Yippee like 4 (Ypel4) is essential for normal mouse red blood cell membrane integrity

The YPEL family genes are highly conserved across a diverse range of eukaryotic organisms and thus potentially involved in essential cellular processes. Ypel4, one of five YPEL family gene orthologs in mouse and human, is highly and specifically expressed in late terminal erythroid differentiation (TED). In this study, we investigated the role of Ypel4 in murine erythropoiesis, providing for the first time an in-depth description of a Ypel4-null phenotype in vivo. We demonstrated that the Ypel4-null mice displayed a secondary polycythemia with macro- and reticulocytosis. While lack of Ypel4 did not affect steady-state TED in the bone marrow or spleen, the anemia-recovering capacity of Ypel4-null cells was diminished. Furthermore, Ypel4-null red blood cells (RBC) were cleared from the circulation at an increased rate, demonstrating an intrinsic defect of RBCs. Scanning electron micrographs revealed an ovalocytic morphology of Ypel4-null RBCs and functional testing confirmed reduced deformability. Even though Band 3 protein levels were shown to be reduced in Ypel4-null RBC membranes, we could not find support for a physical interaction between YPEL4 and the Band 3 protein. In conclusion, our findings provide crucial insights into the role of Ypel4 in preserving normal red cell membrane integrity.

Vesiculation red blood cells. Its role in donor erythrocytes components

The formation of microvesicles by blood cells: monocytes, platelets, granulocytes, erythrocytes and endothelial cells is the most important feature of intercellular interactions. Red blood cells form microvesicles to remove damaged cell components, such as oxidized hemoglobin and damaged membrane components, and thus extend their functioning. Two hypotheses have been put forward for the formation of microvesicles: programmed cell death (eryptosis) and clustering of the band 3 protein as a result of disruption of intercellular interactions. In the process of eryptosis, damage to hemoglobin and a change in the pathways of phosphorylation of membrane proteins, primarily protein of strip 3, weaken the strong bonds between the lipid bilayer and the cytoskeleton, which is accompanied by the transformation of the membrane, the formation of protrusions and their transformation into microvesicles. It was found that the formation of microvesicles by red blood cells is impaired in patients suffering from various pathologies of red blood cells: sickle cell anemia, glucose-6-dehydrogenase deficiency, spherocytosis, and malaria. Studies of the last decade show that a violation of the interaction between the membrane and the cytoskeleton is probably the main mechanism, since it is confirmed by data obtained in the study of structural changes in red blood cells of donor hemocomponents stored in a blood bank. Currently, studies on the effect of microvesicles on the safety of erythrocyte-containing blood components have become widespread. A discussion was resumed on the relationship between the number of accumulated microvesicles in blood components and the effectiveness of donor components for patients during transfusion, depending on the shelf life of the components. Detailed data on proteomic, lipidomic and immunogenic comparisons of microvesicles obtained from various sources are convincing in the identification of trigger stimuli causing the generation of microvesicles. Elucidation of the contribution of microvesicles obtained from red blood cells to inflammation, thrombosis, and autoimmune reactions confirms the need to further study the mechanisms and consequences of the generation of microvesicles by red blood cells of donor components used for transfusion medicine.

Atypical hereditary spherocytosis phenotype associated with pseudohypokalaemia and a new variant in the band 3 protein

Red blood cell (RBC) membrane disorders are predominantly caused by mutations resulting in decreased RBC deformability and permeability. We present a family in which, the proband and his daughter presented with pseudohypokalaemia. Studies on the temperature dependence of pseudohypokalaemia suggested a maximum decrease in serum potassium when whole blood is stored at 37°C. Routine haematology suggested mild haemolysis with a hereditary spherocytosis phenotype. These two cases present a novel variant in temperature-dependent changes in potassium transport. A new variant was identified in the SLC4A1 gene which codes for band 3 protein (anion exchanger 1) in RBC membrane which may contribute to the phenotype. This is the first report of familial pseudohypokalaemia associated with changes in RBC membrane morphology. The clinical implications of pseudohypokalaemia are that it can lead to inappropriate investigation or treatment. However, many questions remain to be solved and other RBC membrane protein genes should be studied.

Thermodynamic Characterization of Red Blood Cell Suspension and Band 3 Protein Oxy-Deoxygenating Functionality: Comparative Study

The importance of studying storage alterations in erythrocytes is highlighted by the need to understand changes that could potentially serve to optimize the storage system. With this aim, the non-equilibrium thermodynamic theory with internal variables was introduced, and perturbing the erythrocyte samples with a harmonic electric field some functions of the theory have been determined varying in the perturbation frequency. A frequency has been noted that acts as a separator element between two states showing a different entropy production above and below this frequency. In stored red blood cells compared to fresh ones, the increase in entropy production measured shows a greater state of disorder in the latter. Further alterations have been highlighted on the surface charge density of the stored erythrocyte membrane and on the speed of anionic kinetics. All these observations highlight the image of membrane structural and functional alterations of the stored erythrocytes and lead to the elaboration of a technique able to correlate a specific perturbation frequency with the aging time of red blood cells.

d-Galactose Decreases Anion Exchange Capability through Band 3 Protein in Human Erythrocytes

d-Galactose (d-Gal), when abnormally accumulated in the plasma, results in oxidative stress production, and may alter the homeostasis of erythrocytes, which are particularly exposed to oxidants driven by the blood stream. In the present investigation, the effect of d-Gal (0.1 and 10 mM, for 3 and 24 h incubation), known to induce oxidative stress, has been assayed on human erythrocytes by determining the rate constant of SO42− uptake through the anion exchanger Band 3 protein (B3p), essential to erythrocytes homeostasis. Moreover, lipid peroxidation, membrane sulfhydryl groups oxidation, glycated hemoglobin (% A1c), methemoglobin levels (% MetHb), and expression levels of B3p have been verified. Our results show that d-Gal reduces anion exchange capability of B3p, involving neither lipid peroxidation, nor oxidation of sulfhydryl membrane groups, nor MetHb formation, nor altered expression levels of B3p. d-Gal-induced %A1c, known to crosslink with B3p, could be responsible for rate of anion exchange alteration. The present findings confirm that erythrocytes are a suitable model to study the impact of high sugar concentrations on cell homeostasis; show the first in vitro effect of d-Gal on B3p, contributing to the understanding of mechanisms underlying an in vitro model of aging; demonstrate that the first impact of d-Gal on B3p is mediated by early Hb glycation, rather than by oxidative stress, which may be involved on a later stage, possibly adding more knowledge about the consequences of d-Gal accumulation.