Rosette formation of concanavalin A-treated erythrocytes around polymorphonuclear leucocytes and lymphocytes.

Concanavalin A (Con A) induces rosette formation of erythrocytes around polymorphonuclear leucocytes and lymphocytes in cell suspensions of autologous human blood cells. The effect which is most characteristic in a concentration between 25 and 50 microgram/ml is due to Con A bound on the erythrocyte membrane. A similar effect, although less pronounced, was observed with phytohaemagglutinin at concentrations of 10 and 25 microgram/ml. The treated erythrocytes showed a higher affinity to polymorphonuclears when compared with lymphocytes. At the contact area, the membrane of the erythrocyte became highly folded while its free surface was smooth and spherical. The effect of the local concentration and immunobilization of the lectin on the erythrocyte membrane and the similarity of the contact pattern to that of erythrophagocytosis are discussed.     

Plasmodium iophurae: cationized ferritin staining, an electron microscope cytochemical method for differentiating malarial parasite and host cell membranes

The surface membranes of erythrocyte-free Plasmodium lophurae and its host cell, the duckling erythrocyte, stain differentially when exposed to cationized ferritin (CF). At low CF concentrations (0.18 mg/ml) only the outer surface of the red cell stains, whereas at the standard concentration (0.7 mg/ml) both the red cell and the parasitophorous vacuolar membranes (PVM) were stained on their outer faces. By using a high CF concentration (3.7 mg/ml), the parasite's plasma membrane (PM) could be distinguished from that of the PVM: The former did not bind CF, whereas the latter was stained on its outer surface. At this level of CF the red cell membrane stained on both faces if these surfaces were exposed to stain. Although the PVM is formed by red cell endocytosis of the parasite, it can be distinguished from the membrane of the erythrocyte as well as that of the PM.     

Adhesivity and rigidity of erythrocyte membrane in relation to wheat germ agglutinin binding

Binding of the plant lectin wheat germ agglutinin (WGA) to erythrocyte membranes causes membrane rigidification. One of our objectives has been to directly measure the effects of WGA binding on membrane rigidity and to relate rigidification to the kinetics and levels of WGA binding. Our other objective has been to measure the strength of adhesion and mechanics of cell separation for erythrocytes bound together by WGA. The erythrocyte membrane rigidity was measured on single cells by micropipette aspiration. The slope of the suction pressure-length data for entry into the pipette provided the measure of the membrane extensional modulus. Data were collected for cells equilibrated with WGA solutions in the range of concentrations of 0.01- 10 micrograms/ml. Erythrocyte-erythrocyte adherence properties were studied by micropipette separation of two-cell aggregates. First, a "test" cell was selected from a WGA solution by aspiration into a small micropipette, then transferred to a separate chamber that contained erythrocytes in WGA-free buffer. Here, a second cell was aspirated with another pipette and maneuvered into close proximity of the test cell surface, and adhesive contact was produced. The flaccid cell was separated from the test cell surface in steps at which the force of attachment was derived from the pipette suction pressure and cell geometry. In addition, we measured the time-dependent binding and release of fluorescently labeled WGA to single erythrocytes with a laser microfluorometry system. The results showed that the stiffening of the erythrocyte membrane and binding of fluorescently labeled WGA to the membrane surface followed the same concentration and time dependencies. The threshold concentration for membrane stiffening was at approximately 0.1 microgram/ml where the time course to reach equilibrium was close to 1 h. The maximal stiffening (almost 30-fold over the normal membrane elastic modulus) occurred in concentrations greater than 2 micrograms/ml where the time to reach equilibrium took less than 1 min. The WGA binding also altered the normal elastic membrane behavior into an inelastic, plastic-like response which indicated that mechanical extension of the membrane caused an increase in cross-linking within the surface plane. Similar to the stiffening effect, we observed that the membrane adhesivity of cells equilibrated with WGA solutions greatly increased with concentration greater than 0.1 microgram/ml.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of wheat germ agglutinin on the viscoelastic properties of erythrocyte membrane

The elasticity and viscosity of the human erythrocyte membrane were measured as a function of the concentration of wheat germ agglutinin (WGA) in a suspending solution containing 1 mg/ml albumin, approximately 5 X 10(5) cells/ml and between 0.0 and 0.2 microgram/ml WGA. Membrane elasticity was characterized by the elastic shear modulus, which provided a measure of the resistance of the membrane to constant-area elastic deformations that occurred in the membrane plane. The elastic shear modulus was determined by aspirating a portion of the membrane into a micropipette and measuring the extension of the membrane into the pipette as a function of the suction pressure. The results indicated no significant change in shear modulus for concentrations of WGA between 0.0 and 0.2 microgram/ml. Membrane viscosity was characterized by the coefficient of surface viscosity, which, in effect, was a measure of the membrane's resistance to rates of deformation. This coefficient was determined from the time required for an erythrocyte to recover its undeformed shape after it had been elongated by the application of an equal and opposite force applied at diametrically opposite points on the erythrocyte rim. The value for the coefficient of surface viscosity was found to increase by a factor of almost three when the WGA concentration was increased from 0.0 to 0.2 microgram/ml. These results indicated that, in the presence of albumin, WGA can increase membrane dissipation (viscosity) without altering the structural rigidity (elasticity) of the membrane.     

Influence of ethanolic extract of Tephrosia purpurea Linn. on mast cells and erythrocytes membrane integrity

The ethanolic extract of T. purpurea Linn. was studied for its in vitro effect on rat mast cell degranulation and erythrocyte membrane integrity in vitro. The extract in concentration of 25-200 microg/ml showed a dose-dependant inhibition of rat mast cell degranulation induded by compound 48/80 and egg albumin. T. purpurea extract was found to inhibit haemolysis of erythrocytes induced by hypotonic solution but accelerated haemolysis induced by heat at a concentration of 100 microg/ml. The studies reveal that the ethanolic extract of T. purpurea may inhibit degranulation of mast cells by a mechanism other than membrane stabilization.     

Interactions of glycolytic enzymes with erythrocyte membranes

Partition equilibrium experiments have been used to characterize the interactions of erythrocyte ghosts with four glycolytic enzymes, namely aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase and lactate dehydrogenase, in 5 mM sodium phosphate buffer (pH 7.4). For each of these tetrameric enzymes a single intrinsic association constant sufficed to describe its interaction with erythrocyte matrix sites, the membrane capacity for the first three enzymes coinciding with the band 3 protein content. For lactate dehydrogenase the erythrocyte membrane capacity was twice as great. The membrane interactions of aldolase and glyceraldehyde-3-phosphate dehydrogenase were mutually inhibitory, as were those involving either of these enzymes and lactate dehydrogenase. Although the binding of phosphofructokinase to erythrocyte membranes was inhibited by aldolase, there was a transient concentration range of aldolase for which its interaction with matrix sites was enhanced by the presence of phosphofructokinase. In the presence of a moderate concentration of bovine serum albumin (15 mg/ml) the binding of aldolase to erythrocyte ghosts was enhanced in accordance with the prediction of thermodynamic nonideality based on excluded volume. At higher concentrations of albumin, however, the measured association constant decreased due to very weak binding of the space-filling protein to either the enzyme or the erythrocyte membrane. The implications of these findings are discussed in relation to the likely subcellular distribution of glycolytic enzymes in the red blood cell.     

Plasma membrane calcium pump activity is affected by the membrane protein concentration: evidence for the involvement of the actin cytoskeleton

Plasma membrane calcium pumps (PMCAs) are integral membrane proteins that actively expel Ca(2+) from the cell. Specific Ca(2+)-ATPase activity of erythrocyte membranes increased steeply up to 1.5-5 times when the membrane protein concentration decreased from 50 microg/ml to 1 microg/ml. The activation by dilution was also observed for ATP-dependent Ca(2+) uptake into vesicles from Sf9 cells over-expressing the PMCA 4b isoform, confirming that it is a property of the PMCA. Dilution of the protein did not modify the activation by ATP, Ca(2+) or Ca(2+)-calmodulin. Treatment with non-ionic detergents did not abolish the dilution effect, suggesting that it was not due to resealing of the membrane vesicles. Pre-incubation of erythrocyte membranes with Cytochalasin D under conditions that promote actin polymerization abolished the dilution effect. Highly-purified, micellar PMCA showed no dilution effect and was not affected by Cytochalasin D. Taken together, these results suggest that the concentration-dependent behavior of the PMCA activity was due to interactions with cytoskeletal proteins. The dilution effect was also observed with different PMCA isoforms, indicating that this is a general phenomenon for all PMCAs.     

Effect of the membrane potential on the fusion of the Sendai viral envelope with the membrane of chick erythrocytes and on virus-induced erythrocyte hemolysis

The ESR data on the influence of membrane potential of the fusion of Sendai virus envelope with erythrocyte membrane are presented. The hyperpolarization of cell membrane takes place at low concentration of KCl (1-5 mM) in extracellular medium in the presence of valinomycin, while at high concentration of KCl (125-150 mM) its depolarization occurs. The hyperpolarization of erythrocyte plasma membrane is accompanied by the increase of its fusion with viral envelope and virus-induced hemolysis. At the same time depolarization of erythrocyte membrane leads to the decrease of virus fusion activity. This evidence together with previously obtained by patch-clamp method data on potential-dependence of virus-induced increase of cell membrane conductivity provide us an opportunity to make a proposal that the electric field membrane damage may be the initial stage of the virus-induced membrane fusion.     

Structural and dynamic states of actin in the erythrocyte

Analysis of the nucleotide tightly associated with isolated erythrocyte cytoskeletons show it to be ADP, rather then ATP. This confirms that at least a major part of the erythrocyte actin is in the F-form. A re-evaluation of the stoichiometry of spectrin and actin in the erythrocyte (taking account of a gross difference between the color responses of the two proteins on staining of electrophoretic gels) leads to values of 1x10(5) and 5x10(5) for the number of molecules of spectrin tetramer and actin respectively per cell. It has been found possible to perform spectrophotometric DNAase I assays fro actin on lysed whole cells. The concentration of monomeric actin at 0 degrees C is approximately 16 μg/ml packed cells. After washing the lysed cells the monomer pool is not re-established, indicating that only a small proportion of the actin subunits are free to dissociate. The actin monomer concentration in the cytosol remains unchanged after equilibration of the cells with cytochalasin E. The ability of actin-containing complexes in the membrane to nucleate the polymerization of added G-actin was measured fluorimetrically; it was found that membranes incubated with cytochalasin E were completely inert with respect to nucleating activity under conditions that favor appreciable growth at the slowly-growing (“pointed”) ends of free actin filaments. This suggests that these ends of the actin “protofilaments” in the red cell are blocked or sterically obstructed. After treatment of the membranes with guanidine hydrochloride under conditions that dissociate F-actin, the measured concentration of actin monomer rises to approximately 180 μg/ml of packed cells, which is nearly 70 percent of the total actin content. On treatment with trypsin in the presence of DNAase, the spectrin and 4.1 are extensively degraded, but the actin remains undamaged. This treatment, followed by exposure to guanidine hydrochloride, causes a further rise in the concentration of actin responsive to the DNAase assay to 250 μg/ml of cells, compared with 270 μg/ml estimated by densitometry of stained gels. The oligomeric complex, consisting of actin, spectrin, and 4.1, that is extracted from the membrane at low ionic strength, generates no detectable actin monomer after the same treatment. From literature data on the number of cytochalasin binding sites per cell and our value for the total actin content, we obtain a number-average degree of polymerization for actin in the membrane of 12-17. The results lead to a model for the structure of the cytoskeletal network and suggest some consequences of metabolic depletion.     

Plasma membrane calcium pump activity is affected by the membrane protein concentration: Evidence for the involvement of the actin cytoskeleton

Plasma membrane calcium pumps (PMCAs) are integral membrane proteins that actively expel Ca²⁺ from the cell. Specific Ca²⁺-ATPase activity of erythrocyte membranes increased steeply up to 1.5-5 times when the membrane protein concentration decreased from 50 μg/ml to 1 μg/ml. The activation by dilution was also observed for ATP-dependent Ca²⁺ uptake into vesicles from Sf9 cells over-expressing the PMCA 4b isoform, confirming that it is a property of the PMCA. Dilution of the protein did not modify the activation by ATP, Ca²⁺ or Ca²⁺-calmodulin. Treatment with non-ionic detergents did not abolish the dilution effect, suggesting that it was not due to resealing of the membrane vesicles. Pre-incubation of erythrocyte membranes with Cytochalasin D under conditions that promote actin polymerization abolished the dilution effect. Highly-purified, micellar PMCA showed no dilution effect and was not affected by Cytochalasin D. Taken together, these results suggest that the concentration-dependent behavior of the PMCA activity was due to interactions with cytoskeletal proteins. The dilution effect was also observed with different PMCA isoforms, indicating that this is a general phenomenon for all PMCAs.     

Interactions of antibiotics of the iturin group with human erythrocytes

The peptidolipid antibiotics, iturin A and bacillomycin L have a disrupting effect on erythrocyte membrane leading to a simultaneous release of K+ ions and hemoglobin. The formation of ghosts is accompanied by a partial solubilisation of lipid components. Binding experiments with radioactive antibiotics show that about 7 X 10(7) molecules of iturin A and 4 X 10(7) molecules of bacillomycin L are bound to one erythrocyte at the concentration giving 100% hemolysis. This concentration is reduced by about 20% after treatment of erythrocytes by phospholipase A2. Scatchard plots show that the affinity for erythrocyte membrane is higher with bacillomycin L than with iturin A. This difference is probably correlated to the differences in their peptide moieties. The substitution of tyrosyl residue leads to a ten-fold increase of the concentrations giving 100% hemolysis, probably due to a low distribution coefficient of derivatives between the membrane and the solvent. This result and the humped Scatchard curves obtained with both antibiotics may be related to the self-association of the compounds in aqueous solutions.     

Relationship between membrane ATPase and shape changes in the dog erythrocyte

The effects of heating blood to 57 degrees 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.     

Characterization of ATP-dependent Ca2+ uptake by canine brain microsomes with saponin

ATP-dependent Ca2+ uptake by brain microsomes was classified into two fractions according to the sensitivity to saponin. Properties of each fraction of Ca2+ uptake were examined and compared with those of inside-out membrane vesicles of erythrocyte and cardiac sarcoplasmic reticulum. The concentration of saponin for 50% inhibition (IC50) of major saponin-sensitive Ca2+ uptake was 11 micrograms/ml, and this uptake was enhanced by calmodulin. The minor saponin-insensitive Ca2+ uptake fraction (IC50; 90 micrograms/ml) was not affected by calmodulin but was enhanced by oxalate or 0.1 M KCl. The IC 50 of saponin for inside-out membrane vesicles of erythrocyte and cardiac sarcoplasmic reticulum was 11.3 and 114.8 micrograms/ml, respectively. A characteristic ring-like saponin-cholesterol micellar structure was observed electron microscopically in most membrane vesicles of brain microsomes and erythrocyte membrane vesicles but not in the cardiac sarcoplasmic reticulum. These observations indicate that saponin-sensitive and insensitive Ca2+ uptake was derived from plasma membranes and endoplasmic reticulum, respectively. Saponin proved useful for distinguishing the Ca2+ transport activity of plasma membrane from the Ca2+ uptake of other cellular organelles in the membrane preparations.     

Influence of flow velocity and cell concentration on dynamic adhesion of erythrocytes to glass surface

Comparative studies were carried out on dynamic adhesion of 51Cr-labelled erythrocytes to the surface of glass beads in the presence of serum in the medium (50 microng of protein/ml) and in protein-free medium. The influence of cell concentration (within the range 4 X 10(5) to 8 X 10(6)/ml) and of cellular flow velocity (within the range 1.5-0.4 cm/min) on the value of adhesion was investigated. It was found that when serum was present in the medium, the decisive influence on erythrocyte adhesion was exerted by the velocity with which the cells pass though the glass bead layer. Cell concentration under these conditions has only a very slight effect. When the medium does not contain serum, erythrocyte adhesion to the bead layer seems to depend on both cell concentration and flow velocity. Preliminary data were obtained concerning the release of 51Cr from the bead layer after erythrocyte adhesion.     

Regulatory control of complement on blood platelets. Modulation of platelet procoagulant responses by a membrane inhibitor of the C5b-9 complex

Antibody against a membrane inhibitor of the C5b-9 complex has been used to investigate regulatory control of the terminal complement proteins on blood platelets. Monospecific rabbit antibody (alpha-P18) was raised against the purified 18-kDa erythrocyte membrane inhibitor of C5b-9 (Sugita, Y., Nakano, Y., and Tomita, M. (1988) J. Biochem. (Tokyo) 104, 633-637). In addition to its interaction with erythrocytes, this antibody (and its Fab) bound specifically to platelet membranes. In immunoblots of cell membrane proteins prepared under non-reducing conditions, alpha-P18 bound specifically to an 18-kDa erythrocyte membrane protein and to a 37-kDa platelet membrane protein. Absorption of this antibody by platelet membranes competed its binding to the purified 18-kDa erythrocyte protein, suggesting that epitopes expressed by the erythrocyte 18-kDa C5b-9 inhibitor are common to the platelet. When bound to the platelet surface, the Fab of alpha-P18 increased C9 activation by membrane C5b-8, monitored by exposure of a complex-dependent C9 neo-epitope. Although alpha-P18 caused little increase in the cytolysis of platelets treated with C5b-9 (total release of lactate dehydrogenase less than 5%), it markedly increased the cell stimulatory responses induced by these complement proteins, including, secretion from platelet alpha- and dense granules, conformational activation of cell surface GP IIb-IIIa, release of membrane microparticles from the platelet surface, and exposure of new membrane binding sites for components of the prothrombinase enzyme complex. Prior incubation of C5b67 platelets with 100 micrograms/ml alpha-P18 (Fab) lowered by approximately 10-fold the half-maximal concentration of C8 required to elicit each of these responses (in the presence of excess C9). Incubation with alpha-P18 (Fab) alone did not activate platelets, nor did incubation with this antibody potentiate the stimulatory responses of platelets exposed to other agonists. These data indicate that a membrane inhibitor of the C5b-9 complex normally serves to attenuate the procoagulant responses of blood platelets exposed to activated complement proteins, and suggest the mechanism by which a deletion or inactivation of this cell surface component would increase the risk of vascular thrombosis.     

Effects of an antimalarial quinazoline derivative on human erythrocytes and on cell membrane molecular models

Plasmodium, the parasite which causes malaria in humans multiplies in the liver and then infects circulating erythrocytes. Thus, the role of the erythrocyte cell membrane in antimalarial drug activity and resistance has key importance. The effects of the antiplasmodial N(6)-(4-methoxybenzyl)quinazoline-2,4,6-triamine (M4), and its inclusion complex (M4/HPβCD) with 2-hydroxypropyl-β-cyclodextrin (HPβCD) on human erythrocytes and on cell membrane molecular models are herein reported. This work evidences that M4/HPβCD interacts with red cells as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a 10μM concentration; b) in isolated unsealed human erythrocyte membranes (IUM) a concentration as low as 1μM induced sharp DPH fluorescence anisotropy decrease whereas increasing concentrations produced a monotonically decrease of DPH fluorescence lifetime at 37°C; c) X-ray diffraction studies showed that 200μM induced a complete structural perturbation of dimyristoylphosphatidylcholine (DMPC) bilayers whereas no significant effects were detected in dimyristoylphosphatidylethanolamine (DMPE) bilayers, classes of lipids present in the outer and inner monolayers of the human erythrocyte membrane, respectively; d) fluorescence spectroscopy data showed that increasing concentrations of the complex interacted with the deep hydrophobic core of DMPC large unilamellar vesicles (LUV) at 18°C. All these experiments are consistent with the insertion of M4/HPβCD in the outer monolayer of the human erythrocyte membrane; thus, it can be considered a promising and novel antimalarial agent.     

The effect of erythrocyte associated light scattering on membrane fluorescence polarization

Summary The apparent membrane fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene has been reported to be lower in intact erythrocytes than in isolated erythrocyte membranes. Although this difference was once suggested to be caused by the fluidizing effect associated with the loss of erythrocyte proteins during membrane isolation, it is currently thought to be an artifact resulting from intense light scattering properties of intact erythrocytes which overwhelm extrapolation methods of correcting for light scattering. This study confirmed that, at erythrocyte concentrations greater than 10⁷ cells/ml, this difference was caused by intense light scattering; however, at erythrocyte concentrations less than 4.0 × 10⁶ cells/ml, the anisotropy values for erythrocytes and isolated membranes are identical, demonstrating that intense light scattering can be overcome with dilute suspensions of cells.     

Isolation of microtubule protein from chicken erythrocytes and determination of the critical concentration for tubulin polymerization in vitro and in vivo

Microtubule protein isolated from nucleated chicken erythrocytes was examined with respect to composition and assembly properties to determine its significance in a microtubule bundle called the marginal band. 1) The protein contains greater than 95% tubulin with small amounts of tau polypeptides and no high molecular weight polypeptides. 2) Microtubule assembly in vitro at 37 degrees C is characterized by low levels of nucleation, despite an abundance of ring oligomers at 5 degrees C, as indicated by long lag times, slow assembly rates, and microtubules that are twice as long as brain microtubules assembled under the same conditions. 3) By radioimmunoassay and sodium dodecyl sulfate gel analysis we determined that 0.6% of erythrocyte protein is tubulin of which three-quarters is in a nonextractable form and is associated with the microtubule bundle and the cell cortex. From these values the in vivo concentrations of total tubulin and tubulin dimer subunits are 2.4 and 0.7 mg/ml, respectively. The value of 0.7 mg/ml is close to the range of values of 0.1-0.6 mg/ml for the critical concentration of erythrocyte microtubule protein in vitro, suggesting that the assembly properties of tubulin in vitro and in vivo are similar.     

The effect of Pycnogenol on the erythrocyte membrane fluidity

In the present study, the in vitro effect of polyphenol rich plant extract, flavonoid--Pycnogenol (Pyc), on erythrocyte membrane fluidity was studied. Membrane fluidity was determined using 1-[4-trimethyl-aminophenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), 1,6-diphenyl-1,3,5-hexatriene (DPH) and 12-(9-anthroyloxy) stearic acid (12-AS) fluorescence anisotropy. After Pyc action (50 microg/ml to 300 microg/ml), we observed decreases in the anisotropy values of TMA-DPH and DPH in a dose-dependent manner compared with the untreated erythrocyte membranes. Pyc significantly increased the membrane fluidity predominantly at the membrane surface. Further, we observed the protective effect of Pyc against lipid peroxidation, TBARP generation and oxidative hemolysis induced by H2O2. Pyc can reduce the lipid peroxidation and oxidative hemolysis either by quenching free radicals or by chelating metal ions, or by both. The exact mechanism(s) of the positive effect of Pyc is not known. We assume that Pyc efficacy to modify effectively some membrane dependent processes is related not only to the chemical action of Pyc but also to its ability to interact directly with cell membranes and/or penetrate the membrane thus inducing modification of the lipid bilayer and lipid-protein interactions.     

Atomic force microscopy of the erythrocyte membrane in obstructive jaundice

The erythrocyte intracellular pressure was calculated using a biomechanical model of the erythrocyte and atomic-force-microscopy (AFM) data. The intracellular pressure was characterized as a function of the model erythrocyte morphology. Based on numerical modeling and data of erythrocyte imaging by AFM, a method was developed to estimate the erythrocyte intracellular pressure by comparing experimental data and the results of numerical calculations. Calculations were performed for erythrocytes of dwarf domestic pigs with and without obstructive jaundice that varied in severity. The erythrocyte membrane was affected with increasing disease severity and blood bilirubin concentration, i.e., the erythrocyte volume increased on average, and substantial changes were observed for erythrocyte intracellular pressure relative to its normal value. As an example, an increase in bilirubin concentration from 5 to 96 μmol/L was associated with an increase in intracellular pressure from 0 to 2.2 kPa. An examination of the erythrocyte membrane surface by high-resolution AMF showed that the membrane is disrupted with an increase in bilirubin concentration and displays lesions and an increasing rupture length.