Sickle cell rheology is determined by polymer fraction–not cell morphology

Sickle cell disease pathophysiology is mediated by acute and chronic impairment of cell flexibility due to the formation of intracellular sickle hemoglobin (Hb S) polymer as cells are partially deoxygenated in the microcirculation. We have recently developed a method to measure the relationship between the formation of intracellular polymerized Hb S and cell filtration. In this study, we have used this method to examine whether sickle cell morphology, independent of Hb S polymer fraction, had an effect on cell rheology. We primarily use sickle trait (AS) and Hb S-β⁺-thalassemia (S-β⁺-thal) erythrocytes with low hemoglobin F levels, which have normal membranes and few or no dense cells, to remove these confounding effects. We find that the relationship between filtration and the percentages of each “type” of morphological deformation of AS erythrocytes was different from that of the S-β⁺-thal erythrocytes. In addition, we find that while the filtration of AS erythrocytes as a function of oxygen saturation was similar, whether measured during deoxygenation or reoxygenation, the relationship between the percentages of each type of deformed erythrocyte and oxygen saturation demonstrated hysteresis during oxygenation-deoxygenation experiments. Transmission electron microscopy, for both elongated and irregularly shaped cells, showed that similarly distorted cells could have very different amounts and alignment of polymer. These results suggests that cell morphology per se is not strongly related to filtration, whereas calculated intracellular Hb S polymer fraction predicts loss of filtration of AS and S-β⁺-thal erythrocytes well. Measured or calculated polymer fraction values would appear to be a better parameter for the study of sickle cell disease pathophysiology and response to treatment than cell morphology studies. ©1995 Wiley-Liss, Inc.     

Effects of hemoglobin concentration on deformability of individual sickle cells after deoxygenation

To assess the role of intracellular hemoglobin concentration in the deformability of sickle (HbSS) cells after deoxygenation, rheologic coefficients (static rigidity E and dynamic rigidity eta) of density- fractionated individual sickle erythrocytes (SS cells) were determined as a function of oxygen tension (pO2) using the micropipette technique in a newly developed experimental chamber. With stepwise deoxygenation, E and eta values showed no significant increase before morphologic sickling but rose sharply after sickling. In denser cells, continued deoxygenation led to steep rises of E and eta toward infinity, as the cell behaved as a solid. The pO2 levels at which rheologic and morphologic changes occurred for individual SS cells during deoxygenation varied directly with the cell density. The extent of recovery in E and eta during reoxygenation varied inversely with the cell density. These results provide direct evidence that the intracellular sickle hemoglobin (HbS) concentration of SS cells plays an important role in their rheologic heterogeneity in deoxygenation and reoxygenation. The elevations of eta during pO2 alteration were greater than those of E, especially for the denser cells, suggesting the importance of the elevated dynamic rigidity in initiating microcirculatory disturbances in sickle cell disease.     

Alterations in sensitivity to calcium and enzymatic hydrolysis of membranes from sickle cell disease and trait erythrocytes

Normally, human erythrocytes display several responses to elevated intracellular calcium levels. These include a shape transition from discocyte to spherocyte, shedding of microvesicles into the extracellular fluid, and enhanced susceptibility to the hydrolytic action of secretory phospholipase A(2). These responses to elevated intracellular calcium were all blunted in erythrocytes containing hemoglobin S. The reduction of both the shape transition and the shedding of microvesicles were greater than the impairment of phospholipase susceptibility, and both correlated strongly with the intracellular content of hemoglobin S. In contrast to the response to elevated intracellular calcium, erythrocytes containing hemoglobin S displayed a 2.5-fold increase in basal susceptibility to phospholipase A(2) compared to control erythrocytes in the absence of ionophore. The effect was more prominent among samples from patients heterozygous for hemoglobin S than in samples from homozygous individuals. These results reveal additional abnormalities in the membranes of sickle cell erythrocytes beyond those described previously and demonstrate that red blood cells from both heterozygous and homozygous are affected. Furthermore, they suggest a possible means by which sickle cell disease and trait patients may display enhanced vulnerability to inflammatory stimuli.     

Hydroxylamine Treatment Increases Glutathione-Protein and Protein-Protein Binding in Human Erythrocytes

ABSTRACT: Hydroxylamine is a direct-acting hematotoxic agent leading to hemolytic anemia in animals and man. The effect of hydroxylamine on the morphology, sulfhydryl status and membrane skeletal proteins of human erythrocytes were studied. Loss of reduced glutathione (GSH) from the red blood cells was directly proportional to the hydroxylamine concentration used. This loss of GSH was larger than the sum of the increase in the amounts of extracellular glutathione and intracellular oxidized glutathione (GSSG). The extracellular glutathione is mainly present as GSSG, which is in agreement with the fact that only GSSG is exported from the erythrocytes by membrane bound ATPases. Lack of GSSG export was not limited by decreased ATP levels in the erythrocytes and we concluded that the GSH that disappeared did not become available as intracellular GSSG. After reduction of the erythrocyte incubates the lost GSH was almost completely recovered indicating that the lost GSH is present in the cell as protein-glutathione mixed disulfides. Glutathione thus stored within the cell can be quickly recovered by combined thioltransferase and glutathione reductase activity when conditions become more favorable again. SDS-polyacrylamide gel electrophoresis of membrane ghosts from human red cells revealed changes in skeletal proteins with a smearing of bands 1, 2 and 3 to the higher molecular weight end of the gel and the appearance of new monomeric and dimeric hemoglobin bands at about 16 and 30 kD. The observed alterations are probably a consequence of disulfide bridge formation between cellular proteins (mainly hemoglobin) and skeletal proteins as well as between hemoglobin monomers. Exposure of hydroxylamine to erythrocytes caused severe Heinz body formation but the outside morphology of the cells was only marginally altered. The described changes in sulfhydryl status of the red blood cells are likely to play a major role in the premature splenic sequestration of hydroxylamine-damaged erythrocytes.     

Identification of hemoglobins in single erythrocytes by electrophoresis

We present here a method for electrophoretic identification of hemoglobin variants in many individual erythrocytes simultaneously, which is made possible by instruments developed in our laboratory. The system spaces and aligns the erythrocytes along a common origin and prevents 'doubling' of cells. Unambiguous separation of different hemoglobins (Hbs) occurred from single erythrocytes, such as Hbs A, C, F. Staged micrographs showed that hemoglobins of differing mobility from a single cell arise from a single point source of hemoglobin. In principle this capability is applicable to other proteins and could facilitate investigation at a cellular level into a variety of questions pertaining to gene action.     

Distributions of hemoglobins A and S among erythrocytes of heterozygotes

The recently developed capability to separate and quantify each of several proteins concurrently in single red cells presents an opportunity to test for biological variations in intercellular distribution of a protein as well as the extent of correlation between quantities of gene products derived from a single cell genome. In this preliminary study, erythrocytes from 30 sickle trait subjects were subjected to single cell electrophoresis and the resulting hemoglobin electropherograms were scanned by a recording densitometer. There was found to be heterogeneity among subjects in the form of the intercellular distribution of Hb S fraction, as tested by g statistics for skewness and kurtosis. Additionally, in all subjects there was statistically significant correlation between relative quantities of cellular Hb A and Hb S as measured concurrently in the same cell. These observations provide a basis for future research on the hypothesis that the form of the distribution of hemoglobin among erythrocytes is a heritable variable.     

Sickle-cell hemoglobin: fall in osmotic pressure upon deoxygenation.

Macromolecules such as hemoglobin exert both kinetic and matrix effects on osmotic pressure. The kinetic osmotic pressure of sickle-cell hemoglobin is lost upon deoxygenation at physiological erythrocyte concentrations. The non-kinetic or matrix component of osmotic pressure remains relatively unchanged. Loss of thermal-osmotic activity during deoxygenation occurs throughout a hemoglobin concentration range between 2.5 and 35 g/100 ml. Deoxygenation of sickle-cell hemoglobin causes aggregation such that the matrix effect is unchanged but the kinetic (van't Hoff) effect nearly vanishes. A loss of intracellular osmotic pressure during deoxygenation could dehydrate the erythrocyte sufficiently to promote more rapid sickle-cell hemoglobin aggregation. Subsequently, complete gelation of these aggregates could cause additional water loss and thrust the sickled cell into an irreversible cycle. The osmotic pressure of normal hemoglobin does not change appreciably during deoxygenation and is essentially the same as the osmotic pressure of oxygenated sickle-cell hemoglobin.     

Calcium-induced Damage of Haemoglobin SS and Normal Erythrocytes

An ionophore specific for divalent cations has been used to load normal erythrocytes and erythrocytes from patients with sickle cell anaemia (Hb SS disease), with small amounts of calcium. Such calcium accumulation leads to decreased cellular water, potassium, adenosine triphosphate (ATP), and osmotic fragility, all characteristics of irreversibly sickled cells (ISCs). In addition, calcium loading of Hb SS, but not normal, erythrocytes causes a marked decrease of haemoglobin oxygen affinity; another, and specific, hallmark of ISCs. Ionophore-induced accumulation of calcium by deoxygenated Hb SS erythrocytes also leads to temporary retention of sickled shape following reoxygenation, despite the absence of detectable intracellular haemoglobin S fibres. All these effects require calcium in the incubation medium and support the idea that increased intracellular calcium is important in the formation of ISCs in patients with Hb SS disease.     

Interrelationships between ATP levels,enzyme leakage,gluconate uptake,trypan blue exclusion and length/width ratio of isolated rat cardiac myocytes subjected to anoxia and reoxygenation

Rat cardiac myocytes were isolated by perfusion of the heart with collagenase and subsequently incubated in the absence or presence of oxygen. As a result of anoxia, there was a gradual increase in plasma membrane permeability, as indicated by a decrease in trypan blue exclusion, leakage of cytosolic lactate dehydrogenase, and intracellular accumulation of the isotope [⁹⁹Tc^m]gluconate. These changes in plasma membrane permeability were preceded by a marked decrease in cellular ATP levels and an increased proportion of contracted myocytes. After an initial anaerobic incubation for 10 min, there was complete replenishment of ATP followed by signs of recovery from anoxic cell injury upon reoxygenation.Cell viability could also be followed on the basis of changes in the length/width ratio of myocytes upon anoxia and reoxygenation. The ability of myocytes to resynthesize ATP and to recover from anoxic injury upon reoxygenation decreased in proportion to the length of the initial anaerobiosis during the first 25 min and disappeared completely after 30 min of anoxia.The present model system is a convenient tool for evaluation of interrelationships between energy metabolism, plasma membrane integrity, and the morphology of myocytes subjected to anoxia and reoxygenation.     

Non-opsonising aggregates of IgG and complement in haemoglobin C erythrocytes

Haemoglobin C (HbC) differs from normal HbA by a lysine for glutamate substitution at position 6 of beta-globin. Heterozygous AC and homozygous CC phenotypes are associated with shortened erythrocyte life spans and mild anaemia. AC and CC erythrocytes contain elevated amounts of membrane-associated haemichromes, band 3 clusters, and immunoglobulin G (IgG) in vivo. These findings led us to investigate whether AC and CC erythrocytes might expose elevated levels of IgG and complement, two opsonins that have been implicated in the phagocytic clearance of senescent and sickle erythrocytes. Surprisingly, we found IgG, complement, and other plasma proteins co-localised in aggregates beneath the membrane of circulating AC and CC erythrocytes. These observations, and our finding of similar aggregates in erythrocytes heterozygous or homozygous for haemoglobin S (sickle-cell haemoglobin), suggest that the vast majority of membrane-associated IgG and complement detected in these abnormal erythrocytes is intracellular and does not contribute to the eventual opsonic clearance of these cells. Phagocytosis studies with macrophages provide evidence in support of this suggestion. Studies of erythrocyte clearance that involve the detection of membrane-associated IgG and complement as putative opsonins should investigate the possibility that these plasma proteins reside in the erythrocyte interior, and not on the cell surface.     

Isolation, characterization, and immunoprecipitation studies of immune complexes from membranes of beta-thalassemic erythrocytes.

beta-Thalassemia, a hemoglobinopathy that results in the precipitation of denatured alpha-globin chains on the membrane, is characterized by erythrocytes with significantly reduced lifespans. We have demonstrated previously that hemoglobin denaturation on the membrane can promote clustering of integral membrane proteins, and that this clustering in turn leads to autologous antibody binding, complement fixation, and rapid removal of the cell by macrophages. To evaluate whether this pathway also occurs in beta-thalassemic cells, we have isolated and characterized the immune complexes from the membranes of these cells. We observe that autologous IgG-containing complexes obtained by either immunoprecipitation or simple centrifugation of nondenaturing detergent extracts of beta-thalassemic cell membranes contain globin, band 3, IgG, and complement as major components. Absorption spectra of these complexes demonstrate that the globin is, indeed, mainly in the form of hemichromes. Immunoblotting studies further show that much of the band 3 protein in the aggregates is covalently cross-linked to a dimeric or tetrameric form, consistent with the preference of the autologous IgG for clustered band 3. Although the insoluble aggregates constitute only approximately 1.6% of the total membrane protein, they still contain 27% of the total IgG and 35% of the total complement C3 on the thalassemic cell surface. Because cell surface IgG and complement component C3 are thought to trigger removal of erythrocytes from circulation, the hemichrome-induced clustering of band 3 may contribute to the beta-thalassemic cell's shortened lifespan.     

Inhibition of K transport by divalent cations in sickle erythrocytes

We report experiments on the effect of intracellular divalent cations (Mg, Ca, Mn) on K transport and cell volume in erythrocytes from patients with homozygous hemoglobin S disease (SS cells). When CO- treated SS erythrocytes are exposed to the ionophore A23187, removal of cell Mg markedly stimulates K efflux, whereas increasing cell Mg inhibits K efflux. The Ki for the inhibition by internal free Mg is 0.38 +/- 0.10 mmol/L, a value comparable to the concentration of free Mg in normal cells (0.3 to 0.4 mmol/L). When swollen SS cells with increased Mg content were incubated in plasma-like medium, they shrunk much less than swollen SS cells with normal Mg content. Thus, elevation of cell Mg produces inhibition of swelling-induced K movement from SS cells. Internal Ca and Mn also inhibit K movement from SS cells. The inhibition of volume regulation by divalent cations suggests that increases in intracellular divalent ions, especially Mg, could induce a persistent degree of cell swelling in SS RBCs and thereby inhibit intracellular polymerization.     

Acetaldehyde-Dependent Changes in Hemoglobin and Oxygen Affinity of Human Erythrocytes

In the presence of acetaldehyde, metabolizing human erythrocytes accumulate an altered hemoglobin product showing chromatographic similarity to hemoglobin A_Ia or A_Ib. The adduct is stable to overnight dialysis with an intracellular half-life of about 5.5 days. Adduct formation is accompanied by proportional changes in cell oxygen affinity (decrease in P₅₀ of 3 mm Hg/mM adduct). Little unaltered hemoglobin remains after overnight incubation in 15 mM acetaldehyde, with significant adduct formation and marked reduction of cell ATP occurring after prolonged incubation in as little as 0.5 mM acetaldehyde.     

Determination of deoxyhemoglobin S polymer in sickle erythrocytes upon deoxygenation.

We have used 13C/1H magnetic double-resonance spectroscopy to measure the amount of sickle hemoglobin polymer within sickle erythrocytes as a function of oxygen saturation. We previously showed that the methods of cross-polarization and scalar decoupling could be used to measure accurately the polymer fraction in deoxygenated sickle hemoglobin solutions [Noguchi, C.T., Torchia, D.A. & Schechter, A.N. (1979) Proc. Natl. Acad. Sci. USA 76, 4936-4940]. Our measurements show that the amount of intracellular deoxyhemoglobin S polymer increases monotonically with decreasing oxygen saturation. Polymer can be detected at oxygen saturation values above 90%. This result can be theoretically explained by the excluded volume effect of the oxyhemoglobin S in the cell. The very high total intracellular hemoglobin concentration (34 g/dl) reduces the amount of soluble deoxyhemoglobin S to about 3 g/dl at 90% oxygen saturation. The agreement between theory and experiment indicates that the equilibrium properties of intracellular polymerization can be described by the analyses resulting from studies of concentrated sickle hemoglobin solutions. The curve for polymer formation as a function of oxygen saturation is roughly hyperbolic whereas that for cell sickling is sigmoidal; the difference is most apparent for measurements at pH 7.65. Intracellular polymer formation may in general have a different relationship to oxygen saturation than cell sickling and may be a more meaningful parameter of the pathophysiological process in sickle cell anemia than cell morphology. In addition, measurements of intracellular polymer should be useful in evaluating potential therapeutic agents.     

Fibers, crystals, and other forms of HbS polymers in deoxygenated sickle erythrocytes

We have examined by electron microscopy the formation of fibers and crystals from sickle hemoglobin within sickle erythrocytes following deoxygenation during capillary storage from 1 to 132 days. Intracellular fibers were found on the first day and throughout the period of study. The fibers exhibited a diameter (mean +/- SD) of 17.4 +/- 0.62 nm and were aligned in the cell with a fiber-to-fiber spacing of 18.6 nm (x-axis) by 22.7 nm (y-axis). Between 65 and 132 days, extracellular hemoglobin crystals developed, with a lattice periodicity of 9.63 +/- 0.6 nm. Fibers and crystals coexist as separate structures. These results suggest that crystal formation upon storage of packed deoxygenated sickle erythrocytes may proceed via a phase of fiber dissolution followed by hemoglobin reassembly into extracellular crystals, rather than by a progressive alignment and direct fusion of existing fibers.     

Deformability of erythrocytes in iron deficiency anemia

Summary The rheological properties of erythrocytes of 14 patients with iron deficiency anemia were studied by filtration of cells through polycarbonate filters with a nominal pore diameter of 5 m and by viscosity measurements of erythrocyte suspensions with a hematocrit of 80%. Erythrocytes of the patients passed through the filter pores more slowly than the cells from controls. The diminished deformability of the erythrocytes of the patients was solely due to an unfavorable ratio of cell surface area to microcytic cell volume. The viscosity of the ghost suspensions of the patients showed a normal flexibility. The hemoglobin content of the isolated ghosts was diminished, indicating an in-increased hemoglobin fluidity in the interior of the intact cells. The viscosity of erythrocytes of the patients was slightly increased at low shear rates but was normal at intermediate and high shear rates. We suggest that the decreased erythrocyte flexibility of microcytosis at low shear rates is no longer present at higher shear rates because of an increased fluidity of the intracellular hemoglobin. We discuss whether or not this mechanism also operates in vivo. The in vitro diminished deformability of erythrocytes explains the shortened survival of the patients' erythrocytes in vivo.This work was supported by the Deutsche Forschungsgemeinschaft     

Hemoglobin aggregation and pseudosickling in vitro of hemoglobin setif-containing erythrocytes

Erythrocytes from individuals heterozygous for hemoglobin Setif (α⁹⁴ Asp→Tyr) sickle in vitro without deoxygenation when incubated in chloride buffer due to hemoglobin aggregation. We now report quantitative studies of hemoglobin polymerization and deformability in these cells. Hemoglobin polymer gradually increased in intact cells during a 24 h incubation period at 24°C. After 24 hr, about 80% of the cells in 290 mosm sodium chloride buffer contained polymer which appeared as short rods compared to >99% containing polymer at 450 mOsm. Similar proportions of cells were morphologically sickled. Deformability of erythrocytes with 40% hemoglobin Setif incubated in 290 mOsm buffer at 37°C decreased to 80% of normal by 210 min but in 450 mOsm decreased to 50% after only 30 min as measured by the ektacytometer. However, at 4°C deformability remained normal even in 450 mOsm buffer. The solubility of gelled hemolysate containing 40% hemoglobin Setif was 24 g/dl and 21 g/dl at 290 and 459 mOsm buffer respectively. The gel persisted at 4°C with a solubility of 25 g/dl, but melted when dialyzed into sodium phosphate or potassium phosphate buffer. These data suggest that hemoglobin polymerization, reduced deformability, and sickling of hemoglobin Setif-containing erythrocytes are related to reduced hemoglobin solubility. The rate and extent of intracellular polymerization in vitro are considerably reduced (as in the case of sickle trait) compared with erythrocytes from individuals with sickle cell anemia. Hence, the slower kinetics of hemoglobin aggregation in hemoglobin Setif-containing cells provide an alternate system for studying hemoglobin aggregation in hemoglobin Setif-containing cells provide an alternate system for studying hemoglobin polymerization and abnormal rhelogy.     

Detection of hemin release during hemoglobin S denaturation

Sickle hemoglobin is relatively unstable upon oxidation or mechanical shaking. During denaturation, it generates oxygen radicals and hemichromes and ultimately precipitates in the form of micro-Heinz bodies. It is not clear, however, whether the degradation product hemin, which is a potent hemolytic agent and a potential perturbant to protein-protein interactions in the red cell membrane skeleton, is also generated during sickle hemoglobin denaturation. By specific absorption of hemin with Dowex AG 1-X8 anion-exchange resin at high-ionic strength conditions, we now separate hemin for quantitation from the bulk hemoglobin and its derivatives. We demonstrate that upon mechanical shaking oxyhemoglobin S denatures much faster than oxyhemoglobin A and that a considerably higher level of hemin is detected in the shaken hemoglobin S as compared with hemoglobin A. By using the same method to measure the hemin content in the hemolysate of fresh red cells from patients with sickle cell disease, we detect a three- to fivefold increase in the hemin content in these patients (0.4 to 0.75 mumol/L) as compared with normal individuals (0.1 to 0.15 mumol/L). These data suggest that the instability of sickle oxyhemoglobin leads to increased intracellular precipitation of hemoglobin and the release of hemin, which may play a role in the membrane lesion of sickle red cells.     

Alternative aspirins as antisickling agents: acetyl-3,5-dibromosalicylic acid.

Acetyl-3,5-dibromosalicylic acid (dibromoaspirin) is shown to be a potent acylating agent of intracellular hemoglobin in vitro. Transfer of the actyl group of dibromoaspirin to amino groups of hemoglobins A and S seems to occur predominantly at just two or three sites on these proteins. This acetylation produces moderate increases in the oxygen affinities of normal and sickle erythrocytes. Furthermore, treatment of intracellular hemoglobin S with dibromoaspirin directly inhibits erythrocyte sickling. This antisickling effect is paralleled by an increase in the minimum gelling concentration of deoxy hemoglobin S extracted from sickle erythrocytes that had been exposed to low concentrations of dibromoaspirin. These observations suggest that dibromoaspirin might be an effective antisickling agent in vivo.     

Membrane-associated sickle hemoglobin: a major determinant of sickle erythrocyte rigidity

Micropipette aspiration tests on single erythrocytes have previously shown that the static rigidity (membrane shear modulus) of oxygenated sickle cells increased with increasing hemoglobin concentration, whereas the rigidity of normal cells was independent of hemoglobin concentration. Moreover, it was observed that after mechanical extension, sickle cells exhibited persistent deformation more frequently and to a greater extent than normal cells. To ascertain if differences in association of normal and sickle hemoglobin with the membrane could account for these observations, we measured rheologic properties of normal membranes reconstituted with sickle hemoglobin and sickle membranes reconstituted with normal hemoglobin. The static rigidity of normal ghosts reloaded with sickle hemoglobin was higher than those of either normal ghosts reloaded with normal hemoglobin or native normal cells. On the other hand, the increased rigidity of native sickle cells decreased to near-normal values following reconstitution with normal hemoglobin. Furthermore, we observed that normal ghosts reconstituted with sickle hemoglobin exhibited persistent bumps after mechanical extension, but no bumps formed on normal ghosts reconstituted with normal hemoglobin. Moreover residual bumps were not produced on sickle cells reloaded with normal hemoglobin. Since mechanical characteristics peculiar to sickle cells could be induced in normal cells by incorporation of sickle hemoglobin, and since normal characteristics could be restored to sickle cells by incorporation of normal hemoglobin, we suggest that the interaction of sickle hemoglobin with the cell membrane is responsible for augmented static rigidity of oxygenated sickle erythrocytes.