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Splenectomised β-thalassaemia/haemoglobin E (HbE) patients have increased levels of circulating microparticles or medium extra-cellular vesicles (mEVs). The splenectomised mEVs play important roles in thromboembolic complications in patients since they can induce platelet activation and endothelial cell dysfunction. However, a comprehensive understanding of the mechanism of mEV generation in thalassaemia disease has still not been reached. Thalassaemic mEVs are hypothesised to be generated from cellular oxidative stress in red blood cells (RBCs) and platelets. Therefore, a proteomic analysis of mEVs from splenectomised and non-splenectomised β-thalassaemia/HbE patients was performed by liquid chromatography with tandem mass spectrometry. A total of 171 proteins were identified among mEVs. Interestingly, 72 proteins were uniquely found in splenectomised mEVs including immunoglobulin subunits and cytoskeleton proteins. Immunoglobulin G (IgG)-bearing mEVs in splenectomised patients were significantly increased. Furthermore, complement C1q was detected in both mEVs with IgG binding and mEVs without IgG binding. Interestingly, the percentage of mEVs generated from RBCs with IgG binding was approximately 15-20 times higher than the percentage of RBCs binding with IgG. This suggested that the vesiculation of thalassaemia mEVs could be a mechanism of RBCs to eliminate membrane patches harbouring immune complex and may consequently prevent cells from phagocytosis and lysis., (© 2024 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

The Kg-antigen was first discovered in an investigation of a mother whose infant had haemolytic disease of the newborn (HDN). The antibody against the Kg-antigen is believed to be responsible for HDN. The Kg-antigen is provisionally registered under the number 700045, according to the Red Cell Immunogenetics and Blood Group Terminology. However, the molecular nature of the Kg-antigen has remained a mystery for over 30 years. In this study, a monoclonal antibody against the Kg-antigen and the recombinant protein were developed that allowed for the immunoprecipitation analysis. Immunoprecipitants from the propositus' red blood cell ghosts were subjected to mass spectrometry analysis, and DNA sequence analysis of the genes was also performed. A candidate for the Kg-antigen was molecularly isolated and confirmed to be a determinant of the Kg-antigen by cell transfection and flow cytometry analyses. The Kg-antigen and the genetic mutation were then screened for in a Japanese population. The molecular nature of the Kg-antigen was shown to be RhAG with a Lys164Gln mutation. Kg phenotyping further clarified that 0.22% of the Japanese population studied was positive for the Kg-antigen. These findings provide important information on the Kg-antigen, which has been clinically presumed to give rise to HDN., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Vaso-occlusive crisis (VOC) is the main acute complication in sickle cell anaemia (SS) and several clinical trials are investigating different drugs to improve the clinical severity of SS patients. A phase III study is currently exploring the profit of Velopoloxamer in SS during VOCs. We analysed, in-vitro, the effect of poloxamer (P188) on red blood cell (RBC) properties by investigating haemorheology, mechanical and adhesion functions using ektacytometry, microfluidics and dynamic adhesion approaches, respectively. We show that poloxamer significantly reduces blood viscosity, RBC aggregation and adhesion to endothelial cells, supporting the beneficial use of this molecule in SS therapy., (© 2016 John Wiley & Sons Ltd.)

Protein 4.1R plays an important role in maintaining the mechanical properties of the erythrocyte membrane. We analysed the expression of Kell blood group protein in erythrocytes from a patient with hereditary elliptocytosis associated with complete 4.1R deficiency (4.1(-) HE). Flow cytometry and Western blot analyses revealed a severe reduction of Kell. In vitro pull down and co-immunoprecipitation experiments from erythrocyte membranes showed a direct interaction between Kell and 4.1R. Using different recombinant domains of 4.1R and the cytoplasmic domain of Kell, we demonstrated that the R(46) R motif in the juxta-membrane region of Kell binds to lobe B of the 4.1R FERM domain. We also observed that 4.1R deficiency is associated with a reduction of XK and DARC (also termed ACKR1) proteins, the absence of the glycosylated form of the urea transporter B and a slight decrease of band 3. The functional alteration of the 4.1(-) HE erythrocyte membranes was also determined by measuring various transport activities. We documented a slower rate of HCO3 (-) /Cl(-) exchange, but normal water and ammonia transport across erythrocyte membrane in the absence of 4.1. These findings provide novel insights into the structural organization of blood group antigen proteins into the 4.1R complex of the human red cell membrane., (© 2015 John Wiley & Sons Ltd.)

Red cells with the D-- phenotype do not express the RHCE protein because of mutations in both alleles of the RHCE gene. At present, little is known of the effect this has on the normal function of erythrocytes. In this study a group of five families belonging to a nomadic tribe in Malaysia were identified as carriers of the D-- haplotype. Analysis of homozygous individuals' genomic DNA showed two separate novel mutations. In four of the families, RHCE exons 1, 9 and 10 were present, while the 5th family possessed RHCE exons 1-3 and 10. Analysis of cDNA revealed hybrid transcripts, suggesting a gene conversion event with RHD, consistent with previously reported D-- mutations. Immunoblotting analysis of D-- erythrocyte membrane proteins found that Rh-associated glycoprotein (RHAG) migrates with altered electrophoretic mobility on sodium dodecyl sulphate polyacrylamide gel electrophoresis, consistent with increased glycosylation. Total amounts of Rh polypeptide in D-- membranes were comparable with controls, indicating that the exalted D antigen displayed by D-- red cells may be associated with altered surface epitope presentation. The adhesion molecules CD44 and CD47 are significantly reduced in D--. Together these results suggest that absence of RHCE polypeptide alters the structure and packing of the band 3/Rh macrocomplex., (© 2012 Blackwell Publishing Ltd.)

Red blood cells (RBCs) from individuals with Southeast Asian ovalocytosis (SAO) contain a mutant band 3 protein that causes the formation of unique linear oligomers in the RBC membrane. We used single-particle tracking to measure the lateral diffusion of individual glycophorin C (GPC), band 3, and CD58 proteins in membranes of intact SAO RBCs and normal RBCs (nRBCs). GPC, an integral protein that binds with high affinity to the RBC membrane skeleton, showed oscillatory motion within confinement areas that were smaller in SAO RBCs than in nRBCs. The additional confinement in SAO RBCs could be due to membrane stiffening associated with the SAO phenotype. Band 3 in both SAO RBCs and nRBCs also showed confined motion over short times (ms) and distances (nm), and the area of confinement was smaller in SAO RBCs than in nRBCs. These data presumably reflect the constraints imposed by band 3 oligomerization. Similarly, the glycosylphosphatidylinositol-linked protein CD58 showed loosely confined diffusion in nRBCs and a substantially higher degree of confinement in SAO RBCs. Restricted protein mobility could contribute to the altered adherence of parasite-infected RBCs to vascular endothelium that is thought to protect individuals with SAO from severe manifestations of malaria., (© 2011 Blackwell Publishing Ltd.)

Erythrocytes are remarkably dynamic structures, possessing multiple and complex pathways for regulating cell membrane properties to compensate for the absence of a nucleus and internal membranes. Unlike the invasion strategies of many viruses and bacteria into their eukaryotic hosts, however, the accepted model for malaria parasite entry into human erythrocytes casts the host cell in a largely passive role. This is in contrast to mounting evidence for a suite of dynamic alterations that the erythrocyte membrane undergoes during the rapid process of invasion by the blood stage malaria parasite - the merozoite. Here we review the cellular and molecular basis for merozoite invasion of the erythrocyte and explore the idea that radical changes in the erythrocyte membrane protein and lipid architecture probably accompany this key step in the establishment of human malaria disease., (© 2011 Blackwell Publishing Ltd.)

Residence in the human erythrocyte is essential for the lifecycle of all Plasmodium that infect man. It is also the phase of the life cycle that causes disease. Although the red blood cell (RBC) is a highly specialized cell for its function of carrying oxygen to and carbon dioxide away from tissues, it is devoid of organelles and lacks any cellular machinery to synthesize new protein. Therefore in order to be able to survive and multiply within the RBC membrane the parasite needs to make many modifications to the infected RBC (iRBC). Plasmodium falciparum (P. falciparum) also expresses parasite-derived proteins on the surface of the iRBC that enable the parasite to cytoadhere to endothelial and other intravascular cells. These RBC modifications are at the root of malaria pathogenesis and, in this ancient disease of man, have formed the epicentre of a genetic 'battle' between parasite and host. This review discusses some of the critical modifications of the RBC by the parasite and some of the consequences of these adaptations on disease in the human host, with an emphasis on advances in understanding of the pathogenesis of severe and cerebral malaria (CM) from recent research., (© 2011 Blackwell Publishing Ltd.)

Spherocytosis is one of the most common inherited disorders, yet presents with a wide range of clinical severity. While several genes have been found mutated in patients with spherocytosis, the molecular basis for the variability in severity of haemolytic anaemia is not entirely understood. To identify candidate proteins involved in haemolytic anaemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in red blood cells (RBCs) from normal and β-adducin (Add2) knock-out mice. We detected seven proteins that were decreased and 48 proteins that were increased in β-adducin null RBC ghosts. Since haemolytic anaemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Among the 48 proteins increased in Add2 knockout RBCs, only 11 were also increased in reticulocytes. Of the proteins decreased in Add2 knockout RBCs, α-adducin showed the greatest intensity difference, followed by SLC9A1, the sodium-hydrogen exchanger previously termed NHE1. We verified these mass spectrometry results by immunoblot. This is the first example of SLC9A1deficiency in haemolytic anaemia and suggests new insights into the mechanisms leading to fragile RBCs., (© 2011 Blackwell Publishing Ltd.)

During definitive erythropoiesis, erythroid precursors undergo differentiation through multiple nucleated states to an enucleated reticulocyte, which loses its residual RNA/organelles to become a mature erythrocyte. Over the course of these transformations, continuous changes in membrane proteins occur, including shifts in protein abundance, rates of expression, isoform prominence, states of phosphorylation, and stability. In an effort to understand when assembly of membrane proteins into an architecture characteristic of the mature erythrocyte occurs, we quantitated the lateral diffusion of the most abundant membrane protein, band 3 (AE1), during each stage of erythropoiesis using single particle tracking. Analysis of the lateral trajectories of individual band 3 molecules revealed a gradual reduction in mobility of the anion transporter as erythroblasts differentiated. Evidence for this progressive immobilization included a gradual decline in diffusion coefficients as determined at a video acquisition rate of 120 frames/s and a decrease in the percentage of compartment sizes >100 nm. Because complete acquisition of the properties of band 3 seen in mature erythrocytes is not observed until circulating erythrocytes are formed, we suggest that membrane maturation involves a gradual and cooperative assembly process that is not triggered by the synthesis of any single protein.

Hereditary Spherocytosis (HS) is a haemolytic anaemia caused by erythrocyte protein membrane defects - spectrin, ankyrin, band 3 or protein 4.2 - that lead to membrane destabilization. This study aimed to evaluate the prevalence of protein deficiencies and the role of membrane proteins or membrane-linked proteins in membrane disturbance and in HS clinical outcome. A total of 215 Portuguese individuals were studied - 203 from 71 families plus 12 individual unrelated subjects; 160 of them were diagnosed with HS. They were classified as presenting mild, moderate or severe forms of HS according to the degree of haemolytic anaemia. Standardized electrophoretic erythrocyte membrane protein analysis was used to identify and quantify protein deficiencies. Band 3 and ankyrin were found to account for the majority of the erythrocyte protein defects underlying HS. Increasing isolated protein deficiency or increasing imbalance between combined protein deficiencies seemed to underlie HS severity, by increasing membrane destabilization. There was an increased membrane linkage of the cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and peroxiredoxin 2, and of denatured haemoglobin, suggesting that this linkage could interfere with membrane structure. Our data suggest that the quantification and the analysis of RBC membrane proteins may be helpful in predicting the clinical outcome of HS.

Lu/BCAM, the unique erythroid receptor for laminin 511/521, interacts with the erythrocyte membrane skeleton through spectrin binding. It has been reported that Hereditary Spherocytosis red blood cells (HS RBC) exhibit increased adhesion to laminin. We investigated the role of Lu/BCAM-spectrin interaction in the RBC adhesion properties of 2 splenectomised HS patients characterized by 40% spectrin deficiency. Under physiological flow conditions, HS RBC exhibited an exaggerated adhesion to laminin that was completely abolished by soluble Lu/BCAM. Triton extraction experiments revealed that a greater fraction of Lu/BCAM was unlinked to the membrane skeleton of HS RBC, as compared to normal RBC. Disruption of the spectrin interaction site in Lu/BCAM expressed in the transfected K562 cell line resulted in a weakened interaction to the skeleton and an enhanced interaction to laminin. These results demonstrated that the adhesion of HS RBC to laminin was mediated by Lu/BCAM and that its interaction with the spectrin-based skeleton negatively regulated cell adhesion to laminin. Finally, the results of this study strongly suggest that the reinforced adhesiveness of spectrin-deficient HS RBC to laminin is partly brought about by an impaired interaction between Lu/BCAM and the membrane skeleton.

Mouse Rhd* and Rhag* genes were targeted using insertional vectors; the resulting knockout mice, and double-knockout descendants, were analysed. Rhag glycoprotein deficiency entailed defective assembly of the erythroid Rh complex with complete loss of Rh and intercellular adhesion molecule 4 (ICAM-4), but not CD47, expression. Absence of the Rh protein induced a loss of ICAM-4, and only a moderate reduction of Rhag expression. Double knockout phenotype was similar to that of Rhag targeted mice. Rhd and Rhag deficient mice exhibited neither the equivalent of human Rh(null) haemolytic anaemia nor any clinical or cellular abnormalities. Rhd-/- and Rhag-/- erythrocytes showed decreased basal adhesion to an endothelial cell line resulting from defective ICAM-4 membrane expression. There was no difference in recovery from phenylhydrazine-induced haematopoietic stress for double knockout mice as compared to controls, suggesting that ICAM-4 might be dispensable during stress erythropoiesis. Ammonia and methylammonia transport in erythrocytes was severely impaired in Rhag-/- but only slightly in Rhd-/- animals that significantly expressed Rhag, supporting the view that RhAG and Rhag, but not Rh, may act as ammonium transporters in human and mouse erythrocytes. These knockout mice should prove useful for further dissecting the physiological roles of Rh and Rhag proteins in the red cell membrane.

Phosphatidylserine (PS) externalization may contribute to Sickle Cell Disease (SCD) characteristics including thrombogenesis, endothelial adhesion and shortened red blood cell (RBC) lifespan. Aminophospholipid translocase (APLT) returns externalized PS to the inner membrane, and phospholipid scramblase (PLSCR) equilibrates phospholipids (PL) across the membrane. APLT inhibition and PLSCR activation appear to be important for PS externalization. We examined relationships between APLT, PLSCR and external PS in mature sickle RBC and reticulocytes. Normally-hydrated sickle RBC without external PS had active APLT and inactive PLSCR. PS-exposing sickle RBC had inhibited APLT and active PLSCR. Sickle reticulocytes had active APLT and active PLSCR independent of external PS. Sickle RBC dehydrated in vivo had the highest proportion of PS-exposing RBC and markedly inhibited APLT. Normal and sickle RBC dehydrated in vitro had moderately decreased APLT. Rehydration resulted in significant recovery of APLT in RBC previously dehydrated in vitro, but not in sickle RBC dehydrated in vivo. These findings indicate that (i) PS externalization in mature sickle RBC depends on the balance between APLT and PLSCR activities, (ii) PS externalization in sickle reticulocytes depends primarily on PLSCR activation and (iii) APLT inhibition in sickle RBC dehydrated in vivo is due to dehydration itself and other factors.

Studies during the last three decades have enabled the development of detailed molecular insights into the structural basis of altered function in various inherited red cell membrane disorders. This review highlights our current understanding of molecular and mechanistic insights into various inherited red cell membrane disorders involving either altered membrane structural organization (hereditary spherocytosis, hereditary elliptocytosis and hereditary ovalocytosis) or altered membrane transport function (hereditary stomatocytosis). The molecular basis for the vast majority of cases of hereditary spherocytosis, elliptocytosis and ovalocytosis have been fully defined while little progress has been made in defining the molecular basis for hereditary stomatocytosis. Mutations in a number of distinct genes account for hereditary spherocytosis and elliptocytosis, while a single genetic defect accounts for all cases of hereditary ovalocytosis. Based on these molecular insights, a comprehensive understanding of the structural basis for altered membrane function has been developed. Loss of vertical linkage between membrane skeleton and lipid bilayer leads to membrane loss in hereditary spherocytosis, while weakening of lateral linkages between skeletal proteins leads to membrane fragmentation and surface area loss in hereditary elliptocytosis. Importantly, the severity of anaemia in both these disorders is directly related to extent of membrane surface area loss. Splenectomy results in amelioration of anaemia.

This study aimed to investigate any correlation between the extent of phosphatidylserine (PS) asymmetry and sialylated glycoconjugate levels with the faster clearance of circulating erythrocytes in haemoglobin E (HbE) beta-thalassaemia. Erythrocytes from peripheral blood samples of different HbEbeta-thalassaemia patients showed loss of PS asymmetry measured by annexin V binding using flow cytometry. Maximum PS exposure was found when HbE was 50-60% and HbF was <20% indicating a possible correlation with severity of the disease. Separation of erythrocytes into aged and younger cells showed higher loss of PS asymmetry in the younger erythrocytes of HbEbeta-thalassaemia patients when compared with normal blood, where PS asymmetry was lost only in the older cells. Sialylated glycoconjugate measurement using the lectins wheatgerm agglutinin and pokeweed mitogen showed loss of sialic acid and N-acetyl-D-glucosamine-bearing glycoproteins in the order normal

Increased oxidant stress has been suggested to play a role in the process of phosphatidylserine (PS) externalization in the red blood cells of sickle cell patients. Inhibition of the ATP-driven translocation from outer to inner monolayer (flippase) by sulphydryl modification has been established. The present study showed that phospholipid scrambling was also sensitive to protein sulphydryl modification. Treatment with N-ethylmaleimide lead to enhanced PS exposure and a lower Ca(++) requirement for scrambling. In contrast, pyridyldithioethylamine treatment inhibited PS exposure. Red blood cells from a murine model for sickle cell disease exhibited a reduced response to both reagents, suggestive of previous sulphydryl modifications to the protein(s) involved in phospholipid scrambling. We conclude that sulphydryl modifications to both scramblase and flippase underlie the enhanced formation of PS-exposing cells in sickle cell disease.

We tested the hypothesis that dehydration-induced alterations in red blood cell (RBC) membrane organisation or composition contribute to sickle cell adhesion in sickle cell disease (SCD). To examine the role of RBC hydration in adhesion to the subendothelial matrix protein thrombospondin-1 (TSP), normal and sickle RBCs were incubated in buffers of varying tonicity and tested for adhesion to immobilised TSP under flow conditions. Sickle RBCs exhibited a decrease in TSP binding with increasing cell hydration (P<0.005), suggesting that cellular dehydration may contribute to TSP adhesion. Consistent with this hypothesis, normal RBCs showed an increase in TSP adhesion with increasing dehydration (P<0.01). Furthermore, increased TSP adhesion of normal RBCs could also be induced by isotonic dehydration using nystatin-sucrose buffers. Finally, TSP adhesion of both sickle RBCs and dehydrated normal RBCs was inhibited by the anionic polysaccharides, chondroitin sulphate A and high molecular weight dextran sulphate, but not by competitors of CD47-, band 3-, or RBC phosphatidylserine-mediated adhesion. More importantly, we found increased adhesion of nystatin-sucrose dehydrated normal mouse RBCs to kidney capillaries following re-infusion in vivo. In summary, these findings demonstrate that changes in hydration can significantly impact adhesion, causing normal erythrocytes to display adhesive properties similar to those of sickle cells and vice versa.

Erythropoietin (EPO), the main growth factor responsible for the regulation of red blood cell production, may be overproduced when blood loss or haemolysis occurs. Patients with mild hereditary spherocytosis (HS) are able to maintain normal haemoglobin concentration, whereas typical and severe HS patients develop an anaemic state. Splenectomy usually reverses anaemia. We aimed to clarify the role of EPO in the response to enhanced spherocyte destruction, and to look for a linkage with the broad clinical spectra of HS. EPO levels, reticulocyte count and production index (RPI), other parameters used to classify HS and the protein deficiencies underlying HS were evaluated in previously diagnosed unsplenectomised (n = 24) and splenectomised (n = 10) patients presenting mild, typical or severe HS. A significant increase in EPO was observed in all unsplenectomised HS patients. In the mild form, a significant correlation of EPO with reticulocyte count and RPI was observed; however, this correlation disappeared in typical HS patients. Splenectomised HS patients presented a correction in EPO levels in all forms of HS, although the reticulocyte count and RPI sustained slightly higher values. Our data show HS as a disease linked to an overproduction of EPO, according to the severity of the disease; however, a disturbance in erythropoiesis seems to occur in typical HS. Moreover, splenectomy leads to a correction in the EPO levels.

Glycophorin-C (GPC) is a 40 kDa glycoprotein expressed on erythrocytes and is a receptor for the malarial parasite Plasmodium falciparum to invade these cells. A link between GPC binding (ligation) and phosphatidylserine (PS) expression on erythrocytes has been suggested by its appearance on P. falciparum-infected erythrocytes. Phosphatidylserine expression has also been shown to be a marker of cellular death in a number of biological pathways including some in erythrocytes. Using Annexin V binding, we demonstrated that ligation of GPC with mouse mAb (BRIC-10) induced PS expression on normal erythrocytes. Phosphatidylserine exposure was prevented following tryptic digestion of intact erythrocytes. In addition, GPC variant phenotypes Yus (Delta exon 2) and Gerbich (Delta exon 3), which express a truncated extracellular domain, did not express PS following BRIC-10 binding, whereas PS was exposed on Ls(a) erythrocytes (duplication of exon 3). GPC ligation was also shown to result in a concomitant loss of erythrocyte viability in wild-type erythrocytes after 24 h in vitro. These results identify a potential pathway linking GPC to PS exposure on erythrocytes that may have a role in regulating red cell turnover. Further characterization of this pathway may also identify new targets for the treatment of P. falciparum malaria.

We report four pedigrees of the group of Na(+)-K(+)-leaky red cell disorders of the 'hereditary stomatocytosis' class. Each showed pseudohyperkalaemia because of temperature-dependent loss of K(+) from red cells on storage of whole blood at room temperature. All pedigrees showed an abnormality in the temperature dependence of the 'passive leak' of the membrane to K(+). Two pedigrees, both of which showed a compensated haemolytic state with dehydrated red cells and target cells on the blood film, showed a novel pattern, in which the profile was flat between 37 degrees C and about 32 degrees C then dropped as the temperature was reduced to zero. The third showed the 'shallow slope' profile, with stomatocytes on the blood film and very markedly abnormal intracellular Na(+) and K(+) levels. Minimal haemolysis was present. The fourth pedigree, of Asian origin, showed the shoulder pattern (minimum at 32 degrees C, maximum at 12 degrees C) with essentially normal haematology. Both of these latter two forms have previously been seen in other pedigrees. The first variant represents a novel kind of temperature dependence of the passive leak found in these pedigrees presenting with pseudohyperkalaemia.

'Cryohydrocytosis' is an unusual human haemolytic anaemia of the 'hereditary stomatocytosis' group, in which the red cell membrane is abnormally permeable to Na and K+ at both body and (even more prominently) refrigerator temperatures. If whole cryohydrocytosis blood is anticoagulated in heparin or EDTA and stored on ice overnight, about 50% of the cells will lyse. Citrate phosphate dextrose adenine (CPDa) anticoagulant, empirically verified as an optimal anticoagulant for storage of normal blood before transfusion, very markedly ameliorated this overnight lysis, suggesting that these cells might form an informative model in which cold storage of the red cell could be studied in a short time scale. Accordingly, we conducted studies of ion flux, cell swelling and lysis in different media used historically for blood preservation and compared the experimental data with an 'integrated red cell model', which seeks mathematically to model the osmotic behaviour of red cells under different conditions. Upon experiment, lysis in these cells was reduced by additives that could be regarded as impermeant extracellular solutes (citrate, mannitol) and by low pH, but not by those agents that are regarded as protecting the cell against energy depletion or oxidation (adenine, glucose, nicotinic acid). The protective effects of these extracellular additives were all reproduced by the computer simulation, confirming the validity of this model, although the effect of pH could be simulated only semi-quantitatively, possibly because Na+ permeability itself depends on pH.

Nonsense/stop mutations in the ankyrin-1 gene (ANK1) are a major cause of dominant HS (dHS) (frequency of 23% in German dHS patients). To date, no common mutation has been found and therefore a simple mutation screening is not feasible. The reduced expression of one cDNA allele in the (AC)n microsatellite polymorphism of the ankyrin-1 gene, as seen in about 20% of Czech patients with dHS, may identify candidates with a possible frameshift/nonsense mutation. In order to verify the efficiency of this screening we screened the ankyrin-1 gene of 22 Czech dHS patients for both the reduced cDNA allele expression in the frequent (AC)n and the common exonic 26/39 polymorphisms, as well as for polymerase chain reaction (PCR) single-stranded conformation polymorphisms in any one of the 42 exons of ANK1. Anomalous PCR products were sequenced. We found seven new ANK1 frameshift/nonsense mutations in nine patients with, but in none of six patients without, a reduced cDNA allele expression (efficiency of 78%). We conclude that screening of dHS patients for such a reduced allele expression in common ANK1 polymorphisms is an efficient procedure for the identification of candidates for frameshift/nonsense mutations in the ankyrin-1 gene.

The tendency for thrombosis to occur if haemolysis persists after splenectomy is especially marked in "hereditary stomatocytosis", in which the red cell membrane "leaks" Na and K. A 21-year-old woman, who was splenectomized in childhood for a congenital haemolytic state, presented with major pulmonary embolism that recurred despite anticoagulation. Tests showed a significant cation leak with a "shallow-slope" abnormality in temperature dependence. Allogeneic bone marrow transplantation caused the thrombophilic state to cease and subsequently anticoagulation was stopped without recurrence of thromboembolism. However, she died 9 months after transplantation: iron overload, intensified by the transfusion demands of the transplant, was a major factor.

The hereditary stomatocytoses are a group of dominant haemolytic anaemias that show two main features: invaginated, 'stomatocytic' morphology; and a membrane leak to the univalent cations Na and K. A patient with the most severe variant of these conditions was reported to show a defect in an in vitro process of ATP-dependent endocytic vesiculation (ADEV), which is found in normal red cells. We have examined this endocytosis process in 11 leaky red cell pedigrees available to us in the UK. ADEV in broken membranes was absent only in the two most severely affected, 'overhydrated' pedigrees studied, both of which showed a deficiency in the membrane raft protein, stomatin. The process was present, although typically diminished by about 10-20% compared with normal red cells, in all others. The cross-linker dimethyl adipimate (DMA), which could correct the cation leak in some of these patients, also corrected the ADEV defect in the same patients. In those patients in whom DMA had no effect on the ion leak, ADEV was not absent. In normal cells, this process of vesiculation was inhibited by inhibitors of membrane 'raft' function, by an antistomatin antibody and by vanadate and N-ethyl maleimide, but not by inhibitors of a number of kinases. These data highlight the heterogeneity of these conditions. A mechanism is discussed by which a defect in raft-based endocytosis could lead to the exaggerated surface exposure of an ion channel, which could then function constitutively, i.e. 'leak'.

The inexplicable severity of anti-Pr autoimmune haemolytic anaemia led us to test the hypothesis that the haemolysis was primarily due to a change in the function of glycophorin A, on which the Pr antigen is located. The lectins Maclura pomifera and wheat germ agglutinin that bind to glycophorin A induced the haemolysis of normal erythrocytes in vitro. Lectin binding led to an increase in erythrocyte membrane permeability to sodium and potassium, the former resulting in an influx of water and subsequent haemolysis. The response was glycophorin A specific as Concanavalin A, which binds to band 3, did not cause haemolysis and peanut agglutinin only did so after removal of erythrocyte sialic acid. The lectin-induced cation leak was not mediated by activation of cation channels as the inhibitors, tetrodotoxin, amiloride and 4,4' disothiocyanate stilbene 2,2'disulphonate, had no effect, suggesting that the haemolysis was due to exacerbation of the inherent cation permeability of the erythrocyte membrane. A human IgAK anti-Pr autoantibody and a mouse anti-human glycophorin A antibody increased erythrocyte permeability to sodium. The role of glycophorin A in stabilizing and, upon aggregation, destabilizing the phospholipid bilayer is discussed. Our findings may help explain the severity of anti-Pr autoimmune haemolytic anaemia and other pathophysiological changes in human erythrocytes.

The structure of red blood cell (RBC) membranes in homozygous sickle cell disease (SCD) is significantly disturbed, with an increased exposure of aminophospholipids (phosphatidylserine and phosphatidylethanolamine) at the outer surface, responsible for a procoagulant activity of SS RBCs. Aminophospholipids are known not only to promote procoagulant reactions, but also to support inhibition of blood coagulation by the protein C system. The aim of the present study was to examine whether SS RBCs could serve as a catalytic surface for the inactivation of factor Va by activated protein C (APC). Venous blood was obtained from 19 consecutive SS patients and 13 controls (AA). In all SS patients, the amount of phosphatidylserine exposed at the outer surface of RBCs was increased compared with controls, as demonstrated by a prothrombinase assay. In addition, SS RBCs significantly (P < 0.0001) increased the rate of FVa inactivation by APC: the mean values (and ranges) of the factor Va inactivation rates were 30 (0-57) vs 9.5 (0-32) mmol Vai/min/mol APC for SS RBCs and normal RBCs respectively. Our results indicate that SS RBCs provide a catalytic surface for the negative control of blood coagulation, which may partially control the procoagulant activity of these cells.

We have investigated a Welsh pedigree showing the 'familial pseudohyperkalaemia' phenotype of dominantly inherited, red-cell-based, temperature-dependent pseudohyperkalaemia associated with normal haematology. The 'passive leak' to K across the membrane of these abnormal red cells showed a 'U-shaped' temperature dependence, with a minimum at about 23 degrees C, qualitatively similar to that seen in the frankly haemolytic 'cryohydrocytosis' variant of the hereditary stomatocytosis group. Like three previous pedigrees with cryohydrocytosis, these patients show an excess of ether lipids in the membrane. However, these patients differ from other 'familial pseudohyperkalaemia' pedigrees, in which the leak showed different temperature profiles.

In this study, we present evidence that red blood cell (RBC) membrane p68 in the Belgrade (b/b) rat is similar if not identical to rat serum albumin (RSA). Structural homology between RSA and the RBC p68 has been determined by a variety of biochemical and immunological criteria. This albumin-like protein is a normal constituent of rat RBC and it is partially exported by exosomes during erythroid differentiation. The endogenous origin of rat RBC albumin-like protein was demonstrated by monitoring protein synthesis in reticulocytes and by identification of reticulocyte mRNA for albumin. Haemolytic anaemia, either hereditary (b/b rat) or drug-induced (phenylhydrazine-treated normal rat), results in increased accumulation of the albumin-like protein in rat RBCs as a result of its induced synthesis.

Congenital dyserythropoietic anaemia type II (CDA II) is well known for glycosylation abnormalities affecting erythrocyte membrane glycoconjugates that encompass hypoglycosylation of band 3 glycoprotein and accumulation of glycosphingolipids: lactotriaosylceramides, neolactotriaosylceramide and polyglycosylceramides. These abnormalities were not observed in erythrocytes from patients with CDA of either type I or III. Recently, however, we have described a CDA type I patient in Poland with identical, though less pronounced, glycoconjugate abnormalities to those observed in patients with CDA type II. The abnormalities included partial unglycosylation of O-linked glycosylation sites in glycophorin A. These abnormalities are now reported in three Bedouin patients from Israel with CDA type I. In addition, the erythrocyte membranes of these patients exhibited highly increased globotetraosylceramide content. Glycoconjugate abnormalities were also present in erythrocyte membranes from three patients from Northern Sweden with CDA type III but they almost exclusively affected glycosphingolipids. In erythrocytes of all patients examined including one with CDA type II, polyglycosylceramides were significantly hypoglycosylated although, on a molar basis, their contents in erythrocyte membranes were increased. Thus, glycoconjugate abnormalities of varying intensity occur in erythrocyte membranes from all patients with CDA that were investigated.

Two families with inherited abnormalities in Na and K transport across the red cell membrane are described. Both presented with 'pseudohyperkalaemia' as a result of loss of K from the red cells on storage at room temperature. Routine haematology was essentially normal, except for macrocytosis in one family. Studies of the temperature dependence of the passive leak to K showed a novel shoulder pattern with a minimum at 25 degrees C, a maximum at 10 degrees C, followed by a further fall. As in other cases of red cell-based pseudohyperkalaemia, the abnormal temperature dependence of this 'leak' flux could be held to account for the loss of K from the cells at room temperature. These cases represent a novel variant of the temperature dependence of the passive leak of K and Na across the red cell membrane, and can be classified as a mild, non-haemolytic form of the group known as the hereditary stomatocytosis and allied disorders'.

Iron-dependent oxidative reactions in beta-thalassaemic erythrocyte membranes are involved in premature cell removal and anaemia. We studied 22 beta-thalassaemia intermedia patients (i) to assess if membrane iron accumulation influences the oxidative damage to thalassaemic cells, and (ii) to see whether the mechanisms of haemoglobin destabilization described in vitro have indicators in circulating erythrocytes. Serum non-transferrin-bound iron as potentially toxic iron for erythrocytes was also evaluated. Membrane-bound free iron significantly correlated to bound haemichromes, suggesting a causal relation, but was poorly related to serum non-transferrin iron, which seems to contribute little to damage from outside the cells. The spleen played an important role in the removal of cells with more membrane iron.

The flow cytometric test measures the fluorescence intensity of intact red cells labelled with the dye eosin-5-maleimide, which reacts covalently with Lys-430 on the first extracellular loop of band 3 protein. In this study, red cells from patients with hereditary spherocytosis (HS), congenital dyserythropoietic anaemia type II, South-east Asian ovalocytosis and cryohydrocytosis have produced a greater degree of reduction of mean channel fluorescence readings than those for other patient groups and normal controls. The predictive value of this test for membrane abnormality was compared with the results obtained from the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method, which is currently the reference laboratory test for the identification of membrane protein deficiencies in hereditary spherocytosis and for the detection of spectrin variants in hereditary elliptocytosis. The dye method is a reliable, speedy diagnostic test (2 h from sample collection to result) for HS with a sensitivity of 92.7% and a specificity of 99.1%. Thus, it will serve well as a first-line screening test for the diagnosis of hereditary spherocytosis in routine haematology.

Congenital dyserythropoietic anaemias (CDAs) are rare hereditary disorders characterized by ineffective erythropoiesis and multinuclearity of erythroblasts. Three main types of the disease have been described. Glycoconjugate abnormalities in erythrocyte membrane glycoconjugates, consisting of hypoglycosylation of band 3 and accumulation of certain glycosphingolipids including lactotriaosylceramide, neolactotriaosylceramide and polyglycosylceramides, have been described only in patients with CDA type II (CDA-II). We report on identical, although less pronounced, abnormalities in erythrocyte glycoconjugates from a patient with CDA-I. A low degree of hypoglycosylation of band 3 in our patient with CDA-I suggests that hypoglycosylation is not a cause, but, most probably, a consequence of dyserythropoiesis.

Haematological data, genotype, transfusion requirements, metabolic indicators of oxidative stress (flux via hexose-monophosphate shunt (HMPS); steady state level of GSH and GSSG, NADPH and NADP; activity of anti-oxidant enzymes), parameters of membrane damage (aggregated band 3; membrane-bound haemichromes, autologous immunoglobulins (Igs) and C3 complement fragments) and erythrophagocytosis were measured in erythrocytes (RBC) of 15 beta-thalassaemia intermedia patients (nine splenectomized) with low, if any, transfusion requirements. Patients presented increased aggregated band 3, bound haemichromes, Igs and C3 complement fragments, and increased erythrophagocytosis. Bound haemichromes strongly correlated with aggregated band 3. Anti-band 3 Igs were predominantly associated with aggregated band 3. Erythrophagocytosis positively correlated with aggregated band 3, haemichromes and Igs, suggesting the involvement of haemichrome-induced band 3 aggregation in phagocytic removal of beta-thalassaemic RBC. Splenectomized patients showed higher degrees of membrane damage and phagocytosis, significantly higher numbers of circulating RBC precursors, and tendentially higher numbers of reticulocytes. Basal flux via HMPS was increased twofold, but HMPS stimulation by methylene blue was decreased, as was the glucose flux via HMPS. GSH was remarkably decreased, whereas NADPH was increased. Except for unchanged catalase and glutathione reductase, anti-oxidant enzymes had increased activity. Negative correlation between HMPS stimulation by methylene blue and bound haemichromes indicated that the ability to enhance HMPS may counteract haemichrome precipitation and limit consequent membrane damage leading to erythrophagocytosis.

K562 cells undergoing differentiation induced by 1-beta-D-arabino-furanosyl-cytosine (ara-C) were examined as a model for studying the biosynthesis and regulation of Rh and other blood group active membrane proteins. Untreated and ara-C-induced K562 cells were analysed for the expression of these proteins using monoclonal antibodies in combination with flow cytometry. The major membrane proteins glycophorins A and C remained unaltered upon induction by ara-C. The display of LFA-3 (CD58) and DAF (CD55) by uninduced K562 was one order of magnitude lower than that of the glycophorins; following ara-C treatment there was a 50% rise in LFA-3 but a modest decrease in the level of DAF expression. The expression by untreated K562 cells of Rh, Lutheran and Kell proteins as well as the Rh D antigen was low, whereas that of CD44 and band 3 protein was negligible. Following induction by ara-C the levels of Rh and Kell proteins rose up to 7- and 3.5-fold respectively, and there was an increase in RhD-antigen expression. In contrast, ara-C induction of K562 cells failed to augment their display of Lutheran, CD44 and band 3 proteins. Analysis of Rh transcripts following the purification and RT-PCR analysis of K562 mRNA showed that uninduced K562 cells contain two distinct mRNAs corresponding to Rh Ce (1.8 kb) and Rh D (3.5 kb). The apparent concentration of each mRNA increased following induction with ara-C. K562 plasma membranes also contained Rh polypeptides as determined by immunoblot analysis using anti-Rh polypeptide rabbit polyclonal sera raised to Rh synthetic peptides. A novel hybrid Rh transcript corresponding to exons 1-4 of RHD and exons 5-10 of RHCE has been cloned and sequenced from ara-C induced K562 cells, and may have arisen by general recombination between the RHD and RHCE genes.

A Calabrian family (Southern Italy) with Sp alpha(I/74) hereditary elliptocytosis (HE) in the heterozygous state was studied. Sp alpha(I/74) HE is associated with asymptomatic elliptocytosis, a defect in spectrin dimer self association and an increase of the alpha(I/74) kD fragment from the alpha chain after partial tryptic digestion of spectrin. To identify the underlying molecular defect, we analysed exons V, W, X, Y, Z of the beta gene and exon 2 of the alpha gene by single-strand conformational polymorphism (SSCP) of the amplification products. Direct DNA sequencing of the mutant exon showed a C-->G substitution at position 6284 of the beta gene. The corresponding substitution at the protein level was Arg-->Pro in the 2064 position of the beta-spectrin chain.

The Kx protein is an erythrocyte membrane polypeptide which is deficient in rare individuals suffering from the McLeod syndrome. The gene encoding this protein has been recently cloned and the Kx protein independently purified as a covalent complex with the Kell blood group protein. To further study the Kx membrane protein, antisera raised in rabbits against six synthetic peptides derived from the primary sequence of this protein were characterized. All antisera but two precipitated the recombinant Kx protein synthesized in coupled transcription-translation in vitro. Three antisera reacted on immunoblots with the 37 kD Kx protein present in the purified Kell-Kx complex and in SDS red cell membrane lysates from variants with different Kell blood group phenotypes, including Ko, which lack the Kell protein of 93 kD. However, no reactivity was found with McLeod preparations lacking Kx protein, thus clearly indicating that these antibodies have a Kx specificity. Unexpectedly, the relative amount of Kx protein in Ko cells was found to be lower than in red cells with common Kell phenotypes, suggesting that the absence of the Kell protein may alter the amount of Kx in the membrane.

Alpha-thalassaemic erythrocytes develop a specific membrane skeletal defect that is manifest as a loss of normal spectrin-binding sites on the inner surface of the thalassaemic membranes. To test whether this lesion could be caused by the excess free beta-globin chains that accumulate in alpha-thalassaemic red cells, we incubated normal red cell membranes with native, haem-containing alpha or beta globin chains or with haemoglobin A. Spectrin-depleted inside-out membrane vesicles (IOVs) derived from membranes incubated with beta-globin chains bound only 9 +/- 3% as much spectrin as IOVs from control membranes incubated with bovine serum albumin. In contrast. IOVs from membranes incubated with alpha-globin chains or haemoglobin A were nearly normal (79 +/- 3% and 86 +/- 5% of controls, respectively). This differential effect of globin chains was not seen when membranes were first transformed into spectrin-depleted IOVs and then incubated with the isolated globin chains. Under these conditions, both alpha and beta globin chains reduced the spectrin-binding capacity of the IOVs by approximately 45% (alpha 46 +/- 7%, beta 43 +/- 6%) whereas haemoglobin A had no effect. Unlike IOVs, spectrin isolated from membranes exposed to alpha or beta globin chains bound normally to IOVs and to actin (in the presence of protein 4.1). These studies show that isolated beta-globin chains (but not alpha-globin chains) can produce a spectrin-binding defect in normal red cell membranes similar to that seen in alpha thalassaemia. The existence of similar defects in the membrane skeletons of red cells from other diseases with unstable beta globins suggests a common pathophysiology for the premature destruction of these cells.

We report a case of apparently recessive hereditary spherocytosis in an Italian child. The proband exhibited a reduction of overall ankyrin in the red cell membrane. The parents were free of any haematological manifestations. The VNDR associated with the ankyrin gene (ANK1) were consistent with the following diplotypes: AC11/ AC14 (father), AC14/AC14 (mother) and AC11/AC14 (child). The cDNA of the patient disclosed the expression of the AC11 allele only. As a consequence, we put forward the hypothesis of a de novo inactivation affecting the ankyrin allele of maternal origin (AC14) and accounting for the disease. PCR amplification of exons, SSCP analysis and nucleotide sequencing disclosed a polymorphism: GAC --> AAC; Asp --> Asn in codon 328 of exon 10, and a one-nucleotide deletion : CTG --> CG in codon 573 of the exon 16. This frameshift mutation placed in phase the TGA triplet that normally overlaps codons 636 and 637. Termination of translation near the middle of ankyrin mRNA coding sequence resulted, presumably, in its premature degadation. The present allele has been designated allele Napoli.

We studied a large Swiss family with dominantly inherited hereditary spherocytosis and band 3 (anion exchanger 1, AE1) deficiency. Band 3 cDNA was analysed by single-strand conformation polymorphism analysis and nucleotide sequencing. A new point mutation was found: G771D (GGC-->GAC). This change was present in all eight investigated patients but absent in four healthy members of the family. It is located at a highly conserved position in the middle of transmembrane segment 11, introducing a negative charge in a stretch of 16 apolar or neutral residues. None of the six amino-acid substitutions already known in this region as being associated with band 3 deficiency were recorded. To rule out any major transcriptional or post-transcriptional defect, we evaluated the amount of band 3 mRNA by RNase mapping using a band 3-protein 4.1 chimaeric probe. Similar mRNA amounts were present in patients and controls. Our results strengthen the view that some amino-acids, that are well conserved throughout the AE family, may be crucial for the insertion and/or the stabilization of band 3 within the lipid bilayer. At the present time, most of the mutations altering such residues are located in the C-terminal region of band 3.

Both beta-thalassaemic red blood cells and normal red blood cells (RBC) artificially loaded with unpaired alpha-haemoglobin chains exhibit increased amounts of membrane-bound haem and iron. In the model beta-thalassaemic RBC the amount of free haem and iron was as much as 20 times that which could have been contributed by the entrapped alpha-haemoglobin chains alone. This excess haem/iron arises from destabilization of haemoglobin via reactions between ferric iron (Fe3+), initially contributed by the unpaired alpha chains, and cytoplasmic constituents, primarily reduced glutathione (GSH). Indeed, in the presence of Fe3+ (100 microM) addition of even small amounts of GSH (0.5 mM) to dilute RBC haemolysates (0.15 mg haemoglobin/dl) greatly accelerated methaemoglobin formation. In contrast, lysates from GSH-depleted RBC demonstrated a significantly reduced rate of iron-mediated haemoglobin oxidation which was reversible by addition of GSH. The initiation, and subsequent propagation, of Fe(3+)-mediated haemoglobin oxidation was significantly inhibited by iron chelators. Finally, Fe(3+)-driven haemoglobin oxidation was synergized by low amounts of H2O2, an oxidant spontaneously generated in thalassaemic RBC. To summarize, the release of small amounts of free iron from unpaired alpha-haemoglobin chains in the beta-thalassaemic RBC can initiate self-amplifying redox reactions which simultaneously deplete cellular reducing potential (e.g. GSH), oxidize additional haemoglobin, and accelerate the red cell destruction.

A novel compound heterozygous mutation of 317 CGC-->TGC with 142 GCT-->ACT in human red cell band 4.2 deficiency is described. A proband and his son suffered from compensated haemolysis with nearly complete deficiency of red cell band 4.2. Their red cell morphology exhibited microspherocytosis resembling classic hereditary spherocytosis (HS). Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed band 4.2 to be nearly missing (< 1% of normal controls) with the presence of 74 kD and 72 kD isoforms in trace amounts. Other family members (daughters older and younger than the son) exhibited nearly normal amounts of 72kD as a wild form of band 4.2 on SDS-PAGE with the presence of the 74kD isoform in a trace amount. The proband and his son demonstrated two compound heterozygous mutations in trans: i.e. nucleotide (nt) 949 CGC-->TGC (codon 317 Arg-->Cys) in exon 7 and nt 424 GCT--ACT (codon 142 Ala-->Thr) in exon 3 of the band 4.2 gene. The two daughters demonstrated only the mutation of nt 949 CGC-->TGC in exon 7 in heterozygous states, but no 142 mutation. Therefore the proband and his son were compound heterozygotes of these two mutations in trans. It is interesting to note that the 74 kD isoform of band 4.2 protein existed in a trace amount in the two daughters in spite of the absence of the 142 Ala-->Thr mutation. In addition, even in the presence of the 142 mutation in one allele in the proband and his son, their red cell morphology demonstrated classic HS with microspherocytosis, although a homozygous state of the 142 mutation known as the Nippon type of band 4.2 deficiency exhibits ovalostomatocytosis.

Hereditary xerocytosis (HX) is a rare haemolytic disease due to dehydrated red blood cells (RBCs). A unique feature of this syndrome is that affected members often show normal or near normal haemoglobin levels despite clinical and laboratory evidence of mild to moderate haemolysis. The diagnostic clue is the association of markedly increased RBC Na+ + K+ fluxes with low total cation (Na+ + K+) content. 11 patients of six unrelated families of Spanish origin with HX have been studied from clinical, genetical and biological points of view. In addition, we have investigated the sensitivity of RBC membrane to heat at three different incubation times (15, 30 and 60 min) and two different temperature values (46 degrees C and 49 degrees C). Under these conditions control RBCs (50 normal subjects) exhibited at 49 degrees C and 30 min a maximum of 30% fragmented RBCs. This value increased to 80% after 60 min of incubation. In contrast, patients with HX showed significantly lower percentages of fragmented RBCs at both 30 and 60 min of incubation (maximum 10% and 30%, respectively). In an attempt to determine if increased heat stability was unique to HX RBCs, several other congenital membranopathies with haemolytic anaemia were also studied. The degree of fragmentation, except in one case of HPP (which was strongly increased), did not differ from the control group. Electrophoretic studies of membrane proteins performed in RBCs of all the patients with HX did not explain any qualitative nor quantitative abnormality. In addition to its physiopathological interest, study of RBC heat stability, together with other haematological parameters (increased MCHC and decreased RBC osmotic fragility), may be useful for HX diagnosis, especially in laboratories which are not equipped to evaluate RBC membrane permeability.

Hereditary spherocytosis (HS) is a very heterogenous condition both at clinical and biochemical level. To establish the relationship between these aspects we performed a clinical and biochemical study in 87 Italian HS subjects. Patients were divided into three groups based on clinical severity (mild, typical and severe) and into five subgroups based on specific membrane abnormalities identified by polyacrylamide gel electrophoresis (isolated spectrin deficiency, spectrin deficiency combined with mild ankyrin reduction, spectrin deficiency combined with severe ankyrin reduction, band 3 reduction and isolated protein 4.2 reduction). We were not able to assess any alteration in six HS patients. A good correlation between clinical HS forms and membrane protein defects is shown. We conclude that erythrocyte membrane analysis should be carried out after diagnosis of HS in order to predict the clinical course of the disease.

To examine the characteristics of 'young' human red cells, we studied blood from seven healthy male volunteers who developed systemic reticulocytosis during a 3-week blood donation period. Each of these subjects donated a total of 6 units (450 ml/unit) of blood (2 units/week for 3 weeks) with subcutaneous recombinant erythropoietin (SC rEPO; 200 U/kg daily for 3 weeks). Two of these subjects were also studied with a similar protocol in the absence of rEPO (4.5 +/- 0.5 units donated). SC rEPO administration was associated with an increased K content of the erythrocyte and with the appearance of hypochromic cells, which were initially normocytic and then became normochromic and microcytic. Measurements of cation transport revealed that, with the exception of the Na-K-Cl cotransport, all the systems studied increased their activities following blood donations with or without SC rEPO. The increase was highest in the K-Cl cotransport (2- and 5-fold for control and rEPO parts of the study, respectively), while the Na-K pump increased slightly in control and 40% with rEPO. The Na-Li countertransport increased 40% and 100% in the control and rEPO parts of the study, respectively. Concomitant with increased ion transport activity, electron microscopic studies of plasma and red cells of subjects receiving SC rEPO showed the presence of circulating exosomes and cytoplasmic multivesicular bodies. The transferrin receptor was detected in the circulating exosomes, thereby providing evidence that, as do nonhuman red cells, maturing human reticulocytes shed exosome-associated transferrin receptors.

The alpha- and beta-thalassaemias are characterized by decreased erythrocyte deformability. To determine what effects excess alpha- and beta-haemoglobin (globin) chains have on cellular and membrane deformability, purified haem-containing alpha- and beta-chains were entrapped within normal erythrocytes. Entrapment of purified alpha-chains in normal erythrocytes resulted in a significant decrease in cellular and membrane deformability similar to that observed in beta-thalassaemia intermedia. The decreased deformability was correlated with alpha-chain membrane deposition, an alteration in membrane proteins and a decrease in membrane reactive thiol groups. These changes in membrane and cellular deformability were time dependent and closely correlated with membrane alpha-chain deposition. The membrane changes and the loss of membrane deformability appeared to account for the loss of cellular deformability in the alpha-chain loaded cells. While both beta-chain loaded and Hb H erythrocytes demonstrated a significant loss of cellular deformability, this loss was less pronounced than in the alpha-chain loaded and beta-thalassaemic cells and may arise from either the increased intracellular viscosity of the beta-chain loaded cells or to the smaller amount of membrane bound globin. In summary, these studies demonstrate that alteration of cellular and membrane deformability occurs very rapidly and as a direct consequence of the autoxidation and membrane binding of the unpaired globin chains.