygenated derivatives and/or at the membrane phospholipid level.     

Low affinity binding of taurine to phospholiposomes and cardiac sarcolemma

A sarcolemma-enriched membrane fraction was prepared from the hearts of Sprague-Dawley rats and its ability to bind taurine (0.5-150 mM) was measured. In the absence of cations, the sarcolemma bound a maximum of 661 nmol taurine/mg protein, with a dissociation constant of 19.2 mM and a Hill coefficient of 1.9, indicating positive cooperativity. Scatchard analysis of taurine binding to sarcolemma gave a bell-shaped curve. Neither beta-alanine nor guanidinoethane sulfonate, inhibitors of taurine transport, affected the degree of taurine binding to sarcolemma. However, hypotaurine was an effective antagonist. Equimolar concentrations of Ca2+, Na+ or K+ also reduced taurine binding. Heterogeneous phospholipid vesicles of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine (18:19:2:1) also bound taurine with positive cooperativity, yielding a bell-shaped Scatchard curve. The affinity of taurine for these mixed phospholipid vesicles was enhanced by the inclusion of cholesterol (50%). Taurine associated in a maximum ratio of 1:1 with homogeneous vesicles of phosphatidylcholine or phosphatidylserine. Vesicles of phosphatidylethanolamine bound taurine in a maximum ratio of 2:1, whereas those of phosphatidylinositol bound insignificant amounts of taurine. These studies demonstrate a low affinity binding to sarcolemma of taurine at concentrations normally present in rat heart. Similar levels of binding were observed in phospholipid vesicles, suggesting that the interaction of taurine with biological membranes involves phospholipids.     

Insufficient mobilization of calcium by early breakdown of phosphatidylinositol 4,5-bisphosphate for aggregation of human platelets by collagen

When human platelets (5 X 10(8)/ml) were stimulated by a threshold concentration of collagen (2 micrograms/ml), a lag period of about 60 s was seen before the initiation of release reaction and aggregation. Breakdown of [32P]phosphatidylinositol 4,5-bisphosphate was seen within 10 s after the addition of collagen. The concentration of intracellular free Ca2+ (monitored by Quin II) rose from 80 nM to 145 nM within 10 s after stimulation by collagen. However, a lag period of about 50 s remained. The rise was not blocked by indomethacin. It was supposed that the initial Ca2+ mobilization by myo-inositol 1,4,5-trisphosphate was too small to cause aggregation. Thromboxane A2 was gradually accumulated during the lag period and then abruptly increased in parallel with aggregation. These events were completely inhibited by 10 microM indomethacin. Thus, aggregation appeared to be dependent on the generation of thromboxane A2. Addition of 25 nM A23187 at 10 s after stimulation by collagen shortened the lag period before initiation of the abrupt thromboxane A2 generation, secretion and aggregation, whereas 25 nM A23187 could not cause these reactions in the absence of collagen. Accordingly, the lag period is assumed to be required for accumulation of free Ca2+ to the threshold for aggregation of platelets. It is considered that thromboxane A2 plays a central role in Ca2+ mobilization during stimulation of human platelets by collagen.     

Selective plasmalogen substrate utilization by thrombin-stimulated Ca(2+)-independent PLA(2) in cardiomyocytes

Thrombin stimulation of rabbit ventricular myocytes activates a membrane-associated, Ca(2+)-independent phospholipase A(2) (PLA(2)) capable of hydrolyzing plasmenylcholine (choline plasmalogen), plasmanylcholine (alkylacyl choline phospholipid), and phosphatidylcholine substrates. To identify the endogenous phospholipid substrates, we quantified the effects of thrombin stimulation on diradyl phospholipid mass and arachidonic acid and lysophospholipid production. Thrombin stimulation resulted in a selective decrease in arachidonylated plasmenylcholine, with no change in arachidonylated phosphatidylcholine. The decrease in arachidonylated plasmenylcholine was accompanied by an increase in plasmenylcholine species containing linoleic and linolenic acids at the sn-2 position. A decrease in arachidonylated plasmenylethanolamine was also observed after thrombin stimulation, with no concomitant change in arachidonylated phosphatidylethanolamine. Thrombin stimulation resulted in the selective production of lysoplasmenylcholine, with no increase in lysophosphatidylcholine content. There was no evidence for significant acetylation of lysophospholipids to form platelet-activating factor. Arachidonic acid released after thrombin stimulation was rapidly oxidized to prostacyclin. Thus thrombin-stimulated Ca(2+)-independent PLA(2) selectively hydrolyzes arachidonylated plasmalogen substrates, resulting in production of lysoplasmalogens and prostacyclin as the principal bioactive products.     

Characterization of phosphoinositide-specific phospholipase C from human platelets.

Phosphoinositide-specific phospholipase C (PI-PLC) from human platelet cytosol was purified 190-fold to a specific activity of 0.68 mumol of phosphatidylinositol (PI) cleaved/min per mg of protein. It hydrolyses PI and phosphatidylinositol 4,5-bisphosphate (PIP2), but not phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. The enzyme exhibits an acid pH optimum of 5.5 and has a molecular mass of 98 kDa as determined by Sephacryl S-200 gel filtration. It required millimolar concentrations of Ca2+ for PI hydrolysis, whereas micromolar concentrations are optimal for PIP2 hydrolysis. Mg2+ could substitute for Ca2+ when PIP2, but not PI, was used as the substrate. EDTA was more effective than EGTA in inhibiting the basal PI-PLC activity towards PIP2. Sodium deoxycholate strongly inhibits the purified PI-PLC activity with either PI or PIP2 as substrate. Ras proteins, either alone or in the form of liposomes, have no effect on PI-PLC activity.     

Mass changes in myoinositol trisphosphate in human platelets stimulated by thrombin. Inhibitory effects of phorbol ester

Myoinositol trisphosphate (IP3) is formed when phosphatidylinositol 4,5-bisphosphate (PIP2) is hydrolyzed by phospholipase C. At micromolar concentrations, IP3 is a stimulus for Ca2+ release in both platelet membranes and various permeabilized cells. We have utilized a combination of ion exchange and capillary gas chromatography to quantitate the mass of IP3 produced by human platelets stimulated by thrombin. Accumulations of IP3 are transient and detectable within 5 s of exposure to thrombin. Within 15 s, thrombin (1 unit/ml) promotes the formation of 134 pmol of IP3/10(9) platelets, the equivalent of an intracellular concentration of 13.4 microM. Incubation of platelets with a stimulus for protein kinase C, 12-O-tetradecanoyl phorbol 13-acetate, prior to the addition of thrombin impairs the hydrolysis of PIP2 and the increase in IP3, with 50% inhibition occurring at 60 nM TPA. We conclude that platelets produce sufficient quantities of IP3 to cause Ca2+ release from membrane stores. TPA inhibits the activation of phospholipase C and consequently the generation of IP3. The decreased accumulation of IP3 in platelets exposed to TPA may account for the inhibited rise in cytoplasmic Ca2+ which has been observed in such platelets.     

Lipid composition of rat liver microsomes under various experimental conditions

Cholesterol and phospholipid content, and phospholipid composition (sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethaolamine) were assayed in rat liver microsomes during regeneration, foetal development and pregnancy. Cholesterol was assayed using Liebermann-Buchard reagent; the phospholipid extract was separated by thin-layer chromatography. While in pregnancy no changes were observed, during foetal development and liver regeneration there was a significative decrease of cholesterol/phospholipid ratio, and of phosphatidylcholine content. Moreover, in developing liver microsomes, there is also a significative increase of sphingomyelin and phosphatidylserine + phosphatidylinositol.     

The effect of chlorpromazine and dibucaine on phospholipid metabolism in the frog sartorius muscle

The effect of cationic amphiphilic drugs, chlorpromazine and dibucaine, on phospholipid metabolism in the frog sartorius muscle was studied at concentrations affecting excitation-contraction coupling. Labelling patterns of phospholipids using 32P orthophosphate were determined. The drugs at a concentration of 3.10(-4) mol/l inhibited the synthesis of phosphatidylcholine and phosphatidylethanolamine and stimulated the synthesis of phosphatidylinositol and phosphatidylserine. At the 3.10(-3) mol/l concentration the drugs blocked the synthesis of all phospholipids without appreciably affecting their degradation. The effect of cationic amphiphilic drugs was independent of the presence of Ca2+ or K+ ions in the media, however, basal labelling of phospholipids was affected by withdrawal of Ca2+ or K+ ions.     

Phosphatidylinositol turnover in platelet activation; calcium mobilization and protein phosphorylation

Ca2+-activated, phospholipid-dependent protein kinase (C-kinase) in platelets is normally activated by diacylglycerol, which is derived from phosphatidylinositol through its receptor-linked breakdown. Under appropriate conditions this enzyme can also be activated by synthetic diacylglycerol which is directly added to intact platelets. C-Kinase thus activated preferentially phosphorylates an endogenous platelet protein having a molecular weight of approximately 40,000. This protein phosphorylation is merely a prerequisite but not a sufficient requirement for the release of serotonin. Evidence is presented suggesting that Ca2+ mobilization and C-kinase activation are synergistically involved in the physiological response of platelets to extracellular messengers, such as thrombin, collagen and platelet-activating factor.     

Effect of phospholipase C (Bacillus cereus) on freshly isolated and 4-day-stored human platelets.

Phospholipase C (from Bacillus cereus) was used to study fresh and stored human platelets. Provided that the enzyme was inactivated before lipid extraction, no significant degradation of phospholipid in fresh cells was noted, even when platelets were activated or induced to change shape by ADP, collagen or thrombin. With platelets isolated from concentrates stored for transfusion for 4 days at 22 degrees C, membrane phospholipids were degraded by the enzyme to an extent depending on the pH in the platelet concentrate at day 4 of storage. The extent of phospholipid hydrolysis in platelets correlated well with the extent of release of lactate dehydrogenase during storage, with both being minimal for platelets from concentrates of final pH 6.5-6.9. Under non-lytic conditions, phosphatidylcholine was the phospholipid most degraded (40%), with no significant degradation of phosphatidylserine being detected. Storage does not seem to alter the distribution of phospholipids at the external leaflet of the plasma membrane.