Red cell membrane disorders: structure meets function.

The mature red blood cell (RBC) lacks a nucleus and organelles characteristic of most cells, but it is elegantly structured to perform the essential function of delivering oxygen and removing carbon dioxide from all other cells while enduring the shear stress imposed by navigating small vessels and sinusoids. Over the past several decades, the efforts of biochemists, cell and molecular biologists, and hematologists have provided an appreciation of the complexity of RBC membrane structure, while studies of the RBC membrane disorders have offered valuable insights into structure-function relationships. Within the last decade, advances in genetic testing and its increased availability have made it possible to substantially build upon this foundational knowledge. Although disorders of the RBC membrane due to altered structural organization or altered transport function are heterogeneous, they often present with common clinical findings of hemolytic anemia. However, they may require substantially different management depending on the underlying pathophysiology. Accurate diagnosis is essential to avoid emergence of complications or inappropriate interventions. We propose an algorithm for laboratory evaluation of patients presenting with symptoms and signs of hemolytic anemia with a focus on RBC membrane disorders. Here, we review the genotypic and phenotypic variability of the RBC membrane disorders in order to raise the index of suspicion and highlight the need for correct and timely diagnosis.

Biochemical, Cellular, and Proteomic Characterization of Hereditary Spherocytosis Among Tunisians.

BACKGROUND/AIMS: Hereditary Spherocytosis (HS) is the most common erythrocyte membrane disorder causing hemolytic anemia. The wide heterogeneity of both clinical and laboratory manifestations of HS contributes to difficulties associated with the diagnosis of this disorder. Although massive data previously reported worldwide, there is yet no data on HS among the Tunisian population. Here we aim to characterize HS in Tunisian patients at biochemical and cellular levels, identify the membrane protein deficiency, and compare the accuracy of the diagnostic tests to identify the most appropriate assay for HS diagnosis. METHODS: We investigated 81 patients with hemolytic anemia and 167 normal controls. The exploration of HS based on clinical and family history, physical examination, and the results of laboratory tests: blood smear, osmotic fragility test (OFT), cryohemolysis test (CT), pink test (PT), eosine-5'-maleimide (EMA) test, and erythrocyte membrane protein electrophoresis. RESULTS: We identified 21 patients with HS, classified as severe (6/21;28.5%), moderate (10/21;47.6%), and mild (5/21;23.8%). The most prevalent protein deficiency was the band 3 protein detected in ten Tunisian HS patients. The EMA test showed a high specificity (97.5%) and sensitivity (94.7%) for HS diagnosis compared to the other screening tests. Interestingly, fourteen among sixteen patients presenting with homozygous sickle cells HbSS showed an increase of EMA fluorescence intensity compared to other anemic patients. CONCLUSION: Our study highlights the efficiency of the EMA dye for the detection of HS whatever the nature of the involved protein deficiency. We report for the first time, the most prevalent protein deficiency among Tunisians with HS. Moreover, we found that the combination of the EMA-binding test with PT or incubated OFT improves the diagnosis sensitivity while maintaining a good specificity.

Plasmodium falciparum maturation across the intra-erythrocytic cycle shifts the soft glassy viscoelastic properties of red blood cells from a liquid-like towards a solid-like behavior.

The mechanical properties of erythrocytes have been investigated by different techniques. However, there are few reports on how the viscoelasticity of these cells varies during malaria disease. Here, we quantitatively map the viscoelastic properties of Plasmodium falciparum-parasitized human erythrocytes. We apply new methodologies based on optical tweezers to measure the viscoelastic properties and defocusing microscopy to measure the erythrocyte height profile, the overall cell volume, and its form factor, a crucial parameter to convert the complex elastic constant into complex shear modulus. The storage and loss shear moduli are obtained for each stage of parasite maturation inside red blood cells, while the former increase, the latter decrease. Employing a soft glassy rheology model, we obtain the power-law exponent for the storage and loss shear moduli, characterizing the soft glassy features of red blood cells in each parasite maturation stage. Ring forms present a liquid-like behavior, with a slightly lower power-law exponent than healthy erythrocytes, whereas trophozoite and schizont stages exhibit increasingly solid-like behaviors. Finally, the surface elastic shear moduli, low-frequency surface viscosities, and shape recovery relaxation times all increase not only in a stage-dependent manner but also when compared to healthy red blood cells. Overall, the results call attention to the soft glassy characteristics of Plasmodium falciparum-parasitized erythrocyte membrane and may provide a basis for future studies to better understand malaria disease from a mechanobiological perspective.

Usage of atomic force microscopy for detection of the damaging effect of CdCl2 on red blood cells membrane.

The possibility of detecting the damaging effect of cadmium salts on red blood cells (RBC) membrane by atomic force microscopy and light microscopy was studied. White wistar rats RBC were incubated with cadmium chloride in concentrations of 1 µg/l, 10 µg/l, 100 µg/l, and 1000 µg/l for the research. A comparison of sample preparation methods proposed by other authors in previous studies is made. The optimal method that does not significantly affect the change in the morphological features of the cell is selected. The quantitative assessment of damaged and destroyed RBC depending on the concentration of cadmium was performed by optical microscopy. The study showed that CdCl2 has a damaging effect on the RBC membrane, which leads to the formation of non-specific cell forms. A comparative assessment was made between the methods of optical microscopy and atomic force microscopy for the suitability of studying the morphological characteristics of abnormal forms of the RBC. It is shown that the method of atomic force microscopy allows registering morphological changes in the RBC that cannot be registered by optical microscopy. It is pointed that CdCl2 has effect on destruction of the RBC and the formation of specific bulges on the RBC membrane. Influence of CdCl2 on the RBC mechanical properties was studied using atomic force microscopy. The possibility of using atomic force microscopy in studies of morphology and mechanical properties of the RBC under toxicity effect of cadmium is shown.

The effect of myeloperoxidase isoforms on biophysical properties of red blood cells.

Myeloperoxidase (MPO), an oxidant-producing enzyme, stored in azurophilic granules of neutrophils has been recently shown to influence red blood cell (RBC) deformability leading to abnormalities in blood microcirculation. Native MPO is a homodimer, consisting of two identical protomers (monomeric MPO) connected by a single disulfide bond but in inflammatory foci as a result of disulfide cleavage monomeric MPO (hemi-MPO) can also be produced. This study investigated if two MPO isoforms have distinct effects on biophysical properties of RBCs. We have found that hemi-MPO, as well as the dimeric form, bind to the glycophorins A/B and band 3 protein on RBC's plasma membrane, that lead to reduced cell resistance to osmotic and acidic hemolysis, reduction in cell elasticity, significant changes in cell volume, morphology, and the conductance of RBC plasma membrane ion channels. Furthermore, we have shown for the first time that both dimeric and hemi-MPO lead to phosphatidylserine (PS) exposure on the outer leaflet of RBC membrane. However, the effects of hemi-MPO on the structural and functional properties of RBCs were lower compared to those of dimeric MPO. These findings suggest that the ability of MPO protein to influence RBC's biophysical properties depends on its conformation (dimeric or monomeric isoform). It is intriguing to speculate that hemi-MPO appearance in blood during inflammation can serve as a regulatory mechanism addressed to reduce abnormalities on RBC response, induced by dimeric MPO.

Hematological effects of ultrafine carbon black on red blood cells and hemoglobin.

The harmful effects of ultrafine particles (UFPs) in the atmosphere have caused widespread concern. Ultrafine carbon black (UFCB) is an important component of UFPs. In this study, we explored the impact of UFCB on the structure, the antioxidant defense system, and the ATPase activity of human red blood cells (hRBCs). It was found that UFCB decreased the activity of SOD (73.58%), CAT (89.79%), and GSH-Px (81.02%), leading to oxidative stress in hRBCs. UFCB had no destructive effect on the structure of hRBCs in 4 hours. ATPase activity increased (119.34%) and UFCB had weakly stimulated the cell membrane. On the molecular level, spectroscopic experiments showed that bovine hemoglobin (BHb) can bind to the UFCB by electrostatic force, leading to the shrinking of the BHb skeleton and increase in microenvironment polarity. This study demonstrates the negative hematological effect of UFCB on hemoglobin and hRBCs and reveals the potential risks in animals and humans.

Hereditary spherocytosis is associated with decreased pyruvate kinase activity due to impaired structural integrity of the red blood cell membrane.

Hereditary spherocytosis (HS) is characterised by increased osmotic fragility and enhanced membrane loss of red blood cells (RBC) due to defective membrane protein complexes. In our diagnostic laboratory, we observed that pyruvate kinase (PK) activity in HS was merely slightly elevated with respect to the amount of reticulocytosis. In order to evaluate whether impaired PK activity is a feature of HS, we retrospectively analysed laboratory data sets from 172 unrelated patients with HS, hereditary elliptocytosis (HE), glucose-6-phosphate dehydrogenase (G6PD) or PK deficiency, sickle cell or haemoglobin C disease, or ß-thalassaemia minor. Results from linear regression analysis provided proof that PK activity decreases with rising reticulocyte counts in HS (R2  = 0·15; slope = 9·09) and, less significantly, in HE (R2  = 0·021; slope = 8·92) when compared with other haemolytic disorders (R2  ≥ 0·65; slopes ≥ 78·6). Reticulocyte-adjusted erythrocyte PK activity levels were significantly lower in HS and even declined with increasing reticulocytes (R2  = 0·48; slope = -9·74). In this report, we describe a novel association between HS and decreased PK activity that is apparently caused by loss of membrane-bound PK due to impaired structural integrity of the RBC membrane and may aggravate severity of haemolysis in HS.

MYH9-related disease mutations cause abnormal red blood cell morphology through increased myosin-actin binding at the membrane.

MYH9-related disease (MYH9-RD) is a rare, autosomal dominant disorder caused by mutations in MYH9, the gene encoding the actin-activated motor protein non-muscle myosin IIA (NMIIA). MYH9-RD patients suffer from bleeding syndromes, progressive kidney disease, deafness, and/or cataracts, but the impact of MYH9 mutations on other NMIIA-expressing tissues remains unknown. In human red blood cells (RBCs), NMIIA assembles into bipolar filaments and binds to actin filaments (F-actin) in the spectrin-F-actin membrane skeleton to control RBC biconcave disk shape and deformability. Here, we tested the effects of MYH9 mutations in different NMIIA domains (motor, coiled-coil rod, or non-helical tail) on RBC NMIIA function. We found that MYH9-RD does not cause clinically significant anemia and that patient RBCs have normal osmotic deformability as well as normal membrane skeleton composition and micron-scale distribution. However, analysis of complete blood count data and peripheral blood smears revealed reduced hemoglobin content and elongated shapes, respectively, of MYH9-RD RBCs. Patients with mutations in the NMIIA motor domain had the highest numbers of elongated RBCs. Patients with mutations in the motor domain also had elevated association of NMIIA with F-actin at the RBC membrane. Our findings support a central role for motor domain activity in NMIIA regulation of RBC shape and define a new sub-clinical phenotype of MYH9-RD.

Thermal sensitivity and haemolysis of erythrocytes with membranopathy.

Measuring the impedance of heated suspensions of erythrocytes and erythrocyte ghost membranes, two thermally-induced alterations are registered in the plasma membrane at TA (denaturation of spectrin with inducing temperature at 49,5 °C) and TG (hyperthermic activation of basal ion permeability with inducing temperature at 60.7 °C). In this study erythrocytes from 9 healthy patients and 15 patients with hemolytic anemia were studied and divided into four groups depending on their TA and TG top temperatures. The TA and TG of erythrocytes with hemoglobinopathy were the same as those of control erythrocytes while those of erythrocytes with membranopathy were significantly reduced. In erythrocytes with severe membranopathy, the TG was decreased by about 5 °C. In latter cells the normal value of TG was restored and the resistance to thermal haemolysis was increased by 90% after the specific stabilization of band 3 protein by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS). Obtained results indicate the involvement of band 3 in the membrane alteration at TG and in the heat target responsible for thermal haemolysis.

[Red cell membrane disorders and thalassemia].

Congenital hemolytic anemias are classified into three major categories: red cell membrane disorders, hemoglobinopathies, and red cell enzyme disorders. The membrane disorders are caused by abnormalities in erythrocyte membrane proteins and are often associated with disease-specific deformations of red blood cells. Historically, membrane disorders have been classified according to morphology. In recent years, however, comprehensive genetic analysis with next-generation sequencing has been performed in patients with hemolytic anemia for whom making an accurate diagnosis is difficult. These studies have led to the identification of new causative genes, but there have been inconsistent associations in some cases between the diagnosed disease and the patient's clinical manifestations. Thalassemia is a hemoglobinopathy caused by a quantitative abnormality of one or the other of the globin chains in hemoglobin. Most Japanese patients with thalassemia have mild forms of the disease, which is different from reports in other countries. However, with globalization, the proportion of Japanese patients with intermediate or severe anemia is increasing. Therefore, it is incumbent on hematologists in Japan to be knowledgeable regarding prenatal diagnosis of and gene therapy for thalassemia.

Stimulation of Eryptosis by Afatinib.

BACKGROUND/AIMS: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is primarily utilized for the treatment of non-small cell lung carcinoma. The drug is at least partially effective by triggering suicidal tumor cell death. Side effects of afatinib treatment include anemia. At least in theory, afatinib induced anemia could be secondary to stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), induction of oxidative stress, and increase of ceramide abundance. The present study explored, whether afatinib induces eryptosis and, if so, whether its effect involves Ca2+ entry, oxidative stress, and/or ceramide. METHODS: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to afatinib (≥ 4 µg/ml) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Afatinib significantly increased Fluo3-fluorescence, DCFDA fluorescence and ceramide abundance. The effect of afatinib on annexin-V-binding and forward scatter was significantly blunted by removal of extracellular Ca2+. CONCLUSIONS: Afatinib triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, and ceramide.

Specific oxygenation of plasma membrane phospholipids by Pseudomonas aeruginosa lipoxygenase induces structural and functional alterations in mammalian cells.

Pseudomonas aeruginosa is a gram-negative pathogen, which causes life-threatening infections in immunocompromized patients. These bacteria express a secreted lipoxygenase (PA-LOX), which oxygenates free arachidonic acid to 15S-hydro(pero)xyeicosatetraenoic acid. It binds phospholipids at its active site and physically interacts with lipid vesicles. When incubated with red blood cells membrane lipids are oxidized and hemolysis is induced but the structures of the oxygenated membrane lipids have not been determined. Using a lipidomic approach, we analyzed the formation of oxidized phospholipids generated during the in vitro incubation of recombinant PA-LOX with human erythrocytes and cultured human lung epithelial cells. Precursor scanning of lipid extracts prepared from these cells followed by multiple reaction monitoring and MS/MS analysis revealed a complex mixture of oxidation products. For human red blood cells this mixture comprised forty different phosphatidylethanolamine and phosphatidylcholine species carrying oxidized fatty acid residues, such as hydroxy-octadecadienoic acids, hydroxy- and keto-eicosatetraenoic acid, hydroxy-docosahexaenoic acid as well as oxygenated derivatives of less frequently occurring polyenoic fatty acids. Similar oxygenation products were also detected when cultured lung epithelial cells were employed but here the amounts of oxygenated lipids were smaller and under identical experimental conditions we did not detect major signs of cell lysis. However, live imaging indicated an impaired capacity for trypan blue exclusion and an augmented mitosis rate. Taken together these data indicate that PA-LOX can oxidize the membrane lipids of eukaryotic cells and that the functional consequences of this reaction strongly depend on the cell type.

Gene disruption of dematin causes precipitous loss of erythrocyte membrane stability and severe hemolytic anemia.

Dematin is a relatively low abundance actin binding and bundling protein associated with the spectrin-actin junctions of mature erythrocytes. Primary structure of dematin includes a loosely folded core domain and a compact headpiece domain that was originally identified in villin. Dematin's actin binding properties are regulated by phosphorylation of its headpiece domain by cyclic adenosine monophosphate-dependent protein kinase. Here, we used a novel gene disruption strategy to generate the whole body dematin gene knockout mouse model (FLKO). FLKO mice, while born at a normal Mendelian ratio, developed severe anemia and exhibited profound aberrations of erythrocyte morphology and membrane stability. Having no apparent effect on primitive erythropoiesis, FLKO mice show significant enhancement of erythroblast enucleation during definitive erythropoiesis. Using membrane protein analysis, domain mapping, electron microscopy, and dynamic deformability measurements, we investigated the mechanism of membrane instability in FLKO erythrocytes. Although many membrane and cytoskeletal proteins remained at their normal levels, the major peripheral membrane proteins spectrin, adducin, and actin were greatly reduced in FLKO erythrocytes. Our results demonstrate that dematin plays a critical role in maintaining the fundamental properties of the membrane cytoskeleton complex.

Sickle cell disease diagnosis based on spatio-temporal cell dynamics analysis using 3D printed shearing digital holographic microscopy.

We present a spatio-temporal analysis of cell membrane fluctuations to distinguish healthy patients from patients with sickle cell disease. A video hologram containing either healthy red blood cells (h-RBCs) or sickle cell disease red blood cells (SCD-RBCs) was recorded using a low-cost, compact, 3D printed shearing interferometer. Reconstructions were created for each hologram frame (time steps), forming a spatio-temporal data cube. Features were extracted by computing the standard deviations and the mean of the height fluctuations over time and for every location on the cell membrane, resulting in two-dimensional standard deviation and mean maps, followed by taking the standard deviations of these maps. The optical flow algorithm was used to estimate the apparent motion fields between subsequent frames (reconstructions). The standard deviation of the magnitude of the optical flow vectors across all frames was then computed. In addition, seven morphological cell (spatial) features based on optical path length were extracted from the cells to further improve the classification accuracy. A random forest classifier was trained to perform cell identification to distinguish between SCD-RBCs and h-RBCs. To the best of our knowledge, this is the first report of machine learning assisted cell identification and diagnosis of sickle cell disease based on cell membrane fluctuations and morphology using both spatio-temporal and spatial analysis.

Sublethal mechanical trauma alters the electrochemical properties and increases aggregation of erythrocytes.

Circulation of blood depends, in part, on the ability of red blood cells (RBCs) to aggregate, disaggregate, and deform. The primary intrinsic disaggregating force of RBCs is derived from their electronegativity, which is largely determined by sialylated glycoproteins on the plasma membrane. Given supraphysiological shear exposure - even at levels below those which induce hemolysis - alters cell morphology, we hypothesized that exposure to supraphysiological and subhemolytic shear would cleave membrane-bound sialic acid, altering the electrochemical and physical properties of RBCs, and thus increase RBC aggregation. Isolated RBCs from healthy donors (n = 20) were suspended in polyvinylpyrrolidinone. Using a Poiseuille shearing system, RBC suspensions were exposed to 125 Pa for 1.5 s for three duty-cycles. Following the first and third shear duty-cycle, samples were assessed for: RBC aggregation; the ability of RBCs to aggregate independent of plasma ("aggregability"); disaggregation shear rate; membrane-bound sialic acid content, and; cell electrophoretic mobility. Initial shear exposure significantly increased RBC aggregation, aggregability, and the shear required for rouleaux dispersion. Sialic acid concentration significantly decreased on isolated RBC membranes ghosts, and increased in the supernatant following shear. Initial shear exposure decreased the electrophoretic mobility of RBCs, decreasing the electronegative charge from -15.78 ±â€¯0.31 to -7.55 ±â€¯0.21 mV. Three exposures to the shear duty-cycle did not further compound altered RBC measures. A single exposure to supraphysiological and subhemolytic shear significantly decreased the electrochemical charge of the RBC membrane, concurrently increasing cell aggregation/aggregability. The cascading implications of hyperaggregation appears to potentially explain the ischemia-associated complications commonly reported following mechanical circulatory support.

Patient-specific modeling of individual sickle cell behavior under transient hypoxia.

Sickle cell disease (SCD) is a highly complex genetic blood disorder in which red blood cells (RBC) exhibit heterogeneous morphology changes and decreased deformability. We employ a kinetic model for cell morphological sickling that invokes parameters derived from patient-specific data. This model is used to investigate the dynamics of individual sickle cells in a capillary-like microenvironment in order to address various mechanisms associated with SCD. We show that all RBCs, both hypoxia-unaffected and hypoxia-affected ones, regularly pass through microgates under oxygenated state. However, the hypoxia-affected cells undergo sickling which significantly alters cell dynamics. In particular, the dense and rigid sickle RBCs are obstructed thereby clogging blood flow while the less dense and deformable ones are capable of circumnavigating dead (trapped) cells ahead of them by choosing a serpentine path. Informed by recent experiments involving microfluidics that provide in vitro quantitative information on cell dynamics under transient hypoxia conditions, we have performed detailed computational simulations of alterations to cell behavior in response to morphological changes and membrane stiffening. Our model reveals that SCD exhibits substantial heterogeneity even within a particular density-fractionated subpopulation. These findings provide unique insights into how individual sickle cells move through capillaries under transient hypoxic conditions, and offer novel possibilities for designing effective therapeutic interventions for SCD.

Rolling Adhesion of Schizont Stage Malaria-Infected Red Blood Cells in Shear Flow.

To avoid clearance by the spleen, red blood cells infected with the human malaria parasite Plasmodium falciparum (iRBCs) adhere to the vascular endothelium through adhesive protrusions called "knobs" that the parasite induces on the surface of the host cell. However, the detailed relation between the developing knob structure and the resulting movement in shear flow is not known. Using flow chamber experiments on endothelial monolayers and tracking of the parasite inside the infected host cell, we find that trophozoites (intermediate-stage iRBCs) tend to flip due to their biconcave shape, whereas schizonts (late-stage iRBCs) tend to roll due to their almost spherical shape. We then use adhesive dynamics simulations for spherical cells to predict the effects of knob density and receptor multiplicity per knob on rolling adhesion of schizonts. We find that rolling adhesion requires a homogeneous coverage of the cell surface by knobs and that rolling adhesion becomes more stable and slower for higher knob density. Our experimental data suggest that schizonts are at the border between transient and stable rolling adhesion. They also allow us to establish an estimate for the molecular parameters for schizont adhesion to the vascular endothelium and to predict bond dynamics in the contact region.

Characterizing pathology in erythrocytes using morphological and biophysical membrane properties: Relation to impaired hemorheology and cardiovascular function in rheumatoid arthritis.

The inflammatory burden of the complex rheumatoid arthritis (RA) disease affects several organ-systems, including rheological properties of blood and its formed elements. Red blood cells (RBCs) are constantly exposed to circulating dysregulated inflammatory molecules that are co-transported within the vasculature; and their membranes may be particularly vulnerable to the accompanying oxidative stress. In the current study, we investigate biophysical and ultrastructural characteristics of RBCs obtained from a cohort of patients using atomic force microscopy (AFM), scanning electron microscopy (SEM) and confocal microscopy (CM). Statistical analyses of AFM data showed that RA RBCs possessed significantly reduced membrane elasticity relative to that of RBCs from healthy individuals (P-value <0.0001). SEM imaging of RA RBCs revealed increased anisocytes and poikilocytes. Poikilocytes included knizocytes, stomatocytes, dacryocytes, irregularly contracted cells, and knot cells. CM imaging of several RA RBCs, spectrin, and band 3 protein networks portrayed the similar morphological profiles. Analyses of CM images confirmed changes to distribution of band-3 skeletal protein, a protein critical for gaseous exchange functions of the RBC and preventing membrane surface loss. Decreased membrane deformability impairs the RBC's capacity to adequately adapt its shape to navigate blood vessels, especially microvasculature, and this decrease is also reflected in the cell's morphology. Changes to morphology and deformability may also indicate loss of functional domains and/or pathological protein and lipid associations. These findings suggest that RA disease and/or its concomitant factors impact on the RBC and its membrane integrity with potential for exacerbating pathological cellular function, hemorheology, and cardiovascular function.

The structural and functional changes of blood cells and molecular components in diabetes mellitus.

It is known fact that diabetes mellitus (DM) affects blood cells. Changes in the erythrocyte membrane, disorder in hemoglobin oxygen-binding and modification in mechanical characteristics, are effects of hyperglycemia on red blood cells. Altered susceptibility infection of patients with diabetes has been ascribed to a depression in the function of polymorphonuclear leukocytes. Neutrophil function in patients with diabetes with good glucose control is slightly different than in healthy ones. DM causes significant changes in lymphocytes metabolism and their functions. Patients with diabetes, presenting with acute coronary syndrome, are at higher risk of cardiovascular complications and recurrent ischemic events in comparison to non-diabetic counterparts. Various mechanisms, including endothelial dysfunction, platelet hyperactivity, and abnormalities in coagulation and fibrynolysis have been implicated for this increased atherothrombotic risk. There are many other alterations of blood cells due to DM. In the present review we focused on modifications of blood cells due to DM. Then, as a second point, we explored how the changes affect functions of red blood cells, white blood cells and platelets.

Drug-induced endovesiculation of erythrocytes is modulated by the dynamics in the cytoskeleton/membrane interaction.

Recent studies on erythrocyte membrane fluctuations revealed that the erythrocyte cytoskeleton actively modulates its membrane association thereby regulating crucial membrane properties. Cationic amphiphilic drugs like chlorpromazine are known to induce a cup-like cell shape and vesicle formation into the cell interior, effectors of this process, however, are largely unknown. Using flow cytometry, this study explored conditions that influence endovesiculation induced by chlorpromazine. We found that inhibitors of membrane fluctuations, like ATP depletion, vanadate or fluoride, also inhibited endovesiculation whereas activation of PKC, known to decrease cytoskeleton association and increase membrane fluctuations, also enhanced endovesicle formation. This indicates that endovesicle formation and membrane fluctuations are modulated by the same cytoskeleton-regulated membrane properties. Further, acanthocytic erythrocytes of chorea acanthocytosis (ChAc) patients that lack the VPS13A/chorein protein - likely a crucial organizer at the erythrocyte cytoskeleton/membrane interface - showed a strong decrease in chlorpromazine-induced endovesiculation. The responses of ChAc erythrocytes to effectors of endovesiculation were similar to that of control erythrocytes, yet at drastically reduced levels. This suggests a more rigid and less dynamic interaction at the membrane-cytoskeleton interphase of ChAc erythrocytes.