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.     

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.     

Opposite effect of albumin on the erythrocyte aggregation induced by immunoglobulin G and fibrinogen

The effect of albumin on the immunoglobulin G (IgG)-induced and fibrinogen-induced aggregation of human erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyzer and a computer. As albumin concentration in the medium was increased, the IgG-induced erythrocyte aggregation was inhibited, while the fibrinogen-induced erythrocyte aggregation was accelerated (albumin itself was not able to aggregate erythrocytes). These relations were empirically expressed by the equations, v = aG1.8/A and v = a'F1.5 (A + b'), respectively (v, the velocity of erythrocyte aggregation; A, G and F, the concentrations of albumin, IgG and fibrinogen, respectively; a, a' and b', constant). The IgG-induced erythrocyte aggregation was remarkably inhibited by the addition of poly(glutamic acid), but the fibrinogen-induced erythrocyte aggregation was not. A mechanism for the interaction of immunoglobulin G and fibrinogen with the surface of erythrocytes was proposed.     

A study of the aggregation of human red blood cells induced by picric acid

The effect of picric acid on the aggregation of human erythrocytes was studied. It was shown that the addition of picric acid to a suspension of washed erythrocytes leads to a decrease in pH of medium to 1.5-2 and the formation of echinocytes. Stirring the suspension of echinocytes at low pH values results in a strong aggregation of cells. Increasing the pH value to 7.4 leads to a desaggregation of echinocytes. It was found that picric acid does not induce the aggregation of cells fixed by glutaraldehyde. A substantial decrease in the aggegation of spheric erythrocytes obtained after heating the cells at 50 degrees C was observed.     

Erythrocyte spectrin alteration induced by low-density lipoprotein

Addition of human plasma low-density lipoproteins (LDL) to intact human erythrocytes induces the erythrocytes to undergo morphologic transition from biconcave disks to echinocytes and spherocytes. The transformation is time-dependent. Two hours are required before echinocytes are detected by scanning electron microscopy. After two hours, LDL also decrease the phosphate content of spectrin by 40% relative to the control, suggesting that these lipoproteins modulate cell shape by influencing phosphorylationdephosphorylation of a membrane-associated cytoskeletal protein. LDL do not induce depletion of intracellular adenosine triphosphate (ATP), nor do they inhibit cyclic adenosine monophosphate-independent protein kinases which phosphorylate spectrin. LDL stimulate membrane-bound phosphatases by a factor of two, thereby reducing the amount of phosphate covalently bound to membrane proteins. The observed effects are specific for LDL. High-density lipoproteins (HDL) do not stimulate dephosphorylation of spectrin or alter erythrocyte morphology. However, HDL protect the erythrocytes against LDL-induced alterations. These data suggest that the circulating lipoproteins have a role in maintaining erythrocyte morphology by regulating the extent of phosphorylation of spectrin.     

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.     

Roles of plasma proteins and surface negative charge of erythrocytes in erythrocyte aggregation

The effect of plasma proteins (and IgG fragments) and sialic acid content of erythrocytes on the aggregation of human erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyser and a computer. (1) The velocity of erythrocyte aggregation by plasma proteins was increased with increasing in their molecular weight, i.e., IgG less than IgA less than fibrinogen less than IgM. F(ab')2. Fab and Fc could not induce the aggregation. (2) The aggregation induced by fibrinogen was accelerated by IgG and its peptic fragment, F(ab')2, but was unaffected by the plasmic fragments, Fab and Fc. The accelerating effect by IgG and F(ab')2 was inhibited by Fab and Fc. (3) The aggregation of erythrocytes was accelerated by decreasing the sialic acid content (due to the reduction of the electrostatic repulsive force among erythrocytes), and the effect of desialylation on the IgG-induced aggregation was greater than that of desialylation on the fibrinogen-induced aggregation. (4) The roles of plasma proteins and of sialic acid content of erythrocytes on the aggregation of erythrocytes were discussed.     

Effect of metabolic state on phytohemagglutinin-P agglutination of normal human erythrocytes.

A reproducible quantitative assay for the lectin-mediated agglutination of human erythrocytes, depending on different rates of settling of agglutinated and nonagglutinated erythrocytes, was developed. This assay was used to study the aggregation of human erythrocytes by phytohemagglutinin-P. The aggregation of human erythrocytes by phytohemagglutinin-P was found to depend upon the metabolic state of the cells. Metabolically depleted erythrocytes agglutinated much less readily than did similar cells supplied with adenosine. This was not due to swelling and rigidity of the cells, since erythrocytes in hypotonic solution did not exhibit significantly altered phytohemagglutinin-P agglutination. Metabolically depleted erythrocytes, or erythrocytes from blood stored 8 weeks, lysed and resealed in the presence of ATP, were agglutinated by phytohemagglutinin-P to a much greater extent than control samples without ATP. The presence of Mg2+, either alone or with ATP, had little effect on the agglutinability of the resealed membranes. Low concentrations of Ca2+ (0.2 mM) had little effect on agglutinability, although high Ca2+ (5 mM) inhibited agglutinability of the resealed membranes somewhat. Both metabolically depleted erythrocytes and depleted erythrocytes, previously treated with adenosine, when treated with trypsin released similar amounts of sialic acid. The agglutinability of the trypsinized adenosine-supplemented cells increased more readily than did that of trypsinized metabolically depleted cells. The agglutination of erythrocytes was not affected by cytochalasin B (40 mug/ml). Vinblastine (0.2 mM) caused depleted erythrocytes to agglutinate similarly to adenosine-supplemented erythrocytes, but had no effect on the agglutination of adenosine-supplemented erythrocytes. It is concluded that ATP in the human erythrocyte probably participates in the modulation of phytohemagglutinin-P agglutinability. This is not a consequence of the more rigid membrane known to accompany ATP depletion in the erythrocyte, or of the effect of ATP levels on Ca2+ or Mg2+ content. It appears likely that ATP modulates human erythrocyte phytohemagglutinin-P agglutinability through interaction, direct or indirect, with a membrane-associated component, which might also be sensitivie to vinblastine.     

Erythrocyte aggregation: bridging by macromolecules and electrostatic repulsion by sialic acid.

Relation between aggregating force (of fibrinogen and IgG) and disaggregating force (due to electrostatic repulsion among erythrocytes) in erythrocyte aggregation was investigated with a rheoscope combining a video camera, an image analyzer and a computer. (i) Erythrocyte aggregation was augmented with the increase of molecular weight of bridging macromolecules as far as examined for fibrinogen and the degradation products and IgG and the related macromolecules, and the augmentation seemed to be dependent on the molecular length of macromolecules. In accelerating the erythrocyte aggregation, fibrinogen was more effective than IgG, and some interaction between fibrinogen and IgG in their coexistence was suggested. (ii) The decrease of sialic acid content on the erythrocyte surface accelerated IgG-induced erythrocyte aggregation much greater than fibrinogen-induced one. (iii) Counteraction between aggregating force and disaggregating force in leading to erythrocyte aggregation was discussed relating to molecular length of bridging macromolecule and electrostatic repulsive force by sialic acid.     

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.     

Effect of metabolic state on phytohemagglutinin-P agglutination of normal human erythrocytes

A reproducible quantitative assay for the lectin-mediated agglutination of human erythrocytes, depending on different rates of settling of agglutinated and non-agglutinated erythrocytes, was developed. This assay was used to study the aggregation of human erythrocytes by phytohemagglutinin-P. The aggregation of human erythrocytes by phytohemagglutinin-P was found to depend upon the metabolic state of the cells. Metabolically depleted erythrocytes agglutinated much less readily than did similar cells supplied with adenosine. this was not due to swelling and rigidity of the cells, since erythrocytes in hypotonic solution did not exhibit significantly altered phytohemagglutinin-P agglutination.Metabolically depleted erythrocytes, or erythrocytes from blood stored 8 weeks, lysed and resealed in the presence of ATP, were agglutinated by phytohemagglutinin-P to a much greater extent than control samples without ATP. The presence of Mg²⁺, either alone or with ATP, had little effect on the agglutinability of the resealed membranes. Low concentrations of Ca²⁺ (0.2 mM) had little effect on agglutinability, although high Ca²⁺ (5 mM) inhibited agglutinability of the resealed membranes somewhat.Both metabolically depleted erythrocytes and depleted erythrocytes, previously treated with adenosine, when treated with trypsin released similar amounts of sialic acid. The agglutinability of the trypsinized adenosine-supplemented cells increased more readily than did that of trypsinized metabolically depleted cells.The agglutination of erythrocytes was not affected by cytochalasin B (40 μg/ml). Vinblastine (0.2 mM) caused depleted erythrocytes to agglutinate similarly to adenosine-supplemented erythrocytes, but had no effect on the agglutination of adenosine-supplemented erythrocytes.It is concluded that ATP in the human erythrocyte probably participates in the modulation of phytohemagglutinin-P agglutinability. This is not a consequence of the more rigid membrane known to accompany ATP depletion in the erythrocyte, or of the effect of ATP levels on Ca²⁺ or Mg²⁺ content. It appears likely that ATP modulates human erythrocyte phytohemagglutinin-P agglutinability through interaction, direct or indirect, with a membrane-associated component, which might also be sensitive to vinblastine.     

Influence of new hybrid antioxidants ichphans on erythrocyte morphology

Scanning electron microscopy was used to study the effects of the new generation of compounds ICHFANs, which have a combined antioxidant and acetylcholine esterase inhibitory effect on the surface architectonics of erythrocytes. The incorporation of each of the studied compounds with the positively charged quaternary ammonium in the erythrocyte membrane and their distribution in the itramembraneous space were accompanied by the formation of echinocytes, stomatocytes, and compensative effects on erythrocyte shape. The time-dependent morphological transformation of erythrocytes apparently is determined by changes in the distribution of the compounds between the outer and inner monolayers of the erythrocyte membrane. A difference in the morphological effects of compounds with different hydrophobic properties was revealed.     

Interaction of plasma lipoproteins with erythrocytes. I. Alteration of erythrocyte morphology.

Intact erythrocytes incubated in the presence of low density lipoproteins (LDL) undergo a time-dependent morphologic transformation from biconcave discs to spherocytes within 4 h. No shape change is observed when erythrocytes are incubated with high density lipoproteins (HDL). The LDL-induced change in erythrocyte morphology occurs without concomitant leakage of hemoglobin from the cell or depletion of intracellular ATP; no change in the distribution of the major lipids of the erythrocyte membranes was detected. The alteration of morphology does require attachment of LDL to the erythrocyte surface. The LDL-induced morphologic alteration is inhibited by HDL, but not by serum albumin. HDL prevent the attachment of LDL to the cell membrane; however, the HDL subfractions, HDL2 and HDL3, are only partially effective. These data suggest that normal erythrocyte morphology and cell function may depend on the concentration and composition of the circulating lipoproteins.     

Erythrocyte morphology reflects the transbilayer distribution of incorporated phospholipids

The transbilayer distribution of exogenous phospholipids incorporated into human erythrocytes is monitored through cell morphology changes and by the extraction of incorporated 14C-labeled lipids. Dilauroylphosphatidylserine (DLPS) and dilauroylphosphatidylcholine (DLPC) transfer spontaneously from sonicated unilamellar vesicles to erythrocytes, inducing a discocyte-to-echinocyte shape change within 5 min. DLPC-induced echinocytes revert slowly (t1/2 approximately 8 h) to discocytes, but DLPS-treated cells revert rapidly (10-20 min) to discocytes and then become invaginate stomatocytes. The second phase of the phosphatidylserine (PS)-induced shape change, conversion of echinocytes to stomatocytes, can be inhibited by blocking cell protein sulfhydryl groups or by depleting intracellular ATP or magnesium (Daleke, D. L., and W. H. Huestis. 1985. Biochemistry. 24:5406-5416). These cell shape changes are consistent with incorporation of phosphatidylcholine (PC) and PS into the membrane outer monolayer followed by selective and energy-dependent translocation of PS to the membrane inner monolayer. This hypothesis is explored by correlating cell shape with the fraction of the exogenous lipid accessible to extraction into phospholipid vesicles. Upon exposure to recipient vesicles, DLPC-induced echinocytes revert to discoid forms within 5 min, concomitant with the removal of most (88%) of the radiolabeled lipid. On further incubation, 97% of the foreign PC transfers to recipient vesicles. Treatment of DLPS-induced stomatocytes with acceptor vesicles extracts foreign PS only partially (22%) and does not affect cell shape significantly. Cell treated with inhibitors of aminophospholipid translocation (sulfhydryl blockers or intracellular magnesium depletion) and then incubated with either DLPS or DLPC become echinocytic and do not revert to discocytic or stomatocytic shape for many hours. On treatment with recipient vesicles, these echinocytes revert to discocytes in both cases, with concomitant extraction of 88-99% of radiolabeled PC and 86-97% of radiolabeled PS. The accessibility of exogenous lipids to extraction is uniformly consistent with the transbilayer lipid distribution inferred from cell shape changes, indicating that red cell morphology is an accurate and sensitive reporter of the transbilayer partitioning of incorporated exogenous phospholipids.     

Interaction of plasma lipoproteins with erythrocytes. II. Modulation of membrane-associated enzymes

Intact erythrocytes incubated in the presence of low density lipoproteins (LDL) undergo a time-dependent morphologic transformation from biconcave discs to spherocytes within 4 h. No shape change is observed when erythrocytes are incubated with high density lipoproteins (HDL). The LDL-induced change in erythrocyte morphology occurs without concomitant leakage of hemoglobin from the cell or depletion of intracellular ATP; no change in the distribution of the major lipids of the erythrocyte membranes was detected. The alteration of morphology does require attachment of LDL to the erythrocyte surface. The LDL-induced morphologic alteration is inhibited by HDL, but not by serum albumin. HDL prevent the attachment of LDL to the cell membrane; however, the HDL subfractions, HDL₂ and HDL₃, are only partially effective. These data suggest that normal erythrocyte morphology and cell function may depend on the concentration and composition of the circulating lipoproteins.     

Interaction of plasma lipoproteins with erythrocytes. I. Alteration of erythrocyte morphology

Intact erythrocytes incubated in the presence of low density lipoproteins (LDL) undergo a time-dependent morphologic transformation from biconcave discs to spherocytes within 4 h. No shape change is observed when erythrocytes are incubated with high density lipoproteins (HDL). The LDL-induced change in erythrocyte morphology occurs without concomitant leakage of hemoglobin from the cell or depletion of intracellular ATP; no change in the distribution of the major lipids of the erythrocyte membranes was detected. The alteration of morphology does require attachment of LDL to the erythrocyte surface. The LDL-induced morphologic alteration is inhibited by HDL, but not by serum albumin. HDL prevent the attachment of LDL to the cell membrane; however, the HDL subfractions, HDL₂ and HDL₃, are only partially effective. These data suggest that normal erythrocyte morphology and cell function may depend on the concentration and composition of the circulating lipoproteins.     

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.     

Effect of membrane cholesterol on dimyristoylphosphatidylcholine-induced vesiculation of human red blood cells

During incubation of intact human erythrocytes with sonicated dimyristoylphosphatidylcholine (DMPC) vesicles, the cells change their discoid morphology to form echinocytes and finally give rise to the release of membrane vesicles. In this process, the red cell membrane accumulates DMPC and loses up to 15% of its cholesterol. On the other hand, replacement of 25% of the endogenous phosphatidylcholine species by DMPC without affecting the cholesterol level of the erythrocytes can be achieved by incubation with DMPC/cholesterol (1:1, mol/mol) sonicated vesicles in the presence of the phosphatidylcholine-specific phospholipid-transfer protein from bovine liver. This replacement also gives rise to an echinocytic cell morphology, but no membrane vesiculation can be observed. However, the vesiculation process can as yet be initiated upon a subsequent decrease of the cholesterol level, by incubation of those modified cells in the presence of sonicated vesicles of pure egg phosphatidylcholine. Incubation of native erythrocytes with pure egg phosphatidylcholine vesicles, on the other hand, results in cholesterol depletion, but does neither induce the formation of echinocytes nor the release of membrane vesicles. Cellular ATP levels are not affected during these incubations. From these results, it can be concluded that a decrease in cholesterol content of the erythrocyte membrane is essential for the DMPC-induced vesiculation of those cells.     

Membrane bilayer balance and erythrocyte shape: a quantitative assessment

When human erythrocytes are incubated with certain phospholipids, the cells become spiculate echinocytes, resembling red cells subjected to metabolic starvation or Ca2+ loading. The present study examines (1) the mode of binding of saturated phosphatidylcholines and egg lysophosphatidylcholine to erythrocytes and (2) the quantitative relationship between phospholipid incorporation and red cell shape. We find that the phospholipids studied become intercalated into erythrocyte membranes, not simply adsorbed to the cell surface. Spin-labeling and radiolabeling data show that the incorporation of (4 +/- 1) X 10(6) molecules of exogenous phosphatidylcholine per cell converts discocytes to stage 3 echinocytes with about 35 conical spicules. This amount of lipid incorporation is estimated to expand the red cell membrane outer monolayer by 1.7% +/- 0.6%. Calculations of the inner and outer monolayer surface areas of model discocytes and stage 3 echinocytes yield an estimated difference of 0.7% +/- 0.2%.