pH dependency of potassium efflux from sickled red cells

Potassium efflux from deoxygenated, hemoglobin S-containing red cells is often used as an "objective" in vitro measure of aed cell sickling, particularly during tests with antisickling agents. Since varying pH is known to affect both the extent of sickling and passive K+-flux across the red cell membrane, in opposite directions, we measured the sickling-related K+-efflux in sickle cell anemia (SS) and sickle cell trait (AS) red cells as a function of extracellular and intracellular pH. The sickling-related K+-efflux was found to show the same direction of pH dependence as normal red cells, so that as the extracellular pH was reduced below 7.6, sickling and K+-efflux were increasingly dissociated. A similar dissociation was observed between sickling and K+-efflux when the intracellular pH was lowered by increasing red cell organic phosphate levels. The sickling-related K+-efflux from osmotically shrunken AS cells (whose sickling tendency resembles that of SS cells) was similar in magnitude and pH dependency to that of the SS cells. The findings suggest that measurement of K+-efflux may be an accurate estimate of the extent of intracellular polymerization in sickled red cells, provided that both the intracellular and extracellular pH levels are carefully controlled, and the experimental conditions produce no independent effects on K+ permeability.     

2,3-Diphosphoglycerate and intracellular pH as interdependent determinants of the physiologic solubility of deoxyhemoglobin S

We have established that 2,3-diphosphoglycerate (2,3-DPG) content and intracellular pH exert separate, but interdependent, effects on the equilibrium solubility (csat) of deoxyhemoglobin S (deoxy-Hb S) that act in concert to modulate intraerythrocytic polymer formation. In a nonphysiologic csat assay system, a steep dependence of csat on pH in the physiologic range 7.0 to 7.6 was shown for both stripped (Hb) and DPG-saturated deoxy-Hb S (Hb-DPG). The solubility-pH profile for Hb under near-physiologic buffer conditions also showed that csat increased steeply in the same pH range (6.8 to 7.6). The effect of 2,3- DPG on csat under near-physiologic conditions was evaluated separately. At pH 7.20, the pH of the human red blood cell, csat values for Hb and Hb-DPG were 19.56 +/- 0.14 and 17.95 +/- 0.45 g/dL, respectively, indicating that the solubility of Hb-DPG is lower than that of Hb by 8.2% +/- 2.3%. Thus, binding of 2,3-DPG in the beta-cleft promotes the polymerization of deoxy-Hb S, the ultimate determinant of cell sickling. Furthermore, because of the abnormal Bohr effect of sickle blood (approximately double that of normal blood), the intracellular pH of deoxygenated sickle erythrocytes should be approximately 0.28 pH unit higher than that of oxygenated cells (ie, 7.41 v 7.13). At the higher pH, the corresponding csat for Hb-DPG is 20.22 g/dL, which is the best estimate of the intrinsic solubility of T-state Hb S under conditions that approximate closely those of pH, temperature, ionic strength, and 2,3-DPG saturation in the fully desaturated sickle erythrocyte.     

Functional characteristics of red cells of sickle cell anemia patients with and without α-thalassemia-2

Oxygen equilibrium curves, red cell indices, 2,3-DPG levels, and the percentages of ISC and Hb F were determined for red cell fractions isolated by Dextran 40 density gradient centrifugation from blood of a sickle cell anemia (SS) patient, an SS patient with an additional homozygosity for α-thalassemia-2, and a normal control. In the SS patient, the MCHC and P₅₀ values increased and 2,3-DPG levels decreased with an increase in red cell density; the P₅₀ values were directly related to the MCHC values and inversely related to the 2,3-DPG levels. The bottom fraction contained about 80% ISC, and had the highest MCHC and P₅₀ values and the lowest 2,3-DPG level. In the SS α-thalassemia-2 patient, a decrease in the P₅₀ value corresponded with a decrease in 2,3-DPG levels because no change in MCHC value was observed. The bottom fraction contained about 10% ISC and had the lowest P₅₀ value. These data confirm that both MCHC and 2,3-DPG levels influence the oxygen affinity of SS red cells; an increase in MCHC decreases the oxygen affinity to a great extent while a concomitant decrease in 2,3-DPG level partially abolishes this effect.     

Distribution of dehydration rates generated by maximal Gardos-channel activation in normal and sickle red blood cells

The Ca(2+)-activated K+ channels of human red blood cells (RBCs) (Gardos channels, hIK1, hSK4) can mediate rapid cell dehydration, of particular relevance to the pathophysiology of sickle cell disease. Previous investigations gave widely discrepant estimates of the number of Gardos channels per RBC, from as few as 1 to 3 to as many as 300, with large cell-to-cell differences, suggesting that RBCs could differ extensively in their susceptibility to dehydration by elevated Ca2+. Here we investigated the distribution of dehydration rates induced by maximal and uniform Ca2+ loads in normal (AA) and sickle (SS) RBCs by measuring the time-dependent changes in osmotic fragility and RBC volume distributions. We found a remarkable conservation of osmotic lysis and volume distribution profiles during Ca(2+)-induced dehydration, indicating overall uniformity of dehydration rates among AA and SS RBCs. In light of these results, alternative interpretations were suggested for the previously proposed low estimates and heterogeneity of channel numbers per cell. The results support the view that stochastic Ca2+ permeabilization rather than Gardos-channel variation is the main determinant selecting which SS cells dehydrate through Gardos channels in each sickling episode.     

Calcium accumulated by sickle cell anemia red cells does not affect their potassium (86Rb+) flux components

We investigate here the hypothesis that the high Ca content of sickle cell anemia (SS) red cells may produce a sustained activation of the Ca2+-dependent K+ permeability (Gardos effect) and that the particularly high Ca levels in the dense SS cell fraction rich in irreversibly sickled cells (ISCs) might account for the Na pump inhibition observed in these cells. We measured active and passive 86Rb+ influx (as a marker for K+) in density-fractionated SS cells before and after extraction of their excess Ca by exposure to the Ca ionophore (A23187) and ethylene glycol tetra-acetic acid and with or without adenosine triphosphate depletion or addition of quinine. None of these maneuvers revealed any evidence of a Ca2+-dependent K leak in SS discocytes or dense cells. Na pump inhibition in the dense SS cells was associated with normal activation by external K+ and a low Vmax that persisted after Ca extraction from the cells. These results are consistent with our recent findings that the excess Ca in these cells is compartmentalized in intracellular inside-out vesicles and unavailable as free Ca2+ to the inner membrane surface. Although the steady-state free cytoplasmic Ca2+ in oxygenated SS cells must be below the levels needed to activate the K+ channel, possible brief activation of the channels of some SS cells resulting from transient elevations of cell Ca2+ during deoxygenation-induced sickling cannot be excluded. The dense, ISC-rich SS cell fraction showed a Ca2+-independent increase in the ouabain-resistant, nonsaturable component of 86Rb+ influx that, if uncompensated by Na+ gain, could contribute to the dehydration of these cells.     

A new sickle cell disease phenotype associating Hb S trait,severe pyruvate kinase deficiency (PK Conakry), and an α2 globin gene variant (Hb Conakry)

A Guinean woman, hetererozygous for haemoglobin (Hb) S, was studied because of episodes of marked anaemia, repeated typical metaphyseal painful crises and haemosiderosis. Her sickling syndrome resulted from the association of Hb S trait with a severe pyruvate kinase deficiency leading to a 2,3-DPG concentration of twice normal levels. Sequence of the PK-R gene revealed an undescribed mutation in the homozygous or hemizygous state within exon 5 (nucleotide 2670 C → A), leading to the interchange of Ser 130 into Tyr (PK Conakry). In addition, the patient carried a new haemoglobin variant, Hb Conakry [α80(F1) Leu → Val], which seemed to have a mild effect. The high intraerythrocytic 2,3-DPG concentration induced by the PK deficiency resulted in a decreased oxygen affinity which favoured sickling to a level almost similar to that of Hb S/C compound heterozygous patients. This was confirmed by oxygen binding measurements of Hb A/Hb S erythrocytes in which 2,3-DPG content was modified in vitro. Hysteresis between deoxy- and reoxygenation curves, as well as increase in the n_max value, demonstrated that the extent of HbS polymerization in the propositus was almost the same as that of RBCs from a homozygous sickle cell patient or those of an A/S heterozygous patient with an artificial in vitro increase of 2,3-DPG concentration.     

Rapid and reproducible characterization of sickling during automated deoxygenation in sickle cell disease patients

In sickle cell disease (SCD), sickle hemoglobin (HbS) polymerizes upon deoxygenation, resulting in sickling of red blood cells (RBCs). These sickled RBCs have strongly reduced deformability, leading to vaso-occlusive crises and chronic hemolytic anemia. To date, there are no reliable laboratory parameters or assays capable of predicting disease severity or monitoring treatment effects. We here report on the oxygenscan, a newly developed method to measure RBC deformability (expressed as Elongation Index - EI) as a function of pO₂. Upon a standardized, 22 minute, automated cycle of deoxygenation (pO₂ median 16 mmHg ± 0.17) and reoxygenation, a number of clinically relevant parameters are produced in a highly reproducible manner (coefficients of variation <5%). In particular, physiological modulators of oxygen affinity, such as, pH and 2,3-diphosphoglycerate showed a significant correlation (respectively R = ‑0.993 and R = 0.980) with Point of Sickling (PoS_5%), which is defined as the pO₂ where a 5% decrease in EI is observed during deoxygenation. Furthermore, in vitro treatment with antisickling agents, including GBT440, which alter the oxygen affinity of hemoglobin, caused a reproducible left-shift of the PoS, indicating improved deformability at lower oxygen tensions. When RBCs from 21 SCD patients were analyzed, we observed a significantly higher PoS in untreated homozygous SCD patients compared to treated patients and other genotypes. We conclude that the oxygenscan is a state-of-the-art technique that allows for rapid analysis of sickling behavior in SCD patients. The method is promising for personalized treatment, development of new treatment strategies and could have potential in prediction of complications.     

Characterization of hyaluronan synthase expression and hyaluronan synthesis in bone marrow mesenchymal progenitor cells: predominant expression of HAS1 mRNA and up-regulated hyaluronan synthesis in bone marrow cells derived from multiple myeloma patients

Interaction of hemoglobin S polymers with the red blood cell (RBC) membrane induces a reversible increase in permeability ("P(sickle)") to (at least) Na(+), K(+), Ca(2+), and Mg(2+). Resulting changes in [Ca(2+)] and [H(+)] in susceptible cells activate 2 transporters involved in sickle cell dehydration, the Ca(2+)-sensitive K(+) ("Gardos") channel (K(Ca)) and the acid- and volume-sensitive K:Cl cotransport. We investigated the distribution of P(sickle) expression among deoxygenated sickle cell anemia (SS) RBCs using new experimental designs in which the RBC Ca(2+) pumps were partially inhibited by vanadate, and the cells' dehydration rates were detected as progressive changes in the profiles of osmotic fragility curves and correlated with flow cytometric measurements. The results exposed marked variations in (sickling plus Ca(2+))-induced dehydration rates within populations of deoxygenated SS cells, with complex distributions, reflecting a broad heterogeneity of their P(sickle) values. P(sickle)-mediated dehydration was inhibited by clotrimazole, verifying the role of K(Ca), and also by elevated [Ca(2+)](o), above 2 mM. Very high P(sickle) values occurred with some SS discocytes, which had a wide initial density (osmotic resistance) distribution. Together with its previously shown stochastic nature, the irregular distribution of P(sickle) documented here in discocytes is consistent with a mechanism involving low-probability, reversible interactions between sickle polymers and membrane or cytoskeletal components, affecting only a fraction of the RBCs during each deoxygenation event and a small number of activated pathways per RBC. A higher participation of SS reticulocytes in P(sickle)-triggered dehydration suggests that they form these pathways more efficiently than discocytes despite their lower cell hemoglobin concentrations.     

Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca²⁺) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1–10 μM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca²⁺ influx. Yoda1 induced Ca²⁺-dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo−/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease.     

Deformation of swollen erythrocytes provides a model of sickling- induced leak pathways, including a novel bromide-sensitive component

Deoxygenation-induced red blood cell (RBC) sickling probably activates multiple cation leak pathways. In an attempt to model this, we examined the net passive K efflux ("K leak") from normal and sickle RBCs undergoing elliptical deformation in hypotonic media (200 mOsmol/L). This hypotonic deformation activates two deformation-dependent K leak pathways that are not detectable during the balanced leak (Kefflux = Nainflux) resulting from deformation of RBCs in isotonic medium. These are (1) a calcium-dependent leak component and (2) a novel leak pathway that is inhibited by substitution of bromide (but not sulfamate) for chloride, which converts the unbalanced K leak (Kefflux > Nainflux) of hypotonic deformation to a residual balanced leak. This dramatic effect of hypotonic deformation is reversible, is detected in both normal and sickle RBCs, and is inhibited significantly by 4,4'-diisothiocyano-2,2'- stilbene disulfonate. Remarkably, bromide also inhibits by 55% the K leak resulting from authentic deoxygenation-induced RBC sickling and, thereby, blunts the imbalance of accompanying monovalent cation leaks. The unique effect of bromide is not readily explainable on the basis of known behaviors of known ion leak/transport pathways. The mechanical threshold for triggering K leak during hypotonic deformation is at applied shear stress of 164 dyne/cm2, a value similar to the abnormal susceptibility we previously found for oxygenated sickle RBCs during isotonic deformation. These data suggest that membrane stretch accompanying hypotonic deformation activates the same multiple leak pathways that contribute to net K leak during authentic RBC sickling, including a previously unknown bromide-sensitive leak.     

In vitro effects of NIPRISAN (Nix-0699): a naturally occurring,potent antisickling agent

Among the various potential antisickling agents tested, hydroxyurea (HU) has been the most effective compound used for the treatment of patients with sickle cell disease (SCD). Although HU is effective in many patients, not all patients respond to this drug. In addition, some patients reveal adverse effects, including myelosuppression. In an attempt to find other effective agents with less adverse effects, we investigated the antisickling effect of NIPRISAN (Nix-0699). We found that Nix-0699, an ethanol/water extract from indigenous plants, has a strong antisickling effect. The concentration of Nix-0699 required to inhibit 50% of erythrocyte sickling was about 0.05 mg/ml. As for the kinetics of polymerization, addition of 0.05 microg/ml Nix-0699 caused a sixfold prolongation of the delay time prior to deoxy-Hb S polymerization when compared with that of untreated Hb S samples. The solubility of deoxy-Hb S significantly increased upon treatment with Nix-0699. Analysis of the effect of Nix-0699 on the Hb S oxygen affinity indicated that the drug slightly shifted the oxygen-dissociation curve of Hb S toward the left without any apparent change in the Hill coefficient. These results suggest that the antisickling properties of Nix-0699 may involve direct interaction with Hb molecules. Incubation of red blood cell (RBC) suspensions with various concentrations of Nix-0699 did not dehydrate RBCs, cause haemolysis, increase the amount of denatured Hb, nor form met-Hb. In view of the outcome of this study, Nix-0699 may be a promising option for the treatment of patients with SCD.     

Dipyridamole inhibits sickling-induced cation fluxes in sickle red blood cells

Sickling-induced cation fluxes contribute to cellular dehydration of sickle red blood cells (SS RBCs), which in turn potentiates sickling. This study examined the inhibition by dipyridamole of the sickling-induced fluxes of Na(+), K(+), and Ca(++) in vitro. At 2% hematocrit, 10 microM dipyridamole inhibited 65% of the increase in net fluxes of Na(+) and K(+) produced by deoxygenation of SS RBCs. Sickle-induced Ca(++) influx, assayed as (45)Ca(++) uptake in quin-2-loaded SS RBCs, was also partially blocked by dipyridamole, with a dose response similar to that of Na(+) and K(+) fluxes. In addition, dipyridamole inhibited the Ca(++)-activated K(+) flux (via the Gardos pathway) in SS RBCs, measured as net K(+) efflux in oxygenated cells exposed to ionophore A23187 in the presence of external Ca(++), but this effect resulted from reduced anion conductance, rather than from a direct effect on the K(+) channel. The degree of inhibition of sickling-induced fluxes was dependent on hematocrit, and up to 30% of dipyridamole was bound to RBC membranes at 2% hematocrit. RBC membrane content of dipyridamole was measured fluorometrically and correlated with sickling-induced flux inhibition at various concentrations of drug. Membrane drug content in patients taking dipyridamole for other clinical indications was similar to that producing inhibition of sickling-induced fluxes in vitro. These data suggest that dipyridamole might inhibit sickling-induced fluxes of Na(+), K(+), and Ca(++) in vivo and therefore have potential as a pharmacological agent to reduce SS RBC dehydration. (Blood. 2001;97:3976-3983)     

Clinical and molecular correlations in the sickle/beta+-thalassemia syndrome

Sickle/beta thalassemia is a sickling disorder of varying severity which results from compound heterozygosity for sickle cell trait and beta-thalassemia trait. Clinical and genetic studies have shown an inverse correlation between the level of hemoglobin A and the severity of the disease. It has been suggested that the level of hemoglobin A may be a function of the severity of the beta-thalassemia defect. In this study, we use molecular biological techniques to test this hypothesis. We show that the interaction of the mildest of the beta+-thalassemia genes with the sickle gene results in a high level of hemoglobin A. However, the interaction in this case resulted in a severe sickling disorder in the absence of significant anemia. We hypothesize that a mild beta+-thalassemia gene may have two opposite effects on the clinical course of sickle/beta+ thalassemia: (1) A high level of hemoglobin A which probably confers a favorable antisickling effect and (2) decreased hemolysis leading to increased numbers of total circulating red cells, thereby increasing the blood viscosity and the propensity for sickling. The inheritance of heterozygous alpha thalassemia 2 in conjunction with the mild beta+-thalassemia gene and sickle gene in this patient may have further enhanced the latter effect and resulted in a severe sickling disorder.     

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.     

Freeze preservation of sickle erythrocytes

The effect of short-term cryopreservation on metabolic, functional, and survival characteristics of erythrocytes from patients with sickle cell disease was examined. Post-thaw hemolysis of glycerolized sickle (SS) erythrocytes was greater (mean 12.9%) than in hemoglobin-AA cells (mean 4.7%). Freeze preservation had no apparent effect on red cell morphology, percent irreversibly sickled cells, and fetal hemoglobin content. There were modest reductions in ATP and 2,3-diphosphoglycerate in thawed, washed sickle erythrocytes (12.7% and 29.7%, respectively). However, the autologous survival of ⁵¹ Cr-labelled SS red cells was not shortened by cryopreservation. The safety and efficacy of autotransfusion of cryopreserved red cells in alloimmunized sickle cell disease patients with anemic episodes unrelated to sickling need to be determined.     

The Gardos channel is responsible for CDNB-induced dense sickle cell formation

The red blood cells (RBCs) derived from blood taken from homozygous sickle cell (SS) patients demonstrate densities that are inversely proportional to the intracellular reduced glutathione (GSH) content. Addition of 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) to low-density sickle cells (LDSS), at 4 degrees C, results in a shift of LDSS erythrocytes to high-density sickle cells (HDSS), with corresponding decreases in GSH. We have previously demonstrated that this CDNB effect was due to increased K(+) leakage and that dense cell formation could be inhibited by clotrimazole (specific for the Gardos channel) but not DIOA (specific for the K(+)-Cl(-) co-transport system) at pH 7.4 (Shartava et al. Am. J. Hematol. 1999;62:19-24). Here we demonstrate that clotrimazole (10 microM) inhibits dense cell formation at pH 7.1 and 6.8, while DIOA (1 mM) has no effect. As pH 6.8 is the optimal pH for the K(+)-Cl(-) co-transport system, we can now reasonably conclude that damage to the Gardos channel is responsible for CDNB-induced dense cell formation.     

Identification and characterization of a newly recognized population of high-Na+, low-K+, low-density sickle and normal red cells

We describe a population of sickle cell anemia red cells (SS RBCs) ( approximately 4%) and a smaller fraction of normal RBCs (<0.03%) that fail to dehydrate when permeabilized to K(+) with either valinomycin or elevated internal Ca(2+). The nonshrinking, valinomycin-resistant (val-res) fractions, first detected by flow cytometry of density-fractionated SS RBCs, constituted up to 60% of the lightest, reticulocyte-rich (R1) cell fraction, and progressively smaller portions of the slightly denser R2 cells and discocytes. R1 val-res RBCs had a mean cell hemoglobin concentration of approximately 21 g of Hb per dl, and many had an elongated shape like "irreversibly sickled cells," suggesting a dense SS cell origin. Of three possible explanations for val-res cells, failure of valinomycin to K(+)-permeabilize the cells, low co-ion permeability, or reduced driving K(+) gradient, the latter proved responsible: Both SS and normal val-res RBCs were consistently high-Na(+) and low-K(+), even when processed entirely in Na-free media. Ca(2+) + A23187-induced K(+)-permeabilization of SS R1 fractions revealed a similar fraction of cal-res cells, whose (86)Rb uptake showed both high Na/K pump and leak fluxes. val-res/cal-res RBCs might represent either a distinct erythroid genealogy, or an "end-stage" of normal and SS RBCs. This paper focuses on the discovery, basic characterization, and exclusion of artifactual origin of this RBC fraction. Many future studies will be needed to clarify their mechanism of generation and full pathophysiological significance.     

Exaggerated cation leak from oxygenated sickle red blood cells during deformation: evidence for a unique leak pathway.

An abnormal susceptibility of the sickle red blood cell (RBC) membrane to deformation could compromise its permeability barrier function and contribute to the exuberant cation leakiness occurring during the sickling phenomenon. We examined this hypothesis by subjecting RBCs at ambient oxygen tension to elliptical deformation, applying shear stress in a viscous medium under physiologic conditions. Compared with normal and high-reticulocyte control RBCs, sickle RBCs manifest an exaggerated K leak response to deformation. This leak is fully reversible, is both Cl and Ca independent, and at pHe 7.4 is fully balanced so that Kefflux equals Nainflux. This abnormal susceptibility is also evident in that the K leak in response to deformation occurs at an applied shear stress of only 141 dyne/cm2 for sickle RBCs, as compared to 204 dyne/cm2 for normal RBCs. Fresh sickle RBC membranes contain elevated amounts of lipid hydroperoxide, the presence of which is believed to provide the biochemical basis for enhanced deformation susceptibility. When examined at pHe 6.8, oxygenated sickle RBCs acquire an additional, unbalanced (Kefflux > Nainflux) component to the K leak increment specifically ascribable to deformation. Studies with inhibitors suggest that this additional component is not caused by a known leak pathway (eg, either K:Cl cotransport or the Gardos channel). This abnormal susceptibility of the sickle membrane to development of cation leakiness during deformation probably contributes to the exuberant cation leak taking place during RBC sickling.     

An increased Bohr effect in sickle cell anemia.

Recent findings that hemoglobin S gelation and sickling are pH-dependent and also influence oxygen affinity suggested that the red cells containing this hemoglobin variant might show an abnormal Bohr effect. We therefore studied the effects of pH variation on the in vitro oxygen affinity of whole blood from persons with sickle cell anemia (SS) and normal donors (at 37 degrees C and constant carbon dioxide tension of 40 mm Hg). The Bohr effect in SS blood was greatly increased only between blood pH 7.4 and 7.2 (cell pH 7.2 and 7.0, a shift that strongly affects gelation), with delta log p50/deltapH= - 0.92 to -0.99 (normal = -0.42 to -0.46). Thus a drop in SS blood pH below 7.4 in tissue capillaries yields twice the normal decrease in oxygen affinity and a large release of oxygen from red cells, whose risk of sickling is high. Even mild transient acidosis would seem hazardous for patients with sickling disorders.     

Erythrocytic mechanism of sickle cell resistance to malaria.

The physiological basis for the resistance to falciparum malaria individuals with sickle cell trait has not been understood. Recent advances in erythrocytic Plasmodium falciparum culture have made possible a direct investigation of the development of the malaria parasite in cells with sickle cell homoglobin. In a high (18%) oxygen atmosphere, there is no apparent sickling of cells, and the growth and multiplication of P. falciparum is identical in normal (AA), hemoglobin S homozygous (SS), and hemoglobin S heterozygous (SA) erythrocytes. Cultures under low (1-5%) oxygen, however, showed clear inhibition of growth. The sickling of SS red cells killed and lysed most or all of the intracellular parasites. Parasites in SA red cells were killed primarily at the large ring stage, probably as a result of a disruption of the parasite metabolism. Incubation in cyanate prior to culture reversed the resistance of SA erythrocytes to plasmodium growth, but had no effect on SS red cell sickling or resistance. Thus, the mechanism of resistance in vivo may be due solely to intraerythrocytic conditions.