Membrane components in the red cells of patients with sickle cell anemia. Relationship to cell aging and to irreversibility of sickling

Cholesterol, phospholipid and sialic acid were measured in red cells from patients with sickle cell anemia to determine whether the cells had abnormal concentrations of these components and whether the amounts of these compounds differed in irreversibly sickled cells as compared to non-irreversibly sickled cells. Sickle cells had significantly higher levels of both lipids than similar populations of normal cells, however, comparisons to populations of young control cells showed that the differences were generally not significant. Sialic acid levels in sickle cells were not significantly different from normal cells. Irreversibly sickled cells had lower lipid and sialic acid concentrations than those not irreversibly sickled, but the differences were either not significant or did not occur when compared to young control cells. The studies show that the increased lipid concentrations in the membrane of sickle cells are not abnormal but are related to cell age and that the decrease in membrane components in irreversibly sickled cells is no greater than would be predicted for similarly aged populations of cells.     

Membrane components in the red cells of patients with sickle cell anemia Relationship to cell aging and to irreversibility of sickling

Cholesterol, phospholipid and sialic acid were measured in red cells from patients with sickle cell anemia to determine whether the cells had abnormal concentrations of these components and whether the amounts of these compounds differed in irreversibly sickled cells as compared to non-irreversibly sickled cells. Sickle cells had significantly higher levels of both lipids than similar populations of normal cells, however, comparisons to populations of young control cells showed that the differences were generally not significant. Sialic acid levels in sickle cells were not significantly different from normal cells. Irreversibly sickled cells had lower lipid and sialic acid concentrations than those not irreversibly sickled, but the differences were either not significant or did not occur when compared to young control cells. The studies show that the increased lipid concentrations in the membrane of sickle cells are not abnormal but are related to cell age and that the decrease in membrane components in irreversibly sickled cells is no greater than would be predicted for similarly aged populations of cells.     

The anti-sickling drug lawsone (2-OH-1,4-naphthoquinone) protects sickled cells against membrane damage

The ability of an anti-sickling drug lawsone, 2-OH-1,4-naphthoquinone, and two related compounds to inhibit the haematoporphyrin-sensitised photohaemolysis of normal and sickle cell erythrocytes has been investigated. The compounds appear to protect the erythrocyte membranes by reaction with transient oxidative species. Differential effects between normal and sickle cells are shown and these are attributed to the different membrane composition of irreversibly sickled erythrocytes. This report describes a possible basis for the decreased formation of irreversibly sickled cells in the presence of lawsone.     

Differences in erythrocyte membrane proteins and glycoproteins in sickle cell disease

Erythrocyte membrane proteins obtained from individuals with sickle cell anemia show an SDS polyacrylamide gel pattern that differs in five regions from the normal pattern. These membranes when compared with membranes from normal individuals also show a marked decrease in sialic acid content which correlates with a marked reduction of the periodic acid-Schiff staining of the three major glycoprotein components. The observed membrane protein and glycoprotein changes are a characteristic of all the red cells in sickle cell anemia and do not correlate with the proportion of irreversibly sickled cells.     

Inhibition of the in vitro formation of dense cells and of irreversibly sickled cells by charybdotoxin, a specific inhibitor of calcium-activated potassium efflux

Charybdotoxin, a specific inhibitor of the calcium-activated potassium channel, was found to inhibit the in vitro formation of irreversibly dehydrated cells and of irreversibly sickled cells, which occur as a result of repeated cycles of sickling and unsickling of sickle red blood cells. The degree of formation of dense cells was measured by Percoll-renografin density gradient centrifugation. 50% inhibition of the formation was achieved at a concentration of 30 nM of charybdotoxin. The approximate half-life of this compound in the circulation of the guinea pig was determined to be 4 h. Charybdotoxin did not inhibit the sickling of sickle cells under deoxygenation. The effects of charybdotoxin in preventing the irreversible changes of sickle cell membranes may be related to the inhibition of calcium-activated potassium efflux in sickle red blood cells.     

Red blood cell [14C]cholesterol exchange and plasma cholesterol esterifying activity of normal and sickle cell blood

The present study performed on density fractions of sickle and normal erythrocytes prepared on Stractan density gradient shows that dense erythrocytes have consistently decreased uptake of [¹⁴C]cholesterol from plasma in comparison to young, less dense erythrocytes. Plasma of sickle cell patients also shows a reduction in cholesterol-esterifying activity in comparison to normal controls. A possible effect of these processes in the increased cholesterol to phospholipid molar ratio of irreversibly sickled cells has been suggested.     

The role of Ca2+-dependent biochemical changes in the ageing process in normal red cells and in the development of irreversibly sickled cells

During the ageing process of normal red cells and in the formation of irreversibly sickled cells (ISCs) there is a progressive increase in the intracellular concentration of Ca2+. This is parallelled by the development of a variety of morphological and biochemical changes in older fractions of normal cells and in ISCs which are similar to those seen in normal cells exposed to Ca2+ ionophore. These changes include cell shrinkage, loss of membrane lipid and degradation of cytoskeletal proteins and polyphosphoinositides. In this paper we consider the ways in which the Ca2+-dependent biochemical changes may be related to the morphological alterations which are characteristic of ageing and irreversible sickling.     

Ubiquitination of spectrin regulates the erythrocyte spectrin-protein-4.1-actin ternary complex dissociation: implications for the sickle cell membrane skeleton.

It has been demonstrated by our laboratory that the irreversibly sickled cell (ISC) spectrin-4.1-actin complex dissociates slowly as compared to ternary complexes formed out of control (AA) and reversibly sickle cell (RSCs) core skeletons. These studies indicated that the molecular basis for the inability of irreversibly sickled cells (ISCs) to change shape is a skeleton that disassembles, and therefore reassembles, very slowly. The present study is based on the following observations: a) alpha-spectrin repeats 20 and 21 contain ubiquitination sites, and b) The spectrin repeats beta-1 and beta-2 are in direct contact with spectrin repeats alpha-20 and alpha-21 during spectrin heterodimer formation, and contain the protein 4.1 binding domain. We demonstrate here that alpha-spectrin ubiquitination at repeats 20 and 21 increases the dissociation of the spectrin-protein-4.1-actin ternary complex thereby regulating protein 4.1's ability to stimulate the spectrin-actin interaction. Performing in vitro ternary complex dissociation assays with AA control and sickle cell SS spectrin (isolated from high-density sickle cells), we further demonstrate that reduced ubiquitination of alpha-spectrin is, in part, responsible for the locked membrane skeleton in sickle cell disease.     

The binding of hemoglobin to membranes of normal and sickle erythrocytes.

The binding of hemoglobins A, S, and A2 to red cell membranes prepared by hypotonic lysis from normal blood and blood from persons with sickle cell anemia was quantified under a variety of conditions using hemoglobin labelled by alkylation with 14C-labelled Nitrogen Mustard. Membrane morphology was examined by electron microscopy. Normal membranes were found capable of binding native hemoglobin A and hemoglobin S in similar amounts when incubated at low hemoglobin: membrane ratios, but at high ratios hemoglobin saturation levels of the membranes increased progressively for hemoglobin A, hemoglobin S and hemoglobin A2, respectively, in order of increasing electropositivity. Binding was unaffected by variations in temperature (4-22 degrees C) and altered little by the presence of sulfhydryl reagents, but was inhibited at pH levels above 7.35; disrupted at high ionic strength; and dependent on the ionic composition of the media. These findings suggest that electrostatic, but not hydrophobic or sulfhydryl bonds are important in membrane binding of the hemoglobin under the conditions studied. An increased retention of hemoglobin in preparations of membranes from red cells of patients with sickle cell anemia (homozygote S) was attributable to the dense fraction of homozygote S red cells rich in irreversibly sickled cells, and the latter membranes had a smaller residual binding capacity for new hemoglobin. This suggests that in homozygote S cells which have become irreversibly sickled cells in vivo, there are membrane changes which involve alteration and/or blockade of hemoglobin binding sites. These findings support the notion that hemoglobin participates in the dynamic structure of the red cell membrane in a manner which differs in normal and pathological states.     

The chelation of nonheme iron within sickle erythrocytes by the hydroxypyridinone chelator CP094.

Nonheme, nonferritin iron has been detected in membrane preparations from sickle erythrocytes and has been suggested to catalyze free radical reactions in these cells contributing to the development of membrane oxidation. In this study the hydroxypyridinone iron chelator, CP094, currently being evaluated as a potentially therapeutic chelator, and desferrioxamine have been studied for their abilities to chelate the nonheme iron within intact sickle erythrocytes under physiological conditions. The results suggest that CP094 can enter sickle erythrocytes, chelate nonheme iron and suppress membrane lipid peroxidation within a timescale in which desferrioxamine does not enter the cells. Suppression of lipid peroxidation showed no protective effect in an in vitro system inducing the formation of irreversibly sickled cells.     

Viscoelastic properties of the oxygenated sickle erythrocyte membrane

Although most apparent in permanently misshapen irreversibly sickled erythrocytes (ISC), biochemical and structural alterations are present in the majority of sickle cell membranes. The relationship of membrane rigidity to cell shape and its dependence upon the internal hemoglobin cytosol are not clarified. We therefore examined the frequency dependent viscoelasticity of oxygenated, packed sickle red cell and ghost suspensions and hemoglobin solutions prepared from density gradient separated ISC and reversibly sickled cell (RSC) fractions. Low amplitude, oscillatory shear was applied in a Weissenberg cone and plate viscometer and the resultant viscoelastic signals provided a dynamic viscosity (eta') and elastic storage modulus (G') which varied with frequency of deformation. The viscoelastic response of the cell and ghost suspensions reflected the material properties of the membrane over most of the frequency range tested. Sickle erythrocyte, red ghost, and white ghost suspensions demonstrated greater viscocoelasticity than comparable normal suspensions. The viscoelastic magnitude of ISC was several-fold greater than normal, with little variation of viscoelasticity with frequency. RSC samples which were characterized by normal shape, size, and internal hemoglobin concentration were also significantly harder than normal, although similar in frequency dependence. Red ghosts prepared from ISC manifested 80% of the viscoelasticity of intact ISC despite diminution of the internal hemoglobin concentration by 90%. Under conditions of low amplitude shear, the behavior of the RSC membrane is compatible with a cytoskeleton possessing an increased number of molecular associations. The mechanical stability of the ISC membrane is related to a substantial, intrinsic reorganization of the cytoskeleton.     

The binding of hemoglobin to membranes of normal and sickle erythrocytes

The binding of hemoglobins A, S, and A₂ to red cell membranes prepared by hypotonic lysis from normal blood and blood from persons with sickle cell anemia was quantified under a variety of conditions using hemoglobin labelled by alkylation with ¹⁴C-labelled Nitrogen Mustard. Membrane morphology was examined by electron microscopy. Normal membranes were found capable of binding native hemoglobin A and hemoglobin S in similar amounts when incubated at low hemoglobin: membrane ratios, but at high ratios hemoglobin saturation levels of the membranes increased progressively for hemoglobin A, hemoglobin S and hemoglobin A₂, respectively, in order of increasing electropositivity. Binding was unaffected by variations in temperature (4–22 °C) and altered little by the presence of sulfhydryl reagents, but was inhibited at pH levels above 7.35; disrupted at high ionic strength; and dependent on the ionic composition of the media. These findings suggest that electrostatic, but not hydrophobic or sulfhydryl bonds are important in membrane binding of the hemoglobin under the conditions studied.An increased retention of hemoglobin in preparations of membranes from red cells of patients with sickle cell anemia (homozygote S) was attributable to the dense fraction of homozygote S red cells rich in irreversibly sickled cells, and the latter membranes had a smaller residual binding capacity for new hemoglobin. This suggests that in homozygote S cells which have become irreversibly sickled cells in vivo, there are membrane changes which involve alteration and/or blockade of hemoglobin binding sites.These findings support the notion that hemoglobin participates in the dynamic structure of the red cell membrane in a manner which differs in normal and pathological states.     

Photoaffinity labeling of procaine-binding sites in normal and sickle cell membranes

A photoaffinity probe, procaine azide, was employed to determine the sites of interaction of procaine in normal and sickle cell erythrocytes. Studies show that the number of binding sites and affinity of procaine to membranes derived from normal and sickled cell erythrocytes were similar, although procaine retards the in vitro formation of irreversibly sickled cells from cells. The results show that procaine azide, a photoaffinity analogue of procaine, is covalently incorporated into both protein (60–70%) and lipid (40–30%) components of the membrane. Sodium dodecyl sulfate-gel electrophoresis of the labeled ghosts show that procaine binds specifically to band 3 and periodic acid-Schiff staining bands in membranes derived from labeled erythrocytes. Binding of procaine or covalent incorporation of procaine azide into membrane proteins does not affect the phosphate transport. Moreover, pre-treatment of intact erythrocytes with 4,4′-diisothiocyano-2,2′-stilbene disulfonate, an anion transport inhibitor, did not affect either the binding or covalent incorporation of procaine azide into erythrocytes. These results indicate that the binding of procaine azide to Band 3 protein occurs at a locus different than that involved in anion translocation process.     

The effect of thyroid hormones on the membrane adenylate cyclase activity in sickle red blood cells in vitro

A comparative study of thyroid hormone responsive membrane adenylate cyclase activity has been carried out on normal (Hb-AA) and sickle cell (Hb-SS) subjects. Thyroid hormones (T3 and T4) and selected analogues (TRIAC and DLT) enhanced adenylate cyclase activity in Hb-AA membranes. The interaction of Hb-SS membranes with the hormones and analogues was not significant except for T3 which moderately stimulated Hb-SS membrane adenylate cyclase. We suggest that circulating irreversibly sickled cells, in view of their membrane phospholipid defect, probably contribute to the low response of membrane adenylate cyclase to effector stimulation.     

Electron microscopic studies of the intracellular polymerization of sickle hemoglobin

Transmission electron microscopy has been used to study intracellular sickle hemoglobin polymer in unfractionated cells from the arterial and venous blood of patients and after external deoxygenation. We detect polymerized hemoglobin in up to 10% of the cells in the venous circulation, especially in cells that are "cigar-shaped" and appear to be irreversibly sickled. We could not see well-defined polymer in mixed arterial samples; nevertheless, we found electron opaque spots, which could be ferritin granules, hemosiderin, or small aggregates of hemoglobin S. However, upon sequential chemical deoxygenation using 1.0% sodium metabisulphite, polymer formation was seen at oxygen saturation values of 75%-85%. Cells that were physically deoxygenated using gas mixtures containing nitrogen-carbon dioxide-oxygen mixtures were found to contain distinct polymers of deoxyhemoglobin S at oxyhemoglobin saturation values of 50%-75%. As deoxygenation increases, we detect short, randomly arranged polymer in a loose network, with occasional long polymers. Upon further deoxygenation, the length and number of polymer forms increased. Between 0% and 50% saturation, most erythrocytes were full of long, parallel, closely packed polymers that tend to align and run parallel to the cell membrane. In both chemical and physically deoxygenated blood samples, cells were seen at 50%-75% oxyhemoglobin saturation that retained their normal biconcave disc shape, although they contained significant amounts of polymer. The structural changes in sickle erythrocytes seen in vitro due to physical or chemical deoxygenation of cells, may reflect in vivo intracellular changes in the sickle cell patient.     

Preparation of irreversibly sickled cell beta-actin from normal red blood cell beta-actin.

We have previously demonstrated that an oxidative change, the formation of a disulfide bridge between two cysteine residues, in the membrane protein beta-actin is primarily responsible for locking the irreversibly sickled red blood cells (ISCs) of sickle cell anemic patients into the sickle shape. To support studies on biological and chemical characterization of the oxidized beta-actin and pharmacological research toward the reversal of the oxidation, we attempted to prepare oxidized beta-actin from normal red blood cell (RBC) beta-actin by a chemical reaction, expecting a product equivalent to that found in ISCs. 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB, or Ellman's reagent) was used for the oxidation. We proved the absence of accessible sulfhydryl groups in the oxidized product using liquid chromatography (LC) with both UV and fluorescence detection. Polymerization assays indicated that the chemically produced ISC actin demonstrated the same kinetics as ISC actin obtained from patients with sickle cell disease. The effect of the oxidation could be reversed by the use of the reducing agent tris(carboxyethyl)phosphine (TCEP).     

Surface properties of irreversibly sickled cells differ from those of the bulk of sickle cells Studies by partitioning in aqueous phase systems

Counter-current distribution (CCD) of red blood cells (RBC) from individuaks with homozygous sickle cell (HbSS) disease in a charge-sensitive aqueous dextran-poly(ethylene glycol) phase system, which fractionates cells on the basis of surface properties, indicates that the percentage of irreversibly sickled cells (ISC) increases and the percentage of reticulocytes decreases with increasing cell partition ratios. The high partition ratios of ISC correspond to those of older RBC when RBC from normal individuals are subjected to CCD. Our results thus indicate that ISC differ in surface properties from those of the bulk of sickle RBC (including reticulocytes) in the population and that the difference is, most likely, charge-related. While the question as to whether ISC are indeed old cells has not yet been unequivocally answered, this view finds support in the fact that the independent parameters of ISC surface properties, as reflected by partition ratios, and densities correlate as they do in older RBC from normal individuals.     

Sickling-induced binding of autologous IgG to erythrocytes heterozygous for sickle hemoglobin

This study reports that sickling-induced increased autoantibody binding can be demonstrated in varying degrees for deoxygenated S/beta-thalassemic (2-fold) and hemoglobin-SC (1.2-fold) erythrocytes as compared with oxygenated paired samples. In contrast, HbAS erythrocytes deoxygenated in autologous plasma exhibited less than 2% morphologic sickling and no increased IgG binding as compared with control samples. Sickling in the presence or absence of plasma increased the IgG binding capacity of S/beta-thalassemic erythrocytes, comparable to previous findings for HbSS erythrocytes, while increased IgG binding to HbSC erythrocytes was detected only after deoxygenation in plasma. It is concluded that specific IgG binding to deoxygenated S/beta-thalassemic RBCs results from subtle permanent sickling-induced alterations of the membrane surface, while IgG binding to HbSC erythrocytes sickled in plasma results from transitory membrane changes. These findings suggest that sickling in vivo will produce cumulative autoantibody binding to S/beta-thalassemic erythrocytes, a process which could lead to immune-mediated erythrocyte destruction. In contrast, comparatively small fractions of the autoantibody bound to HbSC erythrocytes in vivo would result from sickling-induced membrane alterations. These studies indicate that sickling-associated autoantibody binding in vivo will not occur for sickle cell trait (HbAS) erythrocytes protected by plasma.     

Differential turnover of methyl groups on methyl-accepting membrane proteins of irreversibly sickled erythrocytes

Methyl group turnover rates for specific methyl-accepting membrane proteins in intact irreversibly sickled cells (ISCs) have been determined. The turnover of methyl groups on all methyl acceptor membrane proteins carboxylmethylated in ISCs is not concerted but proceeds in an ordered sequence which is markedly different from that exhibited by unfractionated normal erythrocytes (AA). In ISCs methyl group turnover based on initial demethylation rate constants is most rapid for membrane polypeptides migrating in sodium dodecyl sulfate at 30,000-39,000 Da, 40,000-55,000 Da, polypeptides comigrating with cytoskeletal component band 4.1, and band 4.5. In contrast, initial methyl group turnover rates obtained after less than 20% of the total methyl groups are turned over in (AA) cells show most rapid demethylation rates for membrane polypeptides migrating at 40,000-55,000 Da, polypeptides comigrating with band 4.5, cytoskeletal components bands 2.1 and 4.1. Results also show significant differences between ISCs and most dense fractions from normal (AA) and nonsickle hemolytic anemias in the demethylation of cytoskeletal proteins, bands 2.1 and 4.1. These findings indicate qualitative differences in accessibility of methyl acceptor substrates to the methylating-demethylating enzyme activity in the cytosol of irreversibly sickled cells compared to discocytic control erythrocytes.     

Identification of an endothelial cell surface protein that binds plasminogen

To identify and characterize endothelial cell surface components that bind plasminogen, we used ligand-blotting to study binding of plasminogen to sodium dodecyl sulphate solubilized extracts of human umbilical vein endothelial cells. It was observed that glu-plasminogen bound predominantly to a 45 kDa endothelial cell polypeptide. The interaction of labelled glu-plasminogen with this polypeptide was reversible and specific as the binding could be inhibited by both excess cold lysine and unlabelled glu-plasminogen but not by unrelated proteins. Binding of glu-plasminogen to cell extracts prepared from endothelial cells that had been pretreated with proteinase K was significantly reduced indicating that the 45 kDa polypeptide is a cell-surface protein. The cell-surface localization of the 45 kDa polypeptide was also indicated by the positive interaction of glu-plasminogen with membrane fractions of endothelial cells. Lys-plasminogen also interacted with the 45 kDa polypeptide in a specific manner and reversibility experiments indicated that lysplasminogen could also displace the bound glu-plasminogen. Since binding of plasminogen to the 45 kDa endothelial cell surface polypeptide was very similar to plasminogen binding to intact endothelial cells, we propose that the 45 kDa protein represents one of the major receptors for plasminogen on human endothelial cells.