Erythrocyte Shape Changes Research Articles

The influence of erythrocyte shape on suspension viscosities.

Erythrocyte shape changes are known to occur in vivo and can readily be induced in vitro. We have analysed the influence of increasing stomatocytosis produced by 0-0.64 mmol l-1 chlorpromazine and increasing echinocytosis induced with 0-120 mmol l-1 salicylate or 0-8 mmol l-1 2,4-dinitrophenol on suspension viscosities. The morphological index of each sample was determined and related to the suspension viscosity. It was found that the viscosity was increased by echinocytosis in dextran-free solutions, where no aggregation occurred. The viscosity could be normalized by retransforming echinocytes into discocytes. Under conditions with erythrocyte aggregation (suspension with 4 g dl-1 dextran 70, low shear rate: 0.1 s-1) a small degree of echinocytosis produced the highest viscosity, whereas at higher degrees of echinocytosis the ability to aggregate was reduced and the viscosity was similar to that of discocytes or stomatocytes. Erythrocytes incubated in hypotonic medium (constant cell number/volume) had a higher viscosity than cells in iso- or hypertonic medium. Severely hypotonic medium led to sphering of erythrocytes which reduced the ability of these cells to aggregate and hence decreased the viscosity of suspensions with dextran at low shear rate. The results indicate that discocytes have the lowest viscosity and thus the best oxygen transport efficiency and that iso- to hypertonicity provides a lower viscosity and better oxygen transport efficiency than hypotonicity. These results may contribute to the understanding of blood flow in health and disease.

Activation of intracellular calcium-activated neutral proteinase in erythrocytes and its inhibition by exogenously added inhibitors

Intracellular calcium-activated neutral proteinase (CANP) in rabbit erythrocytes was activated by an influx of Ca 2+ into the cells. The catalytic large subunit changed from the original 79 kDa form to the 77 kDa and 76 kDa forms on activation just in the same manner as occurs in the autolytic activation of purified CANP in vitro. The activation required both extracellular Ca 2+ and A23187, and was accompanied by the degradation of some membrane proteins and morphological changes in erythrocyte shape from discocytes to echinodisks, echinocytes, and spherocytes. Exogenously added Cbz-Leu-Leu-Leu-aldehyde the activation of intracellular CANP as well as the degradation of membrane proteins and the morphological changes indicating that the latter two processes are due to the action of CANP. Leupeptin and E64d were without effect on intracellular CANP.

Amphiphile-induced antihaemolysis is not causally related to shape changes and vesiculation

A wide variety of structurally different antihaemolytic amphiphiles were tested for their ability to induce exovesiculation (acetylcholinesterase (AChE) release, transmission electron microscopic (TEM) studies), endovesiculation (fluorescein isothiocyanate conjugated dextran (FITC-dextran) internalization, TEM studies) and shape changes in human erythrocytes at concentrations where they exert maximum protection against hypotonic haemolysis. The results show that vesiculation is a common phenomenon induced by amphiphiles in erythrocytes. Sphero-echinocytogenic amphiphiles induced exovesiculation, whereas stomatocytogenic amphiphiles induced endovesiculation. The antihaemolytic potency of the amphiphiles was not related to their ability to induce exo- or endovesiculation, or to the type or extent of shape changes induced, and it could not be ascribed to any molecular feature of the amphiphiles or to their charge. It is proposed that amphiphiles, when intercalated into the lipid bilayer of the membrane, rapidly induce rearrangements within the bilayer and that these rearrangements are associated with an increase in the permeability of the membrane; it is suggested that a rapid efflux of ions decreases the difference in osmotic pressure between cell interior and hypotonic buffer, thereby protecting cells from being lysed.

Amphiphile-Induced Erythrocyte Shape Change and Simultaneous Tetraethylammonium Ion Uptake.

Amphiphile-induced tetraethylammonium ion (TEA+) uptake into human erythrocytes was examined along with cell shape change. A TEA(+)-sensitive electrode was used to determine the amount of uptake. TEA+ was preferentially incorporated into erythrocytes when amphiphiles changed cell shape to an invaginated form. This was contrasted with the release of acetylcholinesterase outside cells which occurred markedly with the amphiphiles, causing the crenated form. It was suggested that the invagination of erythrocyte membrane stimulated the formation of vacuoles, in which TEA+ existing in an external medium was entrapped.

Erythrocyte shape and volume changes caused by an inhibitor of the glucose and anion transporters

Light scattering measurements and scanning electron microscopy show that p-azidobenzylphlorizin (p-AzBPhz) causes changes in the shape and volume of human erythrocytes by at least two, dose-dependent mechanisms: At nominal concentrations above 5 microM, the azide induces cell swelling by either enlarging a pre-existent channel or by creating pores between phase boundaries of the membrane through which salt and water enter, but sucrose remains excluded. However, over the range 0.03 to 0.3 microM, in either isosmotic NaCl or KCl media, when fewer than 1 million molecules of azide are bound per cell, the ligand causes membrane deformations that convert discocytes into cells resembling stage 2 echinocytes. Whereas a cell volume increase of about 10% accompanies these shape changes, (microhematocrit and electronic cell sizing measurements), no net influx of either Na+ or K+ during this stage of swelling was detectable. These cell alterations take place at p-AzBPhz concentrations which concurrently inhibit both chloride and 3-methoxyglucose equilibrium exchange transport. The results may indicate that when the membrane impermeable p-AzBPhz interacts with the anion and/or sugar transporter, some trans-membrane perturbation occurs which alters the cytoskeleton.

Effective bilayer expansion and erythrocyte shape change induced by monopalmitoyl phosphatidylcholine. Quantitative light microscopy and nuclear magnetic resonance spectroscopy measurements

When human erythrocytes are treated with exogenous monopalmitoyl phosphatidylcholine (MPPC), the normal biconcave disk shape red blood cells (RBC) become spiculate echinocytes. The present study examines the quantitative aspect of the relationship between effective bilayer expansion and erythrocyte shape change by a newly developed method. This method is based on the combination of direct surface area measurement of micropipette and relative bilayer expansion measurement of 13C crosspolarization/magic angle spinning nuclear magnetic resonance (NMR). Assuming that 13C NMR chemical shift of fatty acyl chain can be used as an indicator of lateral packing of membrane bilayers, it is possible for us to estimate the surface area expansion of red cell membrane induced by MPPC from that induced by ethanol. Partitions of lipid molecules into cell membrane were determined by studies of shape change potency as a function of MPPC and red cell concentration. It is found that 8(+/- 0.5) x 10(6) molecules of MPPC per cell will effectively induce stage three echinocytes and yield 3.2(+/- 0.2)% expansion of outer monolayer surface area. Surface area of normal cells determined by direct measurements from fixed geometry of red cells aspirated by micropipette was 118.7 +/- 8.5 microns2. The effective cross-sectional area of MPPC molecules in the cell membrane therefore was determined to be 48(+/- 4) A2, which is in agreement with those determined by x-ray from model membranes and crystals of lysophospholipids. We concluded that surface area expansion of RBC can be explained by a simple consideration of cross-sectional area of added molecules and that erythrocyte shape changes correspond quantitatively to the incorporated lipid molecules.

Erythrocyte protein 4.1 binds and regulates myosin.

Myosin was recently identified in erythrocytes and was shown to partition both with membrane and cytosolic fractions, suggesting that it may be loosely bound to membranes [Fowler, V. M., Davis, J. Q. & Bennett, V. (1985) J. Cell Biol. 100, 47-55, and Wong, A. J., Kiehart, D. P. & Pollard, T. D. (1985) J. Biol. Chem. 260, 46-49]; however, the molecular basis for this binding was unclear. The present studies employed immobilized monomeric myosin to examine the interaction of myosin with erythrocyte protein 4.1. In human erythrocytes, protein 4.1 binds to integral membrane proteins and mediates spectrin-actin assembly. Protein 4.1 binds to rabbit skeletal muscle myosin with a Kd = 140 nM and a stoichiometry consistent with 1:1 binding. Heavy meromyosin competes for protein 4.1 binding with Ki = 36-54 nM; however, the S1 fragment (the myosin head) competes less efficiently. Affinity chromatography of partial chymotryptic digests of protein 4.1 on immobilized myosin identified a 10-kDa domain of protein 4.1 as the myosin-binding site. In functional studies, protein 4.1 partially inhibited the actin-activated Mg2+-ATPase activity of rabbit skeletal muscle myosin with Ki = 51 nM. Liver cytosolic and erythrocyte myosins preactivated with myosin light-chain kinase were similarly inhibited by protein 4.1. These studies show that protein 4.1 binds, modulates, and thus may regulate myosin. This interaction might serve to generate the contractile forces involved in Mg2+-ATP-dependent shape changes in erythrocytes and may additionally serve as a model for myosin organization and regulation in non-muscle cells.

Red blood cell aggregation and sedimentation: the role of the cell shape.

The influence of erythrocyte shape changes on the sedimentation rate was studied in vitro and in vivo. In vitro the highest sedimentation rate was observed with a slight degree of stomatocytosis (morphological index-0.3; i.e. one red cell out of three being a stomatocyte I). With increasing degrees of stomatocytosis the sedimentation rate gradually decreased. Echinocytosis reduced the aggregation and sedimentation very drastically; the sedimentation rate was virtually zero when echinocytosis I or higher degrees were present. The influence of abnormal cell shapes occurring in vivo was studied in patients with an abnormal blood smear. It was found that a severely abnormal red cell morphology reduced the sedimentation rate in a standardized, fibrinogen-rich plasma to about half. These results indicate that the shape plays a crucial role in the aggregation and sedimentation of red cells and they may contribute to the understanding of the interaction of red cells with other cells such as endothelium.

Shape changes in caprine erythrocytes exposed to chlorpromazine and lysolecithin.

Stomatocytic and echinocytic transformations of caprine erythrocytes were studied in vitro using chlorpromazine as a stomatocyyic agent and lysolecithin as an echinocytic agent. Morphologic changes in erythrocytes generally varied with the cell shape and the concentration of the substances used. Discoytic, triangular, and pear-shaped red cells common to normal goats, exhibited classical stomatocytic and echinocytic changes with formation of spherostomatocytes, sphero-echinocytes, and spherocytes. In comparison, fusiform and spindle-shaped red cells found in certain Angora goats seemed less prone to shape changes and required greater concentrations of the inducing agents to effect such changes. Chlorpromazine at higher concentrations also inflicted localized membrane damage in form of tiny pits and lysolecithin likewise induced formation of fragile smooth or beaded filaments.

Mechanism of membrane damage induced by the amphipathic peptides gramicidin S and melittin

The action of gramicidin S and melittin on human erythrocytes, Staphylococcus aureus and Escherichia coli was studied as an extension of the previous study (Katsu, T., Ninomiya, C., Kuroko, M., Kobayashi, H., Hirota, T. and Fujita, Y. (1988) Biochim. Biophys. Acta 939, 57–63). These amphipathic peptides stimulated the release of membrane phospholipids outside cells in a concentration range causing permeability change. The shape change of erythrocytes from normal discoid to spiculate form was observed just prior to the release of membrane components. We have proposed the following action mechanism of gramicidin S and melittin. The peptide molecules were predominantly accumulated in the outer half of the bilayer, deforming the erythrocyte cell into crenature. A large accumulation made the membrane structure unstable, resulting in the release of membrane fragments and the simultaneous enhancement of permeability. The action mechanism of these peptides was compared with that of simple surfactants.

Influence of hypercholesterolemia on morphological and rheological characteristics of erythrocytes

The changes in shape and rheological parameters of erythrocytes in cholesterol-fed rabbits (fed with normal diet plus 0.5% cholesterol for a duration of 1 year) and in vitro cholesterol enrichment of erythrocytes in cholesterol enriched plasma were studied. This process affects the shape of erythrocytes. In rabbits the actual shape change begins after 6 weeks of cholesterol feeding. The shape changes in rabbits and in vitro cholesterol-enrichment are similar. The extent of shape changes depends on the amount of cholesterol incorporated into the membrane. The rheological parameters such as blood viscosity, osmotic fragility and deformability are measured. The whole blood viscosity is increased and deformability decreased. The cells become osmotically more fragile in cholesterol-fed rabbits but are resistant to osmotic lysis in in vitro cholesterol enrichment. The altered lipid composition may be responsible for the observed changes in these parameters.

Factors of the shape change of human erythrocytes induced with lidocaine.

We studied the molecular mechanism of the shape change of erythrocytes with a local anesthetic, lidocaine. The shape of human erythrocytes changed from discocytes to stomatocytes in the presence of lidocaine when ATP was present. But, the shape of resealed cells which were prepared with 10 mM Tris-HCl buffer (pH 7.4) containing 2 mM ATP-MgCl2 and various substances was not changed from discocytes to stomatocytes with lidocaine. When intact cells and resealed cells which were prepared with various concentrations of Tris-HCl buffer (pH 7.4) were incubated with various concentrations of lidocaine and their membrane proteins were analyzed by SDS-PAGE, the densities of bands 62K, 28K and 22K depended on lidocaine concentration: in particular, that of band 28K changed remarkably. These membranous 62K-, 28K- and 22K-proteins agreed with cytoplasmic 62K-, 28K- and 22K-proteins in molecular weight. We propose that not only ATP but also the 62K-, 28K- and 22K-proteins in the cytoplasm are concerned with the shape change of human erythrocytes induced with lidocaine.

Dynamic association of band 3 with Triton shells in human erythrocyte ghosts

To test a possibility that free band 3 and ankyrin-linked band 3 are exchanged in situ, band 3 was labeled with 125I, using intact red blood cells and lactoperoxidase. The cytoplasmic surface of this labeled band 3 was considered to be intact. When Triton shells were incubated with Triton supernatants prepared from 125I-labeled intact erythrocytes at 37° C in the presence of Mg-ATP under isotonic conditions, the incorporation of free 125I-labeled band 3 to shells was observed. This incorporation was affected by the presence of Triton X-100 in the incubation mixture, and significantly decreased when the content of Triton X-100 was less than 0.04% (v/v). On the other hand, ankyrin-linked 125I-labeled band 3 was released when shells prepared from 125I-labeled intact erythrocytes were incubated with the Triton supernatants at 37°C under the same condition as when free 125I-labeled band 3 incorporation was observed. These results strongly suggest that free and ankyrin-linked band 3 exchanged with each other in the presence of Triton X-100. A water-soluble 43 kDa fragment of band 3 inhibited the incorporation of free 125I-labeled band 3 to the shells and also inhibited the Mg-ATP-dependent shape change of ghosts in the absence of Triton X-100. Both of these inhibitory effects remained, even after 10 min of heat treatment at 100°C, but drastically decreased by treatment with trypsin. Our results strongly suggest that a dynamic exchange of the free band 3 for ankyrin-linked band 3 may occur in intact erythrocytes, and it may even contribute to the shape change of erythrocytes.

Increased Resistance to Membrane Deformation of Shape-Transformed Human Red Blood Cells

The study of shape-transformed human erythrocytes is of hematological interest since several clinical conditions are associated with erythrocyte shape changes. Using the micropipette technique, we examined the rheological behaviors of echinocytes induced by sodium salicylate or ATP depletion and of stomatocytes induced by chlorpromazine. Both the discocyte-echinocyte and discocyte-stomatocyte transformations resulted in an increased resistance to extension. Sodium salicylate-treated erythrocytes could be returned to normal morphology and normal membrane rheology by addition of chlorpromazine. We conclude that these morphological changes are associated with detrimental effects on the intrinsic membrane deformability of the human erythrocyte.

Increased resistance to membrane deformation of shape-transformed human red blood cells [published erratum appears in Blood 1987 Sep;70(3):893

The study of shape-transformed human erythrocytes is of hematological interest since several clinical conditions are associated with erythrocyte shape changes. Using the micropipette technique, we examined the rheological behaviors of echinocytes induced by sodium salicylate or ATP depletion and of stomatocytes induced by chlorpromazine. Both the discocyte-echinocyte and discocyte-stomatocyte transformations resulted in an increased resistance to extension. Sodium salicylate-treated erythrocytes could be returned to normal morphology and normal membrane rheology by addition of chlorpromazine. We conclude that these morphological changes are associated with detrimental effects on the intrinsic membrane deformability of the human erythrocyte.

The influence of the antiviral drugs amantadine and rimantadine on erythrocyte and platelet membranes and its comparison with that of tetracaine

The influence of the antivirus drugs amantadine and rimantadine and of the anionic analogue 1-adamantane-carboxylic acid on a range of properties of human erythrocyte membrane and of thrombocytes has been compared with the effect of the local anaesthetic tetracaine. At low antiviral drug concentrations the abilities of the drugs to induce erythrocyte shape change and suppress osmotic haemolysis were quantitatively proportional to their clinical potenty (rimantadine more effective than amantadine at the same concentration). Rimantadine was also more effective than amantadine in suppressing influenza virus-erythrocyte fusion and viral induced haemolysis. The antiviral drug effects were qualitatively similar to those induced by tetracaine. At the quantitative level, tetracaine was more efficient than the antiviral drugs in inhibiting osmotic haemolysis, virus membrane fusion and platelet aggregation. In the absence of any specificity of the antiviral drug effects we argue for a lysosomotropic mode of drug action, i.e. that the drugs modify virus-membrane interactions by changing the endosomal or lysosomal pH.

A novel method for measuring the erythrocyte deformability, under oscillatory shear stress.

A novel apparatus for detecting a subtle decrease in erythrocyte deformability was developed. The oscillatory shape change of erythrocytes was monitored under an oscillatory shear stress (modulating the oscillation frequency in the range of 9-90 dyn/cm2). The ellipsoidal deformation of intact erythrocytes perfectly followed the oscillatory force of up to about 2 Hz, but the diamide-treated cells showed a phase difference of the oscillatory deformation at 1.7 Hz.

Relationship between membrane atpase and shape changes in the dog erythrocyte

The effects of heating blood to 57° C on intraerythrocytic calcium, membrane ATPase activity and cell shape have been studied in canine blood. Intraerythrocytic calcium was determined by use of arsenazo III, membrane ATPase activity was determined by inorganic phosphorous formation and erythrocyte shape was determined by scanning electron microscopy. The results of this study showed that this degree of thermal trauma would cause a 27% increase in intraerythrocytic calcium and a 38% decrease in ATPase activity. During these changes in calcium and ATPase activity the erythrocyte changed form from biconcave to spherical. Addition of catalase (3,200 U/ml) to the blood prior to heating prevented the changes observed in intraerythrocytic calcium, membrane ATPase activity and shape. The addition of the free-radical generating combination of hypoxanthine-xanthine oxidase to blood produced a 20% decrease in membrane ATPase activity and a change in erythrocyte shape, but did not alter intraerythrocytic calcium. These results suggest that free-radicals are responsible for the changes in membrane ATPase activity. The observation that shape change occurs when ATPase activity has been decreased, but calcium has not been increased, suggests that membrane ATPase activity levels are more important in producing changes in erythrocyte shape than are intraerythrocytic calcium levels.

Tangent counting for objective assessment of erythrocyte shape changes.

The resting shape of the human erythrocyte is maintained mainly by the elastic properties of its membrane. There is evidence that negative spontaneous membrane curvature is the cause of the biconcave shape; also negative membrane curvature is thought to be enhanced under "stomatocytogenic" influences and reduced under "echinocytogenic" influences. Experimental tests of these hypotheses are difficult in the absence of objective methods to quantify cell shape and/or membrane curvature. A new method is introduced to estimate the changes in convex and concave curvatures of the cell outlines in larger populations of RBC. It is based on the so-called "tangent count" procedure, carried out on microphotographs of freely suspended, randomly oriented RBCs in isotonic solutions. The method was validated by subjective shape assessment according to BESSIS' classification. It proved to be most sensitive in the range of moderate shape changes (classified subjectively as echinocyte II and stomatocyte II).

Shape change of sickled erythrocytes induced by pulsed rf electrical fields.

The shape change of sickled erythrocytes induced by electrical pulses was investigated under various experimental conditions using different suspending media. The purpose of this research is to find a set of conditions under which sickled erythrocytes from an individual with sickle cell anemia (SS erythrocytes) can be desickled with minimal damage to the membrane. Previously we observed, using 0.9% NaCl solution, that rounding of the SS erythrocytes eventually led to hemolysis even when the field was turned off. Using short pulses, we have now observed that the rounding of sickled erythrocytes does not necessarily lead to hemolysis if buffer solutions such as Hanks' solution are used. The cause of the shape change is believed to be the perforation of the erythrocyte membrane by the potential induced by applied fields. The field-induced membrane potential has been calculated for spherical cells and infinitely long cylinders. Using an ellipsoid of revolution as a model, we generalize the calculation of membrane potential. These calculations indicate that a transverse field is less effective in inducing a potential than a longitudinal field.