Increased erythrocyte adhesion in mice and humans with hereditary spherocytosis and hereditary elliptocytosis

Mice with disruptions of the red blood cell (RBC) cytoskeleton provide severe hemolytic anemia models in which to study multiorgan thrombosis and infarction. The incidence of cerebral infarction ranges from 70% to 100% in mice with alpha-spectrin deficiency. To determine whether mutant RBCs abnormally bind adhesive vascular components, we measured adhesion of mouse and human RBCs to immobilized human thrombospondin (TSP) and laminin (LM) under controlled flow conditions. Mutant RBCs had at least 10-fold higher adhesion to TSP compared with normal RBCs (P <.006). Mutant relative to unaffected RBC adhesion to LM was significantly (P <.01) increased as well. Treatment of RBCs with the anionic polysaccharide dextran sulfate inhibited mutant RBC adhesion to TSP (P <.001). Treatment of RBCs with antibodies to CD47 or the CD47-binding TSP peptide 4N1K did not inhibit TSP adhesion of RBCs. Previously, we have shown that infarcts in alpha-spectrin-deficient sph/sph mice become histologically evident beginning at 6 weeks of age. TSP adhesion of RBCs from 3- to 4- and 6- to 8-week-old sph/sph mice was significantly higher than RBCs from adult mice (> 12 weeks old; P <.005). While the mechanism of infarction in these mice is unknown, we speculate that changes in RBC adhesive characteristics contribute to this pathology.     

Alteration of the erythrocyte membrane skeletal ultrastructure in hereditary spherocytosis, hereditary elliptocytosis, and pyropoikilocytosis

The membrane skeleton of normal erythrocytes is largely organized into a hexagonal lattice of junctional complexes (JC) crosslinked by spectrin tetramers, and occasional double tetramers and hexamers. To explore possible skeletal alterations in hereditary spherocytosis (HS), elliptocytosis (HE), and pyropoikilocytosis (HPP), we have studied the ultrastructure of the spread membrane skeletons from a subpopulation of HS patients with a partial spectrin deficiency ranging from 43% to 86% of normal levels, and in patients with HPP who, in addition to a mild spectrin deficiency, also carried a mutant spectrin that was dysfunctional, thus reducing the ability of spectrin dimers to assemble into tetramers. Membrane skeletons derived from Triton-treated erythrocyte ghosts were examined by negative staining electron microscopy. HS membrane skeletons contained structural elements, consisting of JC and spectrin filaments similar to the normal skeleton. However, less spectrin filaments interconnected the JC, and the decrease of spectrin filaments attached to JC appeared to correlate with the severity of spectrin deficiency. Only in severe HS associated with severe spectrin deficiency was the loss of spectrin sufficient enough to disrupt the overall skeletal architecture. In contrast, membrane skeletons prepared from red blood cells (RBCs) of subjects with HPP were strikingly different from HS RBCs with a comparable degree of spectrin deficiency. Although HPP RBCs were only mildly deficient in spectrin, their skeletal lattice was grossly disrupted, in contrast to only mild ultrastructural abnormalities of HS membrane skeletons with a nearly identical degree of spectrin deficiency. Skeletons from patients with common mild HE or asymptomatic carriers, carrying the mutant spectrin but having normal spectrin content, exhibited a moderate disruption of the skeletal lattice. We propose that the above differences in skeletal ultrastructure may underlie differences in the biomechanical properties and morphology of HS, HE, and HPP RBCs.     

Molecular pathology of inherited erythrocyte membrane disorders: hereditary spherocytosis and elliptocytosis.

Hereditary spherocytosis and elliptocytosis are common genetic defects of the red blood cell membrane skeleton. In recent years rapid advances have been made in the knowledge of the protein structure and assembly of the cytoskeleton. Thanks to the wide use of protein analysis methods several alterations have been discovered in functionally important domains of the different cytoskeletal proteins in these diseases. The cloning of cDNA for the majority of the cytoskeletal proteins allows us to begin elucidating some of these defects at the DNA level. This paper will review the effects of recent advances upon: cytoskeleton structure and assembly; molecular pathology of spherocytosis, elliptocytosis and pyropoikilocytosis.     

Altered erythrocyte membrane protein phosphorylation in an unusual case of hereditary spherocytosis.

Membrane protein phosphorylation was examined in several members from a family with an unusual form of hereditary sperocytosis. After incubation of membrane ghosts with (gamma-32 P) ATP, the phosphorylation of spectrin component II was diminished both in the absence of cAMP. In the presence of this nucleotide, the phosphorylation of components IV5 and IV8 was also decreased. Along with a previously reported alteration of a membrane neutral phosphatase in this family, these abnormalities remove the present condition from the usual form of hereditary spherocytosis.     

Presence of cytosolic peroxiredoxin 2 in the erythrocyte membrane of patients with hereditary spherocytosis

We studied 82 Portuguese individuals, 57 with hereditary spherocytosis (HS) and 25 unaffected controls. We performed standardized diagnosis tests, including electrophoretic membrane protein analysis to identify and quantify protein deficiencies underlying HS. Membrane bound hemoglobin (MBH) and band 3 profiles were determined as oxidative stress and aging markers. A protein of about 22 kDa, present in 21 of 57 HS patients, but not in controls, was identified as peroxiredoxin 2 (Prx2), by mass-spectroscopy and by immunoblotting. Human erythrocyte Prx2 is a peroxiredoxin with thiol-specific antioxidant activity. The presence of Prx2 in erythrocyte membranes was linked to higher levels of oxidative stress, as reflected by significantly increased MBH in those HS patients. No relation with HS clinical severity was observed and Prx2 was detected in all types of membrane protein abnormalities. Prx2 membrane linkage is associated with a higher oxidative stress susceptibility of HS erythrocytes.     

Analysis of erythrocyte membrane proteins in patients with hereditary spherocytosis and other types of haemolytic anaemia

ABSTRACT

Objectives: In order to investigate the pathophysiology of erythrocyte membrane proteins, 10 patients (6 pre- and 4 post-splenectomy) with hereditary spherocytosis (HS) and other patients with haemolytic anaemia were examined.

Methods: The membrane proteins were analysed by biochemical and mass spectrometry.

Results: Reductions in the extracellular membrane of band 3 protein by eosin-5'-maleimide (EMA) binding test were greater in patients with pre-splenectomy HS than in patients with post-splenectomy HS, other types of haemolytic anaemia, and controls. Compared to patients with haemolytic anaemia and healthy controls, the band 3 protein of patients with HS pre- or post-splenectomy was more easily decomposed with N-glycosidase F and by mass spectrometry interactions with degraded low-molecular-weight spectrin and ankyrin. The resulting fragments were observed more frequently in pre-splenectomy than post-splenectomy HS. Haemoglobin-derived peptides were present in patients with haemoglobinopathy (Hb Evans, Hb Sabine) but not in those with haemolytic anaemia and healthy controls.

Conclusion: Haemolysis in patients with HS occurred because the fragile proteins in erythrocytes (band 3, spectrin, and ankyrin) collapsed due to compression during blood circulation in the spleen. Further, haemolysis in patients with haemoglobinopathy occurred owing to membrane damage due to combined spectrin, band 3 with denatured haemoglobin in the vessel during blood circulation.     

Ultracentrifugal analysis of the junction complexes of the red cell membrane cytoskeletal network: application to hereditary spherocytosis and metabolically depleted cells

Summary A method has been developed for the assessment of the number of spectrin dimer units associated with each actin protofilament junction, in the membrane cytoskeletal network (i.e. the degree of branching) of the red cell. Ghosts are first exposed to elevated temperature at low ionic strength to dissociate some 65% of the spectrin tetramers (that link the network junctions) into dimers, without causing their release from the actin filaments. Non-ionic detergent is then added to solubilize the membrane itself with its intrinsic proteins, so as to liberate the cytoskeletal material, and the mixture is immediately examined in the analytical ultracentrifuge. The predominent components observed are isolated junctions (20 S), free spectrin dimers and the residual undissociated cytoskeletal material, with very minor components, probably corresponding to mutiple junctions, linked by spectrin tetramers. The junction boundary is homogeneous within the accuracy of measurement and is taken to correspond to a complex containing six spectrin dimers, known to predominate in situ. About 17% of the total network is liberated in this form and 12% as free spectrin dimers. In hereditary spherocytosis both the size of the junction complex (as reflected by its sedimentation coefficient) and the proportion of the complex and of free spectrin liberated are indistinguishable from normal values. We conclude that the reported deficit of spectrin in hereditary spherocytosis is not reflected by a lower degree of branching of the network, and, if the membrane area is not correspondingly reduced, this must mean that the junctions are more widely spaced and the spectrin tetramers therefore more extended. In metabolically depleted cells, in which the cytoskeletal proteins are known to be extensively dephosphorylated, there is no change in the sedimentation pattern and thus no detectable loss of spectrin from the junctions or weakening in the cohesion of the cytoskeletal network.     

Erythrocyte cellular and membrane deformability in hereditary spherocytosis

In order to determine whether the relative rigidity of the hereditary spherocytosis (HS) red cell is due to membrane rididity or merely to an altered surface/volume ratio, we investigated the deformability of resealed red cell membranes from patients with HS. Whereas the osmotic fragility of intact red cells of HS patients showed the expected increase, the osmotic fragility of resealed HS membranes was normal, thus indicating that their surface/volume ratio was normal. Measurements with an ektacytometer showed that deformability of intact HS cells was markedly diminished, whereas deformability of resealed HS membranes was normal. These findings indicate that the HS red cell membrane is not intrinsically abnormally rigid, as has been suggested, but that the lack of deformability of the erythrocyte is primarily a function of the altered surface/volume ratio.     

Erythrocyte membrane skeleton abnormalities in hereditary spherocytosis

Erythrocyte ghosts from eight individuals with hereditary spherocytosis have been compared with respect to their protein compositions as judged by SDS gel electrophoresis, their ease of spectrin extractability, and their freeze-etch electron microscopic appearance after incubation in condition designed to promote aggregation of the intramembrane particles. Four of these HS cases were unrelated, while the other four represented two generations from a single family, including a pair of identical twins, one of whom had not undergone splenectomy when this investigation was initiated. Of the four unrelated cases, one showed no departures from normal under the conditions of this investigation, whereas the other three exhibited features which suggested a membrane skeleton lesion. In one of these there was a reduced proportion of spectrin tetramers relative to dimers in 4 degrees C extracts, while the two remaining cases exhibited abnormal intramembrane particle aggregation. The four related cases had almost identical variations from normal. Spectrin was not extractable from their ghost membranes during a mild extraction incubation which removed spectrin from normal control ghosts. However, the intramembrane particle aggregation subsequently induced in these ghosts was of a degree unobtainable in normal ghosts without such spectrin extraction. In addition the ghosts from one twin, the only one of these patients who had not undergone splenectomy at the start of this investigation, showed a reduced amount of band 4.2. However, when this patient's blood was re-tested after splenectomy, this protein was found to be at normal levels. Our results support the view that hereditary spherocytosis is not a single disease, but is rather a term used to describe a variety of different molecular lesions of the erythrocyte membrane skeleton with similar clinical manifestations.     

Identification of the molecular defect in the erythrocyte membrane skeleton of some kindreds with hereditary spherocytosis

We have localized the molecular alteration in the membrane skeleton of two of four kindreds with hereditary spherocytosis (HS) to an alteration in the spectrin-protein-4.1 interaction due to a defective spectrin molecule. The defective spectrin-protein-4.1 interaction in these kindreds (referred to as type I HS) leads to a weakened spectrin- protein-4.1-actin ternary complex, which in turn may lead to the friable membrane skeleton and suggested membrane instability related to this disorder. Type I HS spectrin binds approximately 63% as much protein-4.1 as normal spectrin (with equal affinity). This defect does not correlate with splenic function or erythrocyte age in the circulation. However, the approximately 37% reduction in binding of protein-4.1 to HS spectrin approaches the theoretical value of 50% expected in this autosomal dominant disorder. All other type I membrane skeletal interactions (spectrin-syndein, spectrin heterodimer- heterodimer, syndein-band-3) were found to be normal. It would appear therefore that the defective HS spectrin-protein-4.1 interaction in type I hereditary spherocytosis may be the primary molecular defect rather than a secondary phenomena.     

Absence of phosphorylation-induced gelation of erythrocyte membrane skeletons: A diagnostic tool for hereditary spherocytosis

Summary As yet there is no single test specific for the diagnosis of hereditary spherocytosis. In the search for a specific test, a method described by Pinder et al. [14] using a cAMP-independent protein kinase extracted from normal erythrocyte membranes was used. Membrane skeletons were prepared from erythrocyte ghosts by extraction with a non-ionic detergent, i.e., Triton X-100. Upon phosphorylation with c-AMP-independent protein kinase the suspension of normal membrane skeletons set to a gelatinous mass. Membrane skeletons from patients with spherocytosis failed to show this phenomenon. In order to clarify whether this phenomenological difference can be used as a diagnostic tool for hereditary spherocytosis, a semiquantitative method of observing the gelation process was used under definite shear stress conditions. We investigated 33 patients with different hemolytic anemias (spherocytosis, hereditary elliptocytosis, hereditary stomatocytosis, homozygous -thalassemia and enzymopenic hemolytic anemias). With the exception of spherocytosis, all preparations of membrane skeletons showed gelation after 30–50 min. Spherocytosis membrane skeletons did not show a significant gelation even after 12 h of incubation. Thus, the failing gelation is specific for the diagnosis of hereditary spherocytosis. The gelation assay might be a valuable method for defining patients with hemolytic anemias due to erythrocyte membrane defects. Its molecular basis and the possible importance for the pathogenesis of spherocytosis require further investigations.Supported by Grant Eb 99/1-1 from theDeutsche Forschungsgemeinschaft     

Membrane protein phosphorylation and protein kinases in normal and hereditary spherocytosis red cells

Membrane protein phosphorylation by protein kinases in normal red cells takes place mainly on band 2 at the basal activity, and on bands 1 and 2.1 in the presence of cyclic 3':5'-adenosine monophosphate. Calcium precipitates preferentially bands 1 and 2.1 on extracted membrane proteins, and inhibit the membrane protein phosphorylation. Phosphorylation of endogeneous membrane proteins is diminished in red cells of some patients with hereditary spherocytosis (HS), partly corresponding reciprocally to MCHC, % spherocytes and reticulocytosis in peripheral blood of these patients, although the enzymatic activities of glyceraldehyde-3-phosphate dehydrogenase as a marker of the inner surface of red cell membranes are maintained normally in these red cells. The pattern of membrane protein fractions in HS red cells as endogeneous substrates for phosphorylation reactions is almost identical to that in normal red cells. Activities to phosphorylate casein or histone as exogeneous substrates are normal in HS red cell ghosts.     

Abnormal binding of spectrin to the membrane of erythrocytes in some cases of hereditary spherocytosis

Summary In two cases of hereditary spherocytosis that we have examined, spectrin was bound abnormally tightly to the erythrocyte membrane, and could not be released by low ionic strength dialysis. This type of behaviour occurs in normal red cells only after heating above 50 C. It appears that some cases of spherocytosis may be due to the presence of a protein which is abnormally temperature sensitive.This work was supported by an Australian Government Research Grant     

Pocked erythrocyte counts in patients with hereditary spherocytosis before and after splenectomy

The pocked (pitted or vacuolated) erythrocyte count has become increasingly utilized as a simple inexpensive test of splenic reticuloendothelial function. Values are less than 2.0% in normal subjects and 20 to 70% following splenectomy. Because scant and conflicting data are available about pocked erythrocyte measurements in hemolytic anemias other than the hemoglobinopathies, we performed pocked erythrocyte counts in 27 patients with hereditary spherocytosis. Prior to splenectomy patients often had elevated values (mean 4.9%). This unexpected observation suggests that hemolytic anemia may result in congestion of the red pulp and/or induced mild splenic reticuloendothelial blockade. As expected, but contrary to a previous report, pocked erythrocyte values following splenectomy were markedly increased (mean 54.9%).     

A phenomenological difference between membrane skeletal protein complexes isolated from normal and hereditary spherocytosis erythrocytes

S ummary . Membrane skeletons may be obtained from human erythrocytes by extraction with non-ionic detergent. When treated under defined conditions with a cAMP-independent kinase preparation from normal membranes, a suspension of these membrane skeletons sets to a gelatinous mass. Membrane skeletons from the cells of hereditary spherocytosis patients fail to show this response. Those from subjects with some other haemolytic anaemias do not share the abnormality. The gelation process could be shown also to occur with normal membrane skeletons, extracted at high ionic strength, and containing essentially only the structural protein constituents, spectrin, actin, 4·1 and 4·9. It also occurred rapidly when a column-purified kinase preparation was used, so that no significant amounts of contaminating proteins were introduced. Added spectrin, 4·1 or actin in moderate amounts did not induce gelation in the presence of ATP. Cytochalasin E did not perturb the gelation process. Gelation required ATP as well as kinase, and did not occur when the non-hydrolysable analogue, AMP-PNP, was used instead. Gelation was accompanied by phosphorylation of the spectrin alone, and is thus evidently a consequence of the modification of its properties by this means. Inhibition of phosphorylation by added adenosine retarded gelation. It may be inferred that phosphorylation of spectrin generates new, probably weak, non-covalent interactions between cytoskeletal constituents that cause association of the isolated cytoskeletons. A semi-quantitative method of observing the gelation process, based on the time of incubation before the membrane skeleton suspension ceases to flow under gravity a t a low shear. is described.     

A red cell membrane protein abnormality in hereditary spherocytosis

Summary. Dilute acetic acid and n-butanol were used to solubilize red cell membrane protein from patients with hereditary spherocytosis and normal subjects. Electrophoresis of acetic-acid-solubilized membrane protein on urea-starch and polyacrylamide gel revealed a constant absence of one band in the protein of hereditary spherocytosis cells. Red cells from a variety of other haematological disorders were also studied. All but one of the disorders examined demonstrated an electrophoretic pattern identical to that of normal cells, the exception being that of autoimmune haemolytic anaemia in which the pattern was similar to that of hereditary spherocytosis. Electrophoretic studies using sulphydryl binding and reducing agents suggest that the membrane protein abnormality in hereditary spherocytosis is linked to thiol groups in the membrane.     

Red cell membrane protein abnormalities in hereditary spherocytosis in Brazil

Summary. We studied 14 kindred and nine unrelated patients from southeastern Brazil with the typical form of hereditary spherocytosis. Diagnosis was made on the basis of clinical features, presence of spherocytes on the peripheral blood smears and an abnormal osmotic fragility test. By densitometric tracing of SDS-PAGE stained by Coomassie blue, we detected isolated deficiency of spectrin in 39% of our patients, combined spectrin and ankyrion deficiency in 13% and deficiency of band 3 in 13%. One of our patients presented ankyrin deficiency without spectrin reduction. Our data suggest that, despite ethnic differences among the Brazilian and European or North-American populations, these biochemical abnormalities in HS patients may be similar.     

Erythrocyte membrane protein alterations underlying clinical heterogeneity in hereditary spherocytosis

Summary. 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 subjcts. 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, spectin 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 memberane protein defects is shown. We conclude that erythrocyte memberane analysis should be carried out after diagnosis of HS in order to predict the clinical course of the disease.     

Increased adsorption of cytoplasmic proteins to the erythrocyte membrane in ATP-depleted normal and pyruvate kinase-deficient mature cells and reticulocytes

How the metabolic defect of pyruvate kinase deficiency (PK(-)) accelerates red blood cell (RBC) destruction is not established, but may be related to RBC membrane abnormalities associated with altered cellular metabolism. Furthermore, it has been shown that PK(-) reticulocytes are especially sensitive to metabolic depletion. Therefore, we compared the membranes of reticulocyte-rich PK(-) RBC, both fresh and ATP depleted, with membranes of fresh and ATP depleted normal mature RBC and reticulocytes. There was no difference between the specific gravity (SG) of the membranes of normal mature RBC (SG 1.152 +/- 0.004) and membranes of reticulocyte-rich RBC from several anemias (SG 1.150 +/- 0.002). However, membranes from fresh, reticulocyte-rich PK(-) RBC were dense with SG of 1.165 +/- 0.004 which correlated with a corresponding increase of protein to lipid phosphorus ratio of 66 +/- 8 micrograms protein/micrograms lipid phosphorus (normal 52 +/- 6 micrograms/micrograms). The membrane density of PK(-) RBC was further increased when the PK(-) RBC ATP was depleted by anaerobic incubation (SG 1.188 +/- 0.004) or cyanide inhibition (SG 1.182 +/- 0.001). When ATP was depleted in normal RBC and in non-PK(-) reticulocytes, corresponding increases in membrane SG occurred. A distinctive 50,000 MW peptide is adsorbed from the cytoplasm to the membranes of reticulocytes (both normal and PK(-) when these cells were depleted of ATP. The increased membrane adsorption of cytoplasmic proteins by PK(-) RBC was not associated with increased RBC calcium uptake, sulfhydryl oxidation, or altered membrane protein phosphorylation. All the observed abnormalities of PK(-) RBC membranes could by reproduced by ATP depletion of reticulocyte-enriched non-PK(-) RBC.