RESUMO
OBJECTIVES: Blood-processing techniques and preservation conditions cause storage lesions, possibly leading to adverse outcomes after transfusion. The authors investigated the metabolic changes and deformability of red blood cells (RBCs) during storage and determined the effect of storage lesions on circulating RBCs during cardiac surgery. DESIGN: Prospective study. SETTING: Tertiary care center affiliated with a university hospital. PARTICIPANTS: Adults who underwent elective cardiac surgery requiring cardiopulmonary bypass. INTERVENTIONS: The authors collected aliquots of autologous and irradiated allogeneic RBCs and blood samples from seven patients who received autologous whole blood and nine patients who received irradiated allogeneic RBCs before incision (baseline), at the start and end of cardiopulmonary bypass, and at completion of surgery. MEASUREMENTS AND MAIN RESULTS: The authors analyzed RBC deformability, erythrocyte indices, and density distribution to evaluate blood banking-induced alterations of autologous and allogeneic RBCs and changes in circulating RBCs in recipients, after blood transfusion. Time-dependent biochemical changes and significant decreases in deformability during storage occurred in both groups; however, homologous RBCs had significantly lower deformability than autologous RBCs. Trends in mean corpuscular volume and mean corpuscular hemoglobin concentration differed in both groups. In the homologous transfusion group, during cardiac surgery, RBC deformability, mean corpuscular volume, and mean corpuscular hemoglobin concentration showed significant changes compared with baseline values, and a greater number of denser subpopulations was observed at surgery completion. CONCLUSIONS: Blood-processing techniques contribute to storage lesions, suggesting that transfusion of autologous whole blood, rather than allogeneic RBCs, could maintain the ability of circulating RBCs to deform and lead to potentially better transfusion outcomes.
Assuntos
Procedimentos Cirúrgicos Cardíacos , Transplante de Células-Tronco Hematopoéticas , Preservação de Sangue/efeitos adversos , Preservação de Sangue/métodos , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Ponte Cardiopulmonar/efeitos adversos , Deformação Eritrocítica , Eritrócitos , Humanos , Estudos ProspectivosRESUMO
We prospectively studied the dose-dependent effect of transfused stored red blood cells (RBCs) on recipient RBC indices, deformability and cell density in 10 patients administered stored RBCs for blood transfusion during general surgery. There were dose-dependent decreases in mean corpuscular volume and increases in mean corpuscular haemoglobin concentration after completion of 4- and 6-unit stored RBC transfusions. The amount of dense populations increased proportionately with the amount of stored RBCs transfused. The maximal deformability index value was significantly and dose-dependently decreased, suggesting that hemodynamic blood flow, especially the microcirculation may be impaired in patients who receive large amounts of stored RBCs.
Assuntos
Preservação de Sangue/efeitos adversos , Índices de Eritrócitos/fisiologia , Transfusão de Eritrócitos/efeitos adversos , Eritrócitos/fisiologia , Eritrócitos/citologia , Feminino , Humanos , MasculinoRESUMO
OBJECTIVES: During cardiac surgery, circulating red blood cells (RBCs) are at risk of exposure to environmental factors during extracorporeal circulation and transfusion of stored RBCs. For this study, the authors observed morphological differences, deformability, density distribution, and erythrocyte indices of RBCs during cardiac surgery with cardiopulmonary bypass (CPB). DESIGN: Prospective study. SETTING: Tertiary care center affiliated with a university hospital. PARTICIPANTS: Adults who underwent elective cardiac surgery requiring CPB. INTERVENTIONS: Blood samples were obtained from 13 patients before incision (baseline), at initiation of CPB, after separation from CPB, and at completion of surgery. MEASUREMENTS AND MAIN RESULTS: The morphological index (MI) in RBCs using light microscopy and the maximum deformability index (DImax) using an ektacytometer were evaluated. In addition, the fractionation of RBCs and erythrocyte indices were measured. The MI at initiation of CPB was significantly higher without blood transfusion compared with baseline, although the DImax did not significantly decrease simultaneously. The DImax after separation from CPB and at completion of surgery were significantly lower than that at baseline. This lowered DImax was accompanied by a significantly reduced mean corpuscular volume and elevated mean corpuscular hemoglobin concentration compared with baseline. Dense RBC subpopulations increased after initiating CPB. The MI after separation from CPB and at completion of surgery partially recovered. Administered stored RBCs showed a high MI and the lowest DImax. CONCLUSIONS: Morphological changes at initiation of CPB are considered potentially reversible transformations without loss of the membrane surface area and do not have a significant effect on the DImax. A decrease in deformability likely is due to transfusion of stored RBCs.
Assuntos
Ponte Cardiopulmonar/métodos , Deformação Eritrocítica/fisiologia , Eritrócitos/patologia , Cardiopatias/cirurgia , Idoso , Transfusão de Sangue , Feminino , Cardiopatias/sangue , Humanos , Período Intraoperatório , Masculino , Prognóstico , Estudos ProspectivosRESUMO
An asymmetric distribution of phospholipids in the membrane bilayer is inseparable from physiological functions, including shape preservation and survival of erythrocytes, and by implication other cells. Aminophospholipids, notably phosphatidylserine (PS), are confined to the inner leaflet of the erythrocyte membrane lipid bilayer by the ATP-dependent flippase enzyme, ATP11C, counteracting the activity of an ATP-independent scramblase. Phospholipid scramblase 1 (PLSCR1), a single-transmembrane protein, was previously reported to possess scrambling activity in erythrocytes. However, its function was cast in doubt by the retention of scramblase activity in erythrocytes of knockout mice lacking this protein. We show that in the human erythrocyte PLSCR1 is the predominant scramblase and by reconstitution into liposomes that its activity resides in the transmembrane domain. At or below physiological intracellular calcium concentrations, total suppression of flippase activity nevertheless leaves the membrane asymmetry undisturbed. When liposomes or erythrocytes are depleted of cholesterol (a reversible process in the case of erythrocytes), PS quickly appears at the outer surface, implying that cholesterol acts in the cell as a powerful scramblase inhibitor. Thus, our results bring to light a previously unsuspected function of cholesterol in regulating phospholipid scrambling.
Assuntos
Adenosina Trifosfatases/metabolismo , Colesterol/metabolismo , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Animais , Transporte Biológico , Cálcio/metabolismo , Células Cultivadas , Eritrócitos/citologia , Humanos , Camundongos , Fosfatidilserinas/metabolismoRESUMO
The membrane skeleton plays a central role in maintaining the elasticity and stability of the erythrocyte membrane, two biophysical features critical for optimal functioning and survival of red cells. Many constituent proteins of the membrane skeleton are phosphorylated by various kinases, and phosphorylation of ß-spectrin by casein kinase and of protein 4.1R by PKC has been documented to modulate erythrocyte membrane mechanical stability. In this study, we show that activation of endogenous PKA by cAMP decreases membrane mechanical stability and that this effect is mediated primarily by phosphorylation of dematin. Co-sedimentation assay showed that dematin facilitated interaction between spectrin and F-actin, and phosphorylation of dematin by PKA markedly diminished this activity. Quartz crystal microbalance measurement revealed that purified dematin specifically bound the tail region of the spectrin dimer in a saturable manner with a submicromolar affinity. Pulldown assay using recombinant spectrin fragments showed that dematin, but not phospho-dematin, bound to the tail region of the spectrin dimer. These findings imply that dematin contributes to the maintenance of erythrocyte membrane mechanical stability by facilitating spectrin-actin interaction and that phosphorylation of dematin by PKA can modulate these effects. In this study, we have uncovered a novel functional role for dematin in regulating erythrocyte membrane function.
Assuntos
Actinas/metabolismo , Membrana Eritrocítica/metabolismo , Proteínas dos Microfilamentos/metabolismo , Espectrina/metabolismo , Caseína Quinases/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas do Citoesqueleto/metabolismo , Feminino , Humanos , Masculino , Proteínas de Membrana/metabolismo , Multimerização Proteica/fisiologia , Estrutura Terciária de ProteínaRESUMO
Membrane lipid rafts have been implicated in erythrocyte invasion process by Plasmodium falciparum. In this study, we examined the effect of lidocaine, a local anesthetic, which disrupts lipid rafts reversibly without affecting membrane cholesterol content on parasite invasion. In the presence of increasing concentrations of lidocaine in the culture medium, parasite invasion was progressively decreased with complete inhibition at 2mM. Decreased invasion was also seen in erythrocytes pre-treated with lidocaine and cultured in the absence of lidocaine. This inhibitory effect on parasite invasion was reversed following removal of lidocaine from erythrocyte membranes. Our findings show that disruption of lipid rafts in the context of normal cholesterol content markedly inhibits parasite invasion and confirm an important role for lipid rafts in invasion of erythrocytes by P. falciparum.
Assuntos
Anestésicos Locais/farmacologia , Eritrócitos/parasitologia , Lidocaína/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Plasmodium falciparum/fisiologia , Animais , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Humanos , Plasmodium falciparum/efeitos dos fármacosRESUMO
An R664X nonsense mutant AE1 is responsible for dominant hereditary spherocytosis in cattle and is degraded by the proteasomal endoplasmic reticulum-associated degradation. The present study demonstrated that R664X AE1 translated in vitro had the trypsin-sensitve site identical to that of the wild-type AE1. The P661S/R664X mutant containing a possible N-glycosylation site at Asn660 showed an increase in size by 3 kDa both in the cell-free translation system and in transfected HEK293 cells. Moreover, steady state levels of R664X and P661S/R664X in HEK293 cells were markedly increased in the presence of a proteasome inhibitior. These findings indicate that the truncated C-terminal region of R664X AE1 has lumenal localization in the endoplasmic reticulum and is not accessible to proteasomal machineries in the cytosol.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Doenças dos Bovinos/metabolismo , Retículo Endoplasmático/metabolismo , Esferocitose Hereditária/veterinária , Animais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Bovinos , Doenças dos Bovinos/genética , Doenças dos Bovinos/patologia , Mutação Puntual , Esferocitose Hereditária/genética , Esferocitose Hereditária/metabolismo , Esferocitose Hereditária/patologiaRESUMO
Various mutations in the AE1 (anion exchanger 1, band 3) gene cause dominant hereditary spherocytosis, a common congenital hemolytic anemia associated with deficiencies of AE1 of different degrees and loss of mutant protein from red blood cell membranes. To determine the mechanisms underlying decreases in AE1 protein levels, we employed K562 and HEK293 cell lines and Xenopus oocytes together with bovine wild-type AE1 and an R664X nonsense mutant responsible for dominant hereditary spherocytosis to analyze protein expression, turnover, and intracellular localization. R664X-mutant protein underwent rapid degradation and caused specifically increased turnover and impaired trafficking to the plasma membrane of the wild-type protein through hetero-oligomer formation in K562 cells. Consistent with those observations, co-expression of mutant and wild-type AE1 reduced anion transport by the wild-type protein in oocytes. Transfection studies in K562 and HEK293 cells revealed that the major pathway mediating degradation of both R664X and wild-type AE1 employed endoplasmic reticulum (ER)-associated degradation through the proteasomal pathway. Proteasomal degradation of R664X protein appeared to be independent of both ubiquitylation and N-glycosylation, and aggresome formation was not observed following proteasome inhibition. These findings indicate that AE1 R664X protein, which is associated with dominant hereditary spherocytosis, has a dominant-negative effect on the expression of wild-type AE1.