A rotina pré transfusional em Imunohematologia demanda a utilização de reagentes eritrocitários para detecção de anticorpos naturais e irregulares, objetivando a obtenção de maior segurança do evento transfusional para o paciente por minimizar os riscos de reações indesejadas. Para qualificação, padronização e liberação dos reagentes utilizados em imunohematologia, a seleção do perfil fenotípico dos reagentes eritrocitários é crucial. Desta forma, este trabalho demonstra a possibilidade do uso de doações de concentrado de hemácias para produção de reagentes eritrocitários pelos Hemocentros que detêm a matéria prima e o conhecimento para escolha do perfil de doadores correto. Os resultados sugerem que os hemocentros possuem a capacidade de confeccionar \"kits\" empregados para a realização da prova de tipagem sanguínea reversa e detecção de anticorpos irregulares, provas pré-transfusionais. As principais vantagens da produção dos reagentes pelos hemocentros seriam o baixo custo e a regionalização dos antígenos eritrocitários escolhidos, o que conduziria a maior eficácia na identificação de anticorpos irregulares na população de doadores de hemocomponentes / The pre-transfusion routine in Immunohematology requires the use of erythrocyte reagents for the detection of natural and irregular antibodies, aiming to obtain greater safety of the transfusion event for the patient by minimizing the risks of unwanted reactions. For qualification, standardization and release of the reagents used in immunohematology, the selection of the phenotypic profile of erythrocyte reagents is crucial. In this way, this work demonstrates the possibility of the use of donations of red blood cells for the production of erythrocyte reagents by Blood Banks that holds the raw material and the knowledge to choose the correct donor profile. The results suggest that blood banks have the ability to make kits used to perform the reverse blood typing test and the detection of irregular antibodies, pre-transfusion tests. The main advantages of the production of the reagents by the blood banks would be the low cost and the regionalization of the erythrocyte antigens chosen, which would lead to greater efficacy in the identification of irregular antibodies in the donor population of blood components

A malaria humana é uma doença infecciosa transmitida por um mosquito que está atrelada a uma alta taxa de mortalidade. Dentre os parasitas que causam a malaria humana a espécie Plasmodium falciparum é responsável por 90% dos casos letais. Sua virulência é ocasionada por modificações na célula hospedeira provenientes do tráfego de proteínas para a superfície de eritrócitos infectados. Com o intuito de interferir com estas proteínas, aplicamos a técnica Cell- SELEX (do inglês: Systematic Evolution of Ligands by Exponential Enrichment). Após ciclos iterativos de seleção obtivemos moléculas de DNA simples- fita, conhecidas como aptâmeros, que possuem alta afinidade e especificidade contra proteínas alvo presentes na superfície de eritrócitos infectados por P. falciparum. Ensaios de pull- down usando aptâmeros selecionados em sistema de purificação por streptavidina-biotina seguidos por análises de espectrometria de massa revelaram a interação destas moléculas de DNA com proteínas como RIFIN, PfEMP, RAP1, entre outras incorporadas na superfície de eritrócitos infectados. Como consequência destas interações, os aptâmeros selecionados demostraram ser inibidores potenciais na proliferação dos parasitas, como demonstrado por testes in vitro. Outra estratégia usada foi a produção de aptâmeros contra a piridoxal quinase de P. falciparum (PfPdxK) expressa. Usando tanto SELEX-proteína, bem como, ensaios de eletroforese capilar, selecionamos aptâmeros com capacidade para inibir a atividade enzimática da PfPdxK. Nossos dados constituem evidências sobre as aplicações da tecnologia SELEX no desenho racional de compostos contra a malária humana. / The human malaria is a mosquito-borne infectious disease associated with a high risk of mortality. Among the parasite species that causes human malaria, Plasmodium falciparum is responsible by 90% of the lethal cases. The virulence of this pathogen is associated with host cell modifications by trafficking proteins to the surface of infected erythrocytes. Aiming to interfere with these proteins we have applied the Cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology. After iterative cycles of selection we obtained single stranded DNA molecules, known as aptamers, with high binding affinity and specificity against protein targets present in the surface of erythrocytes infected with P. falciparum. Pull-down assays using the selected aptamers in a streptavidin-biotin affinity assay followed by mass spectrometry analysis revealed the interaction of these DNA molecules with proteins such as RIFIN, PfEMP, RAP1, among others embedded in the surface of infected erythrocytes. As consequence of these interactions, the selected aptamers demonstrated to be potent inhibitors of parasite proliferation, as validated by in vitro tests. Another strategy used was the production of aptamers against the recombinantly expressed plasmodial pyridoxal kinase (PfPdxK). Using both protein-SELEX as well as capillary electrophoresis assays, we selected aptamers with capacity to inhibit the enzymatic activity of PfPdxK. Our data constitute evidences about the application of SELEX technology in the rationale design of inhibitors against human malaria.

Introdução: Os hidroxietilamidos (HES) são considerados expansores plasmáticos efetivos em pacientes submetidos a procedimentos cirúrgicos de grande porte. Entretanto, seu uso clínico é limitado principalmente por sua interferência na hemostasia, representada por alterações da função plaquetária e na coagulação. A extensão dessas alterações está relacionada ao seu ipeso molecular ou à sua substituição molar. Este estudo clínico, foi realizado durante cirurgia de artroplastia de quadril em pacientes adultos para comparar os efeitos do HES (130/0,4) e a solução de Ringer lactato em relação ao sangramento intra-operatório, parâmetros hemodinâmicos, alterações na coagulação, necessidade de transfusões e resultados clínicos. Métodos: Quarenta e oito pacientes candidatos à cirurgia de artroplastia total de quadril sob anestesia subaracnoidea foram distribuídos aleatoriamente em dois grupos 24 pacientes foram selecionados para receber HES (30 ml/kg após anestesia) e 24 pacientes para receber solução de Ringer lactato (30ml/kg). O período de observação teve início após a indução da anestesia e terminou 5 horas após o termino do procedimento cirúrgico. Durante esse período o critério para a infusão de doses adicionais de fluido (10ml/kg de Solução de Ringer lactato para ambos os grupos) foi pressão arterial sistólica inferior a 90 mmHg e/ou um decréscimo de 20% da pressão arterial inicial, frequência cardíaca acima de 100 bpm, e/ou débito urinário menor de 0,4ml/kg/h. Vasopressor foi utilizado nos casos em que a hipotensão persistiu, após a reposição de volume. Transfusão de concentrado de hemácias foi administrada nos pacientes que se mantiveram instáveis hemodinamicamente após bolus adicionais de Ringer lactato ou vasopressor, Parâmetros hemodinâmicos foram mensurados em três períodos da cirurgia; dados bioquímicos foram coletados e testes da coagulação realizados e comparados. Os pacientes foram acompanhados durante sua internação hospitalar. Resultados: Os grupos foram uniformes em relação aos dados demográficos, tipo e duração da cirurgia, assim como a doenças pré-existentes. Não foram observadas diferenças significativas em relação aos parâmetros hemodinâmicos ou temperatura corporal durante o estudo. Os testes de coagulação, função plaquetária, análise de gases sanguíneos e dados bioquímicos mostraramse semelhantes entre os grupos. Perdas sanguíneas foram significativamente maiores no grupo HES (1296x890,p=0,04), necessitou de menos unidades de concentrado de hemácias durante o período observacional (17%versus46%, p=0,029) apresentou menores taxas de infecção (0 versus 4 ,p<0,03), comparado ao grupo Ringer lactato. Conclusões: Em cirurgia de artroplastia total de quadrill, a hemodiluição com hidroxietilamido resultou em maiores taxas de sangramento, menos transfusões sanguíneas e menos infecção pós-operatória. / Introduction: Hydroxyethyl starches (HES) are considered effective plasma expanders in patients undergoing major surgeries. However, the clinical use of HES is limited mainly by their affection of hemostasis, detectable by impaired platelet function and altered coagulation. The extent of such alteration has classically been related to the molecular weight or molar substitution of the used HES solution. This prospective, randomized study was performed during hip arthroplasty in adult patients under spinal anesthesia to compare the effects of HES 130/0.4 with lactateds Ringer solution regarding intraoperative bleeding, hemodynamic parameters, coagulation profile, transfusion requirements and clinical outcomes. Methods: Forty eight patients scheduled to hip arthroplasty after spinal anesthesia were randomized in two groups 24 patients were allocated to receive HES 130/0.4 (30 ml/Kg just after anesthesia) and 24 patients were signaled to receive lactateds Ringer solution (30 ml/Kg). The observational period started after the induction of anesthesia and finished 5 hours after the end of the surgery. During this period, the triggers for infusion of additional boluses of fluids (10 ml/Kg of lactateds Ringer for both groups) were a systolic blood pressure lower than 90 mmHg and/or a decrease of 20% from baseline, a heart rate higher than 100 bpm, and/or a urine output lower than 0.4 ml.Kg-1.h-1. Vasopressors were used if there was persistent hypotension despite of fluid reposition. Red blood cell transfusion was administered if patient remained unstable despite of additional boluses of Ringer or vasopressors, according to the preestablished triggers. Hemodynamic measurements were done in three periods of the surgery, biochemical parameters were analyzed and coagulation tests were performed and compared between groups. After surgery, patients were followed during the hospital stay. Results: The groups were well matched regarding demographic data, type of surgery, and duration of surgery, as well as preexisting diseases. No significant differences in hemodynamic or body temperature were seen during the study. Coagulation variables, platelet function, gases analysis and biochemical parameters were not different between groups. Blood losses were significantly higher in HES 130/0.4 group comparing to Ringers group (1296 x 890 ml, p= 0.046). Despite of that, HES group required less units of blood in the observational period comparing to Ringer group (17% versus 46%, p=0.029). HES group presented lower infection rate compared to Ringer group (0 versus 4 cases, p=0.03). Conclusions: During hip arthroplasty, hemodilution with hydroxyethyl starch 130/0.4 resulted in higher rates of bleeding. However, patients treated with hydroxyethyl starch required less transfusion and presented lower rate of infection.

A anemia é a causa primária de indicação de transfusão de glóbulos vermelhos e é especialmente prevalente e esperada em centros de terapia intensiva. Os benefícios das transfusões incluem aumento da oxigenação tissular, diminuição de hemorragias, tratamento da anemia e da hipovolemia. Os poucos estudos publicados têm demonstrado variações significativas nas práticas de transfusões de glóbulos vermelhos e o melhor momento para a indicação destas transfusões ainda não foi identificado. Objetivos Descrever a população de crianças que recebeu transfusão de glóbulos vermelhos com relação à faixa etária, concentração de hemoglobina (Hb) e taxa de hematócrito (HT), uso de saturação venosa central de oxigênio (SvO2), lactato sérico arterial, procedimentos terapêuticos e presença de disfunção de múltiplos órgãos e sistemas (DMOS). Métodos Estudo retrospectivo observacional, realizado no Centro de Terapia Intensiva Pediátrico (CTIP) do Instituto da Criança da Faculdade de Medicina da Universidade de São Paulo, em 2004, com crianças que receberam transfusão de glóbulos vermelhos. Descrevemos o número de pacientes, faixa etária, motivo de internação no CTIP, presença de doença de base e de DMOS, uso de SvO2, lactato sérico arterial, Hb, HT e procedimentos terapêuticos (ventilação mecânica, drogas vasoativas e métodos de substituição renal). Resultados A transfusão de glóbulos vermelhos foi realizada em 50% dos pacientes internados. A idade mediana foi de 18 meses e o principal motivo de internação foi insuficiência respiratória (35% dos casos). Doença de base estava presente em 84% dos casos e DMOS em 47% dos casos. Foram realizadas coletas de Hb e HT em todos os casos e de SvO2 e lactato sérico arterial em 24% dos casos. A mediana de Hb pré-transfusional foi de 7,8 g/dl. Os pacientes transfundidos estavam sendo submetidos a algum procedimento terapêutico em 82% dos casos. Conclusões São realizadas transfusões de glóbulos vermelhos em todas as idades. A concentração de hemoglobina e a taxa de hematócrito são os principais dados utilizados para a indicação destas transfusões. O lactato sérico arterial e a SvO2 são pouco utilizados para se indicar a transfusão de glóbulos vermelhos. A maioria dos pacientes transfundidos recebe algum procedimento terapêutico e, em muitos casos, são realizadas transfusões em pacientes que apresentam DMOS. / Anemia is the primary cause of red blood cell transfusions and it is especially prevalent and even expected in critical care settings. The benefits of red blood cell transfusions include increase in tissue oxygenation, treatment of anemia and hypovolemia. There are substantial variations in transfusion practices and the optimal transfusion practice for various types of critically ill patients with anemia has not been established. Objectives To describe the clinical, hematological and therapeutic characteristics of children who received red blood cell transfusion in a Pediatric Intensive Care Unit (PICU). Methods Retrospective observational study, performed in the PICU of ?Instituto da Criança?, Medicine School of University of São Paulo, in 2004, with one hundred children who received red blood cell transfusion. We described the number of patients included in the study, age, reason for admission to the PICU, presence of baseline illness and multiple organ system failure (MODS), use of oxygen central venous saturation, arterial lactate, hemoglobin concentration (Hb), hematocrit (HT) and therapeutics procedures (mechanical ventilation, use of vasoactive drugs and renal replacement methods). Results The red blood cell transfusion occurred in 50% of the patients. Median age of patients was 18 months. The most common reason for admission was respiratory failure, in 35% of the cases. Baseline illness was present in 84% of the cases and MODS in 47% of the cases. Hb and HT were collected in all the cases and oxygen central venous saturation and arterial lactate were collected in 24% of the cases. The median pre-transfusion hemoglobin concentration was 7.8 g/dl. The patients were receiveing therapeutics procedures in 82% of the cases. Conclusions Red blood cell transfusions are performed in children of all ages. The hemoglobin concentration and hematocrit are the main reason to indicate the red blood cell transfusion. Arterial lactate and oxygen central venous saturation are rarely utilized. The majority of patients receive therapeutic procedures and, in many cases, patients with MODS are given red blood cell transfusions.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O objetivo do presente trabalho foi estudar o comportamento dos potenciais superficiais e do perfil de potencial atraves da membrana de eritr ocito em func ao da forca i onica e das cargas superficiais, usando um modelo que leva em conta as cargas el etricas do glicoc alix e das prote&#305;nas citoplasm aticas, al em das cargas superficiais da bicamada lip&#305;dica e os efeitos dos eletr olitos divalentes. Programas espec&#305;ficos em linguagem C foram elaborados para o c alculo desses potenciais, tomando como dados num ericos resultados experimentais de medidas de mobilidade eletrofor etica de eritr ocitos para diferentes valores de forca i onica. Neste c alculo, o metodo para tratamento dos dados eletrofor eticos indicado por Hsu et al.[57] foi inclu&#305;do em nosso modelo. A equac ao de Poisson-Boltzmann nao linear foi resolvida por computac ao num erica, usando o metodo de Runge-Kutta de quarta ordem, obtendo-se os perfis de potencial. Os resultados mostraram que a estimativa da densidade de carga el etrica na superf&#305;cie de c elulas usando a equac ao cl assica de Helmholtz-Smoluchowski conduz a valores que nao conseguem refletir as forcas que regem o comportamento eletrofor etico das mesmas. O presente modelo gerou valores de potenciais superficiais e perfis de potencial para a membrana do eritr ocito bem distintos daqueles obtidos anteriormente para um modelo descrito por uma equac ao de Poisson-Boltzmann linear. Nossos resultados confirmam que a avaliac ao de parametros el etricos superficiais da membrana de eritr ocito, envolvendo dados oriundos de eletroforese, deve incluir c alculos hidrodin amicos al em de eletroest aticos, como sugerido por Hsu et al. [57]. / The aim of present work was to study the behavior of the surface potentials and the potential profile across erythrocyte membrane in function of ionic strength and surface charge, using a model which takes into account electrical charges on glycocalyx and citoplasmatic proteins, in addition surface charges on lipid bylayer and effects due to mono and divalent electrolytes. Programs in C language were build to estimate the surface potentials, and experimental values of electrophoretic mobilities of erythrocytes for different ionic strength were applied. For this calculation, the method indicated by Hsu et al. [57] for treating electrophoretic data was included in our model. The non linear Poisson-Boltzmann equation was solved by numerical computation, using the forth order Range-Kutta method, to give the potential profiles. Results showed that values of electric charge on cellular surface obtained by applying the classical Helmholtz-Smoluchowski equation were not able to represent the forces involved in the electrophoretic behavior of cells. The present model generate values for surface potentials and potential profiles different from those obtained in previous work for a model described by linear Poisson- Boltzmann equation. According to our results, the estimation of surface electric parameters for the erythrocyte membrane from electrophoretic data must ese hydrodynamics and electrostatics calculations, as suggested by Hsu et al. [57]

A anemia é a causa primária de indicação de transfusão de glóbulos vermelhos e é especialmente prevalente e esperada em centros de terapia intensiva. Os benefícios das transfusões incluem aumento da oxigenação tissular, diminuição de hemorragias, tratamento da anemia e da hipovolemia. Os poucos estudos publicados têm demonstrado variações significativas nas práticas de transfusões de glóbulos vermelhos e o melhor momento para a indicação destas transfusões ainda não foi identificado. Objetivos Descrever a população de crianças que recebeu transfusão de glóbulos vermelhos com relação à faixa etária, concentração de hemoglobina (Hb) e taxa de hematócrito (HT), uso de saturação venosa central de oxigênio (SvO2), lactato sérico arterial, procedimentos terapêuticos e presença de disfunção de múltiplos órgãos e sistemas (DMOS). Métodos Estudo retrospectivo observacional, realizado no Centro de Terapia Intensiva Pediátrico (CTIP) do Instituto da Criança da Faculdade de Medicina da Universidade de São Paulo, em 2004, com crianças que receberam transfusão de glóbulos vermelhos. Descrevemos o número de pacientes, faixa etária, motivo de internação no CTIP, presença de doença de base e de DMOS, uso de SvO2, lactato sérico arterial, Hb, HT e procedimentos terapêuticos (ventilação mecânica, drogas vasoativas e métodos de substituição renal). Resultados A transfusão de glóbulos vermelhos foi realizada em 50% dos pacientes internados. A idade mediana foi de 18 meses e o principal motivo de internação foi insuficiência respiratória (35% dos casos). Doença de base estava presente em 84% dos casos e DMOS em 47% dos casos. Foram realizadas coletas de Hb e HT em todos os casos e de SvO2 e lactato sérico arterial em 24% dos casos. A mediana de Hb pré-transfusional foi de 7,8 g/dl. Os pacientes transfundidos estavam sendo submetidos a algum procedimento terapêutico em 82% dos casos. Conclusões São realizadas transfusões de glóbulos vermelhos em todas as idades. A concentração de hemoglobina e a taxa de hematócrito são os principais dados utilizados para a indicação destas transfusões. O lactato sérico arterial e a SvO2 são pouco utilizados para se indicar a transfusão de glóbulos vermelhos. A maioria dos pacientes transfundidos recebe algum procedimento terapêutico e, em muitos casos, são realizadas transfusões em pacientes que apresentam DMOS. / Anemia is the primary cause of red blood cell transfusions and it is especially prevalent and even expected in critical care settings. The benefits of red blood cell transfusions include increase in tissue oxygenation, treatment of anemia and hypovolemia. There are substantial variations in transfusion practices and the optimal transfusion practice for various types of critically ill patients with anemia has not been established. Objectives To describe the clinical, hematological and therapeutic characteristics of children who received red blood cell transfusion in a Pediatric Intensive Care Unit (PICU). Methods Retrospective observational study, performed in the PICU of ?Instituto da Criança?, Medicine School of University of São Paulo, in 2004, with one hundred children who received red blood cell transfusion. We described the number of patients included in the study, age, reason for admission to the PICU, presence of baseline illness and multiple organ system failure (MODS), use of oxygen central venous saturation, arterial lactate, hemoglobin concentration (Hb), hematocrit (HT) and therapeutics procedures (mechanical ventilation, use of vasoactive drugs and renal replacement methods). Results The red blood cell transfusion occurred in 50% of the patients. Median age of patients was 18 months. The most common reason for admission was respiratory failure, in 35% of the cases. Baseline illness was present in 84% of the cases and MODS in 47% of the cases. Hb and HT were collected in all the cases and oxygen central venous saturation and arterial lactate were collected in 24% of the cases. The median pre-transfusion hemoglobin concentration was 7.8 g/dl. The patients were receiveing therapeutics procedures in 82% of the cases. Conclusions Red blood cell transfusions are performed in children of all ages. The hemoglobin concentration and hematocrit are the main reason to indicate the red blood cell transfusion. Arterial lactate and oxygen central venous saturation are rarely utilized. The majority of patients receive therapeutic procedures and, in many cases, patients with MODS are given red blood cell transfusions.

This study was a follow-up of the Christiansen study which was completed in 1967. Dr. Christiansen's 26 subjects ranged in age from 33 to 60 years. Ten were designated as controls and the other 16 were placed on a high polyunsaturated fatty acid (PUFA) diet for a period of 26 weeks . The purpose of doing a follow-up was to determine what effect the study had on present dietary patterns, serum lipid levels and rate of erythrocyte hemolysis. Eighteen of the original subjects participated in this study. Of these 18, nine were from the control group and nine were from the experimental group. There were nine women and nine men. The serum cholesterol and triglyceride levels, rate of erythrocyte hemolysis and blood pressure reading were determined. General health status and dietary pattern were determined through the use of a questionnaire. The results of the questionnaire indicate that the experimental diet of the original study had influenced the present diet of the study's subjects. The use of vegetable oils was increased and the consumption of eggs and whole milk was decreased. The study had little effect on the consumption of beef, pork, fish and chicken. The rate of erythrocyte hemolysis was greater for the control group (non-instructed) than for the experimental group (instructed). The mean values were 12. 65 and 9. 49 percent, respectively. The results indicate that there was no depletion of tocopherol levels due to continued use of PUFA. Serum triglyceride levels varied from 60 to 72 mg percent. Mean values for men were slightly higher than for the women. The means for the instructed and non-instructed groups were almost the same (6 7. 2 and 6 7. 0 mg percent, respectively). The cholesterol values ranged from 139 to 252 mg percent. The mean values were close to those at the end of the previous study (192 and 188 mg percent, respectively). There was no correlation between cholesterol values and the rate of erythrocyte hemolysis or triglyceride values.

Babesia microti is a tickborne hemoprotozoan parasite that causes the disease babesiosis in humans. Babesia microti Apical Membrane Antigen-1 (AMA-1) is a micronemal protein suspected to play a role in erythrocyte invasion. To investigate interaction between AMA-1 and the host cell, the ectodomain region of the B. microti ama-1 gene was cloned into an expression vector, expressed as a histidine-tagged fusion protein, and used to probe red blood cell membrane proteins in far Western blot assays. The B. microti ama-1 ectodomain, which excludes the signal peptide and the transmembrane region of the open reading frame, was amplified from a cloned gene sequence. The AMA-1 ectodomain is a membrane bound polypeptide that extends into the extracellular space and is most likely to interact or initiate interaction with the host red blood cell surface receptor(s). The amplicon was ligated into a protein expression vector to produce a 58.1 kDa recombinant His-tagged fusion protein, which was confirmed by Western blot analysis. The recombinant B. microti AMA-1 fusion protein was enriched on nickel affinity columns and then used to probe mouse, human and horse red blood cell membrane proteins in far Western blot assays. Babesia microti AMA-1 consistently reacted strongly with a protein migrating at 49 kDa. A similar reaction occurred between the B. microti AMA-1 and horse red blood cell membrane proteins, suggesting that similar interacting proteins of this size are shared by red blood cells from the three species. The B. microti AMA-1 may bind to red blood cell membrane sialic-acid groups, as shown for other Babesia spp. This may explain the signal at the 49 kDa position observed between B. microti AMA-1 and red blood cell membrane proteins from three different species. Further studies may determine if the binding epitopes of the red blood cell binding partner at this position vary and contribute to the specificity of each parasite AMA-1 for their respective host cells.

Anion exchanger 1 (AE1) is a red cell membrane glycoprotein that associates with cytoskeletal protein 4.2 in a complex bridging the cell membrane to the cytoskeleton. Disruption of this linkage results in unstable erythrocytes and hereditary spherocytosis (HS). Three HS mutations (E40K, G130R and P327R) in the cytoplasmic domain of AE1 (cdAE1) result in a decreased level of protein 4.2 in the red cell yet maintain normal amounts of AE1. Biophysical analyses showed the HS mutations had little effect on the structure and conformational stability of the isolated domain. However, the conformation of the cytoplasmic domain of the kidney anion exchanger, lacking the first 65 amino acids including a central -strand, was thermally destabilized relative to cdAE1 and had a more open structure. In transfected human embryonic kidney (HEK)-293 cells the HS mutants had similar expression levels as wild-type AE1, and protein 4.2 expression level was not dependent on the presence of AE1. Protein 4.2 localized to the plasma membrane with wild-type AE1, the HS mutants of AE1, the membrane domain of AE1 and kidney AE1, and to the ER with Southeast Asian ovalocytosis AE1. A fatty acylation mutant of protein 4.2, G2A/C173A, could not localize to the plasma membrane in the absence of AE1. Subcellular fractionation showed wild-type and G2A/C173A protein 4.2 were mostly associated with the cytoskeleton. Co-immunoprecipitation and Ni-NTA pull-down assays revealed impaired binding of protein 4.2 to HS mutants compared to AE1, while the membrane domain of AE1 was unable to bind protein 4.2. These studies show that HS mutations in cdAE1 cause impaired binding of protein 4.2, without causing gross structural changes in the domain. The mutations change the binding surface on cdAE1 by the introduction of positive charges into an otherwise acidic domain. This binding impairment may render protein 4.2 more susceptible to degradation or loss during red cell development.

Anion exchanger 1 (AE1) is a red cell membrane glycoprotein that associates with cytoskeletal protein 4.2 in a complex bridging the cell membrane to the cytoskeleton. Disruption of this linkage results in unstable erythrocytes and hereditary spherocytosis (HS). Three HS mutations (E40K, G130R and P327R) in the cytoplasmic domain of AE1 (cdAE1) result in a decreased level of protein 4.2 in the red cell yet maintain normal amounts of AE1. Biophysical analyses showed the HS mutations had little effect on the structure and conformational stability of the isolated domain. However, the conformation of the cytoplasmic domain of the kidney anion exchanger, lacking the first 65 amino acids including a central -strand, was thermally destabilized relative to cdAE1 and had a more open structure. In transfected human embryonic kidney (HEK)-293 cells the HS mutants had similar expression levels as wild-type AE1, and protein 4.2 expression level was not dependent on the presence of AE1. Protein 4.2 localized to the plasma membrane with wild-type AE1, the HS mutants of AE1, the membrane domain of AE1 and kidney AE1, and to the ER with Southeast Asian ovalocytosis AE1. A fatty acylation mutant of protein 4.2, G2A/C173A, could not localize to the plasma membrane in the absence of AE1. Subcellular fractionation showed wild-type and G2A/C173A protein 4.2 were mostly associated with the cytoskeleton. Co-immunoprecipitation and Ni-NTA pull-down assays revealed impaired binding of protein 4.2 to HS mutants compared to AE1, while the membrane domain of AE1 was unable to bind protein 4.2. These studies show that HS mutations in cdAE1 cause impaired binding of protein 4.2, without causing gross structural changes in the domain. The mutations change the binding surface on cdAE1 by the introduction of positive charges into an otherwise acidic domain. This binding impairment may render protein 4.2 more susceptible to degradation or loss during red cell development.