Permeability characteristics of deoxygenated sickle cells

This study investigated the effect of acute deoxygenation on membrane permeability characteristics of sickle cells. Measured fluxes of Na+ and K+ in ouabain-inhibited cells, of chloride and sulfate exchange in 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS)-inhibited and untreated cells, and of erythritol, mannitol, and arabinose in cytochalasin B-inhibited cells indicated that a deoxygenation-induced permeability change occurred in sickle cells only for cations and chloride. Monovalent cation permeabilities increased five-fold, and chloride influx into DIDS treated cells was enhanced nearly threefold on sickle cell deoxygenation. In contrast, no detectable increase in permeability to the other solutes was found. To gain perspective on these findings, similar measurements were performed in normal cells treated with diamide, an agent shown by others to induce a coupled increase in membrane permeability and phospholipid translocation, reminiscent of deoxygenation-induced changes in sickle cells. Although the increase in cation permeability was no greater than that in sickled cells, treatment with 2 mmol/L diamide also produced a twofold increase in the first order rate constants for sulfate exchange and mannitol efflux, indicating a relatively nonselective permeability increase that permitted flux of larger solutes than in the case of deoxygenated sickle cells. These results suggest that the deoxygenation of sickle cells induces a permeability increase that is relatively insensitive to charge, but is restrictive with respect to solute size.     

The effect of deoxygenation on whole-cell conductance of red blood cells from healthy individuals and patients with sickle cell disease

Red blood cells from patients with sickle cell disease (SCD) exhibit increased electrogenic cation permeability, particularly following deoxygenation and hemoglobin (Hb) polymerisation. This cation permeability, termed P(sickle), contributes to cellular dehydration and sickling, and its inhibition remains a major goal for SCD treatment. Nevertheless, its characteristics remain poorly defined, its molecular identity is unknown, and effective inhibitors have not been established. Here, patch-clamp methodology was used to record whole-cell currents in single red blood cells from healthy individuals and patients with SCD. Oxygenated normal red blood cells had a low membrane conductance, unaffected by deoxygenation. Oxygenated HbS cells had significantly increased conductance and, on deoxygenation, showed a further rise in membrane conductance. The deoxygenation-induced pathway was variable in magnitude. It had equal permeability to Na(+) and K(+), but was less permeable to NMDG(+) and Cl(-). Conductance to Ca(2+) was also of a similar magnitude to that of monovalent cations. It was inhibited by DIDS (100 microM), Zn(2+) (100 microM), and by Gd(3+) (IC(50) of approximately 2 microM). It therefore shares some properties with P(sickle). These findings represent the first electrical recordings of single HbS cells and will facilitate progress in understanding altered red blood cell cation transport characteristics of SCD.     

Deoxygenation-induced cation fluxes in sickle cells: II. Inhibition by stilbene disulfonates

Deoxygenation-induced cation movements in sickle cells were inhibited 80% to 85% by the anion transport inhibitor, 4,4'-diisothiocyano-2,2'disulfostilbene (DIDS). Morphologic sickling was not altered by DIDS treatment, demonstrating that morphologic sickling was not sufficient to produce cation leaks in sickle cells. DIDS inhibition of deoxygenation-induced cation flux was not affected when l- replaced Cl-, indicating that conductive anion movements did not limit cation flux in deoxygenated cells treated with DIDS. Inhibition was irreversible after preincubation with DIDS at 37 degrees C for 20 minutes, and was not affected by the oxygenation state of cells at the time of drug exposure. Sulfate self-exchange was inhibited at lower DIDS concentrations than was deoxygenation-induced flux. Incubation of cells with DIDS at 4 degrees C produced progressive blockade of sulfate exchange, but did not alter deoxygenation-induced cation fluxes. Other stilbene disulfonates, including compounds incapable of covalent reactions, also inhibited deoxygenation-induced cation movements, although several other inhibitors of anion exchange did not. Dissociation of the inhibition of anion exchange and deoxygenation-induced cation flux indicates that the DIDS effect on deoxygenation-induced cation movements does not involve the well-characterized stilbene binding site of the anion exchanger. These data provide evidence for a membrane constituent on the external surface of oxygenated sickle cells capable of interacting with DIDS to prevent the increase in cation permeability associated with sickling.     

Deoxygenation-induced changes in sickle cell-sickle cell adhesion

The tendency for sickle cells to adhere to each other is increased in oxygenated sickle blood in parallel with cell density. The increased adherence of these cells occurred despite their reduced deformability and diminished ability to form rouleaux. Using a method developed in our laboratory, we measured the yield stress: a sensitive index of cell- cell adhesion of deoxygenated suspensions of sickle cells. Deoxygenation of whole sickle blood to 30 to 50 mm Hg caused a significant increase in yield stress of all sickle blood samples. Deoxygenation caused a significant increase in yield stress of both dense and light sickle cells. Deoxygenation-induced increases in yield stress occurred at higher oxygen tensions for dense (> 55 mm Hg) than for light sickle cells (< 45 mm Hg). The increase in yield stress on deoxygenation was correlated with hemoglobin polymerization as assessed morphologically by sickling or by changes in relative viscosity. Thus, deoxygenation-induced cell sticking must involve small areas of strong membrane adhesion because the changes in yield stress occurred despite a reduction in rouleaux formation and surface area of membrane contact. Sickle trait red blood cells also exhibited increased yield stress on deoxygenation but only under hypertonic conditions where sickling occurred. Thus, deoxygenation-induced cell adhesion did not require prior membrane damage because it occurred in sickle trait cells. No change in yield stress was seen when deoxygenated sickle cells were suspended in buffer, but the addition of physiologic amounts of fibrinogen to buffer restored the deoxygenation-induced increase in cell adhesion. We speculate that the increase in sticking among sickle cells on deoxygenation results from spicule formation and may involve interaction of fibrinogen and possibly other plasma proteins with the cell membrane.     

Rate of deoxygenation modulates rheologic behavior of sickle red blood cells at a given mean corpuscular hemoglobin concentration

Although the mean corpuscular hemoglobin concentration (MCHC) plays a dominant role in the rheologic behavior of deoxygenated density-defined sickle red blood cells (SS RBCs), previous studies have not explored the relationship between the rate of deoxygenation and the bulk viscosity of SS RBCs at a given MCHC. In the present study, we have subjected density-defined SS classes (i.e., medium-density SS4 and dense SS5 discocytes) to varying deoxygenation rates. This approach has allowed us to minimize the effects of SS RBC heterogeneity and investigate the effect of deoxygenation rates at a given MCHC. The results show that the percentages of granular cells, classic sickle cells and holly leaf forms in deoxygenated samples are significantly influenced by the rate of deoxygenation and the MCHC of a given discocyte subpopulation. Increasing the deoxygenation rate using high K+ medium (pH 6.8), results in a greater percentage of granular cells in SS4 suspensions, accompanied by a pronounced increase in the bulk viscosity of these cells compared with gradually deoxygenated samples (mainly classic sickle cells and holly leaf forms). The effect of MCHC becomes apparent when SS5 dense cells are subjected to varying deoxygenation rates. At a given deoxygenation rate, SS5 dense discocytes show a greater increase in the percentage of granular cells than that observed for SS4 RBCs. Also, at a given deoxygenation rate, SS5 suspensions exhibit a higher viscosity than SS4 suspensions with fast deoxygenation resulting in maximal increase in viscosity. Although MCHC is the main determinant of SS RBC rheologic behavior, these studies demonstrate for the first time that at a given MCHC, the rate of deoxygenation (hence HbS polymerization rates) further modulates the rheologic behavior of SS RBCs. Thus, both MCHC and the deoxygenation rate may contribute to microcirculatory flow behavior of SS RBCs.     

Membrane transport in sickle cell disease

We have reviewed here a number of membrane transport events in red cells from normal individuals and sickle cell patients which respond to changes in O(2) tension. Some deoxygenation-induced changes in membrane permeability are unique to HbS cells and contribute to their dehydration and subsequent sickling. Polymerization of HbS, or specific oxidant damage (or altered redox potential), is a likely factor underlying the abnormal behavior. The key regulatory sites within the membrane or associated proteins remain uncertain and their identity will form the focus of future research. A model for sickle cell dehydration is presented. Inhibition of these permeability changes represents possible avenues for future chemotherapy to ameliorate the condition.     

Hemoglobin S polymerization: primary determinant of the hemolytic and clinical severity of the sickling syndromes

We examined the extent to which the intracellular polymerization of sickle hemoglobin (HbS) can account for the severity of anemia and of vaso-occlusive manifestations in the various sickling syndromes. Polymer formation in sickle cell disease depends principally on the intraerythrocytic hemoglobin composition and concentration. In our studies, the polymer fraction in sickle red cells was determined from reported mean values for hemoglobin composition and mean corpuscular hemoglobin concentration (MCHC) in 12 groups of patients with sickle hemoglobinopathies (homozygotes for HbS, with and without coexistent alpha-thalassemia or various forms of the hereditary persistence of fetal hemoglobin [HPFH], beta+-, beta 0-, and delta beta-thalassemia, and heterozygotes for HbS with HbA). The calculated HbS polymer fractions at full deoxygenation and at physiologic oxygen saturation values were closely correlated with mean blood hemoglobin concentrations. In addition, polymer fraction correlated with the ranking of the sickling syndromes by vaso-occlusive severity. We find that polymer fraction accounts for about 80% of the variability in hemolytic and clinical severity. The method of analysis presented here provides a quantitative and systematic means of assessing the role of polymer formation in the pathophysiologic manifestations of the sickling syndromes. Our results support the hypothesis that the intracellular polymerization of HbS is the primary determinant of the severity of both anemia and clinical symptomatology in the sickle hemoglobinopathies.     

Influence of red cell water content on the morphology of sickling

The response of sickle cells with varying water content to alterations in oxygen tension has been studied. Cells that were severely dehydrated while sickled retained the characteristic sickled morphology even after prolonged reoxygenation. When the cell water content was increased by reduction of the suspending medium osmolality, the cells unsickled. Cells that were dehydrated before deoxygenation were unable to assume the spiculated morphology typical of sicked cells. This was true both for high mean cell hemoglobin concentration (MCHC) discoid sickle cells and for irreversibly sickled cells. When such cells were resuspended in hypotonic medium before deoxygenation, they sickled with the characteristic morphology of sickle cells with normal MCHC. The morphological behavior of Ca-loaded sickled cells as well as irreversibly sickled cells showed a major influence of increased hemoglobin concentration and extremely high internal viscosity. Constraint on cell morphology by putative membrane rigidity was not observed.     

The effect of N,N'-p-phenylenedimaleimide (PMD) on deoxygenation-induced K loss in sickle erythrocytes

A variety of thiol reactive agents have been found to have antisickling properties thought to be due to the ability of these drugs to bind to hemoglobin, resulting in increased hemoglobin-oxygen affinity. Because thiol reactive agents also influence K movements in red cells and deoxygenation leads to K loss and Na gain in sickle erythrocytes, the authors investigated the possibility that deoxygenation-induced K loss could be influenced by thiol agents, independent of an effect on hemoglobin-oxygen affinity. Experiments were performed with the thiol crosslinking agent N,N'-p-phenylenedimaleimide (PMD). The authors found that PMD inhibited deoxygenation-induced K loss in sickle erythrocytes. This effect was not due to sickling inhibition as PMD-treated cells gained Na with deoxygenation, nor could the effect be explained by monofunctional PMD binding to membrane sulfhydryl groups, as a monofunctional analogue of PMD was not able to retard deoxygenation-induced K loss. These findings support a role for membrane sulfhydryl groups in deoxygenation-induced K movements in sickle red cells and suggest that this K loss may be prevented by crosslinking of certain membrane sulfhydryl groups.     

The effect of erythrocyte membrane on the birefringence formation of sickle cell hemoglobin

The birefringence formation of sickle cell hemoglobin (HbS) in a thin liquid layer was observed while its environment was deoxygenated at different rates, and the effect of membrane was examined. Under slow rate of deoxygenation at 37 degrees C, at pH 7.4, the birefringence of purified HbS appeared at a concentration higher than 24% and its relative magnitude increased as the concentration was increased. Similarly, the partial pressure of oxygen, at which the birefringence formation was evident, increased from 0 to 27 torr as the concentration of HbS was increased from 24 to 28%, but it remained the same above this protein concentration. In all the samples tested relative birefringence was largest at the slow rate of deoxygenation (30 torrO2/min) and the magnitude decreased as the rate of deoxygenation was increased. The samples showed different sensitivity to the rate of deoxygenation. For example, while the total untreated hemolysate made by freeze-thawing of packed sickle cells was most resistant to the increased rates of deoxygenation, purified HbS was not. Washed open ghosts partially restored the birefringence formation pattern of purified HbS. The results indicate that the inner surface of the membranes of erythrocytes could behave as a template for large HbS polymer formation at relatively higher rates of deoxygenation.     

Deoxygenation affects tyrosine phosphoproteome of red cell membrane from patients with sickle cell disease

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder related to the production of a defective form of hemoglobin, hemoglobin S (HbS). One of the hallmarks of SCD is the presence of dense, dehydrate highly adhesive sickle red blood cells (RBCs) that result from persistent membrane damage associated with HbS polymerization, abnormal activation of membrane cation transports and generation of distorted and rigid red cells with membrane perturbation and cytoskeleton dysfunction. Although modulation of phosphorylation state of the proteins from membrane and cytoskeleton networks has been proposed to participate in red cell homeostasis, much still remains to be investigated in normal and diseased red cells. Here, we report that tyrosine (Tyr-) phosphoproteome of sickle red cells was different from normal controls and was affected by deoxygenation. We found proteins, p55 and band 4.1, from the junctional complex, differently Tyr-phosphorylated in SCD RBCs compared to normal RBCs under normoxia and modulated by deoxygenation, while band 4.2 was similarly Tyr-phosphorylated in both conditions. In SCD RBCs we identified the phosphopeptides for protein 4.1R located in the protein FERM domain (Tyr-13) and for α-spectrin located near or in a linker region (Tyr-422 and Tyr-1498) involving protein areas crucial for their functions in the context of red cell membrane properties, suggesting that Tyr-phosphorylation may be part of the events involved in maintaining membrane mechanical stability in SCD red cells.     

Hybrid erythrocytes for membrane studies in sickle cell disease.

A hybrid erythrocyte model for membrane studies in sickle cell disease has been developed. The model consists of normal red cell membranes containing hemoglobin S and sickle cell membranes containing hemoglobin A. In hybrids, complete hemoglobin exchange has been achieved together with restoration of low membrane permeability to potassium. Normal membranes containing HbS sickle upon deoxygenation and assume the characteristic appearance of irreversibly sickled cells (ISC) after prolonged anoxia. It is suggested that the hybrid model will be useful in defining further the process of ISC formation and in studying the influence of sickle hemoglobin upon the function of the surrounding membrane.     

Non-electrolyte permeability of deoxygenated sickle cells compared

The passive permeability pathways of red cells are poorly defined, with the exception of the Gardos channel. Several cation and anion pathways can be induced by a variety of manoeuvres, however, including treatment with oxidants, low ionic strength (LIS), shrinkage, swelling and also infection with the intra-erythrocytic malaria parasite. Several of these stimuli (malaria, swelling, LIS), in addition, also activate a non-electrolyte this permeability. Sickle cells uniquely show a deoxygenation-induced pathway, which is termed P_sickle and is usually considered to be a conductive cationic pathway. In this report, we explore further the extent to which this permeability pathway of deoxygenated sickle cells is available for non-electrolyte transport. We show that a number of solutes are permeable, with greater permeability to sugars (notably lactose and maltose) and smaller molecules, and less to charged or zwitterionic species. Red cells from heterozygous HbSC patients also showed deoxygenation-induced haemolysis in isosmotic sucrose solution, though to a slightly lesser extent than for red cells from homozygous sickle cell patients. In contrast to sickle cells, red cells from β-thalassaemic patients did not show haemolysis in isosmotic sucrose solutions, regardless of the O₂ tension. Of the secondary cellular changes resulting from incubation in non-electrolyte solutions (which include imposition of a highly positive membrane potential, marked intracellular alkalinisation and cell shrinkage), none appear to correlate with activation of the non-electrolyte permeability. Rather, findings indicate that it is low ionic strength per se that is responsible. Normal red cells also show changes in ionic and non-electrolyte permeability in low ionic strength media, and these permeabilities are compared to those found in deoxygenated sickle cells. The extent to which these different permeabilities in normal and sickle red cells can be ascribed to one or more common pathways remains to be determined.     

Ektacytometric measurement of sickle cell deformability as a continuous function of oxygen tension [published erratum appears in Blood 1987 Apr;69(4):1272]

In an effort to study the rheologic effects of small amounts of hemoglobin S (HbS) polymer in sickle red cells, we have used the ektacytometer, a laser diffraction couette viscometer, to measure sickle cell deformability as a function of oxygen tension. Sickle cell populations of defined intracellular hemoglobin concentration (MCHC) were isolated using Stractan density gradients and were resuspended in buffered polyvinylpyrrolidone solutions for deformability measurements. Using a gas-porous, hollow fiber gas exchange system to establish a linear gradient in oxygen tension, deformability was measured over a pO2 range of 76 to 0 mm Hg. Parallel spectroscopic determinations of oxygen saturation permitted determination of cell deformability as a function of oxygen saturation for each discrete MCHC population. From these measurements the level of oxygen saturation at which a loss in cell deformability was first detected could be defined. Then, using the data of Noguchi and Schecter, the amount of polymerized HbS in the cells at that defined level of oxygen saturation was estimated. The results of this analysis suggested that the quantity of polymer that caused a detectable loss in cell deformability increased with increasing MCHC. In addition, for MCHC above 30 g/dL, this represented a substantial fraction of the total HbS in the cell.     

A critical role for altered red cell cation permeability in pathogenesis of sickle cell disease and other haemolytic anaemias

The aetiology of sickle cell disease is well known, but pathogenesis is complicated and details remain uncertain. A thorough understanding may suggest novel ways for designing more effective therapies. One area of importance, covered here in Nader et al., is the altered cation permeability of sickle cells and how the co-ordinated operation of a number of membrane transport proteins contributes to disease progression, all driven by the initial event of HbS polymerisation. There are echoes here of the cation leaks of hereditary stomatocytosis. Nader et al. propose a central role for PIEZO1, a novel mechanosensitive channel found in red cells, which may be aberrantly activated in sickle cells following HbS polymerisation and which may have potential as a novel target for future chemotherapies. Commentary on: Nader et al. Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner. Br J Haematol 2023;202:657–668.     

Rate of deoxygenation and rheologic behavior of blood in sickle cell anemia

To understand the relationship between deoxygenation rate, rheologic behavior, and red blood cell (RBC) morphologic characteristics of blood in sickle (SS) cell anemia, washed oxy SS RBC suspensions (hematocrit, 40%) were subjected to relatively fast and gradual deoxygenation procedures. Relatively fast deoxygenation resulted in 50% decline in percent hemoglobin oxygen saturation (%HbO2) within 1 minute. The SS suspensions following relatively fast deoxygenation showed two distinct phases in viscosity profiles. First, there was a sharp increase in individual viscosities to a peak value at 7 minutes of deoxygenation. Second, prolonged deoxygenation resulted in a 27% to 37% decrease in individual viscosities at 30 minutes as compared with the respective peak values at 7 minutes. Most of the viscosity increase (ie, about fourfold) occurred within the first 3 minutes of relatively fast deoxygenation. Scanning electron microscopy and differential morphologic analysis of deoxy cells showed that at 7 minutes a majority of cells had a granular appearance that was characterized by a bumpy irregular surface and the presence of small spicule-like projections. Prolonged deoxygenation resulted in the appearance of a large percentage of elongated cells that were unlike typical sickle cells. Transmission electron microscopy showed that the elongated shape resulted from the alignment of HbS polymers into long projections. In contrast, gradual deoxygenation over a period of 30 minutes resulted in a progressive increase in viscosity and in the formation of typical sickle shapes and holly leaf cells. The results show that at matching %HbO2, the SS suspensions containing mainly granular shaped cells after 7 minutes of relatively fast deoxygenation are as viscous as the gradually deoxygenated suspensions that contain classic sickle shapes and holly leaf forms, while the suspensions having a large percentage of elongated cells (30 minutes after relatively fast deoxygenation) are the least viscous. The two distinct time-dependent viscosity phases observed after relatively fast deoxygenation probably result from differences in the RBC shape characteristics reflecting physical attributes of the polymer, which could affect cell orientation in the viscometric flow.     

Vanillin, a potential agent for the treatment of sickle cell anemia

Vanillin, a food additive, has been evaluated as a potential agent to treat sickle cell anemia. Earlier studies indicated that vanillin had moderate antisickling activity when compared with other aldehydes. We have determined by high performance liquid chromatography that vanillin reacts covalently with sickle hemoglobin (HbS) both in solution and in intact red blood cells. Hemoscan oxygen equilibrium curves show a dose-dependent left shift, particularly at low oxygen tensions. Rheologic evaluation (pO2 scan Ektacytometry) of vanillin-reacted HbS erythrocytes shows a dose-dependent inhibition of deoxygenation-induced cell sickling. Ektacytometry also suggests that vanillin may have a direct inhibitory effect on HbS polymer formation. Vanillin has no adverse effects on cell ion or water content. X-ray crystallographic studies with deoxyhemoglobin (HbA)-vanillin demonstrate that vanillin binds near His 103 alpha, Cys 104 alpha, and Gln 131 beta in the central water cavity. A secondary binding site is located between His 116 beta and His 117 beta. His 116 beta has been implicated as a polymer contact residue. Oxygen equilibrium, ektacytometry, and x-ray studies indicate that vanillin may be acting to decrease HbS polymerization by a dual mechanism of action; allosteric modulation to a high-affinity HbS molecule and by stereospecific inhibition of T state HbS polymerization. Because vanillin is a food additive on the GRAS (generally regarded as safe) list, and because it has little or no adverse effects at high dosages in animals, vanillin is a candidate for further evaluation as an agent for the treatment of sickle cell disease.     

Ca2+ permeability in deoxygenated sickle cells

Deoxygenation of sickle cells is known to increase cation permeabilities (Na+, K+, and Ca2+). The possible mechanisms involved in the increased uptake of Ca2+ were investigated: activation of Ca2+ channels, involvement of the anion channel, and the formation of endocytic vacuoles. The Ca2+ channel blocker nifedipine reduced the deoxy-stimulated Ca2+ uptake by about 30% to 40%. The anion channel inhibitor DIDS (4,4' diisothiocyanate stilbene 2,2' disulfonate) inhibited the deoxy-stimulated Ca2+ uptake by approximately 50%. Maximal possible endocytic uptake, measured by using an impermeant marker ([3H] inuline), accounted for 6% to 9% of the total Ca2+ uptake. These data indicate that the deoxygenation-induced increase in Ca2+ permeability could result from both the activation of a Ca2+ channel and of a transport system for cations involving interactions between polymerized hemoglobin S, band 3 and other membrane components. Endocytosis appears to play only a minor role in the Ca2+ uptake of deoxygenated sickle cells.     

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.     

Deoxygenation of sickle cells stimulates Syk tyrosine kinase and inhibits a membrane tyrosine phosphatase.

Polymerization of hemoglobin S in sickle red cells, in deoxygenated conditions, is associated with K+ loss and cellular dehydration. It was previously reported that deoxygenation of sickle cells increases protein tyrosine kinase (PTK) activity and band 3 tyrosine phosphorylation and that PTK inhibitors reduce cell dehydration. Here, the study investigates which PTKs are involved and the mechanism of their activation. Deoxygenation of sickle cells induced a 2-fold increase in Syk activity, measured by autophosphorylation in immune complex assays, but had no effect on Lyn. Syk was not stimulated by deoxygenation of normal red cells, and stimulation was partly reversible on reoxygenation of sickle cells. Syk activation was independent of the increase in intracellular Ca++ and Mg2+ associated with deoxygenation. Lectins that promote glycophorin or band 3 aggregation did not activate Syk. In parallel to Syk stimulation, deoxygenation of sickle cells, but not of normal red cells, decreased the activity of both membrane-associated protein tyrosine phosphatase (PTPs) and membrane protein thiol content. In vitro pretreatment of Syk immune complexes with membrane PTP inhibited Syk autophosphorylation. It is suggested that Syk activation in vivo could be mediated by PTP inhibition, itself resulting from thiol oxidation, as PTPs are known to be inhibited by oxidants. Altogether these data indicate that Syk could be involved in the mechanisms leading to sickle cell dehydration.