Band 3 Phosphorylation Induces Irreversible Alteration of Stored Red Blood Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5029-5029
Author(s):  
Slim Azouzi ◽  
Yves Colin Aronovicz ◽  
Catia Pereira ◽  
Marc Romana ◽  
Thierry Peyrard ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Cluster Formation ◽  
Immunoblot Analysis ◽  
Progressive Increase ◽  
Band 3 ◽  
Igg Antibody ◽  
Functional Studies ◽  
Previous Demonstration

Abstract Introduction: Storage of red blood cells (RBCs) for transfusion purposes is accompanied by a number of morphological and biochemical changes (storage lesions) that reduce post-transfusion survival/efficacy and increase risk for adverse reactions in the recipients. The clearance of altered and older RBCs from circulation is triggered by the clustering of Band 3, an aggregate state that is recognized by a low-affinity naturally occurring IgG antibody (Nab). Considering the key role of Band 3 in the maintenance of RBC structure and survival, elucidation of functional and structural modifications of Band 3 during storage should lead to new approaches aiming to improve RBC storage and post-transfusion viability. Results: Immunoblot analysis of RBC membrane proteins using an anti-phosphotyrosine antibody showed a progressive increase in the phosphorylation status of Band 3 during RBC storage (Figure 1). In addition, using the quenching fluorescence of eosin-5-maleimide (EMA), we showed an increase of the mobile fraction of Band 3. These findings are consistent with previous demonstration that tyrosine phosphorylation of Band 3 reduces its affinity for ankyrin, leading to the release of the immobile fraction of Band 3 from the skeleton complex, and enhancement of the lateral mobility of Band 3 into the lipid bilayer. Immunoblot experiments using an antibody that specifically recognizes the clustered form of Band 3 revealed an increase of Band 3 cluster formation from the 28th day of storage. We also showed that the release of microparticles (MPs) that occurs during RBC aging increases from the 28th day of storage (Figure 2). Finally, stopped-flow-based functional studies showed a decrease of the anion exchanger activity of Band 3 from the 28th day of storage. Conclusion: Altogether, our results suggest that the 28th of storage represents a key moment for the molecular processes leading to irreversible lesions of RBCs and allow us to propose a new Band 3 phosphorylation/clustering-based mechanism of RBC aging. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Syk inhibitors interfere with erythrocyte membrane modification during P falciparum growth and suppress parasite egress

Blood ◽  
2017 ◽  
Vol 130 (8) ◽  
pp. 1031-1040 ◽  
Author(s):  
Antonella Pantaleo ◽  
Kristina R. Kesely ◽  
Maria Carmina Pau ◽  
Ioannis Tsamesidis ◽  
Evelin Schwarzer ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Tyrosine Phosphorylation ◽  
Erythrocyte Membrane ◽  
Blood Cells ◽  
Band 3 ◽  
Membrane Modification ◽  
Syk Kinase ◽  
Key Points ◽  
Parasite Egress

Key PointsInhibitors of human Syk kinase suppress parasite egress. Syk inhibitors prevent the tyrosine phosphorylation of band 3 in P falciparum parasitized red blood cells, reducing the release of microparticles.

K562 cell anion exchange differs markedly from that of mature red blood cells

1983 ◽  
Vol 244 (1) ◽  
pp. C68-C74 ◽  
Author(s):  
F. Y. Law ◽  
R. Steinfeld ◽  
P. A. Knauf
Keyword(s):  
Blood Cell ◽  
Red Blood Cells ◽  
Red Blood Cell ◽  
Blood Cells ◽  
Chloride Transport ◽  
K562 Cells ◽  
Band 3 ◽  
Anion Transport ◽  
Leukemic Cells ◽  
Anion Transport System

Human K562 leukemic cells exhibit several erythroid properties, including synthesis and expression of the major red blood cell sialoglycoprotein, glycophorin. This has led us to ask if these cells express a functional anion transport system analogous to that which is associated with the other major erythrocyte glycoprotein, band 3. The chloride-36 exchange flux in K562 cells is less than 0.6% of that which would be expected in mature erythrocytes under similar conditions. Unlike red blood cells, K562 cells do not exhibit a high chloride-sulfate selectivity, and various agents that inhibit red blood cell chloride exchange are all much less effective in K562 cells. On the basis of these flux measurements, K562 cells probably contain less than 600 fully functional red blood cell-like band 3 molecules per cell, in contrast to about a million molecules in the mature red blood cell. The possible-existence of greatly altered band 3 molecules with a reduced turnover rate and/or a reduced affinity for chloride and for various inhibitors is unlikely but cannot be completely excluded. Anion transport was also measured in K562 cells that had been induced to increase hemoglobin synthesis by various chemical agents. Even under these conditions, chloride fluxes indicated no substantial increase in the number of functional anion transport sites or their chloride transport rate.

Semi-Automatic Red Blood Cells Counting in Microscopic Digital Images

2015 ◽  
Vol 73 (2) ◽  
Author(s):  
Naveed Abbas ◽  
Dzulkifli Mohamad ◽  
Abdul Hanan Abdullah
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Digital Images ◽  
Complete Blood Count ◽  
Cluster Formation ◽  
True Positive Rate ◽  
Average Error ◽  
Morphological Operations ◽  
True Negative ◽  
Data Set

The main purpose of this study is to employ the modern technologies and techniques to semi-automate the quantification process of the Red Blood Cells in Microscopic thin blood smear digital images.  The process needs to be more accurate, efficient and universal then the currently practiced methods. The study considers the process to be semi-automated for two reasons, i.e. due to the critical aspect life and due to the diverse nature of the Red Blood Cells in cluster formation. The Methodology of this study involved interactive simple cuts and morphological operations for splitting clusters of Red Blood Cells while counting is carried out through labeling matrix. The Red Blood Cells counting is part of the complete blood count test and is frequently suggested by the Physician to know the number of Red Blood Cells in the patient’s body. The proposed method considers for counting process of the Red Blood Cells first split the clusters and then count the Red Blood Cells. The proposed method achieved an overall True Positive Rate (TPR) of 0.997%, True Negative Rate (TNR) of 0.00265%, accuracy of 0.998% and average error rate of 0.001375% tested on 50 images, data set also on the same number of images linear correlation coefficient R2 is 0.997 between manual and semi-automatic counting of Red Blood Cells.

scholarly journals Role of band 3 in regulating metabolic flux of red blood cells

2009 ◽  
Vol 106 (44) ◽  
pp. 18515-18520 ◽  
Author(s):  
I. A. Lewis ◽  
M. E. Campanella ◽  
J. L. Markley ◽  
P. S. Low
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Metabolic Flux ◽  
Band 3

Abnormal Distribution of Erythroid Progenitors Populations in Mice Lacking p45 NF-E2.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1988-1988
Author(s):  
Jadwiga Gasiorek ◽  
Gregory Chevillard ◽  
Zaynab Nouhi ◽  
Volker Blank
Keyword(s):  
Bone Marrow ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Globin Genes ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Adult Mice ◽  
Deficient Mice

Abstract Abstract 1988 Poster Board I-1010 The NF-E2 transcription factor is a heterodimer composed of a large hematopoietic-specific subunit called p45 and widely expressed 18 to 20-kDa small Maf subunits. In MEL (mouse erythroleukemia) cells, a model of erythroid differentiatin, the absence of p45 is inhibiting chemically induced differentiation, including induction of globin genes. In vivo, p45 knockout mice were reported to show splenomegaly, severe thrompocytopenia and mild erythroid abnormalities. Most of the mice die shortly after birth due to haemorrhages. The animals that survive display increased bone, especially in bony sites of hematopoiesis. We confirmed that femurs of p45 deficient mice are filled with bone, thus limiting the space for cells. Hence, we observed a decrease in the number of hematopoietic cells in the bone marrow of 3 months old mice. In order to analyze erythroid progenitor populations we performed flow cytometry using the markers Ter119 and CD71. We found that p45 deficient mice have an increased proportion of early erythroid progenitors (proerythroblasts) and a decreased proportion of late stage differentiated red blood cells (orthochromatic erythroblasts and reticulocytes) in the spleen, when compared to wild-type mice. We showed that the liver of p45 knockout adult mice is also becoming a site of red blood cell production. The use of secondary sites, such as the spleen and liver, suggests stress erythropoiesis, likely compensating for the decreased production of red blood cells in bone marrow. In accordance with those observations, we observed about 2 fold increased levels of erythropoietin in the serum of p45 knockout mice.Overall, our data suggest that p45 NF-E2 is required for proper functioning of the erythroid compartment in vivo. Disclosures: No relevant conflicts of interest to declare.

Analyzing the Stepwise Developmental Pathway From ES/IPS Cells to Functional Mature Erythrocytes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2534-2534
Author(s):  
Akira Niwa ◽  
Tomoki Fukatsu ◽  
Katsutsugu Umeda ◽  
Itaru Kato ◽  
Hiromi Sakai ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Specific Antigen ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Oxygen Carriers ◽  
Developmental Pathway ◽  
Ips Cell

Abstract Abstract 2534 Poster Board II-511 Induced pluripotent stem (iPS) cells, reprogrammed somatic cells with embryonic stem (ES) cell–like characteristics, are generated by the introduction of combinations of specific transcription factors. Despite the controversy surrounding the gene manipulation, it is expected that iPS cells should contribute to regenerative medicine, disease investigation, drug screening, toxicology, and drug development in future. In the fields of hematology, iPS cells could become used as a new feasible source for transplantation therapy without immunological barrier and for the investigation of various kinds of hematological defects. Previous studies on ES / iPS cells have already demonstrated that they can develop into various lineages of hematopoietic cells including erythrocytes following the similar processes occurred in embryo and fetus. However, it is important to establish the more effective system for developing functional blood cells. Here we present the methods for selectively inducing mature red blood cells from ES / iPS cells in vitro, and show the functional equality of them to natural blood cells. First, Flk1+ mesodermal progenitors were derived from ES / iPS cells on OP9 stromal cells at an efficacy of more than 50% and collected by fluorescence activated cell sorter. Then, those sorted cells were cultured in the presence of exogenous erythropoietin and stem cell factor. They highly selectively developed into erythroid lineages including enucleated red blood cells. Sequential FACS analysis using the antibodies against transferrin receptor CD71 and erythroid specific antigen Ter119 in combination with DNA staining dye Hoechst 33342 demonstrated that ES / iPS cell-derived erythropoiesis in our system follow the normal erythroid developmental pathway occurred in vivo. RT-PCR and Western blot analyses proved the expression of heme biosynthesis enzymes on the produced erythrocytes. Finally, the oxygen dissociation curve showed that ES / iPS cell-derived erythroid cells are functionally virtually equivalent to natural red blood cells as oxygen carriers. Taken together, our system can present the effective methods of investigating the mechanisms of normal erythropoiesis and the deficits in syndromes with disrupted red blood cell production. Disclosures: No relevant conflicts of interest to declare.

The Xla (X-linked anemia) Mouse: A Transient Neonatal Anemia Caused by a Gata1 Splicing Mutation,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3162-3162
Author(s):  
Kyle Miller ◽  
Michael Silvey ◽  
Derek Logsdon ◽  
Frederick Balch ◽  
Ndona Nsumu ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
X Chromosome ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Clonal Selection ◽  
Pcr Analysis ◽  
Nucleotide Change ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Erythroid Precursors

Abstract Abstract 3162 The Xla (X-linked anemia) mutant mouse was generated by N-ethyl-N-nitrosourea (ENU) mutagenesis and results in a severe and transient neonatal anemia. Xla/+ females exhibit severe anemia with 50% the level of red blood cell number, hematocrit and hemoglobin. Male Xla mice die in utero at 10.5 days gestation. The neonatal anemia observed in Xla/+ female pups is resolved by weaning age at 3 weeks by which time the mice present with a normal hematological phenotype. It is unknown how the neonatal anemia in Xla/+ females is alleviated. Previously, we mapped the Xla locus to the proximal end of the X chromosome near candidate gene Gata1 which showed no change in the coding sequence of GATA1 protein. Now we report the identification of a Gata1 mutation in Xla mice that results in an mRNA splicing defect. A nucleotide change (G to A) was identified 5 base pairs downstream of Exon 1E in intron 1 of the Xla Gata1 gene and results in the lack of incorporation of Exon 1E in the Gata1 mRNA expressed from the mutant locus. Therefore, in some erythroid lineage cells in Xla/+ mice, the normal 1E exon of Gata1 mRNA is replaced by Exon 1Eb/c which is known not to impact erythropoeisis since no GATA1 protein is made by this mRNA due to its inability to bind to ribosomes. These data show the Xla mouse results from a single nucleotide change impacting the normal splicing of the Gata1 gene. A second goal of this study was to understand why Xla/+ mice exhibit the neonatal transient anemia. A contributing factor is X chromosome inactivation which occurs in female mice during development. The short-term anemia in Xla mice was thought to be due to clonal selection of erythroid lineage cells characterized by the expression of GATA1 protein from the active X chromosome expressing only from the wild type Gata1 locus. Using an X-linked gene expressed in red blood cells (Pgk1, phosphoglycerate kinase 1) that varies between Xla mice and a wild derived strain, CAST/Ei, we examined the active state of the X chromosomes based on the expression of Pgk1 RNA in reticulocytes from hybrid Xla mice generated by breeding of these different strains. Examining expression of the X-linked Pgk1 SNP variant in the RNA of reticulocytes from hybrid Xla/+ mice reveals red blood cells are generated from two types of erythroid lineage cells. Pgk1 SNP RT-PCR analysis reveals that red blood cells not only derive from erythroid progenitors with the active X chromosome carrying the wild type Gata1 gene but also red blood cells are produced by erythroid lineage cells expressing the Xla mutant Gata1 mRNA on the active X chromosome (which does not make GATA1 protein). Therefore, some Xla erythroid cells derive from progenitors which express Gata1 transcripts using Exon 1Eb/c that does not stimulate erythropoiesis due to lack of GATA1 protein. The question is how these erythroid precursors generate normal red blood cells without the production of GATA1 protein. We hypothesize there is a developmentally expressed compensatory gene or pathway replacing GATA1 expression in GATA1-lacking erythroid precursors and required for the production of red blood cells in Xla mice. Analysis is underway to identify a potential novel gene or pathway impacting erythropoiesis in these mutant mice. Disclosures: No relevant conflicts of interest to declare.

Spatially Organized Structure of Coronary Thrombus in Acute Myocardial Infarction

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 716-716
Author(s):  
Jan E. Dyr ◽  
Tomas Riedel ◽  
Jana Stikarova ◽  
Jiri Suttnar ◽  
Jaroslav Cermak ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Red Blood Cells ◽  
Symptom Onset ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Freeze Fracture ◽  
Ischemic Time ◽  
Internal Structures

Abstract Introduction The use of thromboaspiration in primary percutaneous intervention (PCI) for ST-segment elevation myocardial infarction (STEMI) has offered a unique opportunity to study thrombus composition, its dynamic formation, and architecture in vivo. There has been, however, several limitations, not least the fact that the technique has not yet allowed a precise transversal analysis from one side of the artery to the other, as is done in histological analysis. The dynamic process of intracoronary thrombus formation in STEMI patients is thus still not well understood. Ischemic time was hypothesized to be among the strongest independent correlates of thrombus architecture. In time the platelets are decreasing its proportion and fibrin proportion is increasing (J Silvain, J-P Collet, JW Weisel et al, J Am Coll Cardiol 2011; 57:1359). However, no real report on the internal structures of the in vivo formed thrombi has been shown so far. Therefore, we investigated both the surface and the composition of longitudinally freeze-fractured thrombi. Methods Thrombi were collected by PCI from 119 STEMI patients. Out of the patients there were "early comers " (˃12 h from symptom onset; 23 patients) and "late comers" (more than 720 min; 29 patients). The mean age of all patients was 64 years, 70% of patients were males, 51% were smokers, 50% had arterial hypertension, 20% were diabetics and 23% had chronic renal insufficiency. Scanning electron microscopy; collected thrombi obtained by PCI were thoroughly washed in saline solution and stored in 4% formaldehyde prior dehydration. To reveal the internal structures of the thrombi selected samples were longitudinally freeze fractured in liquid nitrogen and coated with platinum. Samples were examined in SEM Vega Plus TS 5135 (Tescan s.r.o., Brno, Czech Republic). Whole areas of the freeze-fractured thrombi were scanned. Results and discussion The thrombus composition of longitudinally freeze-fractured thrombi was compared between groups of "early-comers" and "late-comers. The distribution of the components in the "early comers" thrombi freeze-fracture seemed to be uniform. Platelets were far the main component (about 75 % in proportion) of the "early comers" thrombus, followed by fibrin and other compounds. The amount of red blood cells was negligible (about 2 - 8 %). We did not observe any significant differences between the thrombi in the group of early comers. Thrombi of the "late-comers" group were composed mainly of red blood cells; platelets and fibrin formed only minority of the thrombi. In contrast to the "early comers" the distribution of the main thrombus components in the "late comers" thrombi was dramatically different between individual parts of the thrombus. The number of platelets and red blood cells varied from 0% to almost 99% and vice versa. It was possible to estimate the initiating place of the thrombus as well as the direction of the growth. Each thrombus could be divided into parts formed mainly either by platelets or by red blood cells. It seems that thrombus develops a regional architecture defined by the extent of platelet activation and packing density. It has been reported that in contracted clots and thrombi, erythrocytes are compressed to close-packed polyhedral structures with platelets and fibrin on the surface demonstrating how contracted clots form an impermeable barrier important for hemostasis and wound healing (D Cines, T Lebedeva, J Weisel et al, Blood 2014; 123:1596). Our investigation of the composition of the in vivo formed thrombi supports these results and helps to explain how fibrinolysis is greatly retarded as clots grow and contract. We have found that on the surfaces of late-comers thrombi fibrin thick fibrils were present. It has been shown that the association of soluble fibrinogen with the fibrin clot results in the reduced adhesiveness of such fibrinogen/fibrin matrices toward leukocytes and platelets (VK Lishko, T Burke, T Ugarova, Blood 2007; 109:1541). Fibrinopeptides A are less accessible for thrombin in surface bound fibrinogen which thus provides additional level of protection of thrombi from premature dissolution (T Riedel, L Medved, JE Dyr, Blood 2011; 117:1700). These findings may have great impact on our knowledge of pathophysiology of the thrombus growth and possible therapeutic consequences related to the time of symptom onset. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document