Effect of phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine on the protein C/protein S anticoagulation system.

Phosphatidylserine is known to significantly accelerate the blood coagulation reaction. In a previous communication submitted for publication, we demonstrated that phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine showed effects on the blood coagulation reaction using the factor Xa-prothrombin reaction system, and discuss a new function of membrane phospholipids. The present study examined the role of phospholipids in the blood coagulation regulatory reaction (anticoagulation system), by studying the effects of phospholipids on the protein C/protein S reaction. We have established quantitative methods for measuring activated protein C activity and protein S activity, and used them to measure their activity after the addition of liposomes with different phospholipid compositions. We found that phosphatidylcholine inhibited activated protein C and protein S activities in a dose-dependent manner, as in the factor Xa-prothrombin reaction system. On the other hand, phosphatidylethanolamine and lysophosphatidylcholine showed no effect on activated protein C activity. Phosphatidylethanolamine inhibited and lysophosphatidylcholine accelerated coagulation activity in the factor Xa-prothrombin system, but such effects were not observed in the protein C/protein S reaction system. The coagulation and anticoagulation reactions are exquisitely balanced by thrombin, with a role both as a procoagulant and anticoagulant. Therefore, it is understandable that phosphatidylethanolamine and lysophosphatidylcholine show different effects in the factor Xa-prothrombin and protein C/protein S reaction systems. It appears that coagulation and anticoagulation reactions are co-ordinated and controlled by changes in phospholipid composition of the cellular membrane where the coagulation reaction takes place.     

Sensitivity of Individual Erythrocyte Membrane Phospholipids to Changes in Fatty Acid Composition in Chronic Alcoholic Patients

The erythrocyte membrane levels of total phospholipids and cholesterol and the fatty acid composition of individual groups of phospholipids (phosphatidylethanolamine, phosphatidylserine plus phosphatidylinositol, and phosphatidylcholine) were studied in 10 patients admitted for ethanol detoxification and in 14 control subjects. The fatty acid composition of the patient phospholipids was modified but the level of cholesterol and the level of phospholipids remained unchanged. The fatty acid changes were mainly confined to phosphatidylcholine. The modifications concerned the levels of the octadecenoic acids (18:1) which rose (p less than 0.01), and linoleic acid (18:2) which fell (p less than 0.05). These results suggest that chronic ethanol ingestion may perturb the cell membrane organization with, in consequence, possible effects on cell morphology and functions.     

Significance of platelet membrane destruction in vasospastic angina pectoris during its exacerbation]

The parameters of the structure and function of platelet membranes were examined in 30 patients with unstable vasospastic angina in relation to the outcome of the disease (myocardial infarction stabilization or development). The fractions of phospholipids (lysophosphatidylcholine, phosphatidylserine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine), the levels of platelet membranous free and esterified cholesterol, plasma malonic dealdehyde and thromboxane B2, the activity of platelet superoxide dismutase and catalase were determined. The exacerbation of vasospastic angina was found to appear as accumulation of cholesterol (mainly of its fraction), an increase in platelet membranous levels of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, accumulation of malonic dealdehyde. These changes were more profound in cases with subsequent myocardial infarctions. There were differences in the dynamics of the examined parameters in relation to the outcome of the disease.     

Measurement of outward translocation of phospholipids across human erythrocyte membrane.

Spin-labeled phospholipids have been used to study the outside----inside and inside----outside transport of phospholipids across the human erythrocyte membrane at 37 degrees C. As already shown, inward transport is much faster for aminophospholipids than for phosphatidylcholine. In addition, we show here that outward transport of the phosphatidylserine and phosphatidylethanolamine analogues is three to four times faster than that of phosphatidylcholine. Magnesium depletion of the erythrocytes considerably decreases the outward rate of both aminophospholipids to values close to that of phosphatidylcholine. These results suggest that the outward aminophospholipid translocation is, at least partly, protein mediated. The protein involved could be identical to the inward Mg-ATP-dependent aminophospholipid carrier.     

Novel high-performance liquid chromatography for determination of membrane phospholipid composition of rat hepatocytes

We have developed a new, quick and efficient method of high-performance liquid chromatography (HPLC) for the isolation and quantitative determination of phospholipids in hepatocyte membranes. A silica gel column was used for the isolation and determination, and an isocratic mixture of acetonitrile, methanol and 85% phosphoric acid (130:5:1.7, v/v/v) was used as a mobile phase. Six kinds of phospholipids, i.e. phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphinogomyelin (SPH), in this order, were completely isolated within 45 min. The phospholipid composition of sinusoidal membrane vesicles (SMV) and canalicular membrane vesicles (CMV) obtained from rat liver, as well as of human erythrocyte ghosts were determined by this HPLC method. The level of SPH in CMV was significantly higher than that in SMV, and the level of PC in CMV was significantly lower than that in SMV. These results were considered attributable to the low fluidity of CMV. The phospholipid composition of human erythrocyte membrane was different from that of rat SMV and CMV. The present technique is suitable for quantitative determination of phospholipids in cell membranes.     

The effects of phospholipid molecular species on cholesterol crystallization in model biles: the influence of phospholipid head groups

Background/Aims: Variations in the molecular species of biliary phospholipids have been shown to exert major effects on cholesterol solubility and carriers in model and human biles. The aim of this study was to explore systematically the effects of various phospholipid head groups on the cholesterol crystallization process in model biles.Methods: Three different control model biles were prepared using varying proportions of egg lecithin, cholesterol and Na taurocholate. In the test biles, 20% of the egg lecithin was replaced with synthetic phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol or phosphatidylcholine, keeping the phospholipid acyl chains and other biliary lipids constant in each experiment.Results: Phosphatidylserine and phosphatidylglycerol significantly prolonged the crystal observation time, from 2 days to 10 and 6 days, respectively (p<0.02), while phosphatidylethanolamine had little and phosphatidylcholine no effect. The crystal growth rate was significantly slowed down with 20% phospholipid replacement in the following order: phosphatidylglycerol> phosphatidylserine> phosphatidylethanolamine. The total crystal mass after 14 days, as measured by chemical analysis, was reduced by 59% with phosphatidylserine (p<0.05), and by 73% with phosphatidylglycerol (p<0.05); while phosphatidylethanolamine had little effect. The precipitable cholesterol crystal fractions after 14 days were significantly reduced with phosphatidylserine (54%) and phosphatidylglycerol (37%), but not with phosphatidylethanolamine or phosphatidylcholine.Conclusions: Variations in the head groups of biliary phospholipids may markedly slow down the cholesterol crystallization process in model biles.     

糖尿病患者红细胞膜磷脂成分的改变

应用高效液相色谱法分析７２例ＮＩＤＤＭ患者红细胞膜磷脂成分。结果显示，ＮＩＤＤＭ患者红细胞膜磷脂酰乙醇胺、磷脂酰肌醇、磷脂酰丝氨酸、磷脂酰胆碱、神经鞘磷脂均显著降低，而溶血磷脂酰胆碱显著增高，并且红细胞膜磷脂成分的改变与空腹血糖、糖化血红蛋白、血脂及过氧化脂质有关。提示糖尿病代谢紊乱与脂质过氧化可能是导致红细胞膜磷脂成分改变的重要因素。     

Phospholipid composition and organization in model β-thalassemic erythrocytes

The membrane phospholipid organization in human red blood cells (RBC) is rigidly maintained by a complex system of enzymes. However, several elements of this system are sensitive to oxidative damage. An important component in the destruction of β-thalassemic RBC is the generation of reactive oxygen species and the release of redox-active iron by the unpaired α-hemoglobin chains. Consequently, we hypothesized that the presence of this oxidative stress to the RBC membrane could lead to alterations in membrane lipid organization. Model β thalassemic RBC, prepared by the introduction of excess α-globin in the cell, have previously been shown to exhibit structural and functional changes almost identical to those observed in β-thalassemic cells. After 24 hr at 37°C, the model β thalassemic cells exhibited a significant loss of deformability, as measured by ektacytometric analysis, indicative of extensive membrane damage. However, a normal steady-state distribution of endogenous phospholipids was found, as evidenced by the accessibility of membrane phospholipids to hydrolysis by phospholipases. Similarly, the kinetics of transbilayer movement of spin-labeled phosphatidylserine (PS) and phosphatidylethanolamine (PE) in all samples was in the normal range and was not affected by the presence of excess α-globin chains. In contrast, a faster rate of spin-labeled phosphatidylcholine (PC) transbilayer movement was observed in these cells. While control RBC exhibited a complete loss of their initial (2 mol%) lysophosphatidylcholine (LPC) levels following 24 hr of incubation at 37°C, 1.5 mol% LPC was still present in model β-thalassemic cells, suggesting an altered phospholipid molecular species turnover, possibly as a result of an increased repair of oxidatively damaged phospholipids. © 1996 Wiley-Liss, Inc.     

糖尿病患者红细胞膜磷脂成分与血液流变学的改变

应用高效液相色谱法分析了４０例Ⅱ型糖尿病患者红细胞膜磷脂成分与血液流变学的有关指标及血糖、血脂水平。结果显示，糖尿病患者红细胞膜磷脂成分有改变，并且全血粘度及血浆粘度增高，红细胞变形指数增高，红细胞过度聚集，纤维蛋白原增高。以上变化与血糖、血脂水平及糖尿病视网膜病变存在相关性。多元逐步回归分析提示糖尿病红细胞膜磷脂成分的改变是影响血液流变学变化的重要因素。     

ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes.

Spin-labeled analogs of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine have been used to study phospholipid transverse diffusion and asymmetry in the human erythrocyte membrane. Ascorbate reduction was used to assess the transbilayer distribution of the labels. All three spin-labeled phospholipids initially incorporated into the outer leaflet of the membrane. On fresh erythrocytes at 5 degrees C, the phosphatidylcholine label remained mainly in the outer leaflet. In contrast, the phosphatidylserine and phosphatidylethanolamine labels underwent rapid transverse diffusion that led to their asymmetric distribution in favor of the inner leaflet. The latter effect was reversibly inhibited after ATP depletion of the erythrocytes and could be reproduced on resealed erythrocyte ghosts only if hydrolyzable Mg-ATP was included in the internal medium. It is suggested that an ATP-driven transport of amino phospholipids toward the inner leaflet could be the major cause of the phospholipid asymmetry in the erythrocyte membrane. It is also proposed that the same mechanism could explain the ATP requirement of the maintenance of the erythrocyte membrane discoid shape.     

The distribution of erythrocyte phospholipids in hereditary spherocytosis demonstrates a minimal role for erythrocyte spectrin on phospholipid diffusion and asymmetry

In the human erythrocyte membrane phosphatidylcholine and sphingomyelin reside mainly in the outer leaflet, whereas the aminophospholipids, phosphatidylethanolamine and phosphatidylserine, are mainly found in the inner leaflet. Maintenance of phospholipid asymmetry has been assumed to involve interactions between the aminophospholipids and the membrane skeleton, in particular spectrin. To investigate whether spectrin contributes to maintaining the phospholipid transbilayer distribution and kinetics of redistribution, we studied erythrocytes from hereditary spherocytosis patients whose spectrin levels ranged from 34% to 82% of normal. The phospholipid composition and the accessibility of membrane phospholipids to hydrolysis by phospholipases were in the normal range. Spin-labeled phosphatidylserine and phosphatidylethanolamine analogues that had been introduced into the outer leaflet were rapidly transported at 37 degrees C to the inner leaflet, whereas the redistribution of spin-labeled phosphatidylcholine was slower. The kinetics of transbilayer movement of these spin-labeled phospholipid in all samples was in the normal range and was not affected by the level of spectrin. Although these erythrocyte membranes contained as little as 34% of the normal level of spectrin and were characterized by several physical abnormalities, the composition, distribution, and transbilayer kinetics of the phospholipids were found to be normal. We therefore conclude that spectrin plays, at best, only a minor role in maintaining the distribution of erythrocyte membrane phospholipid.     

Molecular defects in sarcolemmal glycerophospholipid subclasses in diabetic cardiomyopathy

Although still scarcely studied, the phospholipid component of the cell membrane is of absolute importance for cell function. Experimental evidence indicates that individual molecular species of a given phospholipid can influence specific membrane functions. We have examined the changes in molecular species of diacyl and alkenylacyl choline/ethanolamine glycerophospholipid subclasses and those of phosphatidylserine in purified cardiac sarcolemma of healthy and streptozotocin-induced insulin dependent diabetic rats without or with insulin treatment. The relative content of plasmalogens increased in all the phospholipid classes of diabetic sarcolemma under study. Phosphatidylcholine and phosphatidylethanolamine were mostly enriched with molecular species containing linoleic acid in sn-2 position and deprived of the molecular species containing arachidonic acid. The molecular species of phosphatidylserine containing either arachidonic or docosahexaenoic acid were less abundant in membranes from diabetic rats than in membranes from controls. Insulin treatment of diabetic rats restored the species profile of phosphatidylethanolamine and overcorrected the changes in molecular species of phosphatidylcholine. The results suggest that the high sarcolemmal level of plasmalogens and the abnormal molecular species of glycerophospholipids may be critical for the membrane dysfunction and defective contractility of the diabetic heart.     

Novel high-performance liquid chromatography for determination of membrane phospholipid composition of rat hepatocytes.

We have developed a new, quick and efficient method of high-performance liquid chromatography (HPLC) for the isolation and quantitative determination of phospholipids in hepatocyte membranes. A silica gel column was used for the isolation and determination, and an isocratic mixture of acetonitrile, methanol and 85% phosphoric acid (130:5:1.7, v/v/v) was used as a mobile phase. Six kinds of phospholipids, i.e. phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphinogomyelin (SPH), in this order, were completely isolated within 45 min. The phospholipid composition of sinusoidal membrane vesicles (SMV) and canalicular membrane vesicles (CMV) obtained from rat liver, as well as of human erythrocyte ghosts were determined by this HPLC method. The level of SPH in CMV was significantly higher than that in SMV, and the level of PC in CMV was significantly lower than that in SMV. These results were considered attributable to the low fluidity of CMV. The phospholipid composition of human erythrocyte membrane was different from that of rat SMV and CMV. The present technique is suitable for quantitative determination of phospholipids in cell membranes.     

Insulin stimulation of phospholipid methylation in isolated rat adipocyte plasma membranes.

Partially purified plasma membranes prepared from rat adipocytes contain N-methyltransferase(s) that utilize(s) S-adenosyl-L-methionine to synthesize phosphatidylcholine from phosphatidylethanolamine. The incorporation of [3H]methyl from S-adenosyl-L-[methyl-3H]methionine into plasma membrane phospholipids was linear with incubation time and plasma membrane protein concentration and was inhibited in a dose-dependent manner by both S-adenosyl-L-homocysteine and 3-deazadenosine. The addition of insulin to plasma membranes stimulated the methylation of endogenous phosphatidylethanolamine, as evidenced by an increase in the levels of phosphatidyl-N-monomethylethanolamine, phosphatidyl-N, N-dimethylethanolamine, and phosphatidylcholine. The effect of insulin was rapid and concentration-dependent, with 100 microunits/ml providing near maximal stimulation. The incorporation of [3H]methyl into phospholipids of control and insulin-stimulated plasma membranes was enhanced by the addition of exogenous methyltransferase substrates phosphatidylethanolamine, phosphatidyl-N-monomethylethanolamine, and phosphatidyl-N,N-dimethylethanolamine. The stimulatory effect of insulin on adipocyte plasma membrane phospholipid methylation may have a physiological role in insulin action.     

Interaction between terminal complement proteins C5b-7 and anionic phospholipids

We have recently shown that C5b-6 binds to the erythrocyte membrane via an ionic interaction with sialic acid before the addition of C7 and subsequent membrane insertion. In this study we assessed the role of anionic lipids in the binding of the terminal complement proteins to the membrane and the efficiency of subsequent hemolysis. Human erythrocytes were modified by insertion of dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylserine (DPPS), dipalmitoyl phosphatidylethanolamine (DPPE), or dipalmitoyl phosphatidic acid (DPPA). Lipid incorporation and the hemolytic assays were done in the presence of 100 micromol/L sodium orthovanadate to prevent enzymatic redistribution of lipid. We found that the neutral lipids, DPPC and DPPE, did not affect C5b-7 uptake or hemolysis by C5b-9. In contrast, the two acidic phospholipids, DPPS and DPPA, caused a dose-dependent increase in both lysis and C5b-7 uptake. We conclude that the presence of anionic lipids on the exterior face of the membrane increases C5b-7 uptake and subsequent hemolysis. It is known that sickle cell erythrocytes have increased exposure of phosphatidylserine on their external face and are abnormally sensitive to lysis by C5b-9. The data presented here provide a plausible mechanism for this increased sensitivity.     

Altered plasma membrane phospholipid organization in Plasmodium falciparum-infected human erythrocytes

The intraerythrocytic development of the malaria parasite is accompanied by distinct morphological and biochemical changes in the host cell membrane, yet little is known about development-related alterations in the transbilayer organization of membrane phospholipids in parasitized cells. This question was examined in human red cells infected with Plasmodium falciparum. Normal red cells were infected with strain FCR3 or with clonal derivatives that either produce (K+) or do not produce (K-) knobby protuberances on the infected red cells. Parasitized cells were harvested at various stages of parasite development, and the bilayer orientation of red cell membrane phospholipids was determined chemically using 2,4,6-trinitrobenzene sulphonic acid (TNBS) or enzymatically using bee venom phospholipase A2 (PLA2) and sphingomyelinase C (SMC). We found that parasite development was accompanied by distinct alterations in the red cell membrane transbilayer distribution of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Increases in the exoplasmic membrane leaflet exposure of PE and PS were larger in the late-stage parasitized cells than in the early-stage parasitized cells. Similar results were obtained for PE membrane distribution using either chemical (TNBS) or enzymatic (PLA2 plus SMC) methods, although changes in PS distribution were observed only with TNBS. Uninfected cohort cells derived from mixed populations of infected and uninfected cells exhibited normal patterns of membrane phospholipid organization. The observed alterations in P falciparum-infected red cell membrane phospholipid distribution, which is independent of the presence or absence of knobby protuberances, might be associated with the drastic changes in cell membrane permeability and susceptibility to early hemolysis observed in the late stages of parasite development.     

Specific phospholipids are required to reconstitute adenylate cyclase solubilized from rat brain.

Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] was solubilized from a rat brain homogenate with sodium deoxycholate. This solubilized preparation had no detectable enzymic activity with either Mg-ATP or Mn-ATP as substrate. The activity could be restored by addition of either nonionic detergent or certain specific phospholipids. Maximal restoration of enzyme activity was obtained with Triton X-100, L-alpha-phosphatidylcholine, L-alpha-lysophosphatidylcholine, phosphatidyl-N-monomethylethanolamine, or sphingomyelin. Activity was only partially restored by phosphatidylethanolamine (40-60%) or phosphatidyl-N,N-dimethylethanolamine (10-20%). Other phospholipids tested, including phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid, could not restore enzyme activity but, instead, could inhibit the stimulation of enzyme activity by phosphatidylcholine. The restoration of activity by L-alpha-phosphatidylcholine was inhibited by cholesterol at concentrations above 33 mol %, although this effect was not observed with three different esters of cholesterol. These studies suggest a possible specific role of phospholipids in modulating adenylate cyclase activity.     

Reconstitution of ATP-dependent aminophospholipid translocation in proteoliposomes.

In addition to ion-pumping ATPases, most plasma membranes of animal cells contain a Mg2+ ATPase activity, the function of which is unknown. This enzyme, of apparent molecular mass 110 kDa, was purified from human erythrocyte membranes by a series of column chromatographic procedures after solubilization in Triton X-100. When reincorporated into artificial bilayers formed from phosphatidylcholine, it was able to transport a spin-labeled phosphatidylserine analogue from the inner to the outer membrane leaflet provided Mg2+ ATP was present in the incubation mixture. The ATP-dependent transport of the phosphatidylethanolamine analogue required the presence of an anionic phospholipid (e.g., phosphatidylinositol) in the outer membrane leaflet. In contrast the transmembrane distribution of spin-labeled phosphatidylcholine was unaffected in the same experimental conditions. This transmembrane movement of aminophospholipid analogues was inhibited by treatment of the proteoliposomes with a sulfhydryl reagent. We conclude that the Mg2+ ATPase is sufficient for the biochemical expression of the aminophospholipid translocase activity, which is responsible for the inward transport of phosphatidylserine and phosphatidylethanolamine within the erythrocyte membrane. The presence of this transport activity in many animal cell plasma membranes provides a function for the Mg2+ ATPase borne by these membranes.     

Red blood cells from patients with homozygous sickle cell disease provide a catalytic surface for factor Va inactivation by activated protein C

The structure of red blood cell (RBC) membranes in homozygous sickle cell disease (SCD) is significantly disturbed, with an increased exposure of aminophospholipids (phosphatidylserine and phosphatidylethanolamine) at the outer surface, responsible for a procoagulant activity of SS RBCs. Aminophospholipids are known not only to promote procoagulant reactions, but also to support inhibition of blood coagulation by the protein C system. The aim of the present study was to examine whether SS RBCs could serve as a catalytic surface for the inactivation of factor Va by activated protein C (APC). Venous blood was obtained from 19 consecutive SS patients and 13 controls (AA). In all SS patients, the amount of phosphatidylserine exposed at the outer surface of RBCs was increased compared with controls, as demonstrated by a prothrombinase assay. In addition, SS RBCs significantly (P < 0.0001) increased the rate of FVa inactivation by APC: the mean values (and ranges) of the factor Va inactivation rates were 30 (0-57) vs 9.5 (0-32) mmol Vai/min/mol APC for SS RBCs and normal RBCs respectively. Our results indicate that SS RBCs provide a catalytic surface for the negative control of blood coagulation, which may partially control the procoagulant activity of these cells.     

Transmembrane mobility of phospholipids in sickle erythrocytes: effect of deoxygenation on diffusion and asymmetry

We studied the effect of sickling on the transmembrane reorientation and distribution of phospholipids in the red blood cells of patients homozygous for sickle cell anemia (SS). To this purpose, we followed the redistribution kinetics of trace amounts of spin-labeled analogues of natural phospholipids first introduced in the membrane outer leaflet of normal or sickle erythrocytes exposed to air or nitrogen. Deoxygenation had no effect on the lipid redistribution kinetics in normal (AA) cell membranes. At atmospheric pO2, unfractionated SS cells were not different from normal cells. However, on deoxygenation inducing sickling, phosphatidylcholine passive diffusion was accelerated and the rate of the adenosine triphosphate-dependent transport of aminophospholipids was reduced, especially for phosphatidylserine. The stationary distribution of the aminophospholipids between the two leaflets was slightly less asymmetric, a phenomenon more pronounced with phosphatidylethanolamine. These changes were rapidly reversible on reoxygenation. When SS cells were separated by density, both dense and light cells exhibited the properties cited above. However, dense cells exposed to air possessed a lower aminophospholipid transport rate. These data favor the relationship between aminophospholipid translocase activity and phospholipid transmembrane asymmetry. Sickle cell disease is the first case of aminophospholipid translocase pathology.