Red Blood Cell Membrane Proteins Research Articles

Label-free proteomic analysis of red blood cell membrane fractions from abdominal aortic aneurysm patients.

To test whether red blood cell (RBC) membrane composition is modified in abdominal aortic aneurysms (AAA) patients. RBC membrane extracts from AAA patients (aortic diameter >3 cm, n = 7) and control subjects (n = 4) were analyzed by a label-free quantitative MS-based strategy, using spectral count data. Additional validation was performed by western-blot. Data analysis based on spectral count from MS/MS-based experiments provided us a signature of 39 proteins differentially expressed in RBC membranes between AAA and controls (changes equal/over 1.515-fold; p-values equal/lower 0.05). MS data revealed altered levels of structural membrane proteins (e.g. spectrins and ankyrin), components of the degradation machinery (proteasome subunits), and oxidative stress related proteins (e.g. catalase and peroxiredoxin-2) among others. Decreased catalase and peroxiredoxin-2 expression in RBC membrane of AAA patients compared to controls were further validated by Western blot, confirming the proteomic results. RBCs membrane protein composition is altered in AAA patients, which could be involved in the pathological role of RBCs in aortic tissue and become potential targets to prevent AAA progression.

Effect of Gouania longipetala (Hemsl.) methanolic leaf extract on the kidney and hematology of alloxan-induced hyperglycemic Wistar rats

The kidney is involved in glucose regulation. Disruption in blood parameters in hyperglycemia can lead to anemia due to the increased non-enzymatic glycosylation of red blood cell membrane proteins. The methanol leaf extract of Gouania longipetala was investigated for its effect on the kidney and hematological parameters of hyperglycemic Wistar rats induced by a single intraperitoneal injection of alloxan monohydrate (160 mg/kg). Treatment was done orally using 50, 100 and 150 mg/kg of G. longipetala extract (GLE) with glibenclamide (2 mg/kg) as standard reference drug for 21 days. At the end of the treatment, blood collected from the retro-orbital plexus of the rats’ eyes was used for evaluation of serum urea and creatinine and hematological determinations. Histopathological evaluation of the kidney was also done using standard procedures. GLE and glibenclamide caused a dose-dependent and significant (p 0.05) differences in the mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration of the extract-treated groups and the negative control rats. Histopathology of the kidney revealed no variations in the kidney of both treated and control rats. In conclusion, the results suggest that G. longipetala may be of help in diabetes-induced anemia and nephropathy associated with diabetes mellitus.

Does quercetin protect human red blood cell membranes against γ-irradiation?

ObjectivesRadioprotective potential of quercetin, a powerful free radical scavenger, was investigated in human red blood cells (RBCs) and in isolated RBC membranes exposed to γ-irradiation-induced oxidative stress.MethodsRBCs and RBC membrane suspensions were irradiated (50 Gy) in the presence of quercetin (2–50 µM). Oxidative damage of the membranes was analysed by protein carbonyl measurement (enzyme-linked immunosorbent assay). In RBCs, the concentration of glutathione (GSH) was determined. Lipid peroxidation in RBCs, and for comparison in plasma and peripheral lymphocytes, was quantified by the amount of thiobarbituric acid-reactive substances (TBARS). Radiation-induced damage of the RBC membrane integrity was evaluated by the degree of haemolysis.ResultsQuercetin (50 µM) brought back the level of carbonyls to normal in γ-irradiated RBC membrane proteins and inhibited radiation-induced lipid peroxidation in plasma and lymphocytes, by 75 and 96%, respectively. However, it moderately decreased reduced/oxidized glutathione (GSH/GSSG) ratio and significantly increased TBARS concentrations, by 60 and 28% in irradiated and non-irradiated RBCs, respectively. Haemolysis rate was much higher in RBCs irradiated in the presence of quercetin vs. non antioxidant.DiscussionIn non-cellular systems (RBC membranes or plasma) and in lymphocytes, quercetin shows antioxidative/radioprotective activity but in whole RBCs it acts as a pro-oxidant and a cytotoxic substance. The possible mechanisms of such action are discussed.

Glycophorin C ligation: another biochemical pathway in red blood cell senescence?

In this issue of TRANSFUSION two articles describe the effects on erythroid cells of antibody ligation to the red blood cell (RBC) membrane glycoprotein glycophorin C (GPC).1, 2 GPC is a monotopic high-abundance RBC membrane protein that forms a structural linkage to the membrane skeleton via the proteins 4.1R and p55. GPC derives from the GYPC gene, which additionally expresses GPC in a truncated form, glycophorin D (GPD). Glycophorins are extensively sialyated and contribute significantly to the zeta potential (net negative charge) that prevents systemic aggregation of RBCs by mutual repulsion. RBCs lacking both GPC and GPD are known as the Leach phenotype and have irregular ellipsoid shape. GPC and GPD express the 11 Gerbich blood group antigens,3 and three Gerbich-negative phenotypes are known, Gerbich (Ge:–2,–3,4), Yus (Ge:–2,3,4), and the aforementioned Leach phenotype (Ge:–2,–3,–4). Anti-Gerbich (notably anti-Ge3) has previously been noted as a cause of hemolytic disease of the fetus and newborn (HDFN; and further described here by Pate and colleagues2), so knowledge regarding the immune destruction of the fetal RBCs is of relevance to the clinical management of these cases. The study of Wang and coworkers1 reveals that ligation using anti-GPC induces cell death in the erythroleukemic cell line K562 and erythroid progenitors derived from CD34+ umbilical cord blood stem cells. This process remains only partially characterized biochemically, but involves the exposure of phosphatidylserine (PS) at the extracellular leaflet of the membrane (it is normally polarized to the intracellular face of the membrane). These findings confirm our earlier study,4 which interestingly also found that Gerbich and Yus phenotype cells lacked this pathway, despite binding anti-GPC on their surface. Thus, anti-Ge3 causing the HDFN described in previous studies, most likely induces PS exposure on fetal RBCs and induces their death and is clearly a different mechanism to that induced by anti-D which involves monocyte binding to the Fc region of RBC bound antibody. The GPC pathway induced by anti-Ge3 (and other anti-GPC) most likely is mimicking binding to the protein by an as yet unknown ligand(s), possibly on macrophages or other white blood cells. The mechanism of PS exposure induced by GPC ligation is also intriguing; the previously mentioned GPC-p4.1R-p55 interaction clearly must trigger the PS exposure in some manner. Indeed, p4.1R is known to bind PS and is regulated by calmodulin.5 It is therefore important that this pathway is unraveled as it may play a fundamental part in RBC turnover. It is somewhat puzzling that despite its huge physiologic significance, mechanisms of RBC turnover (dubbed eryptosis) remain relatively poorly characterized; this deficiency no doubt will be rectified based on the identification of the key RBC membrane proteins that appear to be involved. GPC is now among the candidates. The author is a member of the Transfusion Medicine Advisory Board (TMAB) for Grifols SA.

Cell Surface Membrane Proteins as Personalized Biomarkers: Where we Stand and Where we are Headed

Personalized medicine requires the development of a wide array of biomarker diagnostic assays, reflecting individual variations and thus allowing tailored therapeutic interventions. Membrane proteins comprise approximately 30% of total human proteins; they play a key role in various physiological functions and pathological conditions, although, currently, only a limited number of membrane proteins are applied as biomarkers. In many normal tissues, cell surface membrane proteins are not easily accessible for diagnostic sampling, and tumor-derived membrane preparations - while serving as potential tumor biomarkers - may not reflect physiological protein expression. In addition to post-translational modifications, which may include glycosylation, phosphorylation and lipid modifications, the trafficking of membrane proteins is also regulated. Moreover, a tight cellular quality control monitors membrane protein maturation, and continuous removal and reinsertion, involving special signaling systems, occurs in many cases. However, cell surface membrane proteins already serve as valuable prognostic and predicative biomarkers, for example, in hematological and immunological diseases, by the determination of the cluster of differentiation markers. In this review, we demonstrate the relevance of cell surface membrane biomarkers in various diseases and call attention to the potential application of red blood cell (erythrocyte) membrane proteins in this regard. Surprisingly, red blood cells express hundreds of membrane proteins, which seem to reflect a general genetic and regulatory background, and may serve as relatively stable and easily accessible personalized membrane biomarkers. Quantitative membrane protein detection in red blood cells by flow cytometry may bring a breakthrough in this regard.

Effects of a TASER® conducted energy weapon on the circulating red-blood-cell population and other factors in Sus scrofa

In previous studies hematocrit has been consistently increased in an anesthetized animal model after exposures to TASER(®) conducted energy weapons (CEWs). In the present study we analyzed changes in blood cell counts and red blood cell membrane proteins following two 30-s applications of a TASER C2 device (which is designed for civilian use). Hematocrit increased significantly from 33.2 ± 2.4 (mean ± SD) to 42.8 ± 4.6 % immediately after CEW exposure of eleven pigs (Sus scrofa). Red blood cell count increased significantly from 6.10 ± 0.55 × 10(12)/L to 7.45 ± 0.94 × 10(12)/L, and mean corpuscular volume increased significantly from 54.5 ± 2.4 fl to 57.8 ± 2.6 fl. Mean corpuscular hemoglobin concentration decreased significantly from 20.5 ± 0.7 to 18.5 ± 0.6 mM. Thirty protein spots (from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, selected for detailed comparison) exhibited greater densities 30-min post-exposure compared with pre-exposure values. A greater number of echinocytes were observed following CEW exposure. On the basis of these results it appears that, during the strong muscle contractions produced by TASER CEWs, a specific population of red blood cells (RBCs) may be released from the spleen or other reservoirs within the body. The total time of CEW exposure in the present study was relatively long compared with exposures in common law-enforcement scenarios. Despite statistically significant changes in red blood cell counts (and other measures directly related to RBCs), the alterations were short-lived. The transient nature of the changes would be likely to counteract any potentially detrimental effects.

Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein phosphorylation

BackgroundIn sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs). ERK1/2 is involved in activation of ICAM-4-mediated sickle RBC adhesion to the endothelium. However, other effects of the ERK1/2 activation in sickle RBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are unknown.ResultsTo further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Sickle RBC treatment with U0126 decreased thirty-six phosphopeptides from twenty-one phosphoproteins involved in regulation of not only RBC shape, flexibility, cell morphology maintenance and adhesion, but also glucose and glutamate transport, cAMP production, degradation of misfolded proteins and receptor ubiquitination. Glycophorin A was the most affected protein in sickle RBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreases in both anion transport by band 3 and band 3 trafficking. The abundance of twelve of the thirty-six phosphopeptides were subsequently increased in normal RBCs co-incubated with recombinant ERK2 and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2.ConclusionsThese findings expand upon the current model for the involvement of ERK1/2 signaling in RBCs. These findings also identify additional protein targets of this pathway other than the RBC adhesion molecule ICAM-4 and enhance the understanding of the mechanism of small molecule inhibitors of MEK/1/2/ERK1/2, which could be effective in ameliorating RBC hemorheology and adhesion, the hallmarks of SCD.

A new paradigm in the diagnosis of hereditary hemolytic anemia

A new paradigm in the diagnosis of hereditary hemolytic anemia

A Case of Hereditary Spherocytosis Coexisting with Gilbert's Syndrome

We recently encountered a case of hereditary spherocytosis coexisting with Gilbert's syndrome. Patient was initially diagnosed with Gilbert's syndrome and observed, but other findings suggestive of concurrent hemolysis, such as splenomegaly and gallstones were noted during the follow-up period. Therefore, further evaluations, including a peripheral blood smear, osmotic fragility test, autohemolysis test, and red blood cell membrane protein test were performed, and coexisting hereditary spherocytosis was diagnosed. Genotyping of the conjugation enzyme uridine diphosphate-glucuronosyltransferase was used to confirm Gilbert's syndrome. Because of the high prevalence rates and similar symptoms of these 2 diseases, hereditary spherocytosis can be masked in patients with Gilbert's syndrome. In review of a case and other article, the possibility of the coexistence of these 2 diseases should be considered, especially in patients with unconjugated hyperbilirubinemia who also have splenomegaly and gallstones.

Hereditary Hemolytic Anemia in Korea From 1997 to 2011: A Study by the Korean Hereditary Hemolytic Anemia Working Party of the Korean Society of Hematology

AbstractAbstract 5157 Background:With the development of diagnostic technique, an accurate diagnosis of hereditary hemolytic anemia (HHA)- red blood cell (RBC) membranopathy, hemoglobinopathy, RBC enzymopahty – have been made. Therefore, we surveyed the prevalence and characteristics of patients diagnosed as HHA during recent five years in Korea. Methods:Through the use of questionnaires, information on the clinical and laboratory findings of HHA diagnosed from 2007 to 2011 in Korea was collected. The globin gene analysis (direct sequencing) and RBC enzyme analysis was performed at the representative laboratories. A total of 203 cases were collected in this study by the Korean Hereditary Hemolytic Anemia Working Party of the Korean Society of Hematology. Results:Patients number of RBC membranopathy, hemoglobinopathy, and RBC enzymopahty was 125, 47, and 31, respectively. Percentage of patients with dominant family history was 57% in patients with hereditary spherocytosis (n=116) and dominant symptoms were anemia, jaundice, splenomegaly and gallstones. Osmotic fragility test and flow cytometric method for detection of RBC membrane defect were performed about 60% of patients. RBC membrane protein analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed on 59 patients. Of the 47 cases of hemoglobinopathies, 36 cases (77%) were β-thalassemia minor, 10 cases (21%) were α-thalassemia minor and one case (2%) was unstable Hb, Hb M-Saskatoon (beta 64 His-→Tyr). Median age at diagnosis was 7 years (range: 6 months–58 years). Eleven of 47 cases (23%) had family history of HHA. As all thalassemia patients were thalassemia minor, they presented with mild jaundice or pallor. Of the 31 patients were diagnosed as RBC membranopathy, pyruvate kinase deficiency was 3 cases and glucose-6 phosphate dehydrogenase deficiency was 2, and other various forms were reported. Conclusions:We could confirm that accurate diagnosis has been made in more patients using elegant diagnostic technique. However, more defined diagnostic approaches were needed in this rare disease and further systematic supporting systems for patients and their families were warranted in public health aspect. Disclosures:No relevant conflicts of interest to declare.

Interaction of Different Extracts of Primula heterochroma Stapf. with Red Blood Cell Membrane Lipids and Proteins: Antioxidant and Antihemolytic Effects

ABSTRACTIn recent years, considerable attention has been paid to plants as potent natural drugs for their ameliorative roles against free-radical-mediated oxidative stress. Therefore, their interactions with cell membrane lipids and proteins, which generally serve as primary targets of lipid peroxidation, are of much interest. In the current investigation, in vitro and ex vivo studies are performed in order to estimate possible effects of different extracts of Primula heterochroma Stapf. on red blood cell membranes of rat erythrocytes using colorimetric methods. The results indicate that binding of the extracts to lipids and proteins of red blood cell membranes both significantly inhibits lipid peroxidation, and also increases red blood cell integrity against hemolysis. Moreover, a polyphenol extract, in particular, demonstrates notable antihemolytic activity in hydrogen peroxide-induced hemolysis model (IC50 = 199.49 ± 9.1 μg ml−1).

Comprehensive identification of erythrocyte membrane protein deficiency by 2D gel electrophoresis based proteomic analysis in hereditary elliptocytosis and spherocytosis

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1DE) may reveal qualitative or quantitative defects in red blood cell (RBC) membrane proteins, two-dimensional gel electrophoresis (2DE) can be used for determination of the protein changes caused by the disease process in a relatively high-throughput manner, because it permits an analysis of thousands of modified or unmodified proteins simultaneously. The principal aim of this study was to compare hereditary elliptocytosis (HE), hereditary spherocytosis (HS), and control RBC membrane protein profiles and identify proteins as a clinical marker by the sensitive methods. RBC membrane proteins of HE, HS, and control groups were compared by 2DE and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis was obtained using peptide mass fingerprint for protein identification. Twenty proteins were identified with peptide mass fingerprint analysis and different expression levels were found in HE, HS, and controls for some proteins that includes three biomarker proteins (ankyrin, spectrin, band 3) that may have prognostic or predictive importance. 2DE of RBC proteins is a potentially valuable method for studying heritable disorders such as HE and HS. By identifying a deficiency in membrane proteins associated with the RBC cytoskeleton, the diagnosis of HE and HS can be confirmed.

Proteomic analysis shows the upregulation of erythrocyte dematin in zinc-restricted human subjects

Although the importance of adequate zinc intake has been known for decades, the estimated global prevalence of zinc deficiency remains high. This substantiates the need for a specific and sensitive status assessment tool. The objective was to evaluate erythrocyte zinc transporters as candidate molecules with the potential of being a biomarker of dietary zinc status in humans. A 24-d observational study with acclimation (7 d, 10.4 mg Zn/d), zinc-depletion (10 d, 0.3 mg Zn/d), and zinc-repletion (7 d, 29.5 mg Zn/d) phases was conducted in healthy men (n = 9). Proteomic approaches including Western blot analyses and tandem mass spectrometry were implemented to identify the zinc responsiveness of selected red blood cell membrane proteins. Zinc transporter 1 (ZnT1) and Zrt/Irt-like proteins ZIP8 and ZIP10 were detected in human erythrocyte membranes. No effects of short-term dietary zinc depletion were observed on the amounts of these proteins. However, changes in a cytoskeletal protein, dematin, by zinc depletion were identified through the nonspecific signals produced by an anti-ZIP8 antibody. This response was further validated by a dematin-specific antibody and with erythrocytes collected from mice fed a zinc-deficient diet. The presence of ZnT1, ZIP8, and ZIP10 in human red blood cells implicates their role in the regulation of cellular zinc metabolism in the human erythroid system. The zinc responsiveness of membrane dematin suggests its capability to serve as a biomarker for dietary zinc depletion and its involvement in impaired erythroid membrane fragility by zinc restriction. This trial was registered at clinicaltrials.gov as NCT01221129.

Computational Identification of Phospho-Tyrosine Sub-Networks Related to Acanthocyte Generation in Neuroacanthocytosis

Acanthocytes, abnormal thorny red blood cells (RBC), are one of the biological hallmarks of neuroacanthocytosis syndromes (NA), a group of rare hereditary neurodegenerative disorders. Since RBCs are easily accessible, the study of acanthocytes in NA may provide insights into potential mechanisms of neurodegeneration. Previous studies have shown that changes in RBC membrane protein phosphorylation state affect RBC membrane mechanical stability and morphology. Here, we coupled tyrosine-phosphoproteomic analysis to topological network analysis. We aimed to predict signaling sub-networks possibly involved in the generation of acanthocytes in patients affected by the two core NA disorders, namely McLeod syndrome (MLS, XK-related, Xk protein) and chorea-acanthocytosis (ChAc, VPS13A-related, chorein protein). The experimentally determined phosphoproteomic data-sets allowed us to relate the subsequent network analysis to the pathogenetic background. To reduce the network complexity, we combined several algorithms of topological network analysis including cluster determination by shortest path analysis, protein categorization based on centrality indexes, along with annotation-based node filtering. We first identified XK- and VPS13A-related protein-protein interaction networks by identifying all the interactomic shortest paths linking Xk and chorein to the corresponding set of proteins whose tyrosine phosphorylation was altered in patients. These networks include the most likely paths of functional influence of Xk and chorein on phosphorylated proteins. We further refined the analysis by extracting restricted sets of highly interacting signaling proteins representing a common molecular background bridging the generation of acanthocytes in MLS and ChAc. The final analysis pointed to a novel, very restricted, signaling module of 14 highly interconnected kinases, whose alteration is possibly involved in generation of acanthocytes in MLS and ChAc.

Modifications in Erythrocyte Membrane Protein Content Are Not Responsible for the Alterations in Rheology Seen in Sepsis

Red blood cell (RBC) rheology is altered in sepsis and may contribute to the microcirculatory alterations in these patients, but the mechanisms of these changes are not well defined. An increase in the RBC protein band 3/α-spectrin ratio has been observed in a mouse model of septic shock, suggesting a possible alteration in the RBC membrane integral/peripheral protein ratio. This protein modification could contribute to the alterations in RBC rheology observed in sepsis. As there are interspecies differences in membrane composition, these observations need confirmation in humans. We studied RBCs from healthy volunteers (n = 10) and from patients with (n = 15) and without (n = 9) sepsis within 24 h of intensive care unit admission and also on day 3 for the septic patients. Exclusion criteria were recent RBC transfusion, hematologic diseases, cirrhosis, and diabetes mellitus. Procedures included screening for alterations in RBC membrane proteins using cryohemolysis and separation of RBC membrane and skeletal proteins by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The hemogram, including reticulocyte count, was similar between nonseptic and septic patients on day 1. The majority of RBC membrane protein ratios, including band 3/spectrin, were more elevated in critically ill patients (nonseptic and septic) than in volunteers, but RBC membrane skeletal protein content was similar in septic and nonseptic patients. There were no significant differences in cryohemolysis results among groups. Alterations in RBC rheology in sepsis are therefore mainly due to alterations in membrane compounds other than skeletal proteins, like carbohydrates, such as sialic acid and/or lipids.

Apolipoprotein J/clusterin in human erythrocytes is involved in the molecular process of defected material disposal during vesiculation.

BackgroundWe have showed that secretory Apolipoprotein J/Clusterin (sCLU) is down-regulated in senescent, stressed or diseased red blood cells (RBCs). It was hypothesized that sCLU loss relates to RBCs vesiculation, a mechanism that removes erythrocyte membrane patches containing defective or potentially harmful components.Methodology/Principal FindingsTo investigate this issue we employed a combination of biochemical and microscopical approaches in freshly prepared RBCs or RBCs stored under standard blood bank conditions, an in vitro model system of cellular aging. We found that sCLU is effectively exocytosed in vivo during membrane vesiculation of freshly prepared RBCs. In support, the RBCs' sCLU content was progressively reduced during RBCs ex vivo maturation and senescence under cold storage due to its selective exocytosis in membrane vesicles. A range of typical vesicular components, also involved in RBCs senescence, like Band 3, CD59, hemoglobin and carbonylated membrane proteins were found to physically interact with sCLU.Conclusions/SignificanceThe maturation of RBCs is associated with a progressive loss of sCLU. We propose that sCLU is functionally involved in the disposal of oxidized/defected material through RBCs vesiculation. This process most probably takes place through sCLU interaction with RBCs membrane proteins that are implicit vesicular components. Therefore, sCLU represents a pro-survival factor acting for the postponement of the untimely clearance of RBCs.

Elastase release during the hemodialysis procedure seems to induce changes in red blood cell membrane proteins

To the Editor: Elastase is a serine proteinase, expressed mainly by neutrophils, and also by monocytes and mast cells. It is a component of the primary azurophil granules of the neutrophils. Within the cell, it is involved in the degradation of microorganisms that are phagocytosed, and, outside the cell, it may degrade local extracellular matrix proteins, favoring neutrophil migration into or through tissues. 1,2 Recently, we showed that the red blood cell (RBC) membrane protein composition is altered in hemodialysis (HD) patients, particularly in nonresponders to recombinant human erythropoietin (rhEPO) therapy. 3,4 We also showed that the HD procedure induces neutrophil activation. 5,6 Therefore, we hypothesized that the RBC changes could be due to the release of elastase by neutrophils during HD. Our aim was to study the effect of elastase in RBC membrane protein composition from healthy controls and from HD patients under rhEPO therapy (responders and non-responders) to strengthen this hypothesis. We performed in vitro assays using RBC from 18 HD patients (10 responders and 8 nonresponders) and of 8 healthy controls. Classification of the patients, as responders or non-responders, was performed in accordance with the European Best Practice Guidelines, which defines resistance to rhEPO as a failure to achieve target Hb levels (between 11 and 12g/dL) with maintained doses of rhEPO higher than 300IU/kg/wk of epoietin or 1.5mg/kg/wk of darbopoietin-a. The two groups of patients were matched for age, gender, weight, body mass index, mean time under HD, urea reduction ratio, urea Ktv, and parathyroid hormone serum levels. Hemodialysis patients (10 males, 8 females; mean age 62.1 15.7 years) were under therapeutic HD 3 times per week, for 3‐5 hours, for a median period of time of 35 months. All patients used the highflux polysulfone FX-class dialysers of Fresenius (Fresenius Medical Care, Bad Homburg, Germany). The causes of renal failure in patient’s population were as follows: diabetic nephropathy (n=5), chronic glomerulonephritis (n=3), polycystic kidney disease (n=2), hypertensive nephrosclerosis (n=2), obstructive nephropathy (n=1), and chronic renal failure of uncertain etiology (n=5). Patients with autoimmune disease, malignancy, hematological disorders, and acute or chronic infection were excluded. All patients gave their informed consent to participate in this study. In HD patients, this assay used RBCs collected before and immediately after the HD procedure. Red blood cell suspensions (5 10 8 cell/mL; 10mL PBS, pH 7.4) were incubated for 3 hours at 371C, under the following assay conditions: without elastase and with 0.03, 0.1, and 0.5mg/mL of elastase. After incubation, the relative amount of each major RBC membrane protein was evaluated by densitometry. 3 We found no significant differences in the membrane protein composition of the RBCs from healthy controls and from responders and nonresponders after the HD procedure, when incubated without and with different elastase concentrations. However, the RBCs from responders and nonresponders HD patients, collected before the HD procedure, showed some susceptibility to elastase; the RBCs from responders incubated with 0.5mg/mL of elastase showed a significant decrease in ankyrin [7.0 (6.5‐7.5%) vs. 6.0 (5.9‐6.5%), P=0.024] and trends toward a decrease in spectrin [25.6 (25.1‐ 26.9%) vs. 24.7 (24.4‐25.6%), P=0.073) and an increase in band 3 [36.6 (34.8‐37.6%) vs. 39.1 (36.9‐39.4%), P=0.077) as compared with RBCs incubated without elastase. Similar changes were found for the RBCs incubated with 0.1mg/mL of elastase. In nonresponder patients, the RBCs incubated with 0.1 and 0.5mg/mL of elastase showed a significant decrease in spectrin [25.5 (24.9‐25.9%) and 25.3 (24.8‐26.2%), respectively, vs.

Phosphorylation-Dependent Perturbations of the 4.1R-Associated Multiprotein Complex of the Erythrocyte Membrane

The bulk of the red blood cell membrane proteins are partitioned between two multiprotein complexes, one associated with ankyrin R and the other with protein 4.1R. Here we examine the effect of phosphorylation of 4.1R on its interactions with its partners in the membrane. We show that activation of protein kinase C in the intact cell leads to phosphorylation of 4.1R at two sites, serine 312 and serine 331. This renders the 4.1R-associated transmembrane proteins GPC, Duffy, XK, and Kell readily extractable by nonionic detergent with no effect on the retention of band 3 and Rh, both of which also interact with 4.1R. In solution, phosphorlyation at either serine suppresses the capacity of 4.1R to bind to the cytoplasmic domains of GPC, Duffy, and XK. Phosphorylation also exerts an effect on the stability in situ of the ternary spectrin-actin-4.1R complex, which characterizes the junctions of the membrane skeletal network, as measured by the enhanced competitive entry of a β-spectrin peptide possessing both actin- and 4.1R-binding sites. Thus, phosphorylation weakens the affinity of 4.1R for β-spectrin. The two 4.1R phosphorylation sites lie in a domain flanked in the sequence by the spectrin- and actin-binding domain and a domain containing the binding sites for transmembrane proteins. It thus appears that phosphorylation of a regulatory domain in 4.1R results in structural changes transmitted to the functional interaction centers of the protein. We consider possible implications of our findings for the altered membrane function of normal reticulocytes and sickle red cells.

Compound heterozygosity for two novel mutations in the erythrocyte protein 4.2 gene causing spherocytosis in a Caucasian patient

Compound heterozygosity for two novel mutations in the erythrocyte protein 4.2 gene causing spherocytosis in a Caucasian patient

Peroxiredoxin‐2 as a candidate biomarker to test oxidative stress levels of stored red blood cells under blood bank conditions

Several researches on aging red blood cells (RBCs)--performed both in vivo and under blood bank conditions--revealed that RBC membrane proteins undergo a number of irreversible alterations, mainly due to oxidative stress. The individuation of proteins to be used as indicators of irreversible RBC injury and to be proposed as candidate biomarkers of oxidative damage or aging status during blood storage is therefore of great interest. Based on this purpose we performed proteomic analysis of the membranes of RBCs during various storage periods under blood bank conditions. Changes in protein composition of RBC membranes were monitored as a function of the storage period by means of polyacrylamide gel electrophoresis coupled with immunoblotting and mass spectrometry analyses. During storage, a progressive linkage of typical cytosolic proteins to the membrane was detected, including both antioxidant and metabolic enzymes (such as catalase, peroxiredoxin-2 [Prx2], and 2,3-bisphosphoglycerate-mutase), as well as nonreducible cross-linkings of probably oxidized or denatured hemoglobin. This phenomenon was unequivocally related to oxidative stress, since storage of RBCs under anaerobic conditions showed a suppression of these protein recruitments to the membrane. The detailed analysis of these protein associations to the membrane of aged RBCs allowed Prx2 to be suggested as a potential RBC oxidative stress marker for the sake of developing new approaches in quality assurance of blood components.