Lysophosphatidic acid and human erythrocyte aggregation

The effect of lysophosphatidic acid (LPA) on the shape and aggregation of human erythrocytes in autologous plasma was studied. The morphology of erythrocytes and their aggregates were studied by light microscopy. It is shown that the addition of plasma with a high LPA content to erythrocytes leads to a change of their shape: discocytes are transformed into echinocytes. There is practically no aggregation of erythrocytes in the form of rouleaux. At the same time, there is observed a strong aggregation of echinocytes. This is accompanied by the formation of microvesicles. The addition of normal blood plasma to echinocytes restores their shape and aggregation of red blood cells in the form of rouleaux. A possible mechanism of action of lysophosphatidic acid on erythrocytes is discussed.     

Study of the relationship between shape and aggregation change in human erythrocytes

The relationship between shape and spontaneous and fibrinogen-induced aggregation change in human erythrocytes was studied. Spontaneous and fibrinogen-induced erythrocyte aggregation was investigated using a rheoscope designed according to the method of H. Schmid-Schonbein et al. (1973). The erythrocyte shape was studied by means of light microscopy. It was shown that plasma enriched with lysophosphatidic acid and ATP depletion of erythrocytes led to the change of erythrocyte shape: discocytes transformed into echinocytes. It was found that spontaneous aggregation of such cells was considerably decreased. Aggregation of erythrocytes, treated with lysophosphatidic acid, was diminished more markedly. Fibrinogen-induced aggregation of echinocytes, obtained after treatment with lysophosphatidic acid and produced by ATP depletion, was also greatly reduced.     

Effect of adenosine on the shape,aggregates morphology and aggregability of ATP-depleted erythrocytes

The effect of adenosine on the shape, aggregate morphology and aggregability of ATP-depleted erythrocytes was studied. It is shown that the ATP-depletion of erythrocyte leads to the change in their shape: diskocytes transform to echinocytes. It is found that the aggregability of such cells in autologous plasma significantly decreased. Incubation of echinocytes with adenosine largely restored discoid shape and erythrocyte aggregability.     

The apparent viscosity of aggregating and non-aggregating erythrocyte suspensions in the isolated perfused liver

Vasodilated guinea pig livers were perfused with normal erythrocytes and echinocytes suspended in isoviscous high- and low-molecular-weight dextran solutions. The relative flow resistance of these suspensions and the oxygen uptake of the livers were then determined. The relative flow resistance of the echinocytes that were suspended in high-molecular-weight dextran, however, was significantly higher than that of any other red cell suspension. The oxygen uptake was independent of the perfusion media. It is proposed that high-molecular-weight dextran induces echinocytes to attach to one another, and that this clumping together, and shape-transformation of red cells, hinders their flow in the vasodilated liver.     

Electrostatic repulsion among erythrocytes in tube flow, demonstrated by the thickness of marginal cell-free layer

Electrostatic repulsion among erythrocytes in flow was evaluated through measurement of the thickness of the marginal cell-free layer in narrow glass tubes of 20-50 microns in inner diameter. To reduce the electrostatic repulsive force, due mainly to sialic acid of the membrane glycoproteins, human erythrocytes were treated with neuraminidase. The surface negative charge of the erythrocytes, as determined from the electrophoretic mobility using free-flow electrophoresis, was found to be proportional to the sialic acid content. When erythrocytes with decreased sialic acid content flowed through narrow tubes, the thickness of cell-free layer determined using an image processor increased even in the absence of erythrocyte aggregation in the suspension. The effect was more pronounced at acidic pH. The addition of Dextran T-70 (70,400 Mol. Wt.) further increased the cell-free layer thickness due to erythrocyte aggregation. Thus, reducing the negative charge density on the erythrocyte surface by itself accelerates the axial accumulation of erythrocytes in flow due to the decreased electrostatic repulsive force between the cells, even in the absence of erythrocyte aggregation.     

Cytospectrophotometric research on hemoglobin in human erythrocytes. I. The methemoglobin content in intact erythrocytes and its alteration under the influence of chromosmon, ascorbic acid, riboflavin and glutathione

The blood of healthy men and patients with methemoglobinemia of different genesis was incubated with chromosmon, ascorbic acid, riboflavin and glutathione, the percentage of erythrocytes with thorn-shaped protuberances-echinocytes being subsequently determined in the blood smears. The absorbtion spectra at the range 400-650 nm were investigated both in the smooth erythrocytes and in echinocytes. A correlation was found between the percentage of echinocytes and the methemoglobin content in the blood. The methemoglobin amount in the echinocytes was determined to be higher than in the smooth erythrocytes. It is discovered that effects of chromosmon, glutathione and riboflavin on production of methemoglobin depend on the dose, individual peculiarities of erythrocytes and on the illness that caused methemoglobinemia. The calculation of echinocyte percentage may be used as an express-diagnostics of methemoglobinemia and for purposes of studying the effect of methemoglobin-producing substances and drugs.     

Comparison of the effect of phenoxyherbicides on human erythrocyte membrane (in vitro)

The molecular mechanisms of phenoxyherbicides action in animals have been insufficiently studied. Now, we have investigated the interaction of sodium salts of phenoxyherbicides, e.g., 2,4-dichlorophenoxyacetic acid (2,4-D-Na), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T-Na) and 4-chloro-2-methylphenoxyacetic acid (MCPA-Na) with human erythrocytes. In this study, we evaluated the effect of these compounds on erythrocyte membrane fluidity as well as changes in membrane proteins content. It was observed that all of the compounds studied altered membrane fluidity, changed the size and shape of the erythrocytes and provoked echinocytes formation. It was also revealed that 2,4-D-Na and 2,4,5-T-Na changed the content of erythrocyte membrane proteins mainly by a decrease in the level of spectrin and low molecular weight proteins. The comparison of the action of phenoxyherbicides examined showed that 2,4,5-T-Na caused the greatest changes in the erythrocytes membrane, whereas MCPA-Na induced the lowest alterations in the incubated cells. It must be noted that changes of the investigated parameters were observed only at presence of significant concentrations of these compounds that may penetrate human organism only as a result of acute poisoning.     

Modulation of human erythrocyte shape and fatty acids by diet

A semi-synthetic diet (Vivonex) was administered via nasogastric tube to three cystic fibrosis patients with pancreatic exocrine deficiency for 14 days to gain weight. Dietary essential fatty acids were provided as safflower oil, which constituted 1.3% of total calories. Plasma and red blood cells were analyzed for the content and composition of lipids at the start of the diet and at days 7 and 14 of the dietary period, and the results were correlated with the morphology of the cells. Feeding Vivonex to the patients led to an essential fatty acid deficiency, which was manifested in a 50% decrease in the linoleic acid content of the phosphatidylcholine of plasma and red blood cells at days 7 and 14 and in a 20% decrease in the linoleic acid content of red cell phosphatidylethanolamine at day 14. There was no significant alteration in the levels or composition of the other phospholipid classes and in the free cholesterol/phospholipid ratio. The decrease in the linoleic acid content of the erythrocytes was accompanied by a dramatic increase in the proportion of cells as echinocytes. We conclude that restricted linoleic acid availability in cystic fibrosis patients causes a change in red blood cell shape either directly by decreasing the linoleoylphosphatidylcholine content of the membrane or indirectly by affecting enzyme activity.     

A method for the quantitative measurement of cell aggregation

It is demonstrated that formation of cellular aggregates in a slowly rotating suspension is accompanied by a decrease in total cell concentration in the top layer of the suspension. Both the average particle size and the initial cell concentration of the homogeneous suspension, are parameters which determine the magnitude of the effect.The method is exemplified by

1. 1. aggregation of HeLa cells after treatment with neuraminidase;

2. 2. agglutination of HeLa cells with concanavalin A;

3. 3. agglutination of human erythrocytes with poly- -lysine;

4. 4. agglutination of human erythrocytes with poly- -lysine following pretreatment with neuraminidase.

     

Structural-functional state of platelets at the initial stage of aggregation

ADP-induced changes of light transmission of platelet suspension were studied with a laser aggregometer at concentration variation of both cells and aggregation agents. It was shown that a decrease of light transmission of platelet suspension was due to structure-morphological changes of platelets and pseudopodia formation. An investigation method of platelet aggregation with a small content of cells in the solution permits to fix distinctly the stages of the platelet structural states in the processes of stimulation and aggregation.     

Abnormalities of elastic and transporting properties of red blood cells under development of apoptosis

The functional properties of erythrocytes under development of apoptotic process in these cells were investigated by the low angle light scattering technique. Apoptosis induced by ionomycin was associated with an initial decrease of cell volume and caused formation of echinocytes. After that the cells restored their volume forming rounded erythrocytes with rugged membrane capable to agglomerate with each other. At the late stages of apoptosis, small fragmented cells can be revealed. Preapoptotic red blood cells (at all stages of apoptosis) manifested an enormous tolerance to hypotonic loading, whereas control cells hemolyzed just after reaching a critical volume (∼150 fl). Acidic hemolysis cannot differentiate between control and preapoptotic erythrocytes, the cells being hemolyzed not reaching the critical volume. Placing the control erythrocytes to a medium with ammonia ions instead of sodium ions caused an initial increase of cell volume above the critical point, and then it was also followed by hemolysis. Under ammonia loading, an initial rate of the cell volume growth and a ratio of the hemolyzed cells were significantly reduced in preapoptotic cells.     

Membrane potential and human erythrocyte shape.

Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The process is fast and reversible at room temperature, so it seems to involve shifts in weak inter- or intramolecular bonds. This shape change has been reported to depend on changes in membrane potential, but control experiments excluding roles for other simultaneously varying cell properties (cell pH, cell water, and cell chloride concentration) were not reported. The present study examined the effect of independent variation of membrane potential on red cell shape. Red cells were equilibrated in a set of solutions with graduated chloride concentrations, producing in them a wide range of membrane potentials at normal cell pH and cell water. By using assays that were rapid and accurate, cell pH, cell water, cell chloride, and membrane potential were measured in each sample. Cells remained discoid over the entire range of membrane potentials examined (-45 to +45 mV). It was concluded that membrane potential has no independent effect on red cell shape and does not mediate the membrane curvature changes known to occur in red cells equilibrated at altered pH.     

Cytoplasmic pH and human erythrocyte shape.

Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The mechanism of this transformation is unknown. The preceding companion study (Gedde and Huestis) demonstrated that these shape changes are not mediated by changes in membrane potential, as has been reported. The aim of this study was to identify the physiological properties that mediate this shape change. Red cells were placed in a wide range of physiological states by manipulation of buffer pH, chloride concentration, and osmolality. Morphology and four potential predictor properties (cell pH, membrane potential, cell water, and cell chloride concentration) were assayed. Analysis of the data set by stratification and nonlinear multivariate modeling showed that change in neither cell water nor cell chloride altered the morphology of normal pH cells. In contrast, change in cell pH caused shape change in normal-range membrane potential and cell water cells. The results show that change in cytoplasmic pH is both necessary and sufficient for the shape changes of human erythrocytes equilibrated in altered pH environments.     

Formation of large, membrane skeleton-free erythrocyte vesicles as a function of the intracellular pH and temperature

Vesiculation of intact erythrocytes can be induced by decreasing their intracellular pH and then heating the red cell suspension to a critical temperature value. While at intracellular pH 6 vesiculation begins at 45 degrees C, further decrease in the intracellular pH lowers the critical temperature. In addition, the critical temperature value can be modified by varying the length of the interval between titration and heating as well as by changing the temperature during this interval. The vesicles are large (1-3.5 micron in diameter), haemoglobin-containing and completely free of skeletal proteins. Pretreatment of the cells with diamide and 2,4-dinitrophenol had no substantial effect on vesiculation, while N-ethylmaleimide, chlorpromazine and wheat germ agglutinin proved to be inhibitory. Increasing the osmolarity of the incubation medium markedly decreased the critical temperature: red cells suspended in a solution of 600 mosM NaCl vesiculated at 42 degrees C instead of 45 degrees C when the intracellular pH was decreased to 6. We propose that the vesiculation is due to a purely physicochemical molecular mechanism which affects the state and dimension of the membrane skeleton. We also discuss the possible role of an altered haemoglobin-membrane interaction in preventing low pH-induced intramembrane particle aggregation in the membrane skeleton-free vesicles.     

Erythrocyte membrane permeability for univalent cations (Na+, K+) and their transformation in patients with hypertension and symptomatic (renal) hypertension

The form and surface architectonic of erythrocytes studied in 18 teenagers and men with hypertensive disease of I stage (HD) and in 7 men with symptomatic (renal) hypertension (SH). Simultaneously permeability of erythrocyte membranes for Na+ and K+ was studies. The change in the form and surface architectonics was found in the erythrocytes of the patients with hypertensive disease, I stage. The same was true for the patients with renal hypertension but the difference was not so prominent. Rapid shift of correlation of erythrocyte morphological varieties has been revealed during the study of Na+ and K+ ions' transport rate after the treatment by p-chloromercuribenzoate acid. Increase of irreversible transformation of erythrocytes was discovered in patients with HD. Besides in some cases there was decrease in the size of echinocytes. The transformation of erythrocytes into echinocytes is more prominent in healthy subjects and in patients with SH. Our data suggest that the change in erythrocyte form is related to the change of erythrocyte membrane permeability to Na+ and K+ ions and the alteration of membrane structure is the basis for these disturbances.     

Hematotoxicity and blood glutathione levels after cisplatin treatment of tumor-bearing mice

The involvement of glutathione, a major cellular antioxidant, in cisplatin-mediated development of various hematological changes in mice bearing ascites Dalton lymphoma tumor was investigated. With tumor growth, glutathione levels decreased in blood but increased in tumor cells. Cisplatin treatment of tumor-bearing mice caused a decrease in glutathione levels in blood, ascites supernatant, and tumor cells. Blood hemoglobin, erythrocytes, packed cell volume and leukocytes (eosinophils, basophils, and lymphocytes) were also decreased along with the development of various morphological abnormalities in erythrocytes (microcytes, macrocytes, echinocytes, acanthocytes, etc.) after cisplatin treatment. All these hematotoxic features were noted to be increased more when buthionine sulfoximine (a specific glutathione-depleting agent) was also given prior to cisplatin treatment. However, combination treatment of cysteine (precursor for glutathione synthesis) plus cisplatin resulted in an improvement in the glutathione levels and decrease in hematological toxicities. It is noted that the glutathione levels in blood and abnormalities in erythrocytes and other hematological parameters are inversely related in cisplatin-mediated cancer chemotherapy. It is suggested that blood glutathione may play an important role in the development of cisplatin-mediated hematological toxicity in the host. This revised version was published online in July 2006 with corrections to the Cover Date.     

Recovery of human erythrocytes from the echinocyte shape induced by added choline-phospholipids is dependent on the acyl chain length

Addition of an amphiphilic lipid, such as phosphatidylcholine (PC) species with two identical saturated chains or lysophosphatidylcholine (lysoPC) species with one saturated acyl chain of various lengths, into a suspension of intact human erythrocytes resulted in lipid incorporation into the erythrocytes membrane to produce echinocytes (crenated cells). The altered shape gradually reverted on incubation at 37 degrees C until the cells reassumed their normal disc shape. The rate of such recovery of shape increased with decreasing acyl chain length for both PC with C8-C12 acyl chains and lysoPC with a C14-C18 acyl chain, and was strongly influenced by incubation temperature. The identical rate of recovery of shape was observed for cells with normal, decreased or increased ATP content, implying that the metabolic state of the cell had no influence on the recovery process. Recovery of shape is therefore considered to be caused by translocation of the incorporated lipid molecules from the outer to the inner leaflet of the membrane lipid bilayer and the rate of recovery increases with decreasing hydrophobicity of the lipid.     

Influence of erythrocyte shape on the rate of Ca2+-induced scrambling of phosphatidylserine

Membrane-perturbing agents that cause transformation of biconcave erythrocytes into echinocytes or stomatocytes were used to investigate the influence of erythrocyte shape on the rate of Ca(2+)-induced scrambling of phospholipids. Erythrocytes were treated with a variety of lipid-soluble compounds to induce these shape changes, followed by incubation with calcium and ionomycin to activate lipid scramblase. Prothrombinase activity of the cells was used to monitor the rate of surface exposure of phosphatidylserine, which is taken as a measure of scramblase activity. Echinocytes show an enhanced rate of scrambling, whereas stomatocytes show a reduced rate, relative to normocytes. This phenomenon appears to correlate with enhanced and diminished micro-exovesicle shedding from echinocytes and stomatocytes, respectively. It is concluded that the rate of calcium-induced phosphatidylserine exposure (rate of lipid scrambling) in erythrocytes depends for a considerable part on the cells' ability to form microvesicles.     

Influence of erythrocyte shape on the rate of Ca2+-induced scrambling of phosphatidylserine

Membrane-perturbing agents that cause transformation of biconcave erythrocytes into echinocytes or stomatocytes were used to investigate the influence of erythrocyte shape on the rate of Ca²⁺-induced scrambling of phospholipids. Erythrocytes were treated with a variety of lipid-soluble compounds to induce these shape changes, followed by incubation with calcium and ionomycin to activate lipid scramblase. Prothrombinase activity of the cells was used to monitor the rate of surface exposure of phosphatidylserine, which is taken as a measure of scramblase activity. Echinocytes show an enhanced rate of scrambling, whereas stomatocytes show a reduced rate, relative to normocytes. This phenomenon appears to correlate with enhanced and diminished micro-exovesicle shedding from echinocytes and stomatocytes, respectively. It is concluded that the rate of calcium-induced phosphatidylserine exposure (rate of lipid scrambling) in erythrocytes depends for a considerable part on the cells' ability to form microvesicles.     

Cytospectrophotometric study of hemoglobin in human erythrocytes. II. Methemoglobin formation in acute hypobaric hypoxia

The percentage of erthrocytes with thorn-shaped protuberances--echinocytes--was calculated in the blood smears of 10 healthy men before and after a 48 hour sharp hypobaric hypoxia in the climatic chamber of Tabai. The absorbtion spectra at the range 400-650 nm were investigated in the smooth erythrocytes and echinocytes. The methemoglobin content in the echinocytes is higher than in the smooth erythrocytes. The sharp hypobaric hypoxia results in the increase in the percentage of echinocytes and erythrocytes with fetal hemoglobin, in the change of osmotic stability and acidic resistance of erythrocytes, and in the rise of peroxid oxidation of lipids. The role of methemoglobin production upon a sharp hypobaric hypoxia is discussed.