Red cell aging results in a change of cell surface carbohydrate epitopes allowing for recognition by galactose-specific receptors of rat liver macrophages

Binding and uptake studies of in vitro aged or senescent rat erythrocytes by isolated rat liver macrophages suggest recognition by galactose-specific receptors on the cell surface of the macrophages. We analyzed carbohydrates exposed on old erythrocytes by plant lectins in an agglutination assay in comparison with freshly isolated untreated erythrocytes. Rat erythrocytes aged in vitro by storage are agglutinated by a panel of lectins that do not react with freshly isolated erythrocytes. Specificity of agglutination was shown by inhibition with monosaccharides. Antibodies eluted from senescent rat erythrocytes agglutinate in vitro aged as well as senescent rat erythrocytes, but not freshly isolated cells nor human erythrocytes. Galactose-specific lectins isolated from rat liver give similar results; they also agglutinate normal human erythrocytes. Agglutination by the liver lectin is inhibitable by galactose and N-acetylgalactosamine but not by N-acetylglucosamine or mannose. Furthermore, rat liver macrophages devoid of galactose-specific receptors show markedly reduced binding of senescent rat erythrocytes. We conclude that recognition of old rat erythrocytes is mediated by two systems: old erythrocytes expose different terminal sugar residues or a different arrangement of glycans when compared to young erythrocytes, rendering them recognizable by liver lectins and by autoantibodies.     

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.     

Physiological ageing of red blood cells and changes in membrane carbohydrates

Evidence is presented to indicate a generalized role for the terminal sialic acid residues of circulating erythrocytes. After reinjection into their donors, neuraminidase-treated human, rabbit, rat and dog erythrocytes were promptly removed from the circulation : intect erythrocytes, previously incubated under the same conditions but without neuraminidase, were removed after a significantly longer period. The neuraminidase-treated erythrocytes were cleared by the liver and in a little part by the spleen. Old and young human, rabbit, rat erythrocytes contained different quantities of stromal sialic acid, significantly lowered on the old cells. But the half-life of old intact rabbit erythrocytes is sigificantly shorter than that of neuraminidase-treated young erythrocytes with a similar minidase-treated young erythrocytes with a similar sialic acid content. Indeed sialic acid is not the only carbohydrate component of the membrane that is decreased during erythrocyte ageing, the others membranous sugars are decreased too. Theses changes in the carbohydrate moity could have a role in the clearance of the erythrocytes.     

The immunomodulating properties of erythrocytes induced by physical factors]

It is shown that the action of ultraviolet rays (UVR) or magnetic field (MF) on the erythrocytes of intact Wistar rats by weight 140-170 g induces the property to stimulate the immune response on sheep's T-dependent antigen-erythrocytes and on bull serum albumin in allogenic transference. The most expressed immune stimulated effect is induced by the heavy erythrocytes, which are effected by magnetic field. The warming and vibration don't induce the immunomodulating characteristics in erythrocytes. By spleen cells sticking to glass, the modified UVR and MF, the heavy erythrocytes induce factors (differentiated in mass), which stimulate the development of immune response to T-dependent antigens of sheep's erythrocytes and of bull serum albumin. These factors depress the function of antigen-specific T-suppressors and induce the immune-suppressive characteristics in light erythrocytes.     

Glutaraldehyde fixation and the mechanism of erythrocyte agglutination by concanavalin a and soybean agglutinin

To evaluate the importance of lectin receptor mobility and clustering for enhanced cell agglutinability, the effect of glutaraldehyde fixation on the agglutinability of human erythrocytes by concanavalin A and soybean agglutinin was investigated. Agglutinability was evaluated in unperturbed Microtiter^® plates. Fixation increased slightly the agglutinability of the erythrocytes by both lectins. Fixation did not alter trypsin-enhanced agglutinability. Furthermore, when fixed erythrocytes were trypsinized, their agglutinability increased to the level of unfixed, trypsinized erythrocytes.The kinetics of agglutination of fixed and unfixed erythrocytes were monitored in an electronic particle counter. The shear forces associated with the kinetic experiments diminished fixed-cell to fixed-cell agglutination, i.e., both lectins gave slower kinetics of agglutination with fixed erythrocytes than with unfixed erythrocytes. In contrast, the kinetics of concanavalin A-mediated agglutination of trypsinized-fixed erythrocytes mixed with equal numbers of trypsinized-unfixed erythrocytes were indistinguishable from the rapid kinetics of agglutination of trypsinizedunfixed erythrocytes alone. Light microscopy revealed aggregates composed of fixed and unfixed erythrocytes.We conclude that glutaraldehyde fixation does not diminish the agglutinability of human erythrocytes under low-shear conditions. Our results indicate that the enhanced agglutination of trypsinized erythrocytes is not dependent on clustering of lectin receptors. The disruption of agglutination of fixed erythrocytes by shear forces that do not disrupt agglutination of fixed erythrocytes with unfixed erythrocytes suggests that the rigidity of the fixed erythrocyte may prevent stable aggregate formation by fixed erythrocytes alone.     

The recording of ATP in the erythrocytes using luciferase injected into the cells

The luciferase preparation obtained from fireflies Luciola mingrelica has entrapped into the human erythrocytes by means of reversible osmotic lysis. The addition of luciferin to such erythrocytes leads to the appearance of luminescence, conditioned by the entrance of luciferin into the cells. Luciferin is uniformly distributed between cells and external medium. Luciferin transport through the erythrocyte membrane is a result of simple diffusion. Values of rate constant of luciferin transport through the membrane lie between 0.009-0.021 l/s 1 cells for erythrocytes of different donors. The maximum luminescence intensity increases monotonously with rise of temperature and luciferin concentration. The dependence of the maximum luminescence intensity on luciferin concentration is described by Michaelis kinetics. Obtained in different experiments, values of luciferase Michaelis constant for luciferin inside erythrocytes lie between 4.1-21.5 microM. Luminescence intensity of the luciferase containing erythrocytes depends on the intracellular ATP concentration. Under the same luciferin concentration the correlation of luminescence intensities of control erythrocytes with normal ATP level and erythrocytes depleted without glucose is near to correlation of their ATP concentrations. After the addition of glucose to the depleted erythrocytes their ATP concentration rises and luminescence intensity approaches to the level of control erythrocytes. Luciferase entrapment permit one to control rapid ATP concentration changes in the erythrocytes.     

除草剂丁草胺对蟾蜍红细胞微核及核异常的影响

本文研究除卓剂丁革胺对蟾蜍红细胞核的诱变效应。采用体内红细胞微核测定法,观察蟾蜍在不同浓度和不同的染毒时间红细胞微核及核异常的变化。结果表明,在一定的范围内,丁草胺可引起蟾蜍微核细胞率和核异常细胞率等遗传指标发乍明显变化,随着丁草胺浓度的增加和作用时间的延长,蟾蜍红细胞微核和核异常率呈现先上升后下降的规律性变化。丁草胺的作用具有双向性。一定利量的除草剂丁草胺对蟾蜍红细胞具有明显的遗传毒性,除草剂对水体的污染不容忽视。     

Erythrocytes-the 'house elves' of photodynamic therapy.

Photodynamic therapy (PDT) and fluorescence diagnosis (FD) are being developed for a number of clinical applications. Since fluorophores and photosensitising drugs are usually given systemically their effect on blood elements are of significant importance. Photodynamic effects on erythrocytes occur naturally in patients with erythropoietic protoporphyria (EPP). Exposure to small fluences, as obtained by the erythrocytes when they pass capillaries in the skin, leads to transfer of the photosensitiser protoporphyrin IX (PP IX), from EPP erythrocytes to endothelial cells. Thus, the erythrocytes are partly protected while the endothelial cells suffer photodamage. During photodynamic therapy in vivo erythrocytes are regularly photosensitised. This side effect is partly intended but mostly unwanted, and a summary of this topic is given. Furthermore, the effect of UV-A on erythrocytes that is accompanied with the formation of bilirubin is reviewed. Erythrocytes serve as convenient model cells for experimental research. Such use of erythrocytes to screen new photosensitisers may be of limited value. A combination of photohaemolysis and haemoglobin oxygenation may become the basis for an assay for in vitro phototoxicity. Erythrocytes from birds are good model cells for exploration of physiological and molecular mechanisms involved in PDT. A potential mechanism of PDT induced behaviour resembling apoptosis in erythrocytes is provided.PDT for sterilisation of erythrocyte concentrates has a potential for medical use. Photodynamic effects on the erythrocytes themselves should be avoided. This is realised by choosing a virus-selective photosensitiser, low fluences and treatment of the concentrates with agents like dipyridamole and antioxidants. Future aspects of applications of photosensitisation of red blood cells are discussed.     

The role of sialic acid in the determination of survival of rabbit erythrocytes in the circulation

Evidence is presented to indicate a generalized role for the terminal sialic acid residues of circulating erythrocytes of rabbit. Neuraminidase is shown to remove only sialic acid from these erythrocytes. Neuraminidase-treated and intact rabbit erythrocytes have similar in vitro properties, except those of cellular charge and cellular adhesion in their sera. These properties include similar shape, osmotic fragility curve, autohemolysis at 37°, K⁺ retention and pyruvate kinase activity. The D-glucose 6-phosphate dehydrogenase and the cholinesterase activities are higher on the neuraminidase-treated erythrocytes than on the intact ones. After injection into rabbits, the sialic acid-less erythrocytes tested, were promptly removed from the circulation; intact erythrocytes, previously incubated under the same conditions but without neuraminidase, were removed from the circulation after a significantly longer period.     

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.     

Intracellular location of polyamines associated to red blood cells

The polyamines associated to human erythrocytes from healthy donors are mainly localized intracellularly. In fact chromatography of the erythrocytes on a resin which has a high affinity and capacity for polyamines does not affect the amount of polyamines associated to the erythrocytes. The low ability of spermine to adsorb to the external surface of erythrocytes at physiological ionic strength is suggested also by studies conducted with sealed ghosts. Also erythrocytes from patients with hematological and dermatological diseases which contain increased levels of polyamines show an intracellular location of these amines.     

Receptor distribution and the mechanism of enhanced erythrocyte agglutination by soybean agglutinin

We have examined the role of receptor clustering in intact erythrocyte membranes exhibiting enhanced lectin-mediated cell agglutination by analyzing freeze-fracture and freeze-etch images of human erythrocytes labeled with ferritin-conjugated soybean agglutinin. We find that trypsinization and fixation of intact erythrocytes, in either order, causes no alteration of the random distribution of ferritin-conjugated soybean agglutinin on the surfaces of these cells as compared to their distribution on the surfaces of fixed erythrocytes and untreated erythrocyte ghosts. Furthermore, clustering of the intramembranous particles in the membrane of intact erythrocytes was not found with any of the cells described above.We conclude that clustering of the soybean agglutinin receptors is not a major factor involved in the enhanced agglutination of intact trypsinized erythrocytes. Caution is necessary in transferring information obtained with erythrocyte ghosts, where clustering can be induced, to intact erythrocytes.     

ABO Blood Groups Influence Macrophage-mediated Phagocytosis of Plasmodium falciparum-infected Erythrocytes

Erythrocyte polymorphisms associated with a survival advantage to Plasmodium falciparum infection have undergone positive selection. There is a predominance of blood group O in malaria-endemic regions, and several lines of evidence suggest that ABO blood groups may influence the outcome of P. falciparum infection. Based on the hypothesis that enhanced innate clearance of infected polymorphic erythrocytes is associated with protection from severe malaria, we investigated whether P. falciparum-infected O erythrocytes are more efficiently cleared by macrophages than infected A and B erythrocytes. We show that human macrophages in vitro and mouse monocytes in vivo phagocytose P. falciparum-infected O erythrocytes more avidly than infected A and B erythrocytes and that uptake is associated with increased hemichrome deposition and high molecular weight band 3 aggregates in infected O erythrocytes. Using infected A₁, A₂, and O erythrocytes, we demonstrate an inverse association of phagocytic capacity with the amount of A antigen on the surface of infected erythrocytes. Finally, we report that enzymatic conversion of B erythrocytes to type as O before infection significantly enhances their uptake by macrophages to observed level comparable to that with infected O wild-type erythrocytes. These data provide the first evidence that ABO blood group antigens influence macrophage clearance of P. falciparum-infected erythrocytes and suggest an additional mechanism by which blood group O may confer resistance to severe malaria.     

Species specificity of recognition by the alternative pathway of complement

The recognition function of the alternative complement pathway was studied with isolated human and rabbit components. Zymosan and homologous and heterologous erythrocytes were used as representative activators or nonactivators. The binding affinity of Factor B and Factor H for particle-bound C3b was measured. In both species, the average affinity of Factor H for bound C3b on homologous cells (nonactivators) was eight to 10 times higher than on zymosan particles (activators). The interaction between Factor H and C3b on rabbit erythrocytes was species-specific: rabbit Factor H bound strongly to rabbit C3b on rabbit erythrocytes and also on human erythrocytes, which are nonactivators for the rabbit alternative pathway. Human Factor H bound strongly to human C3b on human erythrocytes but seven times weaker on rabbit erythrocytes, which are activators of the human alternative pathway. No substantial differences were found in the binding of Factor B to bound C3b regardless of the nature of the particle to which C3b was bound. The results indicate that in the two species studied, the molecular mechanism of recognition is analogous and that recognition is species-specific.     

L-Cysteine influx and efflux: a possible role for red blood cells in regulation of redox status of the plasma

The objective of this study was to investigate if erythrocytes play a role in the maintenance of redox homeostasis of the plasma. Thus, we studied L-cysteine efflux and influx in vitro in human erythrocytes. In the present study, we exposed the erythrocytes to different concentrations of L-cysteine and then measured the intracellular free -SH concentrations. Erythrocytes treated in the same manner were later utilized for the cysteine efflux studies. The effect of temperature on the influx and the efflux processes were also evaluated. Change in the free -SH content of the buffer was evaluated as a measure for the presence of an efflux process. The effects of free -SH depletion on L-cysteine transport is also investigated. We also determined the rate of L-cysteine efflux in the presence and absence of buthionine sulfoximine (BSO) in erythrocytes that are pretreated with 1-chloro-2,4-dinitro benzene, a glutathione (GSH) depletory. Our L-cysteine influx studies demonstrated that erythrocytes can respond to increases in L-cysteine concentration in the extracellular media and influx L-cysteine in a concentration-dependent manner. Free -SH concentrations in erythrocytes treated with 1 mM L-cysteine reached to 1.64 +/- 0.06 mM in 1 h whereas this concentration reached to 4.30 +/- 0.01 mM in 10 mM L-cysteine treated erythrocytes. The L-cysteine efflux is also determined to be time-and concentration-dependent. Erythrocytes that are pretreated with higher L-cysteine concentrations displayed a higher efflux process. Outside concentration of free -SH in 1 mM L-cysteine pretreated erythrocytes reached to 0.200 +/- 0.005 mM in 1 h whereas this concentration reached to 1.014 +/- 0.002 with 10 mM L-cysteine pretreated erythrocytes. Our results also indicate that the rate of inward and outward transport of L-cysteine is affected by the oxidative status of the erythrocytes. When GSH is depleted and GSH synthesis is blocked, the L-cysteine uptake and the efflux processes are significantly decreased. Depending on our results, it could be concluded that erythrocytes play a role in the regulation of the plasma redox status and intracellular level of GSH determines the rate of the L-cysteine efflux.     

Transfer of 1-pyrroline-5-carboxylate as oxidizing potential from hepatocytes to erythrocytes.

The interconversions of proline and 1-pyrroline-5-carboxylate form an intercellular cycle that is the basis of a metabolic interaction between hepatocytes and erythrocytes. The cycle transfers oxidizing potential from hepatocytes to erythrocytes, which stimulates pentose phosphate pathway in erythrocytes. This interaction depends on the differential metabolism of proline and 1-pyrroline-5-carboxylate in erythrocytes and hepatocytes and consists of the following: in hepatocytes proline oxidase converts proline into 1-pyrroline-5-carboxylate, which is released into the medium and taken up by erythrocytes; erythrocyte 1-pyrroline-5-carboxylate reductase converts 1-pyrroline-5-carboxylate into proline and concomitantly generates NADP+; the generated oxidizing potential drives glucose metabolism through the pentose phosphate pathway in erythrocytes; finally, erythrocytes release proline into the medium, enabling it to re-enter hepatocytes and repeat the cycle. The increased activity of the pentose phosphate pathway in erythrocytes may enhance the production of 5-phosphoribosyl pyrophosphate, a necessary moiety for the processing of purines.     

Comparative characteristics of the properties of erythrocyte Na,K-ATPase in man and the carp Cyprinus carpio

Studies have been made on some of the properties of Na,K-ATPase of a nuclear erythrocytes of man and nuclear erythrocytes of the carp Cyprinus carpio. Human erythrocytes yielded the enzymic activity only after their treatment by a detergent Twin-20; under optimal conditions, it amounted to 2.6 mcmole /ml of erythrocytes per 1 h. In carp erythrocytes, Na,K-ATPase activity could be detected without detergent treatment, being 10-fold higher under optimal conditions than that in human erythrocytes. Repetitive washing of carp erythrocytes from the plasma (for more than 3 times), significantly increased their viscosity and resulted in spontaneous hemolysis. Simultaneously , the activity of Na,K-ATPase increased 2-10 times depending on the composition of incubation media. Under these conditions, the pattern of changes in the enzymatic activity, resulting from shifts in Mg2+ and EDTA concentrations, was altered. The presence of latent Na,K-ATPase activity in the erythrocytes in explained by a low permeability of membranes to ATP and ions. Exogeneous ATP cannot be utilized by the enzyme in the intact human erythrocytes, whereas intact carp erythrocytes exhibit significant permeability to the exogeneous substrate. It is suggested that in vivo this fact may be of physiological importance.     

Response of erythrocytes to heat in the presence of D2O, glycerol, and anisotonic saline

Mammalian erythrocytes that lack cytoplasmic organelles and a nucleus are a useful model for studying the effect of heat on the cell membrane and cytoskeleton. The effect of heat on the membrane bilayer and cytoskeleton of erythrocytes is remarkably similar to that observed in nucleated cells. Some concentrations of D2O and glycerol can effectively protect erythrocytes from heat-induced damage to the membrane and cytoskeleton. These results are similar to observations in nucleated cells. Heating erythrocytes in some concentrations of anisotonic NaCl solutions reduced damage, an observation that does not apply to enhanced killing of nucleated cells. This difference implies that some components of the cytoplasm or nucleus, or both, may contribute to the enhancement of cytotoxicity of nucleated cells when they are heated in the anisotonic NaCl solution. Incremental heating, dividing a heat treatment into two fractions, and preheating of erythrocytes all modify the effect of heat on erythrocytes slightly, but the results suggest little, if any, development of thermotolerance. The response of chicken erythrocytes is similar to that of mammalian erythrocytes, although higher temperatures are required to produce a heat effect in chicken erythrocytes. These observations suggest that the characteristic differences in heat sensitivity in nucleated and enucleated cells involve components other than the cell membrane.     

Distribution of erythrocyte in a model vessel exposed to inhomogeneous magnetic fields

In order to investigate magnetic field effects on blood flow, changes in the flow of erythrocytes in a model branched vessel were observed in an inhomogeneous magnetic field. The magnetic field was applied perpendicular to the straight vessel before branching. When the suspension containing paramagnetic erythrocytes with high spin methemoglobin or deoxygenated hemoglobin flowed in the model vessel, the erythrocytes were attracted towards the stronger magnetic field (i.e. to the side branch) and an excess flow of erythrocytes to the side branch was detected. This excess flow of erythrocytes to the side branch was the highest at a hematocrit of about 5% for the suspension containing erythrocytes with high spin methemoglobin. In the case of mixed suspensions containing erythrocytes with high spin methemoglobin and oxygenated erythrocytes, the excess flow of erythrocytes to the side branch reached its maximum at the "partial hematocrit" for the paramagnetic erythrocyte of around 5% and remained nearly constant with a further increase of the "partial hematocrit." The effect of magnetic field decreased as the flow velocity increased. These results are explained with the paramagnetism of erythrocytes and with the assumption of a hydrodynamic interaction among erythrocytes which are pulled in the direction of the magnetic field. It is suggested that a strong inhomogeneous magnetic field is not totally negligible to the blood circulation.     

Molecular mechanisms of erythrophagocytosis. Characterization of the senescent erythrocytes that are phagocytized by macrophages

We have recently developed a flow cytometric assay for the quantitation of erythrophagocytosis, using PKH 26-labeled erythrocytes as the target cells. Using this assay we have shown that there is extensive phagocytosis of desialylated erythrocytes. Furthermore, we have demonstrated that it is the densest population of erythrocytes obtained on a self-forming gradient of Percoll that shows the greatest susceptibility to phagocytosis. We designate this population of erythrocytes as fraction X; it is even denser than the fraction 5 found previously. This population of erythrocytes corresponds to zone X previously seen in the dot-plot of the flow cytometric analyses of human erythrocytes. Further scrutiny of this fraction indicates that a) it shows the greatest reactivity with annexin V, which is specific for the detection of phosphatidylserine (PS) exposed on the outer leaflet of the erythrocyte membrane, b) it is the most susceptible to erythrophagocytosis by resident murine peritoneal macrophages, and c) this erythrophagocytosis of PKH 26-labeled erythrocytes can be inhibited by annexin V and by liposomes containing PS. Scanning electron microscopy of fraction X shows two populations of erythrocytes: (A) spheroechinocytes with filipodes and (B) echinocytes without filipods. After a 2-h period of phagocytosis, the cells remaining in fraction X show a decrease in population A, commensurate with a decrease in reactivity with FITC-labeled annexin V from 65.5 to 24%.