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(1) Background: When red blood cells are centrifuged in a continuous Percoll-based density gradient, they form discrete bands. While this is a popular approach for red blood cell age separation, the mechanisms involved in banding were unknown. (2) Methods: Percoll centrifugations of red blood cells were performed under various experimental conditions and the resulting distributions analyzed. The age of the red blood cells was measured by determining the protein band 4.1a to 4.1b ratio based on western blots. Red blood cell aggregates, so-called rouleaux, were monitored microscopically. A mathematical model for the centrifugation process was developed. (3) Results: The red blood cell band pattern is reproducible but re-centrifugation of sub-bands reveals a new set of bands. This is caused by red blood cell aggregation. Based on the aggregation, our mathematical model predicts the band formation. Suppression of red blood cell aggregation reduces the band formation. (4) Conclusions: The red blood cell band formation in continuous Percoll density gradients could be explained physically by red blood cell aggregate formation. This aggregate formation distorts the density-based red blood cell age separation. Suppressing aggregation by osmotic swelling has a more severe effect on compromising the RBC age separation to a higher degree.
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Eritrócitos , Povidona , Separação Celular/métodos , Centrifugação com Gradiente de Concentração , Dióxido de SilícioRESUMO
The N-methyl-D-aspartate receptor (NMDAR) provides a pathway for glutamate-mediated inter-cellular communication, best known for its role in the brain but with multiple examples of functionality in non-neuronal cells. Data previously published by others and us provided ex vivo evidence that NMDARs regulate platelet and red blood cell (RBC) production. Here, we summarize what is known about these hematopoietic roles of the NMDAR. Types of NMDAR subunits expressed in megakaryocytes (platelet precursors) and erythroid cells are more commonly found in the developing rather than adult brain, suggesting trophic functions. Nevertheless, similar to their neuronal counterparts, hematopoietic NMDARs function as ion channels, and are permeable to calcium ions (Ca2+). Inhibitors that block open NMDAR (memantine and MK-801) interfere with megakaryocytic maturation and proplatelet formation in primary culture. The effect on proplatelet formation appears to involve Ca2+ influx-dependent regulation of the cytoskeletal remodeling. In contrast to normal megakaryocytes, NMDAR effects in leukemic Meg-01 cells are diverted away from differentiation to increase proliferation. NMDAR hypofunction triggers differentiation of Meg-01 cells with the bias toward erythropoiesis. The underlying mechanism involves changes in the intracellular Ca2+ homeostasis, cell stress pathways, and hematopoietic transcription factors that upon NMDAR inhibition shift from the predominance of megakaryocytic toward erythroid regulators. This ability of NMDAR to balance both megakaryocytic and erythroid cell fates suggests receptor involvement at the level of a bipotential megakaryocyte-erythroid progenitor. In human erythroid precursors and circulating RBCs, NMDAR regulates intracellular Ca2+ homeostasis. NMDAR activity supports survival of early proerythroblasts, and in mature RBCs NMDARs impact cellular hydration state, hemoglobin oxygen affinity, and nitric oxide synthase activity. Overexcitation of NMDAR in mature RBCs leads to Ca2+ overload, K+ loss, RBC dehydration, and oxidative stress, which may contribute to the pathogenesis of sickle cell disease. In summary, there is growing evidence that glutamate-NMDAR signaling regulates megakaryocytic and erythroid cells at different stages of maturation, with some intriguing differences emerging in NMDAR expression and function between normal and diseased cells. NMDAR signaling may provide new therapeutic opportunities in hematological disease, but in vivo applicability needs to be confirmed.
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Mean values of hematological parameters are currently used in the clinical laboratory settings to characterize red blood cell properties. Those include red blood cell indices, osmotic fragility test, eosin 5-maleimide (EMA) test, and deformability assessment using ektacytometry to name a few. Diagnosis of hereditary red blood cell disorders is complemented by identification of mutations in distinct genes that are recognized "molecular causes of disease." The power of these measurements is clinically well-established. However, the evidence is growing that the available information is not enough to understand the determinants of severity of diseases and heterogeneity in manifestation of pathologies such as hereditary hemolytic anemias. This review focuses on an alternative approach to assess red blood cell properties based on heterogeneity of red blood cells and characterization of fractions of cells with similar properties such as density, hydration, membrane loss, redox state, Ca2+ levels, and morphology. Methodological approaches to detect variance of red blood cell properties will be presented. Causes of red blood cell heterogeneity include cell age, environmental stress as well as shear and metabolic stress, and multiple other factors. Heterogeneity of red blood cell properties is also promoted by pathological conditions that are not limited to the red blood cells disorders, but inflammatory state, metabolic diseases and cancer. Therapeutic interventions such as splenectomy and transfusion as well as drug administration also impact the variance in red blood cell properties. Based on the overview of the studies in this area, the possible applications of heterogeneity in red blood cell properties as prognostic and diagnostic marker commenting on the power and selectivity of such markers are discussed.
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Detection of hematopoietic activity in horses is a challenge due to the lack of cells carrying reticulocyte markers such as RNA remnants or CD71 in the circulation. In this study, we fractionated equine red cells according to their density and analyzed the cells forming low (L), medium (M), and high (H) density fractions for markers of aging such as membrane loss, oxidation, and alterations in the intracellular free Ca2+ levels. Cells forming L and M fraction were highly heterogeneous in projected areas and shapes, and had higher propensity to swell in response to hypo-osmotic challenge than the cells from the H fraction. The densest cells were deprived of band 3 protein compared to the cells within L or M fraction. Furthermore, the equine red cells from the H fraction were hyper-oxidized compared to the cells within M and L fractions as follows from an increase in autofluorescence characteristic for oxidized damaged hemoglobin and from thiol oxidation as detected using monobromobimane. The lightest cells showed lower free thiol content compared to the red blood cells from the M fraction, but did not contain oxidized hemoglobin. Finally, the majority of red blood cells forming L, M, and H fraction prominently differed from each other in intracellular free Ca2+ levels and its distribution within the cells. Based on the obtained findings, we suggest that intraerythrocytic Ca2+ levels and its subcellular distribution, eosin-5-maleimide binding test for band 3 abundance, and autofluorescence of cells along with the changes in red blood cell indices, distribution width and creatine levels may become potential markers of regenerative erythropoiesis in horses. Validation of the power of these potential markers of red cell aging is pending.
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Congenital haemolytic anaemias are inherited disorders caused by red blood cell membrane and cytoskeletal protein defects, deviant hemoglobin synthesis and metabolic enzyme deficiencies. In many cases, although the causing mutation might be known, the pathophysiology and the connection between the particular mutation and the symptoms of the disease are not completely understood. Thus effective treatment is lagging behind. As in many cases abnormal red blood cell cation content and cation leaks go along with the disease, by direct electrophysiological measurements of the general conductance of red blood cells, we aimed to assess if changes in the membrane conductance could be a possible cause. We recorded whole-cell currents from 29 patients with different types of congenital haemolytic anaemias: 14 with hereditary spherocytosis due to mutations in α-spectrin, ß-spectrin, ankyrin and band 3 protein; 6 patients with hereditary xerocytosis due to mutations in Piezo1; 6 patients with enzymatic disorders (3 patients with glucose-6-phosphate dehydrogenase deficiency, 1 patient with pyruvate kinase deficiency, 1 patient with glutamate-cysteine ligase deficiency and 1 patient with glutathione reductase deficiency), 1 patient with ß-thalassemia and 2 patients, carriers of several mutations and a complex genotype. While the patients with ß-thalassemia and metabolic enzyme deficiencies showed no changes in their membrane conductance, the patients with hereditary spherocytosis and hereditary xerocytosis showed largely variable results depending on the underlying mutation.
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The Gardos channel is a Ca2+ sensitive, K+ selective channel present in several tissues including RBCs, where it is involved in cell volume regulation. Recently, mutations at two different aminoacid residues in KCNN4 have been reported in patients with hereditary xerocytosis. We identified by whole exome sequencing a new family with two members affected by chronic hemolytic anemia carrying mutation R352H in the KCNN4 gene. No additional mutations in genes encoding for RBCs cytoskeletal, membrane or channel proteins were detected. We performed functional studies on patients' RBCs to evaluate the effects of R352H mutation on the cellular properties and eventually on the clinical phenotype. Gardos channel hyperactivation was demonstrated in circulating erythrocytes and erythroblasts differentiated ex-vivo from peripheral CD34+ cells. Pathological alterations in the function of multiple ion transport systems were observed, suggesting the presence of compensatory effects ultimately preventing cellular dehydration in patient's RBCs; moreover, flow cytometry and confocal fluorescence live-cell imaging showed Ca2+ overload in the RBCs of both patients and hypersensitivity of Ca2+ uptake by RBCs to swelling. Altogether these findings suggest that the 'Gardos channelopathy' is a complex pathology, to some extent different from the common hereditary xerocytosis.
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Anemia Hemolítica Congênita/genética , Canalopatias/genética , Hidropisia Fetal/genética , Mutação/genética , Trifosfato de Adenosina/metabolismo , Adolescente , Adulto , Sinalização do Cálcio , Criança , Eritrócitos/metabolismo , Células Precursoras Eritroides/metabolismo , Família , Feminino , Glicólise , Humanos , Lactente , Padrões de Herança/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Masculino , Modelos Biológicos , Linhagem , Sódio/metabolismoRESUMO
BACKGROUND: Prolonged storage of red blood cells (RBCs) leads to storage lesions, which may impair clinical outcomes after transfusion. A hallmark of storage lesions is progressive echinocytic shape transformation, which can be partially reversed by washing in albumin solutions. Here we have investigated the impact of this shape recovery on biorheologic variables. STUDY DESIGN AND METHODS: RBCs stored hypothermically for 6 to 7 weeks were washed in a 1% human serum albumin (HSA) solution. RBC deformability was measured with osmotic gradient ektacytometry. The viscosity of RBC suspensions was measured with a Couette-type viscometer. The flow behavior of RBCs suspended at 40% hematocrit was tested with an artificial microvascular network (AMVN). RESULTS: Washing in 1% albumin reduced higher degrees of echinocytes and increased the frequency of discocytes, thereby shifting the morphologic index toward discocytosis. Washing also reduced RBC swelling. This shape recovery was not seen after washing in saline, buffer, or plasma. RBC shape normalization did not improve cell deformability measured by ektacytometry, but it tended to decrease suspension viscosities at low shear rates and improved the perfusion of an AMVN. CONCLUSIONS: Washing of stored RBCs in a 1% HSA solution specifically reduces echinocytosis, and this shape recovery has a beneficial effect on microvascular perfusion in vitro. Washing in 1% albumin may represent a new approach to improving the quality of stored RBCs and thus potentially reducing the likelihood of adverse clinical outcomes associated with transfusion of blood stored for longer periods of time.
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Preservação de Sangue/métodos , Transfusão de Eritrócitos , Eritrócitos/efeitos dos fármacos , Albumina Sérica/farmacologia , Adenina/farmacologia , Trifosfato de Adenosina/sangue , Soluções Tampão , Forma Celular , Índices de Eritrócitos , Eritrócitos/citologia , Eritrócitos Anormais/ultraestrutura , Glucose/farmacologia , Glutationa/sangue , Guanosina/farmacologia , Hemorreologia , Humanos , Técnicas In Vitro , Manitol/farmacologia , Modelos Anatômicos , Oxirredução , Perfusão , Plasma , Refrigeração , Cloreto de Sódio/farmacologia , SoluçõesRESUMO
OBJECTIVES: A severe donor organ shortage leads to the death of a substantial number of patients who are listed for transplantation. The use of hearts from donors after circulatory death could significantly expand the donor organ pool, but due to concerns about their viability, these are currently not used for transplantation. We propose short-term ex vivo normothermic machine perfusion (MP) to improve the viability of these ischaemic donor hearts. METHODS: Hearts from male Lewis rats were subjected to 25 min of global in situ warm ischaemia (WI) (37°C), explanted, reconditioned for 60 min with normothermic (37°C) MP with diluted autologous blood and then stored for 4 h at 0-4°C in Custodiol cold preservation solution. Fresh and ischaemic hearts stored for 4 h in Custodiol were used as controls. Graft function was assessed in a blood-perfused Langendorff circuit. RESULTS: During reconditioning, both the electrical activity and contractility of the ischaemic hearts recovered rapidly. Throughout the Langendorff reperfusion, the reconditioned ischaemic hearts had a higher average heart rate and better contractility compared with untreated ischaemic controls. Moreover, the reconditioned ischaemic hearts had higher tissue adenosine triphosphate levels and a trend towards improved tissue redox state. Perfusate levels of troponin T, creatine kinase and lactate dehydrogenase were not significantly lower than those of untreated ischaemic controls. The micro- and macroscopic appearance of the reconditioned ischaemic hearts were improved compared with ischaemic controls, but in both groups myocardial damage and oedema were evident. CONCLUSIONS: Our results indicate that functional recovery from global WI is possible during short-term ex vivo reperfusion, allowing subsequent cold storage without compromising organ viability. We expect that once refined and validated, this approach may enable safe transplantation of hearts obtained from donation after circulatory death.
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Transplante de Coração , Coração/fisiologia , Preservação de Órgãos/métodos , Transplantes/fisiologia , Animais , Frequência Cardíaca , Masculino , Modelos Biológicos , Reperfusão Miocárdica , Estresse Oxidativo , Ratos , Coleta de Tecidos e ÓrgãosRESUMO
Recently we showed that N-methyl D-aspartate receptors (NMDARs) are expressed in erythroid precursors (EPCs) and present in the circulating red blood cells (RBCs) of healthy humans, regulating intracellular Ca(2+) in these cells. This study focuses on investigating the possible role of NMDARs in abnormally high Ca(2+) permeability in the RBCs of patients with sickle cell disease (SCD). Protein levels of the NMDAR subunits in the EPCs of SCD patients did not differ from those in EPCs of healthy humans. However, the number and activity of the NMDARs in circulating SCD-RBCs was substantially up-regulated, being particularly high during haemolytic crises. The number of active NMDARs correlated negatively with haematocrit and haemoglobin levels in the blood of SCD patients. Calcium uptake via these non-selective cation channels was induced by RBC treatment with glycine, glutamate and homocysteine and was facilitated by de-oxygenation of SCD-RBCs. Oxidative stress and RBC dehydration followed receptor stimulation and Ca(2+) uptake. Inhibition of the NMDARs with an antagonist memantine caused re-hydration and largely prevented hypoxia-induced sickling. The EPCs of SCD patients showed higher tolerance to memantine than those of healthy subjects. Consequently, NMDARs in the RBCs of SCD patients appear to be an attractive target for pharmacological intervention.
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Anemia Falciforme/sangue , Cálcio/sangue , Eritrócitos/metabolismo , Receptores de N-Metil-D-Aspartato/sangue , Adulto , Estudos de Casos e Controles , Hipóxia Celular/fisiologia , Células Cultivadas , Volume de Eritrócitos/efeitos dos fármacos , Volume de Eritrócitos/fisiologia , Células Precursoras Eritroides/metabolismo , Eritropoese/fisiologia , Glutationa/sangue , Humanos , Oxirredução , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/fisiologia , Adulto JovemRESUMO
Ca2+ is a universal signalling molecule involved in regulating cell cycle and fate, metabolism and structural integrity, motility and volume. Like other cells, red blood cells (RBCs) rely on Ca2+ dependent signalling during differentiation from precursor cells. Intracellular Ca2+ levels in the circulating human RBCs take part not only in controlling biophysical properties such as membrane composition, volume and rheological properties, but also physiological parameters such as metabolic activity, redox state and cell clearance. Extremely low basal permeability of the human RBC membrane to Ca2+ and a powerful Ca2+ pump maintains intracellular free Ca2+ levels between 30 and 60 nM, whereas blood plasma Ca2+ is approximately 1.8 mM. Thus, activation of Ca2+ uptake has an impressive impact on multiple processes in the cells rendering Ca2+ a master regulator in RBCs. Malfunction of Ca2+ transporters in human RBCs leads to excessive accumulation of Ca2+ within the cells. This is associated with a number of pathological states including sickle cell disease, thalassemia, phosphofructokinase deficiency and other forms of hereditary anaemia. Continuous progress in unravelling the molecular nature of Ca2+ transport pathways allows harnessing Ca2+ uptake, avoiding premature RBC clearance and thrombotic complications. This review summarizes our current knowledge of Ca2+ signalling in RBCs emphasizing the importance of this inorganic cation in RBC function and survival.
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Sinalização do Cálcio , Cálcio/metabolismo , Eritrócitos/metabolismo , Anemia Hemolítica/metabolismo , Anemia Hemolítica/patologia , Animais , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Eritrócitos/patologia , Humanos , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Trombose/metabolismo , Trombose/patologiaRESUMO
Na,K-ATPase is highly sensitive to changes in the redox state, and yet the mechanisms of its redox sensitivity remain unclear. We have explored the possible involvement of S-glutathionylation of the catalytic α subunit in redox-induced responses. For the first time, the presence of S-glutathionylated cysteine residues was shown in the α subunit in duck salt glands, rabbit kidneys, and rat myocardium. Exposure of the Na,K-ATPase to oxidized glutathione (GSSG) resulted in an increase in the number of S-glutathionylated cysteine residues. Increase in S-glutathionylation was associated with dose- and time-dependent suppression of the enzyme function up to its complete inhibition. The enzyme inhibition concurred with S-glutathionylation of the Cys-454, -458, -459, and -244. Upon binding of glutathione to these cysteines, the enzyme was unable to interact with adenine nucleotides. Inhibition of the Na,K-ATPase by GSSG did not occur in the presence of ATP at concentrations above 0.5 mm. Deglutathionylation of the α subunit catalyzed by glutaredoxin or dithiothreitol resulted in restoration of the Na,K-ATPase activity. Oxidation of regulatory cysteines made them inaccessible for glutathionylation but had no profound effect on the enzyme activity. Regulatory S-glutathionylation of the α subunit was induced in rat myocardium in response to hypoxia and was associated with oxidative stress and ATP depletion. S-Glutathionylation was followed by suppression of the Na,K-ATPase activity. The rat α2 isoform was more sensitive to GSSG than the α1 isoform. Our findings imply that regulatory S-glutathionylation of the catalytic subunit plays a key role in the redox-induced regulation of Na,K-ATPase activity.