Leach Phenotype Research Articles

Glycophorin C ligation: another biochemical pathway in red blood cell senescence?

In this issue of TRANSFUSION two articles describe the effects on erythroid cells of antibody ligation to the red blood cell (RBC) membrane glycoprotein glycophorin C (GPC).1, 2 GPC is a monotopic high-abundance RBC membrane protein that forms a structural linkage to the membrane skeleton via the proteins 4.1R and p55. GPC derives from the GYPC gene, which additionally expresses GPC in a truncated form, glycophorin D (GPD). Glycophorins are extensively sialyated and contribute significantly to the zeta potential (net negative charge) that prevents systemic aggregation of RBCs by mutual repulsion. RBCs lacking both GPC and GPD are known as the Leach phenotype and have irregular ellipsoid shape. GPC and GPD express the 11 Gerbich blood group antigens,3 and three Gerbich-negative phenotypes are known, Gerbich (Ge:–2,–3,4), Yus (Ge:–2,3,4), and the aforementioned Leach phenotype (Ge:–2,–3,–4). Anti-Gerbich (notably anti-Ge3) has previously been noted as a cause of hemolytic disease of the fetus and newborn (HDFN; and further described here by Pate and colleagues2), so knowledge regarding the immune destruction of the fetal RBCs is of relevance to the clinical management of these cases. The study of Wang and coworkers1 reveals that ligation using anti-GPC induces cell death in the erythroleukemic cell line K562 and erythroid progenitors derived from CD34+ umbilical cord blood stem cells. This process remains only partially characterized biochemically, but involves the exposure of phosphatidylserine (PS) at the extracellular leaflet of the membrane (it is normally polarized to the intracellular face of the membrane). These findings confirm our earlier study,4 which interestingly also found that Gerbich and Yus phenotype cells lacked this pathway, despite binding anti-GPC on their surface. Thus, anti-Ge3 causing the HDFN described in previous studies, most likely induces PS exposure on fetal RBCs and induces their death and is clearly a different mechanism to that induced by anti-D which involves monocyte binding to the Fc region of RBC bound antibody. The GPC pathway induced by anti-Ge3 (and other anti-GPC) most likely is mimicking binding to the protein by an as yet unknown ligand(s), possibly on macrophages or other white blood cells. The mechanism of PS exposure induced by GPC ligation is also intriguing; the previously mentioned GPC-p4.1R-p55 interaction clearly must trigger the PS exposure in some manner. Indeed, p4.1R is known to bind PS and is regulated by calmodulin.5 It is therefore important that this pathway is unraveled as it may play a fundamental part in RBC turnover. It is somewhat puzzling that despite its huge physiologic significance, mechanisms of RBC turnover (dubbed eryptosis) remain relatively poorly characterized; this deficiency no doubt will be rectified based on the identification of the key RBC membrane proteins that appear to be involved. GPC is now among the candidates. The author is a member of the Transfusion Medicine Advisory Board (TMAB) for Grifols SA.

The Ernest Witebsky memorial lecture. Red but not dead: not a hapless sac of hemoglobin.

The purpose of this presentation was to demonstrate that the red blood cell is not a hapless sac of hemoglobin and that much research is still needed for better understanding of its complexities. After a brief historical introduction the following subjects are presented: 1). Phosphofructokinase is the rate limiting step in the anaerobic glycolytic pathway. Ribose-5-phosphate, a metabolite of the oxidative pentose phosphate pathway is essential for the generation of phosphoribosylpyrophosphate which in turn is needed for the synthesis of adenosine monophosphate from adenine by the action of adenine phosphoribosyl transferase. 2). There are at least 17 blood group systems with more than 400 epitopes expressed on the red cell membrane. The Rh null and the McLeod phenotypes associated with abnormally shaped red cells and hemolytic anemia are briefly described as is the present understanding of the nature of the Rh complex. 3). The structure of the cytoskeleton and the composition and behavior of the lipid bilayer are presented with some discussion of the MN and Ss sialoglycoproteins and the Leach phenotype. 4). Touched upon is the role of phosphoinositides with some emphasis on recent discoveries relating to the glycophosphoinositide protein anchor. 5). The intricacies of the many faceted transport mechanisms are introduced. Briefly mentioned are the mechanisms activated when regulatory volume adjustments occur in fine tuning red cell volume after exposure respectively to hypotonic or hypertonic stress. Sufficient evidence is presented to convince that a cell doesn't have to have a nucleus to be respected even though it is just a corpuscle.

Gerbich blood groups and minor glycophorins of human erythrocytes

Four main glycophorins which can be specifically detected by periodic-acid-Schiff (PAS) staining after separation of red cell membranes by SDS-polyacrylamide gel electrophoresis have been identified and are known under different nomenclatures. Here, the designation of glycophorins A, B and C and glycophorin D will be used. A new member designated glycophorin E (GPE) has been recently identified in the course of molecular genetic studies. These glycophorins represent about 2% of the total erythrocyte membrane protein mass and have been fully characterized both at the protein and at the DNA level. Accordingly, these molecules can be subdivided into two groups that are distinguished by distinct properties such as blood group antigenic properties, apparent M(r), copy number, attached glycans, detergent solubility, and gene structure. GPC and GPD are minor sialoglycoproteins contributing to 4 and 1% to the PAS-positive material and are present at about 2.0 and 0.5 x 10(5) copies/cell, respectively. Both carry blood group Gerbich (Ge) antigens. Protein and nucleic acid analysis indicated that GPD is a truncated form of GPC in its N-terminal region and that both proteins are produced by a unique gene which is present as a single copy on chromosome 2q14-q21. GPC and GPD are produced from the same gene through use of alternative translation initiation sites. These proteins and the GYPC gene share no homology with the GPA, GPB and GPE proteins and the GYPA gene cluster, respectively. Thus, the glycophorin name, which suggests that all these sialoglycopropteins have a common genetic origin, might be now considered as a misnomer. As a further difference between the two groups of membrane proteins, GPC and GPD are expressed both in erythroid and non erythroid tissues, but the level of transcription is much higher in erythroid than in non erythroid tissues and in addition the proteins are differently glycosylated in the two cell types. Increasing evidence suggests a significant role for GPC and GPD in the regulation of the red cell shape and the membrane mechanical properties by providing a membrane linkage site for cytoskeletal proteins, especially proteins 4.1 and p55. The total lack of GPC and GPD in the red cell membrane is associated with hereditary ellyptocytosis in the Leach phenotype and the molecular basis of these defects have been elucidated.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional factors in the red cell membrane: interactions between the membrane and its underlying skeleton.

Recent studies involving two abnormal red cell phenotypes (South-east Asian ovalocytosis and Leach phenotype) provide novel information concerning the nature and significance of interactions of both the anion transport protein AE-1 (syn. band 3) and Glycophorins C and D with the underlying skeleton. The location of Wra and Dia blood group antigens to mutations on AE-1 at residues 658 and 854 respectively, together with the availability of monoclonal antibodies recognising epitopes dependent upon the integrity of the third extracellular loop of AE-1, have allowed us to study the organisation of the membrane domain of the mutant AE-1 found in South-east Asian ovalocytes (AE-1 SAO). The results suggest that the organisation of the whole membrane domain of AE-1 SAO is abnormal and that the organisation of other integral membrane proteins like those involved in expression of Rh blood group antigens may also be affected. Increased homo- and hetero-associations involving AE-1 SAO and other integral proteins may in turn result in reduced membrane flexibility. Purified protein 4.1 binds with 50-fold higher affinity to protein 4.1 depleted normal red cell membranes than to protein 4.1 depleted red cell membranes of Leach phenotype which lack Glycophorin C (GPC) and Glycophorin D (GPD). Experiments using purified protein 4.1 and p55 together with synthetic peptides corresponding to different regions of the cytoplasmic domain of Glycophorins C and D (GPC/D) demonstrate that protein 4.1 interacts directly with GPC through residues 82-98. They also show that p55 binds to GPC through residues 112-128. Since p55 also binds directly to protein 4.1 it is clear that protein 4.1 can bind to GPC through two different sites either directly through residues 82-98 or indirectly through p55. These results show that GPC and GPD provide major attachment sites for the red cell skeleton via protein 4.1 and that p55 is part of this complex.

Molecular basis of glycophorin C variants and their associated blood group antigens.

The normal and variant forms of GPC and GPD molecules carry antigens of the Gerbich blood group system. This blood group system comprises three high-incidence antigens (Ge2, Ge3 and Ge4) and four low-incidence antigens (Wb, Lsa, Dha and Ana). Erythrocytes of the Ge and Yus phenotypes lack normal GPC and GPD molecules but express variant molecules (denoted GPC.Ge, GPC.Yus, respectively) that functionally substitute for normal GPC and GPD in the membrane. Leach phenotype cells lack GPC and GPD molecules and are elliptocytic in shape with a membrane that is less deformable than that of normal cells. The Lsa antigen is expressed on higher molecular-weight variants of GPC (GPC.Lsa) and GPD (GPD.Lsa). Wb, Dha and Ana antigens arise from point mutations in the GYPC gene and are expressed on GPC.Wb, GPC.Dha and GPD.Ana, respectively. The structure of each of the variant GPC and GPD molecules and the location of the Gerbich blood group system antigens is discussed. The GYPC gene, located on chromosome 2q14-q21, is 13.5 kb long and comprises four exons. Exons 1, 2 and most of exon 3 encode the N-terminal extracellular domain while the remainder of exon 3 and exon 4 encode transmembrane and cytoplasmic domains of GPC. Exons 2 and 3 are highly homologous, with less than 5% nucleotide divergence. The molecular basis of generation of variation GPC and GPD molecules, and the structure of the GYPC gene from different Leach phenotype individuals, is discussed.

Localization of the protein 4.1-binding site on human erythrocyte glycophorins C and D

The flexibility of the human erythrocyte membrane is mediated by an underlying network of skeletal proteins which interact with the membrane through ankyrin and protein 4.1. The nature of the membrane attachment site(s) for protein 4.1 has yet to be fully elucidated. In this paper we show that purified protein 4.1 binds much less strongly to alkali-stripped membranes from erythrocytes of individuals with total glycophorin C and D deficiency (Leach phenotype) than to alkali-stripped normal membranes. We further show that a synthetic peptide corresponding to amino acid residues 82-98 of the cytoplasmic domain of glycophorin C specifically binds to purified protein 4.1 and inhibits protein 4.1 binding to alkali-stripped normal membranes. The same synthetic peptide binds directly to membranes from individuals with glycophorin C and D deficiency but not to normal membranes. These results indicate that glycophorins C and D provide major membrane attachment sites for protein 4.1 in normal erythrocytes and that this interaction is mediated by protein 4.1 binding to amino acid residues 82-98 of glycophorin C and 61-77 of glycophorin D.

Absence of high-affinity band 4.1 binding sites from membranes of glycophorin C- and D-deficient (Leach phenotype) erythrocytes

We investigated the role of glycophorins C and D in the association of band 4.1 with the erythrocyte membrane by measuring the binding of band 4.1 to erythrocyte inside-out vesicles stripped of endogenous band 4.1. Vesicles were prepared from either normal erythrocytes or erythrocytes completely lacking glycophorins C and D (Leach phenotype). Band 4.1 binding to vesicles from normal erythrocytes gave rise to a nonlinear Scatchard plot, indicative of two classes of binding sites: a low-capacity, high-affinity class of sites (about 10% of the total) and a high-capacity, low-affinity class of sites. Vesicles prepared from Leach erythrocytes had a binding capacity for band 4.1 that was, on average, 32% lower than that of vesicles from normal erythrocytes. This difference was caused by the complete absence of the high-affinity binding sites as well as by a decrease in the number of low-affinity binding sites. Reduction of membrane phosphatidylinositol 4,5-biphosphate (PIP2) content by adenosine triphosphate depletion or activation of phosphoinositidase C resulted in a decrease in band 4.1 binding capacity to a similar extent in both control and Leach vesicles. The principal effect of PIP2 depletion was a reduction in the number of low-affinity band 4.1 binding sites in control and Leach vesicles. The fact that PIP2 depletion induced a decrease in band 4.1 binding to Leach vesicles shows that glycophorin C or D is not required for the formation of PIP2-sensitive band 4.1 binding sites, and may not be involved in PIP2-sensitive band 4.1 binding sites even when they are present. Our studies give new insights into the involvement of glycophorins and of PIP2 in modulating cytoskeletal-membrane interactions.

Absence of High-Affinity Band 4.1 Binding Sites From Membranes of Glycophorin C- and D-Deficient (Leach Phenotype) Erythrocytes

AbstractWe investigated the role of glycophorins C and D in the association of band 4.1 with the erythrocyte membrane by measuring the binding of band 4.1 to erythrocyte inside-out vesicles stripped of endogenous band 4.1. Vesicles were prepared from either normal erythrocytes or erythrocytes completely lacking glycophorins C and D (Leach phenotype). Band 4.1 binding to vesicles from normal erythrocytes gave rise to a nonlinear Scatchard plot, indicative of two classes of binding sites: a low-capacity, high-affinity class of sites (about 10% of the total) and a high-capacity, low-affinity class of sites. Vesicles prepared from Leach erythrocytes had a binding capacity for band 4.1 that was, on average, 32% lower than that of vesicles from normal erythrocytes. This difference was caused by the complete absence of the high-affinity binding sites as well as by a decrease in the number of low-affinity binding sites. Reduction of membrane phosphatidylinositol 4,5-biphos-phate (PIP2) content by adenosine triphosphate depletion or activation of phosphoinositidase C resulted in a decrease in band 4.1 binding capacity to a similar extent in both control and Leach vesicles. The principal effect of PIP2 depletion was a reduction in the number of low-affinity band 4.1 binding sites in control and Leach vesicles. The fact that PIP2 depletion induced a decrease in band 4.1 binding to Leach vesicles shows that glycophorin C or D is not required for the formation of PIP2-sensitive band 4.1 binding sites, and may not be involved in PIP2-sensitive band 4.1 binding sites even when they are present. Our studies give new insights into the involvement of glycophorins and of PIP2 in modulating cytoskeletal-membrane interactions.

Membrane attachment sites for the membrane cytoskeletal protein 4.1 of the red blood cell [see comments

The identity of the membrane binding sites for the membrane cytoskeletal protein 4.1 of the human red blood cell has been investigated. Exhaustive proteolysis of the membrane with a range of proteases led to the elimination of only some 60% of all binding sites. The predominant integral membrane protein, band 3, as well as glycophorin A, was totally digested at levels of proteolysis that were essentially without effect on the number of 4.1 binding sites. Proteolysis caused scission of the polypeptide chain of glycophorin C (together with the minor product, glycophorin D, of the same gene), but left a fragment from the region of the C-terminus still attached to the membrane. We have found a low-molecular weight protein, possessing an epitope (recognized by an antibody directed against the cytoplasmic domain of glycophorin C) in common with this proteolytic fragment, in cells of a Leach phenotype, which are characterized by lack of extracellular epitopes of glycophorin C. When these membranes were extracted at low ionic strength to dissociate the membrane cytoskeleton, approximately half the content of 4.1 was liberated, compared with only some 25% from normal membranes. Cells of a different variant of the Leach phenotype, which are totally devoid of glycophorin C, lost close to 70% of their 4.1 under these circumstances. The Rh(D) transmembrane protein, which interacts with the membrane cytoskeleton, is also resistant to proteolysis of the cytoplasmic membrane surface, but Rhnull cells, devoid of this protein, showed no decreased retention of 4.1. The results suggest that glycophorin C (with D) may contain two types of binding site for 4.1, which would be sufficient in number to account for all the strong binding of 4.1 on normal membranes; modulation of binding at one of the sites by another protein or by lipid is not excluded. A possible site for reinitiation of translation overlapping the premature stop codon in the mutant expressing the truncated glycophorin C can be discerned.

Membrane Attachment Sites for the Membrane Cytoskeletal Protein 4.1 of the Red Blood Cell

The identity of the membrane binding sites for the membrane cytoskeletal protein 4.1 of the human red blood cell has been investigated. Exhaustive proteolysis of the membrane with a range of proteases led to the elimination of only some 60% of all binding sites. The predominant integral membrane protein, band 3, as well as glycophorin A, was totally digested at levels of proteolysis that were essentially without effect on the number of 4.1 binding sites. Proteolysis caused scission of the polypeptide chain of glycophorin C (together with the minor product, glycophorin D, of the same gene), but left a fragment from the region of the C-terminus still attached to the membrane. We have found a low-molecular weight protein, possessing an epitope (recognized by an antibody directed against the cytoplasmic domain of glycophorin C) in common with this proteolytic fragment, in cells of a Leach phenotype, which are characterized by lack of extracellular epitopes of glycophorin C. When these membranes were extracted at low ionic strength to dissociate the membrane cytoskeleton, approximately half the content of 4.1 was liberated, compared with only some 25% from normal membranes. Cells of a different variant of the Leach phenotype, which are totally devoid of glycophorin C, lost close to 70% of their 4.1 under these circumstances. The Rh(D) transmembrane protein, which interacts with the membrane cytoskeleton, is also resistant to proteolysis of the cytoplasmic membrane surface, but Rhnull cells, devoid of this protein, showed no decreased retention of 4.1. The results suggest that glycophorin C (with D) may contain two types of binding site for 4.1, which would be sufficient in number to account for all the strong binding of 4.1 on normal membranes; modulation of binding at one of the sites by another protein or by lipid is not excluded. A possible site for reinitiation of translation overlapping the premature stop codon in the mutant expressing the truncated glycophorin C can be discerned.

Molecular analysis of glycophorin C deficiency in human erythrocytes

Human erythrocyte glycophorin C plays a functionally important role in maintaining erythrocyte shape and regulating membrane mechanical stability. We report here the characterization of the glycophorins C and D deficiency in erythrocytes of the Leach phenotype. Glycophorin C gene is encoded by 4 exons. Amplification of reticulocyte cDNA from Leach phenotype and normal individuals generated a 140-bp fragment when using primers spanning exons 1 and 2. However, no polymerase chain reaction (PCR) products were detected in the Leach phenotype using primers flanking either exons 1 and 3 or exons 1 and 4, suggesting that the 3' end of the mRNA was missing or altered. Exon 4 also appeared to be missing from Leach genomic DNA, based on both Southern hybridization and PCR. These results indicate that an absence of glycophorin C and glycophorin D in erythrocytes from these Leach phenotype individuals is a consequence of a deletion or marked alteration of exon 3 and exon 4 of their glycophorin C gene. Surprisingly, the mutant gene encodes an mRNA stable enough to be detected in circulating reticulocytes. Although this mRNA could encode an N-terminal fragment of glycophorin C, these protein isoform(s) would not be expressed in the membrane because they lack the transmembrane and cytoplasmic domains.

Molecular Analysis of Glycophorin C Deficiency in Human Erythrocytes

AbstractHuman erythrocyte glycophorin C plays a functionally important role in maintaining erythrocyte shape and regulating membrane mechanical stability. We report here the characterization of the glycophorins C and D deficiency in erythrocytes of the Leach phenotype. Glycophorin C gene is encoded by 4 exons. Amplification of reticulocyte cDNA from Leach phenotype and normal individuals generated a 140-bp fragment when using primers spanning exons 1 and 2. However, no polymerase chain reaction (PCR) products were detected in the Leach phenotype using primers flanking either exons 1 and 3 or exons 1 and 4, suggesting that the 3’ end of the mRNA was missing or altered. Exon 4 also appeared to be missing from Leach genomic DNA, based on both Southern hybridization and PCR. These results indicate that an absence of glycophorin C and glycophorin D in erythrocytes from these Leach phenotype individuals is a consequence of a deletion or marked alteration of exon 3 and exon 4 of their glycophorin C gene. Surprisingly, the mutant gene encodes an mRNA stable enough to be detected in circulating reticulocytes. Although this mRNA could encode an N-terminal fragment of glycophorin C, these protein isoform(s) would not be expressed in the membrane because they lack the transmembrane and cytoplasmic domains.

Molecular Basis for Elliptocytosis Associated With Glycophorin C and D Deficiency in the Leach Phenotype

Abstract Glycophorin C (GPC) and glycophorin D (GPD) are highly glycosylated integral membrane proteins of human erythrocytes encoded by the same gene and associated with expression of Gerbich blood group system antigens. GPC/D deficiency (the Leach phenotype) is a rare condition usually found after identification of Gerbich blood group system antibodies in persons undergoing prenatal or pretransfusion evaluation. In all cases, the Leach phenotype has been associated with elliptocytosis. Characterization of the molecular basis of this phenotype in three previously uninvestigated families has shown that the most common genetic basis of GPC/D deficiency is deletion of exons 3 and 4 of the GPC gene. However, in one family, the Leach phenotype appeared due to a deletion of one nucleotide in exon 3, causing a frameshift mutation in the messenger RNA and premature generation of a stop codon. The GPC and GPD protein sequences are therefore interrupted in the extracellular domain and probably intracellularly degraded.

Molecular basis for elliptocytosis associated with glycophorin C and D deficiency in the Leach phenotype

Glycophorin C (GPC) and glycophorin D (GPD) are highly glycosylated integral membrane proteins of human erythrocytes encoded by the same gene and associated with expression of Gerbich blood group system antigens. GPC/D deficiency (the Leach phenotype) is a rare condition usually found after identification of Gerbich blood group system antibodies in persons undergoing prenatal or pretransfusion evaluation. In all cases, the Leach phenotype has been associated with elliptocytosis. Characterization of the molecular basis of this phenotype in three previously uninvestigated families has shown that the most common genetic basis of GPC/D deficiency is deletion of exons 3 and 4 of the GPC gene. However, in one family, the Leach phenotype appeared due to a deletion of one nucleotide in exon 3, causing a frameshift mutation in the messenger RNA and premature generation of a stop codon. The GPC and GPD protein sequences are therefore interrupted in the extracellular domain and probably intracellularly degraded.

Effects of deficiencies of glycophorins C and D on the physical properties of the red cell

Red cells of the rare Leach phenotype lack the membrane glycophorins C and D, and a proportion of the red cells are elliptocytes. Judging from tests on suspensions of red cell ghosts sheared rotationally in an ektacytometer, it has previously been suggested that these membranes are relatively fragile and poorly deformable. We have carried out analyses of individual red cells to investigate possible factors which underlie the physical changes in these glycophorin-deficient cells. Micropipette analysis of the red cell membrane showed that the rigidity and viscosity were normal, both for elliptocytes and discocytes, for three donors deficient in glycophorins C and D. Red cell transit times through 5 microns pores, measured electronically for 2000 individual cells, showed no differences from controls. It was confirmed that the index of deformation obtained using an ektacytometer was reduced, but our results suggest that this arises from shape rather than membrane changes. The elliptocytes were found to have a lower volume and surface area than discocytes from the same donor (measured by micropipette aspiration of single red cells), and were rarely found in less dense red cell fractions. No reticulocytes were found to be elliptical. These data suggest that the elliptocytes are older red cells, and are formed from red cells which are initially released into the circulation with normal shape. Their elongated shape might arise from permanent distortion of the unstable membrane by shear forces in the circulation.

A Novel Human Alloantibody in the Gerbich System

A Gerbich-negative Leach phenotype individual was identified on the basis of distinct morphological, biochemical, and serological characteristics of her red blood cells. This individual has produced an antibody which was reactive with the various Gerbich phenotypes including Ge:-2,3 (Yus type) and Ge:-2,-3 (Ge type) cells; only her own and Leach phenotype cells were non-reactive. It is suggested that this antibody represents an example of a new specificity, one which defines the Leach phenotype, in the Gerbich system.

Transient reduction in erythrocyte membrane sialoglycoprotein β associated with the presence of elliptocytes

Erythrocyte membranes from an anaemic patient receiving gold therapy for rheumatoid arthritis had reduced beta-sialoglycoprotein (beta-SGP) content but normal expression of sialoglycoproteins alpha, delta and gamma. Elliptocytes were present in the peripheral blood. The serum of the patient contained anti-beta-SGP which did not appear to bind to her own cells. It reacted with all erythrocytes apart from beta-SGP deficient Leach phenotype cells. The antibody was inhibited by purified beta-SGP from normal red cells, bound to beta-SGP on immunoblots and also reacted with the abnormal beta-related-SGP in erythrocyte membranes of both the Gerbich type and Yus type of Gerbich negative. Two years later the patient was no longer anaemic, no elliptocytes were seen in her peripheral blood film and her erythrocyte membranes had normal beta-SGP content. Antibody was no longer present in her serum and antibody from the earlier sample now reacted with the patient's erythrocytes. Erythrocyte membrane beta-SGP is known to be important in the maintenance of normal cell shape. It is likely that the transient occurrence of elliptocytes in the patient resulted from the concurrent temporary reduction in beta-SGP content of her erythrocyte membranes.

Transient reduction in erythrocyte membrane sialoglycoprotein β associated with the presence of elliptocytes

Erythrocyte membranes from an anaemic patient receiving gold therapy for rheumatoid arthritis had reduced β‐sialoglycoprotein (β‐SGP) content but normal expression of sialoglycoproteins α, δ and γ. Elliptocytes were present in the peripheral blood. The serum of the patient contained anti‐β‐SGP which did not appear to bind to her own cells. It reacted with all erythrocytes apart from β‐SGP deficient Leach phenotype cells. The antibody was inhibited by purified β‐SGP from normal red cells, bound to β‐SGP on immunoblots and also reacted with the abnormal β‐related‐SGP in erythrocyte membranes of both the Gerbich type and Yus type of Gerbich negative. Two years later the patient was no longer anaemic, no elliptocytes were seen in her peripheral blood film and her erythrocyte membranes had normal β‐SGP content. Antibody was no longer present in her serum and antibody from the earlier sample now reacted with the patient's erythrocytes.Erythrocyte membrane β‐SGP is known to be important in the maintenance of normal cell shape. It is likely that the transient occurrence of elliptocytes in the patient resulted from the concurrent temporary reduction in β‐SGP content of her erythrocyte membranes.

On the measurement of shear elastic moduli and viscosities of erythrocyte plasma membranes by transient deformation in high frequency electric fields

We present a new method to measure the shear elastic moduli and viscosities of erythrocyte membranes which is based on the fixation and transient deformation of cells in a high-frequency electric field. A frequency domain of constant force (arising by Maxwell Wagner polarization) is selected to minimize dissipative effects. The electric force is thus calculated by electrostatic principles by considering the cell as a conducting body in a dielectric fluid and neglecting membrane polarization effects. The elongation A of the cells perpendicular to their rotational axis exhibits a linear regime (A proportional to Maxwell tension or to square of the electric field E2) at small, and a nonlinear regime (A proportional to square root of Maxwell tension or to the electric field E) at large extensions with a cross-over at A approximately 0.5 micron. The nonlinearity leads to amplitude-dependent response times and to differences of the viscoelastic response and relaxation functions. The cells exhibit pronounced yet completely reversible tip formations at large extensions. Absolute values of the shear elastic modulus, mu, and membrane viscosity, eta, are determined by assuming that field-induced stretching of the biconcave cell may be approximately described in terms of a sphere to ellipsoid deformation. The (nonlinear) elongation-vs.-force relationship calculated by the elastic theory of shells agress well with the experimentally observed curves and the values of mu = 6.1 x 10(-6) N/m and eta = 3.4 x 10(-7) Ns/m are in good agreement with the micropipette results of Evans and co-workers. The effect of physical, biochemical, and disease-induced structural changes on the viscoelastic parameters is studied. The variability of mu and eta of a cell population of a healthy donor is +/- 45%, which is mainly due to differences in the cell age. The average mu value of cells of different healthy donors scatters by +/- 18%. Osmotic deflation of the cells leads to a fivefold increase of mu and 10-fold increase of eta at 500 mosm. The shear modulus mu increases with temperature showing that the cytoskeleton does not behave as a network of entropy elastic springs. Elliptic cells of patients suffering from elliptocytosis of the Leach phenotype exhibit a threefold larger value of mu than normal discocytes of control donors. Cross-linking of the spectrin by the divalent S-H agents diamide (1 mM, 15 min incubation) leads to an eightfold increase of mu whereas eta is essentially constant. The effect of diamide is reversed after treatment with S-S bond splitting agents.

Blood group antigen deficiencies associated with abnormal red cell shape

Rare individuals are known with erythrocytes which show an inherited deficiency of certain blood group antigens and also have abnormal red cell shape. Studies of these cells can give an insight into the functional role of blood group active components in maintaining the shape and membrane properties of the normal erythrocyte. The biochemical characterisation of the red cell membrane alterations occurring in two such rare erythrocyte phenotypes—the Leach phenotype and the Rh null phenotype are reviewed here.