Changes in phosoholipid susceptibility toward phospholipases induced by ATP depletion in avian and amphibian erythrocyte membranes.

About half of the sphingomyelin content of fresh and ATP-depleted chicken erythrocytes is hydrolysed by sphingomyelinase. Removal of spingomyelin exposes the rest of the membrane phospholipids to hydrolysis by phospholipase C only in ATP-depleted but not in fresh cells. Addition of both sphinogomyelinase and phospholipase C to ATP-depleted cells causes about 60-70 percent hydrolysis of the total phospholipids accompanied by extensive (90 percent) hemolysis. The phospholipids of toad erythrocytes are partially available to phospholipase C activity in fresh cells (17-25 percent hydrolysis) without prior sphingomyelinase treatment. However, in ATP-depleted toad cells phospholipase C hydrolyses 66 percent of phospholipids and causes extensive lysis. Treatment of either fresh or ATP-depleted toad erythrocytes by sphingomyelinase together with phospholipase C induces hydrolysis of most of the phospholipds with complete lysis. Restoration of ATP to ATP-depleted cells endows them with resistance to the attack of phospholipase C. The correlation between changes in ATP level and membrane organization as revealed by increased susceptibility toward phospholipases is discussed.     

Action of Phospholipase A2 and Phospholipase C on Bacillus subtilis Protoplasts

Protoplasts prepared from Bacillus subtilis by lysozyme digestion lysed in the presence of pure pancreatic phospholipase A(2). The phospholipids cardiolipin, phosphatidylethanolamine, phosphatidylglycerol and lysylphosphatidylglycerol, which are present in the membrane, are degraded by phospholipase A(2) only after removal of the cell wall, giving free fatty acids and lyso derivatives. The four phospholipids are hydrolyzed equally well at a given enzyme concentration. Differences in the phospholipid composition of the protoplasts were obtained by variations in the growth medium, time of harvesting, and preincubation time with lysozyme. The extent of hydrolysis appeared to depend on the initial phospholipid composition. A relative increase in acidic phospholipids in the membrane facilitated the action of phospholipase A(2), whereas the rate of hydrolysis was diminished when protoplasts were tested which contained a relatively high amount of positively charged phospholipid. Pure phospholipase C from B. cereus preferentially hydrolyzed phosphatidyl-ethanolamine in the B. subtilis membrane. More than 80% of this phospholipid was converted into diglyceride, whereas only 30% of the cardiolipin was hydrolyzed. Such a loss of phospholipids, however, was not followed by lysis of the protoplasts. Liposomes were prepared from the lipid extracts of B. subtilis and incubated with both phospholipases. The hydrolysis pattern of the phospholipids in these model membrane systems was identical to the hydrolysis pattern of the phospholipids in the protoplast membrane. Phospholipase A(2) hydrolyzed all the phospholipids in the liposomes equally well, whereas phospholipase C preferentially degraded phosphatidylethanolamine.     

Phospholipid topology and flip-flop in intestinal brush-border membrane

The topological distribution of the two major phospholipids of brush-border membrane, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), has been investigated using brush-border membrane vesicles from rabbit small intestine. Bee venom phospholipase A2 and phosphatidylcholine exchange protein from bovine liver were used as membrane probes. It is shown that the brush-border membrane retains its integrity under conditions of phospholipase hydrolysis and intermembrane phospholipid exchange. Kinetic analysis of the data of phospholipase hydrolysis and phospholipid exchange at temperatures under 10 degrees C shows that both PC and PE occur in two pools: a minor (about 25%) more readily accessible pool and a major one (about 75%) less readily available. The rate of PC exchange between these two pools is relatively fast. The half-time derived under conditions of phospholipase hydrolysis is of the order of 20 min. Under conditions of phospholipid exchange the exchange rates may be even faster. The difference in exchange kinetics observed with the two methods of probing is probably due to changes in membrane properties such as the bilayer fluidity induced by the probing process itself. It is proposed that the two pools represent the transverse distribution of the phospholipids. The two major phospholipids of brush-border membranes, PC and PE, would be distributed mainly on the inner (cytoplasmic) side of the brush-border membrane. The phospholipid exchange between the brush-border vesicles and unilamellar phosphatidylcholine vesicles in the presence of phosphatidylcholine exchange protein reveals that significant quantities of phospholipid are taken up by brush-border membrane independently, i.e., in a separate process independent of the exchange protein-catalyzed phosphatidylcholine exchange.     

Lipid-protein interactions in mitochondria. The effect of protein interaction on susceptibility of phospholipids to phospholipase A2 and C hydrolysis.

Phospholipase A₂ (Naja naja) and phospholipase C (from either Clostridium welchii or Bacillus cereus) have been tested on phospholipid dispersions and natural or reconstituted membranes; notwithstanding the different substrate specificities, the different enzymes gave comparable behaviors, suggesting that the results were the expression of sterical features in the lipid bilayers, i.e., availability of the phospholipids to enzymatic attack. The hydrolysis of phospholipids (Asolectin) in sonic protein-free vesicles is hindered by ionic interaction with basic proteins (cytochrome c or lysozyme). On the other hand binding of Asolectin to lipid-depleted mitochondria to obtain reconstituted mitochondria does not prevent phospholipase action on the phospholipids; similarly, phospholipids are hydrolyzed at maximal rates in natural membranes (mitochondria or submitochondrial particles). Surprisingly, ionic interaction of RM or natural membranes with basic proteins does not prevent phospholipase hydrolysis of the membrane phospholipids. The interpretation of this phenomenon may be related to the heterogeneity of phospholipid distribution in protein-containing membranes.     

Lipid peroxidation and phospholipase A2 activity in liposomes composed of unsaturated phospholipids: a structural basis for enzyme activation

The effect of lipid peroxidation on membrane structure and phospholipase A2 activity was studied using liposomes composed of bovine liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The phospholipids were mixed at set ratios and sonicated to yield small unilamellar vesicles. The liposome preparations were subjected to lipid peroxidation as induced by cumene hydroperoxide and hematin. Under these conditions, a sharp increase in lipid peroxidation was noted over a 30 min incubation period and was accompanied by loss of polyunsaturated fatty acids (PUFA). Liposomes enriched in PE were most extensively peroxidized with a preferred oxidation of this phospholipid. The extent of PC oxidation was also greater in liposomes containing the largest proportions of PE. Analysis of liposome anisotropy, via steady-state fluorescence polarization of diphenylhexatriene indicated that progressive increases in either PE content or the level of lipid peroxidation increased the apparent microviscosity of the vesicles. Moreover, lipid peroxidation increased anisotropy more effectively than variations in the ratios of PE vs. PC. Thus, peroxidation of 5-10% of the phospholipids produced the same anisotropy increase as a 20% increase in the ratio of PE vs. PC. Analysis of vesicle turbidity suggested that fusion was also more readily achieved through lipid peroxidation. When liposomes were incubated with 0.4 U/ml of snake venom phospholipase A2, a direct correlation was found between the degree of lipid peroxidation and the extent of phospholipid hydrolysis. The more unsaturated phospholipid, PE, was most extensively hydrolyzed following peroxidation. Increasing the proportion of PE also resulted in more extensive phospholipid hydrolysis. These findings indicate that lipid peroxidation produces a general increase in membrane viscosity which is associated with vesicle instability and enhanced phospholipase A2 attack. A structural basis for membrane phospholipase A2 activation as a consequence of lipid peroxidation is discussed in light of these findings.     

Studies on the hemolytic and hydrolytic actions of phospholipases against mammalian erythrocyte membranes.

The hemolytic actions of three kinds of phospholipase C on horse and sheep erythrocytes were studied in relation to their hydrolytic activities on the phospholipid components of these red cells. Clostridium novyi (oedematiens) type A phospholipase C hemolyzed horse red cells by hydrolyzing phosphatidylcholine. However, the enzyme did not lyse sheep cells nor did it hydrolyze any phospholipid under the same conditions, although this enzyme hydrolyzed both sphingomyelin and phosphatidylethanolamine in the phospholipid mixture extracted from sheep red cells. Clostridium perfringens phospholipase C hemolyzed not only horse red cells by hydrolyzing phosphatidylcholine but also sheep red cells by hydrolyzing sphingomyelin. Sphingomyelin on sheep red cell membrane was hydrolyzed 10 times faster by this enzyme than that on horse red cell membrane. Pseudomonas aureofaciens phospholipase C hemolyzed horse red cells by attacking phosphatidylcholine and phosphatidylethanolamine. The enzyme did not attack sheep red cells but it did hydrolyze phosphatidylethanolamine in the extracted phospholipid mixture from sheep cells. The hemolytic activity of phospholipase C depends not only on the enzyme and the asymmetric distribution of phospholipids in the erythrocyte membrane but also on the accessibility of the enzymes to the phospholipids in the surface of the membranes. Hemolysis by phospholipase C belongs to a hot-cold type of lysis.     

Phosphorylation and dephosphorylation of membrane proteins as a possible mechanism for structural rearrangement of membrane components.

A correlation was found between dephosphorylation of chicken erythrocyte membrane proteins, aggregation of intramembrane particles, increase in the lipid bilayer phase of the membrane and exposure of membrane phospholipids toward phospholipase A and trinitrobenzene sulfonic acid. Most of the covalently bound phosphate of the membrane proteins turns over and is associated with 5 major bands. It is suggested that phosphorylation and dephosphorylation of these proteins causes changes in their charge and conformation. Such changes might affect the interaction of these proteins with the neighbouring lipids or lipoprotein complexes and results in the aggregation of intramembrane particles and relative increase in the exposed free lipid bilayer phase of the membrane.     

Induction of ATP depletion, intramembrane particle aggregation and exposure of membrane phospholipids in chicken erythrocytes by local anesthetics and tranquilizers

Incubation of chicken erythrocytes with 1 mM tetracaine, 10 mM lidocaine and 0.24–0.48 mM chlorpromazine significantly reduced the ATP content of the cells, while procaine even at concentrations as high as 10 mM had only a slight effect. When chlorpromazine was used, it was found that the final level of the ATP was dependent on the drug concentration, which at 0.48 mM depletes the cells to about 10% of the initial ATP content. The ATP depletion of chicken erythrocytes was accompanied by dephosphorylation of certain membrane proteins which were identified by acrylamide gel electrophoresis as an 180 000 dalton protein band and peptides with molecular weight of 60 000–100 000. Treatment of chicken and rat erythrocytes with 0.5 mM tetracaine and 1 mM lidocaine or with 0.48 mM chlorpromazine induced significant aggregation of intramembrane particles as revealed by the freeze-etching technique. Procaine (10 mM) had no effect. Incubation of chicken erythrocytes with the above-mentioned drugs induced also exposure of the masked membrane phospholipids to the action of phospholipase-C (Bacillus cereus) and to phospholipase A₂ (bee venom). Negligible amounts of phospholipids were hydrolyzed in the untreated cells, while about 40% of the membrane phosphatidylethanolamine and 50% of the phosphatidylcholine were hydrolyzed by phospholipase A₂ in chicken erythrocytes treated with 0.48 mM chlorpromazine.Treatment of chicken and rat erythrocytes with 0.48 mM chlorpromazine resulted also in an increase in the amount of the phospholipid fraction which could be extracted by dry ether. About 41% and 60% of phospholipids were respectively, as compared to 25% and 35% of phospholipids extracted from the same untreated cells.     

Gonadotropin receptors in plasma membranes of bovine corpus luteum. II. Role of membrane phospholipids.

The role of phospholipids in the binding of 125I-choriogonadotropin to bovine corpus luteum plasma membranes has been investigated with the use of purified phospholipase A and phospholipase C to alter membrane phospholipids. The phospholipase C-digested plasma membrane preparation showed 85 to 90% inhibition of 125I-choriogonadotropin binding activity when 70% of the membrane phospholipid was hydrolyzed. Similarly treatment of plasma membranes with phospholipase A resulted in 45 to 55% hydrolysis of membrane phospholipid and almost 75% inhibition of receptor activity. Both these enzymes hydrolyzed membrane-associated phosphatidylcholine to a greater extent than phosphatidylethanolamine and phosphatidylserine. Phosphorylaminoalcohols of phospholiphase C end products were completely released into the medium, while phospholipase A by-products remained associated with plasma membranes. Addition of a phospholipids suspension or liposomes to plasma membranes pretreated with phospholipase A and C did not restore gonadotropin binding activity. Soluble phosphorylcholine, phosphorylethanolamine, and phosphorylserine and insoluble diglyceride products of phospholipase C action had no effect on receptor activity. In contrast, end products of the phospholipase A action, such as lysophosphatides and fatty acids, inhibited both on the membrane-associated and solubilized receptor activity. Lysophosphatidylcholine was the most effective end product inhibiting the binding of gonadotropin to the receptor, followed by lysophosphatidylethanolamine and lysophosphatidylserine. The inhibitory effects of phospholipase A or lysophosphatides were completely reversed upon removal of membrane-bound phospholipid end products by washing the membranes with defatted bovine serum albumin. However, phospholipase C inhibition could not be overcome by defatted albumin washings. Solubilization of plasma membranes with detergents which had been pretreated with phospholipase C partially restored the inhibited activity. It is concluded that the phospholipase-mediated inhibition of gonadotropin binding activity was due to hydrolysis and alterations of the phospholipid environment in the case of phospholipase C and by direct inhibition by end products in the case of phospholipase A.     

Mitochondrial phospholipase A2 activity and mitochondrial aging

The changes in mitochondrial phospholipid metabolism and energy-linked functions have been followed as coupled mitochondria are allowed to age in isotonic sucrose at 18 degrees C. Analysis of the aging process has provided an approach for studying the structure--function relationships within the mitochondrion without adding external agents to perturb the membrane structure. The initial event observed in this process of deterioration is a loss of respiratory control which is paralleled by diminishing levels of ATP. As ATP levels decline, so do the rates of reacylation of monoacyglycerophosphorylethanolamine and fatty acid oxidation. In most cases the previously inactive phospholipase A2 (EC 3.1.1.4, phosphatide-2-acyl-hydrolase) begins rapid hydrolysis of membrane phosphatidylethanolamine as ATP levels approach zero. The final energy-linked phenomenon observed to decline is the anilinonaphthalenesulfonic acid fluorescence response. Evidence is presented which suggests strongly that the activity of the mitochondrial phospholipase A2 on endogenous phospholipids is suppressed in tightly coupled mitochondria. This suppression is temporally linked to ATP levels in the mitochondria. Furthermore, this study demonstrates that mitochondria which are only slightly damaged have the potential to effect membrane repair through reacylation of monoacyl phospholipids.     

Effect of purified phospholipases on the binding of tetrodotoxin to axon plasma membrane

Summary The role of phospholipids in the binding of [³H] tetrodotoxin to garfish olfactory nerve axon plasma membrane was studied by the use of purified phospholipases. Treatment of the membranes with low concentrations of either phospholipase A₂ (Crotalus adamanteus andNaja naja) or phospholipase C (Bacillus cereus andClostridium perfringens) resulted in a marked reduction in tetrodotoxin binding activity. A 90% reduction in the activity occurred with about 45% hydrolysis of membrane phospholipids by phospholipase A₂, and with phospholipase C the lipid hydrolysis was about 60–70% for a 70–80% reduction in the binding activity. Phospholipase C fromB. cereus andCl. perfringens had similar inhibitory effects. Bovine serum albumin protected the tetrodotoxin binding activity of the membrane from the inhibitory effect of phospholipase A₂ but not from that of phospholipase C. In the presence of albumin about 25% of the membrane phospholipids remained unhydrolyzed by phospholipase A₂. It is suggested that these unhydrolyzed phospholipids are in a physical state different from the rest of the membrane phospholipids and that these include the phospholipids which are directly related to the tetrodotoxin binding component. It is concluded that phospholipids form an integral part of the tetrodotoxin binding component of the axon membrane and that the phospholipase-caused inhibition of the binding activity is due to effects resulting from alteration of the phospholipid components.     

Role of mitochondrial membrane lipid hydrolysis in their swelling induced by thyroxine]

The thyroxin-induced mitochondrial swelling was accompanied by an accumulation in organellas of free fatty acids which level was restored after the mitochondria contraction in the ATP presence. EGTA induced mitochondrial contractions as well, but with no free fatty acids utilization. Apparently, the thyroxin-induced mitochondrial swelling is the result of the membrane phospholipase activation and of the increase in the membrane cationic permeability due to the hydrolysis of membrane phospholipids.     

Promotion of conidia aggregation in Aspergillus niger by cyclic AMP and 5'-GMP

Cholesterol and phospholipids remain tightly associated with the ferroxidase-II protein from human serum following extensive purification. Purified ferroxidase-II preparations show a consistent ratio of protein, phospholipid, and cholesterol. Thin-layer chromatographic analyses indicate that phosphatidyl choline accounts for 70% of the bound phospholipid. Treatment of purified ferroxidase-II with phospholipase C or A results in a loss of ferroxidase activity which parallels the hydrolysis of phospholipid. A lipid-depleted form of ferroxidase-II can be prepared by gel-filtration following treatment with phospholipase C. However, hydrolysis, not removal, of the lipid is sufficient for the loss of ferroxidase activity. These studies indicate that the bound lipid components are essential to the maintainence of the catalytic activity of ferroxidase-II.     

Lipid asymmetry in rabbit small intestinal brush border membrane as probed by an intrinsic phospholipid exchange protein.

All classes of phospholipids present in brush border membrane are exchanged in a 1:1 ratio for egg phosphatidylcholine when brush border membrane vesicles from rabbit small intestine are incubated with small unilamellar vesicles of egg phosphatidylcholine. The exchange reaction exhibits biphasic kinetics similar to those of the hydrolysis of brush border membrane phospholipids by phospholipase A2 and sphingomyelinase C. In both reactions there is an initial fast phase followed by a markedly slower one. The phospholipid exchange appears to be catalyzed by intrinsic brush border membrane protein(s), while the digestion by phospholipases is mediated by externally added enzymes. From a comparison of the kinetics of phospholipid exchange and phospholipid hydrolysis, the following conclusions can be drawn: Both sets of experiments indicate the presence of two phospholipid pools differing in the rate of phospholipid exchange and hydrolysis. Except for sphingomyelin, the size of the two phospholipid pools derived from phospholipid exchange is in good agreement with that derived from phospholipid hydrolysis. This is the main finding of this work, and on the basis of this result the two lipid pools are tentatively assigned to phospholipid molecules located on the outer and inner layer of the brush border membrane. The slow rate of phospholipid exchange reflects the rate of transverse or flip-flop movement of phospholipids. The half-time of this motion is approximately 8 h for isoelectric (neutral) phospholipids such as phosphatidylethanolamine and approximately 80 h for negatively charged phosphatidylserine and phosphatidylinositol. Isoelectric phospholipids (phosphatidylcholine, phosphatidylethanolamine) are preferentially located on the inner (cytoplasmic) side (to about 70%) while the negatively charged phospholipids are more evenly distributed: 55-60% are located on the inner side.     

DNA methylation and development.

(1) Isolated rat liver mitochondria were subjected to catalytic hydrogenation using a water-soluble Pd complex and molecular H2. This treatment resulted in a reduction of double bonds on phospholipid acyl chains as judged by gas chromatography of fatty acid methyl esters and HPLC of dinitrobenzoyldiacylglycerols. (2) After hydrogenation, mitochondria lost their ability to hydrolyze endogenous phospholipids in alkaline, Ca2+ containing medium, while phospholipase A2 retained full activity against exogenous substrates, regardless of whether those substrates were hydrogenated or not. (3) Inhibition by hydrogenation of endogenous phospholipid hydrolysis correlated with the loss of polyunsaturated fatty acyls, rather than with changes of the bulk membrane fluidity as measured by ESR and fluorescence studies. (4) These data suggest that the unsaturation of mitochondrial membrane lipids might be important for regulation of phospholipid breakdown by endogenous phospholipases. In particular, polyunsaturated molecular species seem to be involved in making phospholipids accessible to phospholipase A-mediated hydrolysis.     

Differentiation of phospholipases A in mitochondria and lysosomes of rat liver

Highly purified mitochondria from rat liver contain a phospholipase A that catalyzes removal of 2-fatty acids, with a pH optimum above pH 8.0. Lysosomal preparations appeared to have two phospholipases A associated with them, one with a pH optimum at about pH 4.0, the second between pH 6.0 and 7.0. Mitochondrial phospholipase A hydrolyzed exogenous phospholipid as fast as or faster than endogenous phospholipid. The difference in specific radioactivity of (14)C-ethanolamine-labeled endogenous mitochondrial phospholipid before and after incubation indicates that a fraction of mitochondrial phosphatidyl ethanolamine is hydrolyzed more rapidly than the mitochondrial phospholipids as a whole. Acyl bond hydrolysis of exogenous and endogenous phospholipid by mitochondria was stimulated by free fatty acid, Ca(++), or in certain cases, monoacyl phospholipids or by treatments that disrupt the mitochondrial membrane. Of various fatty acids tested, lauric, myristic, oleic, and linoleic were most effective. ADP and ATP inhibited mitochondrial phospholipase, probably because they compete for Ca(++). Mg(++) also behaved as a competitive inhibitor; the effect was overcome by relatively little Ca(++).     

Lipid levels in sperm, eggs, and during fertilization in Xenopus laevis

Critical developmental periods, such as fertilization, involve metabolic activation, membrane fusion events such as sperm-egg or plasma membrane-cortical granule merger, and production and hydrolysis of phospholipids. However, there has been no large-scale quantification of phospholipid changes during fertilization. Using an enzymatic assay, traditional FA analysis by TLC and gas chromatography, along with a new method of phospholipid measurement involving HPLC separation and evaporative light-scattering detection, we report lipid levels in eggs, sperm, and during fertilization in Xenopus laevis. Sperm were found to contain different amounts of phospholipids as compared with eggs. During fertilization, total phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, and phosphatidylserine decreased, and ceramide increased, whereas there was no change in phosphatidylcholine, cardiolipin, or phosphatidylethanolamine. FA analysis of phospholipids found numerous changes during fertilization. Because there is an increase in sn-1,2-diacylglycerol at fertilization, the FAs associated with this increase and the source of the increase in this neutral lipid were examined. Finally, activation of phospholipase C, phospholipase D, phospholipase A2, autotoxin, and sphingomyelinase at fertilization is discussed.     

Regulation by Lipids of Plant Microsomal Enzymes: III. Phospholipid Dependence of the Cytidine-Diphospho-Choline Phosphotransferase of Potato Microsomes

Cytidine-diphospho-choline diacyl-glycerol phosphorylcholine phosphotransferase activity was demonstrated in potato (Solanum tuberosum L.) microsomes and the incorporation of cytidine-diphospho[¹⁴C]choline into phosphatidylcholine was characterized by the time course of ¹⁴C incorporation and the effect of microsomal protein concentration on choline incorporation.

Potato microsomes were progressively delipidated by treatments (2 min at 0°C) with increasing amounts of phospholipase C from Bacillus cereus. A decrease in choline phosphotransferase activity was observed in parallel with the progressive hydrolysis of membrane phospholipids. A 70% (or more) phospholipid hydrolysis provoked the total inactivation of the enzyme.

Adding back exogenous phospholipids (in the form of liposomes) to phospholipase C-treated membranes restored the enzymic activity. Restoration could be obtained with egg yolk phospholipids as well as with potato phospholipids. Restoration was time dependent and completed after 10 minutes; restoration was also dependent on the quantity of liposomes added to lipid-depleted membranes: the best restorations were obtained with 1 to 2.5 milligrams of phospholipid per mg of microsomal protein; higher phospholipid to protein ratios were less efficient or inhibitory.

These results clearly demonstrate the phospholipid dependence of the cytidine-diphospho-choline phosphotransferase from potato microsomes.

     

Estimation of the phospholipid distribution in the human platelet plasma membrane based on the effect of phospholipase A2 from Naja nigricollis

Human platelets in three physiological states were prepared. These states were the gel-filtered, the thrombin-induced shape-changed, and the thrombin-activated platelets. The phospholipid distributions in these three types of membrane were probed by using the basic phospholipase A2 of Naja nigricollis. This enzyme could penetrate through these membranes to hydrolyze all of their accessible phospholipids and to cause cell lysis. The hydrolytic time-courses displayed three phases. The state of platelet in each lipid hydrolytic phase was examined by: (1) measuring the leakage of lactate dehydrogenase; (2) analyzing the morphology by both scanning and transmission electron microscopy (scanning EM and transmission EM); and (3) estimating the hydrolysis of the [32P]phosphate-labeled platelets. The existence of these three hydrolytic phases may signify that the phospholipase A2 sequentially hydrolyzed its substrates in the membrane outer leaflet, in the inner one, and in the cytosol. The content and the distribution of each phospholipid class in the plasma membranes of the resting and of the shape-changed platelets were similar. These membrane surfaces consisted mainly of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Phosphatidylserine (PS) was not exposed on the surface of the shape-changed platelet. The content of each lipid class in the activated platelet membrane was 10% more than that in the resting platelet. PS was found on the activated platelet cell surface. This implies that PS is exposed only during platelet secretion.