Irreversibly Sickled Cells Research Articles

Regulation of Human Sickle Erythrocyte Hydration Status by Endothelin-1 Is Mediated by Casein Kinase II.

Abstract 3020Poster Board II-996In Sickle Cell Disease, K+ efflux and osmotically induced water loss leads to erythrocyte dehydration, an important step in the formation of hemoglobin S polymers and irreversible sickle cells. We have previously reported that activation of sickle erythrocyte endothelin-1 (ET-1) receptors is partially responsible for dense irreversible sickle cell formation thru mechanisms that are still undefined. Casein Kinase II (CK2), a serine/threonine kinase that phosphorylates acidic proteins, has been shown to regulate calmodulin activity and cytoskeletal proteins. Thus, we posit that CK2 may regulate cellular hydration status. CK2 activity is modulated by apigenin, emodin, heparin, and ornithine decarboxylase. Our results indicate that CK2 activity in erythrocyte membranes increases in the presence of ET-1 (2.8 ± 0.1 to 4.9 ± 0.01 nmol/min/mL * 106 cell) an event that is blunted by pre-incubation with the ET-1 B receptor blocker, BQ788 (2.5 ± 0.1 nmol/min/mL * 106 cell). Our data also indicate that 20 μM apigenin blocks ET-1 induced CK2 activity in sickle erythrocytes (2.8 ± 0.1 to 2.7 ± 0.4 nmol/min/mL * 106 cell). We then examined the role of CK2 activation in cellular dehydration. We incubated sickle erythrocytes for 3 hours in deoxygenation-oxygenation cycles in the presence or absence of 20μM apigenin or 2μM 4,5,6,7-tetrabromobenzotriazole (TBB), another specific CK2 inhibitor, and measured the changes in erythrocyte density by the phthalate oil method. We observed that inhibition of CK2 led to the inhibition of deoxygenation-stimulated cellular dehydration in sickle erythrocytes by apigenin (D50= 1.106 to 1.100 g/mL) or TBB (D50 =1.097 g/mL). Apigenin also inhibited the volume-stimulated K+ efflux in a dose-dependent manner (IC50 = 18 ± 1 μM). These results strongly implicate CK2 as an intermediate of the ET-1 stimulated cellular volume regulatory systems in erythrocytes and suggest that regulation of CK2 activity may lead to reduction of hemoglobin S polymerization in vivo. Supported by NIH R01-HL09632 to AR. DisclosuresNo relevant conflicts of interest to declare.

High oxygen environment during pregnancy rescues sickle cell anemia mice from prenatal death

Several mouse models of sickle cell disease have been developed for the study of the pathophysiology of sickle cell disease and the investigation of drug and gene therapies. In previous years, we produced a sickle cell anemia mouse model in which the endogenous mouse α- and β-globin genes were knocked out and replaced by the human α- and β s-globin transgenes. The β s-globin gene was contained in a 240 kb YAC that preserved the entire native genomic context of the β-globin locus. These mice have hemolytic anemia, reticulocytosis and irreversible sickle cells in the peripheral blood, as well as other pathological features of sickle cell disease. However, in the embryo, the γ-globin, like the mouse embryonic globin, declined quickly, and was replaced by β s-globin expression from 12 days of gestation. The low level of fetal hemoglobin expression in utero led to intrauterine sickling and fetal death so that very few live-born sickle cell anemia mice could be obtained. To rescue these mice from intrauterine death, we investigated the effect of placing the pregnant mothers in a high O 2 environment. From the tenth day of gestation onwards, we placed the mothers into a chamber containing 50% O 2 and kept them with the newborn pups in it for another 10 days after birth. The frequency of sickle cell anemia mice we obtained was increased from less than 2% to 35%. The survived sickle cell anemia mice develop congestion, atrophy, and infarcts in multiple organs similar to those found in patients with sickle cell disease. We conclude that a high oxygen environment can be used to obtain more sickle cell anemia mice in those models that have a high perinatal mortality. The higher yield of these mice has facilitated physiological and therapeutic studies of sickle cell anemia.

Kinetics of uptake and deacetylation of N-acetylcysteine by human erythrocytes

Overproduction of reactive oxygen species associated with several diseases including sickle cell anaemia reduces the concentration of glutathione, a principal cellular antioxidant. Glutathione depletion in sickle erythrocytes increases their conversion to irreversible sickle cells that promote vaso-occlusion. Therapeutically, N-acetylcysteine partially restores glutathione concentrations but its mode of action is controversial. Following glutathione depletion, glutathione synthesis is limited by the supply of cysteine and it has been assumed that deacetylation of N-acetylcysteine within erythrocytes provides cysteine to accelerate glutathione production. To determine whether this is the case we studied the kinetics of transport and deacetylation of N-acetylcysteine. Uptake of N-acetylcysteine had a first order rate constant of 2.40±0.070min−1 and only saturated above 10mM. Inhibition experiments showed that 56% of N-acetylcysteine transport was via the anion exchange protein. Deacetylation, measured using 1H NMR, had a Km of 1.49±0.16mM and Vmax of 2.61±0.08μmolL−1min−1. Oral doses of N-acetylcysteine increase glutathione concentrations in sickle erythrocytes at plasma N-acetylcysteine concentrations of ∼10μM. At this concentration, calculated rates of N-acetylcysteine uptake and deacetylation were ∼5% of the rate required to maintain normal glutathione production. We concluded that on oral administration, intracellular deacetylation of N-acetylcysteine supplies little of the cysteine required for accelerated glutathione production. Instead, N-acetylcysteine acts by freeing bound cysteine in the plasma that then enters the erythrocytes. To be effective, intracellular cysteine precursors must be designed to enter erythrocytes rapidly and employ enzymes with high activity within erythrocytes to liberate the cysteine.

High Oxygen Environment during Pregnancy Rescues Sickle Cell Animia Mouse from Prenatal Death.

Several mouse models for sickle cell disease have been developed for the studies of the pathophysiology of sickle cell disease and the investigations of drug and gene therapies. In previous years, we have also succeeded in producing a sickle cell anemia mouse model. In this model the endogenous mouse α and β globin genes were knocked out and replaced by the human α and βs globin transgenes. The βs globin gene is contained in a 240kb YAC that preserves the entire native genomic context of the β-globin locus. These mice have anemia, reticulocytosis and irreversible sickle cells in the peripheral blood, as well as other pathological features of sickle cell disease. However, their γ globin switches to βs globin expression from around 12 days of gestation. The low level of fetal hemoglobin expression in utero led to intrauterine sickling and fetal death so that very few live-born sickle cell anemia mice could be obtained. To rescue these mice from intrauterine death we investigated the effect of placing the pregnant mothers into a high O2 environment. From the tenth day of gestation onwards, we placed the mothers into a chamber containing 50% O2 and kept the newborn pups in it for another 10 days after birth. The frequency of sickle cell anemia mice we obtained was increased from 0.2% to 32%. Moreover, 55% to 88% of the newborn sickle cell anemia mice survived in the oxygen treated group, whereas none of the sickle cell anemia mice survived in regular air breeding conditions. The survived sickle cell anemia mice develop congestion, atrophy, and infarcts in multiple organs by histological analysis. These pathological finding are very similar to those find in patients with sickle cell disease. We conclude that a high oxygen environment can be used to obtain more sickle cell anemia mice in those models that have a high perinatal mortality. The higher yield of these mice will facilitate physiological and therapeutic studies of sickle cell anemia.

Hematological parameters in sickle cell anemia patients with and without priapism.

BACKGROUNDPriapism was associated with certain hematological parameters in sickle cell anemia (SCA) patients in one report but not in another. We studied differences in haematological parameters between SCA patients with and without priapism.PATIENTS AND METHODSEighteen patients with SCA who presented with acute priapism during the years 2001–2004 were compared with age- and sex-matched SCA patients without priapism with respect to hematocrit, reticulocyte count, level of irreversibly sickled cells (ISC), percentage of haemoglobin F (Hb F), total leukocyte and platelet counts.RESULTSSCA patients with priapism had a mean hematocrit of 0.28 L/L, which was significantly higher than the mean hematocrit value of 0.24 L/L (P<0.05) in patients without priapism. The mean reticulocyte count of 8% in patients with priapism was significantly lower than mean reticulocyte count of 12% (P<0.05) in patients without priapism. The level of ISC of 3% in patients with priapism was significantly lower than the level of 6.5% (P<0.05) in patients without priapism. There was no statistically significant difference in the mean levels of Hb F (7% vs. 6%). Patients with priapism had a mean leukocyte count and mean platelet count that did not significantly differ from values in patients without priapism.CONCLUSIONSSCA patients with priapism had a lower rate of hemolysis, resulting in a higher hematocrit and greater blood viscosity, which increased the risk of corpora cavernosal sickling and blockade. Hence, a relatively high hematocrit is a risk factor for the development priapism in patients with sickle cell anemia.

Splenic changes in sickle cell anaemia

The spleen is one of the most frequently affected organ in Sickle cell anaemia (SCA). This has been attributed to its complex anatomy and prominent reticuloendothelial functions which include clearance of unwanted particulate matter in blood ( culling), defense against infection and reservoir for blood cells. This paper aims to highlight the current information on the changes that occur in the spleen of Sickle Cell Disease patients in this environment. A review of relevant literature on the subject of splenic changes in Sickle Cell Disease sourced by manual library and medline search. The essential splenic change in SCA is splenomegaly and subsequent shrinkage in size (autosplenectomy), which maybe due to several factors. These include: high levels of irreversible sickle cells, decreased HbF associated with increased intravascular sickling and chronic Malaria infection secondary to hyperplasia of the reticulo-endothelial system and increased antibody production especially IgG and IgM. Finally, the clinical complications of these splenic changes such as increased susceptibility to infection, acute splenic sequestration and hypersplenism are also reviewed in this paper. In view of the above changes, it is important to ensure regular monitoring and follow-up in order to prevent complications, recurrent crisis and death.

Blood samples collected under venous oxygen pressure from patients with sickle cell disease contain a significant number of a new type of reversibly sickled cells: Constancy of the percentage of sickled cells in individual patients during steady state

We found various levels of a new type of reversibly sickled cell (RSC) with blunt edges in 44 blood samples obtained from 32 steady-state patients with sickle cell disease (SCD) without exposure to air (UnExp-blood). Because these RSCs could be generated in vitro by partial oxygenation of once-deoxygenated SS cells to venous oxygen pressure, we named them "partially oxygenated sickled cells" (POSCs). These RSCs were classified into elongated and non-elongated RSCs, depending on the ratio of the short axis to long axis. The presence of these cells was previously unknown because the standard blood collection method oxygenates most of the POSCs to discocytes due to oxygen in the air space in the needle, syringe, and blood collection tube (Exp-blood). Although the shape of elongated POSCs is similar to that of irreversibly sickled cells (ISCs), POSCs revert to discocytes upon exposure to air. We found the following: (1) the percentage of total sickled cells (total POSCs + ISCs) in UnExp-blood (29.0 +/- 14.5%) was significantly higher than the percentage of sickled cells (mainly ISCs) in Exp-blood (7.3 +/- 5.7%); (2) the percentage of sickled cells in UnExp-blood was specific to individual patients during steady state, while it decreased at the onset of a vaso-occlusive event; and (3) the percentage of sickled cells in UnExp-blood varied widely among steady-state patients (4-56%). This new type of RSC may be used as an internal biomarker to evaluate the disease state of individual patients.

Clinical significance of glucose-6-phosphate dehydrogenase deficiency in Nigerian patients with sickle cell disease

A total of 75 (45 males, 30 females) patients with sickle cell disease (SCD) at the university of Maiduguri Teaching Hospital were screened for G-6-PD deficiency and their steady state levels of haematocrit, reticulocyte count and irreversibly sickled cells (ISC) as well as the occurrence of vaso-occlusive crises were analysed with respect to their G-6-PD status during a 3 year period (1998-2000). None of the female patients was G-6-PD deficient, while (15.6%) male patients had the enzyme deficiency. The patients with G-6-PD deficiency had significantly lower mean level of haematocrit (0.221/l) with higher level of reticulocyte count (15%) as compared to their non-G-6-PD deficient counter parts who had mean values of 0.271/l and 9% for haematocrit count respectively. However, there was no significant difference in mean levels of ISC between G-6-PD deficient (6%) and non-G-6-PD deficient (5%) patients with SCD. Patients with G-6-PD deficiency had significantly lower episodes of vaso-occlusive crises (2.1 episodes/patient). This data would suggest that co-inheritance of G-6-PD deficiency in SCD reduces frequency of crises and improves prognosis.

Two Distinct Pathways Mediate the Formation of Intermediate Density Cells and Hyperdense Cells From Normal Density Sickle Red Blood Cells

AbstractIn sickle cell anemia (SS), some red blood cells dehydrate, forming a hyperdense (HD) cell fraction (>1.114 g/mL; mean corpuscular hemoglobin concentration [MCHC], >46 g/dL) that contains many irreversibly sickled cells (ISCs), whereas other SS red blood cells dehydrate to an intermediate density (ID; 1.090 to 1.114 g/mL; MCHC, 36 to 46 g/dL). This study asks if the potassium-chloride cotransporter (K:Cl) and the calcium-dependent potassium channel [K(Ca2+)] are participants in the formation of one or both types of dense SS red blood cells. We induced sickling by exposing normal density (ND; 1.080 to 1.090 g/mL; MCHC, 32 to 36 g/dL) SS discocytes to repetitive oxygenation-deoxygenation (O-D) cycles in vitro. At physiologic Na+, K+, and Cl−, and 0.5 to 2 mmol/L Ca2+, the appearance of dense cells was time- and pH-dependent. O-D cycling at pH 7.4 in 5% CO2-equilibrated buffer generated only ID cells, whereas O-D cycling at pH 6.8 in 5% CO2-equilibrated buffer generated both ID and HD cells, the latter taking more than 8 hours to form. At 22 hours, 35% ± 17% of the parent ND cells were recovered in the ID fraction and 18% ± 11% in the HD fraction. Continuous deoxygenation (N2/5% CO2) at pH 6.8 generated both ID and HD cells, but many of these cells had multiple projections, clearly different from the morphology of endogenous dense cells and ISCs. Continuous oxygenation (air/5% CO2) at pH 6.8 resulted in less than 10% dense cell (ID + HD) formation. ATP depletion substantially increased HD cell formation and moderately decreased ID cell formation. HD cells formed after 22 hours of O-D cycling at pH 6.8 contained fewer F cells than did ID cells, suggesting that HD cell formation is particularly dependent on HbS polymerization. EGTA chelation of buffer Ca2+ inhibited HD but not ID cell formation, and increasing buffer Ca2+ from 0.5 to 2 mmol/L promoted HD but not ID cell formation in some SS patients. Substitution of nitrate for Cl− inhibited ID cell formation, as did inhibitors of the K:Cl cotransporter, okadaic acid, and [(dihydroindenyl) oxy]alkanoic acid (DIOA). Conversely, inhibitors of K(Ca2+), charybdotoxin and clotrimazole, inhibited HD cell formation. The combined use of K(Ca2+) and K:Cl inhibitors nearly eliminated dense cell (ID + HD cell) formation. In summary, dense cells formed by O-D cycling for 22 hours at pH 7.4 cycling are predominately the ID type, whereas dense cells formed by O-D cycling for 22 hours at pH 6.8 are both the ID and HD type, with the latter low in HbF, suggesting that HD cell formation has a greater dependency on HbS polymerization. A combination of K:Cl cotransport and the K(Ca2+) activities account for the majority of dense cells formed, and these pathways can be driven independently. We propose a model in which reversible sickling-induced K+ loss by K:Cl primarily generates ID cells and K+ loss by the K(Ca2+) channel primarily generates HD cells. These results imply that both pathways must be inhibited to completely prevent dense SS cell formation and have potential therapeutic implications.

Two Distinct Pathways Mediate the Formation of Intermediate Density Cells and Hyperdense Cells From Normal Density Sickle Red Blood Cells

In sickle cell anemia (SS), some red blood cells dehydrate, forming a hyperdense (HD) cell fraction (>1.114 g/mL; mean corpuscular hemoglobin concentration [MCHC], >46 g/dL) that contains many irreversibly sickled cells (ISCs), whereas other SS red blood cells dehydrate to an intermediate density (ID; 1.090 to 1.114 g/mL; MCHC, 36 to 46 g/dL). This study asks if the potassium-chloride cotransporter (K:Cl) and the calcium-dependent potassium channel [K(Ca2+)] are participants in the formation of one or both types of dense SS red blood cells. We induced sickling by exposing normal density (ND; 1.080 to 1.090 g/mL; MCHC, 32 to 36 g/dL) SS discocytes to repetitive oxygenation-deoxygenation (O-D) cycles in vitro. At physiologic Na+, K+, and Cl-, and 0.5 to 2 mmol/L Ca2+, the appearance of dense cells was time- and pH-dependent. O-D cycling at pH 7.4 in 5% CO2-equilibrated buffer generated only ID cells, whereas O-D cycling at pH 6.8 in 5% CO2-equilibrated buffer generated both ID and HD cells, the latter taking more than 8 hours to form. At 22 hours, 35% +/- 17% of the parent ND cells were recovered in the ID fraction and 18% +/- 11% in the HD fraction. Continuous deoxygenation (N2/5% CO2) at pH 6.8 generated both ID and HD cells, but many of these cells had multiple projections, clearly different from the morphology of endogenous dense cells and ISCs. Continuous oxygenation (air/5% CO2) at pH 6.8 resulted in less than 10% dense cell (ID + HD) formation. ATP depletion substantially increased HD cell formation and moderately decreased ID cell formation. HD cells formed after 22 hours of O-D cycling at pH 6.8 contained fewer F cells than did ID cells, suggesting that HD cell formation is particularly dependent on HbS polymerization. EGTA chelation of buffer Ca2+ inhibited HD but not ID cell formation, and increasing buffer Ca2+ from 0.5 to 2 mmol/L promoted HD but not ID cell formation in some SS patients. Substitution of nitrate for Cl- inhibited ID cell formation, as did inhibitors of the K:Cl cotransporter, okadaic acid, and [(dihydroindenyl) oxy]alkanoic acid (DIOA). Conversely, inhibitors of K(Ca2+), charybdotoxin and clotrimazole, inhibited HD cell formation. The combined use of K(Ca2+) and K:Cl inhibitors nearly eliminated dense cell (ID + HD cell) formation. In summary, dense cells formed by O-D cycling for 22 hours at pH 7.4 cycling are predominately the ID type, whereas dense cells formed by O-D cycling for 22 hours at pH 6.8 are both the ID and HD type, with the latter low in HbF, suggesting that HD cell formation has a greater dependency on HbS polymerization. A combination of K:Cl cotransport and the K(Ca2+) activities account for the majority of dense cells formed, and these pathways can be driven independently. We propose a model in which reversible sickling-induced K+ loss by K:Cl primarily generates ID cells and K+ loss by the K(Ca2+) channel primarily generates HD cells. These results imply that both pathways must be inhibited to completely prevent dense SS cell formation and have potential therapeutic implications.

The Efficacy of Reducing Agents or Antioxidants in Blocking the Formation of Dense Cells and Irreversibly Sickled Cells In Vitro

We show that N-acetylcysteine (NAC) has the ability to cause statistically significant diminishment in the in vitro formation of irreversibly sickled cells (ISCs) at concentrations greater than 250 μmol/L. Other antioxidants, approved for human use (cysteamine, succimer, dimercaprol), were not efficacious. NAC had the ability to cause statistically significant conversion of ISCs formed in vivo back to the biconcave shape. NAC was also shown to reduce the formation of dense cells and increase the available thiols in β-actin. We showed that diminishing reduced glutathione (GSH), by treatment with 1-chloro-2,4-dinitrobenzene, resulted in increased dense cells. We conclude the NAC blocks dense cell formation and ISC formation by targeting channels involved in cellular dehydration and β-actin, respectively. The efficacy of NAC is probably due to its combined antioxidant activity and ability to increase intracellular GSH.

The Efficacy of Reducing Agents or Antioxidants in Blocking the Formation of Dense Cells and Irreversibly Sickled Cells In Vitro

AbstractWe show that N-acetylcysteine (NAC) has the ability to cause statistically significant diminishment in the in vitro formation of irreversibly sickled cells (ISCs) at concentrations greater than 250 μmol/L. Other antioxidants, approved for human use (cysteamine, succimer, dimercaprol), were not efficacious. NAC had the ability to cause statistically significant conversion of ISCs formed in vivo back to the biconcave shape. NAC was also shown to reduce the formation of dense cells and increase the available thiols in β-actin. We showed that diminishing reduced glutathione (GSH), by treatment with 1-chloro-2,4-dinitrobenzene, resulted in increased dense cells. We conclude the NAC blocks dense cell formation and ISC formation by targeting channels involved in cellular dehydration and β-actin, respectively. The efficacy of NAC is probably due to its combined antioxidant activity and ability to increase intracellular GSH.

Irreversibly sickled cell beta-actin: defective filament formation.

It has been demonstrated that cysteine modification in irreversibly sickled cell beta-actin slows down the remodeling of membrane skeletons [Shartava et al.: J Cell Biol 128:805-812, 1995]. This slow remodeling can be due to alterations in spectrin-actin binding and/or actin-actin interactions in irreversibly sickled cell (ISC) membrane skeletons. In these studies we demonstrate that ISC actin binds spectrin normally. However, ISC beta-actin polymerizes and depolymerizes more slowly than control beta-actin, and forms unusual aggregates when placed under polymerizing conditions. Electron microscopic analysis of actin polymers indicated that ISC actin generates a large amount of aggregates which we conclude are due to the structural modification caused by the disulfide bridge between cysteine284 and cysteine373 in beta-actin.

Percentage of Reversibly and Irreversibly Sickled Cells Are Altered by the Method of Blood Drawing and Storage Conditions

ABSTRACT: We previously reported that the percentage of reversibly and irreversibly sickled cells (RSC and ISC, respectively) in the blood of patients with sickle cell disease is strongly influenced by the method of blood drawing (PNAS91:12589, 1994). We now document the effect of blood storage conditions on the percentage of RSC and ISC. The percentage of RSC was lowest when blood was stored at 0°C, while the percentage of RSC was highest in specimens kept at 37°C. At room temperature, the percentage of RSC increased slightly over 8 hours. The percentage of ISC was also temperature dependent and was reduced significantly upon cooling. Our results showed that many ISC reverted to a discoidal shape after 3 hrs of cooling after treatment of blood with oxygen or carbon monoxide. Since no Hb S polymers were detected in ISC treated with oxygen or carbon monoxide, the time required for shape restoration may be attributed to the membrane. We measured ISC levels of 10 patients with consideration of storage temperature and compared the values with those determined by the conventional method and also with those published previously.

A Rat Model for Sickle Cell-Mediated Vaso-occlusion in Retina

Occlusions of the retinal vasculature are the initiating event in sickle cell retinopathy. In order to understand the mechanism(s) of sickle cell-mediated occlusion, a rat model was developed. Red blood cells (RBCs) from patients homozygous for hemoglobin (Hb) S (SS) or double heterozygous for Hb S and Hb C (SC) were separated on Percoll–Larex continuous density gradients, labeled with fluorescein isothiocyanate (FITC), and delivered via the left ventricle to anesthetized, ventilated rats. Blood gas levels were altered by changing inspired gas and monitored via a femoral arterial catheter. After the RBCs circulated for 5 min, animals were perfused with heparinized saline, the eyes enucleated, and the retinas removed and processed by our ADPase flatmount technique. The retinal vasculature was visualized under dark-field illumination and the FITC-RBCs visualized by fluorescence microscopy. Greater numbers of high-density SS cells (SS4, which consist of dense, dehydrated discocytes and irreversible sickled cells) were retained in the normal rat retinal vasculature than normal-density SS cells (SS2, which have the same density as normal AA cells, but consist of reticulocytes and young cells). Retention of SS4 cells was inversely dependent on the arterial oxygen tension. Most SS4s were retained in capillaries, but a few were observed within precapillary arterioles. The retained RBCs occupied the full lumenal diameter of vessels in most cases. In contrast, very few RBCs from SC donors (normal or high density) were retained in the normal retinal vasculature and retention did not increase significantly with hypoxia. This model demonstrates that high-density SS cells, which include irreversibly sickled cells, are retained in normal rat retinal vessels and that the number retained is oxygen dependent. Furthermore, it appears that trapping, not adhesion, is responsible for retention of RBCs in the normal retinal vasculature because there was preferential retention of SS4 cells, which are known to have lower adherence propensity, and the retained RBCs blocked the full diameter of the vessel. These results also demonstrate that the mechanism of vascular obstruction by SS and SC RBCs is different because low retention of SC cells was observed. The well-known propensity of SC patients to have retinal abnormalities must involve extraerythrocytic factors like increased hematocrit, induction of adhesive molecules and integrins, etc.

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

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

Identification of the disulfide-linked peptide in irreversibly sickled cell beta-actin.

We have previously demonstrated that the membrane skeletons of irreversibly sickled cells (ISCs) dissociate more slowly at 37 degrees C, in high ionic strength Triton X-100 buffer, than do the membrane skeletons of reversibly sickled cells or control erythrocytes [Shartava et al. (1995) J. Cell. Biol. 128, 805-818]. Furthermore, we demonstrated that the major cause of this slow dissociation was a single posttranslational modification in ISC beta-actin. Two sulfhydryl groups (Cys284 and Cys373) became inaccessible to thiol reagents because of this modification. We suggested the possibility that the modification was a disulfide bridge between Cys284 and Cys373 since the reducing agent dithiothreitol restored the sulfhydryl groups. In this article, we directly demonstrate the existence of the disulfide bridge between cysteine284 and cysteine373 in ISC beta-actin. We synthesized the associated ISC beta-actin tryptic cystine-peptide (KCF-CDVDIR), characterized it by HPLC, MS. and MSMS, and identified it in the tryptic digest of the ISC beta-actin. These results support our earlier suggestion that the oxidative change in ISC beta-actin is a major cause of the irreversible sickling phenomenon.

Bencyclane as an anti-sickling agent.

A vasodilating Ca2+ channel blocker, bencyclane, was used in 18 patients with homozygous sickle cell anaemia (SCD) to test the possible anti-sickling effect. With bencyclane intervention the Na(+)-K+ ATPase activity increased from 256 +/- 29 to 331 +/- 37 nmol Pi/mg protein/h (P < 0.0001) and the Ca(2+)-Mg2+ ATPase level increased from 172 +/- 12 to 222 +/- 44 nmol Pi/mg protein/h (P < 0.0001). The intracytoplasmic Ca2+ concentration reduced from 3.5 +/- 0.6 to 2.7 +/- 0.25 mumol/l (P < 0.0001). The patient's blood contained fewer irreversibly sickled cells (ISCs) (a reduction from 21.4% to 14.4%) (P < 0.05). At the same time MCHC of the erythrocytes decreased from 34.5 to 33.0 g/dl (P < 0.05). Bencyclane appears to be a promising anti-sickling agent that can be used orally in SCD.

A posttranslational modification of beta-actin contributes to the slow dissociation of the spectrin-protein 4.1-actin complex of irreversibly sickled cells.

Irreversibly sickled cells (ISCs) remain sickled even under conditions where they are well oxygenated and hemoglobin is depolymerized. In our studies we demonstrate that triton extracted ISC core skeletons containing only spectrin, protein 4.1, and actin also retain their sickled shape; while reversibly sickled cell (RSC) skeletons remodel to a round or biconcave shape. We also demonstrate that these triton extracted ISC core skeletons dissociate more slowly upon incubation at 37 degrees C than do RSC or control (AA) core skeletons. This observation may supply the basis for the inability of the ISC core skeleton to remodel its shape. Using an in vitro ternary complex dissociation assay, we demonstrate that a modification in beta-actin is the major determinant of the slow dissociation of the spectrin-protein 4.1-actin complex isolated from the ISC core skeleton. We demonstrate that the difference between ISC and control beta-actin is the inaccessibility of two cysteine residues in ISC beta-actin to labeling by thiol reactive reagents; due to the formation of a disulfide bridge between cysteine284 and cysteine373 in ISC beta-actin, or alternatively another modification of cysteine284 and cysteine373 which is reversible with DTT and adds less than 100 D to the molecular weight of beta-actin.

Partially oxygenated sickled cells: sickle-shaped red cells found in circulating blood of patients with sickle cell disease.

A previously uncharacterized type of sickled cell was found in venous blood of patients with sickle cell disease when blood was collected without exposure to air and fixed immediately with 1% glutaraldehyde solution equilibrated with 5% oxygen. These cells were either elongated, resembling irreversibly sickled cells (ISCs), or nonelongated, with a raisin-like shape. Both types assumed a normal discoidal shape upon full oxygenation. Since these cells exist only under partially oxygenated conditions, they are described as partially oxygenated sickled cells (POSCs). POSCs are morphologically distinct from partially deoxygenated sickled cells formed during deoxygenation by having rounded edges, while the latter have sharp edges. Transmission electron microscopy of POSCs revealed various amounts of misaligned Hb S polymers. Investigations in vitro demonstrated the formation of POSC-like cells by partial oxygenation of deoxygenated cells. Since POSCs contain intracellular fibers and sickle readily upon deoxygenation, they may have clinical and pathological significance.