Mechanical Properties Of Erythrocytes Research Articles

Phosphodiesterase delta governs the mechanical properties of erythrocytes infected with Plasmodium falciparum gametocytes

To persist in the blood circulation and to be available for mosquitoes, Plasmodium falciparum gametocytes modify the deformability and the permeability of their erythrocyte host via cyclic AMP (cAMP) signaling pathway. Cyclic nucleotide levels are tightly controlled by phosphodiesterases (PDE), however in Plasmodium these proteins are poorly characterized. Here, we characterize the P. falciparum phosphodiesterase delta (PfPDEδ) and we investigate its role in the cAMP signaling-mediated regulation of gametocyte-infected erythrocyte mechanical properties. Our results revealed that PfPDEδ is a dual-function enzyme capable of hydrolyzing both cAMP and cGMP, with a higher affinity for cAMP. We also show that PfPDEδ is the most expressed PDE in mature gametocytes and we propose that it is located in parasitophorous vacuole at the interface between the host cell and the parasite. We conclude that PfPDEδ is the master regulator of both the increase in deformability and the inhibition of channel activity in mature gametocyte stages, and may therefore play a crucial role in the persistence of mature gametocytes in the bloodstream.

New Insights into Hemolytic Anemias: Ultrastructural and Nanomechanical Investigation of Red Blood Cells Showed Early Morphological Changes.

Several diseases are characterized by changes in the mechanical properties of erythrocytes. Hemolytic anemias are an example of these diseases. Among the hemolytic anemias, Sickle Cell Disease and Thalassemia are the most common, characterized by alterations in the structure of their hemoglobin. Sickle cell disease has a pathological origin in synthesizing abnormal hemoglobin, HbS. In contrast, thalassemia results in extinction or decreased synthesis of α and β hemoglobin chains. This work presents a detailed study of biophysical and ultrastructural early erythrocytes membrane alterations at the nanoscale using Atomic Force Microscopy (AFM). Cells from individuals with sickle cell anemia and thalassemia mutations were studied. The analysis methodology in the AFM was given by blood smear and exposure of the inner membrane for ghost analysis. A robust statistic was used with 65,536 force curves for each map, ten cells of each type, with three individuals for each sample group. The results showed significant differences in cell rigidity, adhesion, volume, and roughness at early morphological alterations, bringing new perspectives for understanding pathogenesis. The sickle cell trait (HbAS) results stand out. Significant alterations were observed in the membrane properties, bringing new perspectives for the knowledge of this mutation. This work presents ultrastructural and biomechanical signatures of sickle cell anemia and thalassemia genotypes, which may help determine a more accurate biophysical description and clinical prognosis for these diseases.

In vitro alteration on erythrocytes mechanical properties by propofol, remifentanil and vecuronium

Several studies report flow disturbance and microcirculation disorders upon anesthesia treatment. These alterations are often related to blood rheology changes. In this work, it was attempted to make a detailed description of the alterations in erythrocyte mechanical properties by the action of propofol, remifentanil, and vecuronium. For this, an in vitro study was performed on red blood cell samples from healthy donors incubated with solutions of propofol (4 μg/mL whole blood), remifentanil (10 ng/mL plasma), and vecuronium (0.15 μg/mL plasma). Erythrocyte viscoelastic parameters were determined by octuplicate using a Reómetro Eritrocitario. Also, a Wilcoxon signed rank-test with Yates correction for continuity was performed to analyze the overall alteration in the mechanical properties of erythrocytes. Statistical analysis showed that the three studied anesthetics changed the erythrocyte mechanical properties at different parts of the membrane. These results would imply an interaction of these anesthetics with the erythrocyte membrane. Finally, this could conduce to alterations in microcirculation.

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.

Numerical Model for the Determination of Erythrocyte Mechanical Properties and Wall Shear Stress in vivo From Intravital Microscopy.

The mechanical properties and deformability of Red Blood Cells (RBCs) are important determinants of blood rheology and microvascular hemodynamics. The objective of this study is to quantify the mechanical properties and wall shear stress experienced by the RBC membrane during capillary plug flow in vivo utilizing high speed video recording from intravital microscopy, biomechanical modeling, and computational methods. Capillaries were imaged in the rat cremaster muscle pre- and post-RBC transfusion of stored RBCs for 2-weeks. RBC membrane contours were extracted utilizing image processing and parametrized. RBC parameterizations were used to determine updated deformation gradient and Lagrangian Green strain tensors for each point along the parametrization and for each frame during plug flow. The updated Lagrangian Green strain and Displacement Gradient tensors were numerically fit to the Navier-Lame equations along the parameterized boundary to determined Lame's constants. Mechanical properties and wall shear stress were determined before and transfusion, were grouped in three populations of erythrocytes: native cells (NC) or circulating cells before transfusion, and two distinct population of cells after transfusion with stored cells (SC1 and SC2). The distinction, between the heterogeneous populations of cells present after the transfusion, SC1 and SC2, was obtained through principle component analysis (PCA) of the mechanical properties along the membrane. Cells with the first two principle components within 3 standard deviations of the mean, were labeled as SC1, and those with the first two principle components greater than 3 standard deviations from the mean were labeled as SC2. The calculated shear modulus average was 1.1±0.2, 0.90±0.15, and 12 ± 8 MPa for NC, SC1, and SC2, respectively. The calculated young's modulus average was 3.3±0.6, 2.6±0.4, and 32±20 MPa for NC, SC1, and SC2, respectively. o our knowledge, the methods presented here are the first estimation of the erythrocyte mechanical properties and shear stress in vivo during capillary plug flow. In summary, the methods introduced in this study may provide a new avenue of investigation of erythrocyte mechanics in the context of hematologic conditions that adversely affect erythrocyte mechanical properties.

Effect of Cerium on Erythrocyte Mechanical Properties

Lanthanide’s are known as rare earth elements and are used in many areas. Although the place of Cerium in the Lanthanide family in its biological importance in mammalian physiology is not fully known, a human weighing 70 kg contains about 40 mg Cerium. Soluble Ce3+ salts (nitrate, acetate, chloride, etc.) are used for many biomedical purposes and in particular act as calcium analogues in biological systems. In non-physiological flow conditions occurring in extracorporeal circulation areas, increased calcium ions within the cell play a role in reducing erythrocyte deformability. In this study, it is aimed to show the possible effects of Cerium on changes in erythrocyte deformability in shear stress at sub-hemolytic level. For this purpose, blood samples were taken from adult male volunteers. Normal erythrocyte deformability was measured at nine shear rates between 0.5 and 50 Pa, followed by stress application at a slip rate of 290 Pa for 300 seconds without removing the sample from the system, followed by erythrocyte deformability at shear rates of 0.5-50 Pa. There was no significant difference between the erythrocyte deformability values of the control and Cerium incubated blood samples before stress application. However, significant differences were found after stress application (p <0.01). Incubation of erythrocytes with Cerium did not cause any change in erythrocyte deformability, but caused further deterioration of erythrocytes after mechanical stress. Cerium chloride, which we use in our study, should be kept in mind that under normal conditions, even if there is no negative effect on erythrocyte mechanics, conditions change, erythrocytes are exposed to short-term sub-hemolytic mechanical effects and their mechanical properties are deteriorated. Keywords: Lanthanide, Cerium, Erythrocyte, Deformability, Mechanical Stress DOI: 10.7176/JSTR/5-8-11

Erythrocyte Rigidity Affects Blood Clot Contraction and Formation of Polyhedrocytes

Blood clot contraction or retraction has been implicated to play a significant role in hemostasis, reduction of thrombus volume, and wound healing. Clot contraction is driven by forces that are generated by platelets and transmitted by fibrin and results in volume shrinkage followed by the compaction of erythrocytes into the core of the blood clot, resulting in their mechanical deformation towards a polyhedral shape, giving rise to the term polyhedrocytes. Despite the fact that erythrocytes are a major component of blood clots, relatively little is known about the influence of the mechanical properties or deformability of erythrocytes on the process of clot contraction. Increased hematocrit reduces extent of clot contraction due to mechanical resilience of erythrocytes and it is likely that in addition to a volume fraction the stiffness of erythrocytes can also affect the extent and rate of clot contraction. Here we tested this assumption by using artificially or naturally stiffened erythrocytes that have pathophysiological implications. The reduced deformability of erythrocytes is associated with a number of pathological conditions, such as hypertension, diabetes mellitus, atherosclerosis and smoking, but perhaps one of the most well-known diseases associated with increased erythrocyte rigidity is sickle cell disease (SCD). Another example of naturally stiff erythrocyte membrane is that of llama or camel that have red blood cells with increased osmotic resistance.To assess the extent of clot contraction, we used an optical tracking methodology that allows for the quantitative tracking for clot size. To assess the influence of erythrocyte rigidity on clot contraction we also used scanning electron microscopy to evaluate deformations of the erythrocytes, including the presence of polyhedrocytes. Centrifugation of citrated blood can be used to mimic the contractile forces generated by platelets and has been shown to cause polyhedrocyte formation. Increasing the erythrocyte rigidity through their treatment with a low concentration of glutaraldehyde resulted in a decrease in polyhedrocyte formation and the requirement of larger centrifugal forces to observe erythrocyte deformation, suggesting that the mechanical properties of erythrocytes could influence the process of clot contraction. As residual glutaraldehyde may have unwanted effects on platelets, clot contraction experiments were completed using naturally stiffer erythrocytes from SCD patients and llama ovalocytes, which are stiffer than human erythrocytes due to the increased amount of the membrane cytoskeletal protein spectrin. SCD patients were only included in this study if they had Sickle Trait, SCD Hb SS, SCD Hb SC and have not received recent transfusions. The blood samples of SCD patients were examined and on average had a 53% decrease (p<0.0001) in the extent of clot contraction compared to healthy subjects. Likewise, addition of llama ovalocytes to human platelet rich plasma resulted in a 28% decrease in extent of clot contraction compared to human erythrocytes and larger centrifugal forces were needed to see red cell deformation. SCD patients contracted 2.4X slower (p<0.001) during linear contraction (Phase 2) and 2.7X slower (p<0.05) during mechanical stabilization (Phase 3) when compared to healthy subjects. Clot contraction was impaired also by erythrocytes treated with antibodies that bind to the Wright b epitope on the erythrocytes and exert a rigidifying effect on the cells. The binding of the antibody to erythrocytes was determined by flow cytometry and the KD was ~50 nM. Increased red blood cell rigidity following exposure to antibodies was confirmed through mechanical and osmotic resistance and compared to unaltered erythrocytes.Collectively, these results demonstrate that erythrocyte mechanical properties can influence the process of clot contraction so that stiffer cells reduce the rate and extent of clot contraction. A better understanding of the role of erythrocyte deformability in the process of clot contraction has the potential to inform the development of more targeted treatments for limiting bleeding and thrombosis in patients who are prone to having altered erythrocyte content and mechanical properties of these highly abundant cells embedded into blood clots and thrombi. DisclosuresWeisel:Bayer: Research Funding.

Erythrocyte stiffness during morphological remodeling induced by carbon ion radiation.

The adverse effect induced by carbon ion radiation (CIR) is still an unavoidable hazard to the treatment object. Thus, evaluation of its adverse effects on the body is a critical problem with respect to radiation therapy. We aimed to investigate the change between the configuration and mechanical properties of erythrocytes induced by radiation and found differences in both the configuration and the mechanical properties with involving in morphological remodeling process. Syrian hamsters were subjected to whole-body irradiation with carbon ion beams (1, 2, 4, and 6 Gy) or X-rays (2, 4, 6, and 12 Gy) for 3, 14 and 28 days. Erythrocytes in peripheral blood and bone marrow were collected for cytomorphological analysis. The mechanical properties of the erythrocytes were determined using atomic force microscopy, and the expression of the cytoskeletal protein spectrin-α1 was analyzed via western blotting. The results showed that dynamic changes were evident in erythrocytes exposed to different doses of carbon ion beams compared with X-rays and the control (0 Gy). The magnitude of impairment of the cell number and cellular morphology manifested the subtle variation according to the irradiation dose. In particular, the differences in the size, shape and mechanical properties of the erythrocytes were well exhibited. Furthermore, immunoblot data showed that the expression of the cytoskeletal protein spectrin-α1 was changed after irradiation, and there was a common pattern among its substantive characteristics in the irradiated group. Based on these findings, the present study concluded that CIR could induce a change in mechanical properties during morphological remodeling of erythrocytes. According to the unique characteristics of the biomechanical categories, we deduce that changes in cytomorphology and mechanical properties can be measured to evaluate the adverse effects generated by tumor radiotherapy. Additionally, for the first time, the current study provides a new strategy for enhancing the assessment of the curative effects and safety of clinical radiotherapy, as well as reducing adverse effects.

Viscoelasticity as a Biomarker for High-Throughput Flow Cytometry

The mechanical properties of living cells are a label-free biophysical marker of cell viability and health; however, their use has been greatly limited by low measurement throughput. Although examining individual cells at high rates is now commonplace with fluorescence activated cell sorters, development of comparable techniques that nondestructively probe cell mechanics remains challenging. A fundamental hurdle is the signal response time. Where light scattering and fluorescence signatures are virtually instantaneous, the cell stress relaxation, typically occurring on the order of seconds, limits the potential speed of elastic property measurement. To overcome this intrinsic barrier to rapid analysis, we show here that cell viscoelastic properties measured at frequencies far higher than those associated with cell relaxation can be used as a means of identifying significant differences in cell phenotype. In these studies, we explore changes in erythrocyte mechanical properties caused by infection with Plasmodium falciparum and find that the elastic response alone fails to detect malaria at high frequencies. At timescales associated with rapid assays, however, we observe that the inelastic response shows significant changes and can be used as a reliable indicator of infection, establishing the dynamic viscoelasticity as a basis for nondestructive mechanical analogs of current high-throughput cell classification methods.

A role for the membrane proteome in human chronic kidney disease erythrocytes

The molecular basis of the reduced half-life of chronic kidney disease (CKD) erythrocytes is unclear. The erythrocyte membrane plays a key role in the erythrocyte mechanical properties and survival. The aim of the present work is to uncover erythrocyte membrane proteins whose expression could be altered in CKD. The erythrocyte membrane subproteome was analyzed by a non-biased approach where the whole set of proteins was simultaneously investigated by 2D fluorescence difference gel electrophoresis without preselection of potential targets. Proteins significantly altered in CKD were identified by mass spectrometry (MS) and results validation was performed by Western blot and confocal microscopy. Nine differentially expressed spots among healthy individuals, non-dialyzed CKD and erythropoietin/dialysis-treated CKD patients were identified by MS/MS corresponding to 5 proteins (beta-adducin, HSP71/72, tropomodulin-1, ezrin, and radixin). Ezrin and radixin were higher in dialyzed CKD patients than in the other 2 groups. Beta-adducin was increased in CKD patients (dialyzed or not). Three spots were normalized in patients on the dialysis/erythropoietin combination compared with non-dialyzed CKD. Among these, a spot corresponding to tropomodulin 1, was found to be of higher abundance in non-dialyzed CKD patients compared with controls or dialyzed CKD. In conclusion, this study identifies changes in erythrocyte membrane proteins in CKD, which may be relevant for the pathogenesis of red cell abnormalities in uremia.

Flickering Analysis of Erythrocyte Mechanical Properties: Dependence on Oxygenation Level, Cell Shape, and Hydration Level

Erythrocytes (red blood cells) play an essential role in the respiratory functions of vertebrates, carrying oxygen from lungs to tissues and CO 2 from tissues to lungs. They are mechanically very soft, enabling circulation through small capillaries. The small thermally induced displacements of the membrane provide an important tool in the investigation of the mechanics of the cell membrane. However, despite numerous studies, uncertainties in the interpretation of the data, and in the values derived for the main parameters of cell mechanics, have rendered past conclusions from the fluctuation approach somewhat controversial. Here we revisit the experimental method and theoretical analysis of fluctuations, to adapt them to the case of cell contour fluctuations, which are readily observable experimentally. This enables direct measurements of membrane tension, of bending modulus, and of the viscosity of the cell cytoplasm. Of the various factors that influence the mechanical properties of the cell, we focus here on: 1), the level of oxygenation, as monitored by Raman spectrometry; 2), cell shape; and 3), the concentration of hemoglobin. The results show that, contrary to previous reports, there is no significant difference in cell tension and bending modulus between oxygenated and deoxygenated states, in line with the softness requirement for optimal circulatory flow in both states. On the other hand, tension and bending moduli of discocyte- and spherocyte-shaped cells differ markedly, in both the oxygenated and deoxygenated states. The tension in spherocytes is much higher, consistent with recent theoretical models that describe the transitions between red blood cell shapes as a function of membrane tension. Cell cytoplasmic viscosity is strongly influenced by the hydration state. The implications of these results to circulatory flow dynamics in physiological and pathological conditions are discussed.

Influence of tourniquet application on venous blood sampling for serum chemistry, hematological parameters, leukocyte activation and erythrocyte mechanical properties

Venous blood sampling is usually performed using a tourniquet to help locate and define peripheral veins to achieve successful and safe venipuncture. Despite widespread usage of tourniquets for venipuncture by medical and laboratory staff, very few are aware of the effects of tourniquet application on laboratory parameters. In addition, definitive guidelines regarding when and how to use a tourniquet for blood sampling are lacking. The aim of the present study was to define the optimal sampling time after tourniquet removal to avoid adverse impact on laboratory analytes. Blood oxygen and carbon dioxide partial pressure, pH, oxyhemoglobin saturation (satO(2)), hematological parameters, serum electrolyte concentrations, erythrocyte, deformability and aggregation, leukocyte activation and nitrite/nitrate concentrations obtained 180 s after tourniquet release were compared with baseline values for 10 healthy subjects. Blood gases, hematological parameters and serum electrolyte levels were not affected by the application and removal of a tourniquet. However, there were significant decreases in erythrocyte deformability at 90, 120, 180 s, and increases in erythrocyte aggregation at 5 and 30 s following removal of the tourniquet. A significant increase in granulocyte respiratory burst at 60 s was observed, confirming leukocyte activation due to application of the tourniquet. There were no significant alterations of blood nitrite/nitrate levels. Our blood sampling technique which mimicked the application and release of a tourniquet indicated unaltered values for routine blood gases, hematological testing and serum electrolyte levels. Conversely, hemorheological measurements can be affected. Therefore, it is strongly recommended that tourniquet application should be avoided during blood sampling or, if this is not possible, the procedure should be well standardized and details of the sampling method should be reported.

Spectroscopical and mechanical characterization of normal and thalassemic red blood cells by Raman Tweezers

In this work, the effects of thalassemia, a blood disease quite diffuse in the Mediterranean sea region, have been investigated at single cell level using a Raman Tweezers system. By resonant excitation of hemoglobin Raman bands, we have examined the oxygenation capability of beta-thalassemic erythrocytes. A reduction of this fundamental erythrocyte function has been found. The measurements have been performed on a significant number of red blood cells; the relative statistical analysis is presented. Moreover, the response to photo-induced oxidative stress of diseased cells with respect to the normal ones has been analyzed. Finally, the deformability of thalassemic erythrocytes has been quantified by measuring the membrane shear modulus by using a double-trap system: the measurements have revealed an increase in membrane rigidity of more than 40%, giving evidence that the genetic defect associated to thalassemia, which manly relies on hemoglobin structure, also strongly affects the erythrocyte mechanical properties. Our results demonstrate that the developed set-up may have potential for the monitoring of blood diseases and their response to drug therapies.

Short-term effects of atorvastatin on hemorheologic parameters and endothelial dysfunction in patients with hypercholesterolemia. Lower is better?

We have read with interest the paper published recently by Uyuklu et al. in Clinical Hemorheology and Microcirculation [5]. It provides important experimental information on the effect of high dose atorvastatin on erythrocyte mechanical properties. Numerous randomized trials confirmed the benefit of cholesterol-lowering therapy in patients with atherosclerotic cardiovascular diseases (ASCVD). In high-risk persons, the recommended LDL-C goal now is <100 mg/dl, but a question raised by HPS and PROVE IT studies is whether this goal is sufficiently low in very high-risk patients who already have relatively low LDL cholesterol at baseline. In HPS the subgroup whose LDL-C levels at baseline were <100 mg/dl exhibited significant risk reduction when statin therapy was introduced [3]. In PROVE IT, intensive LDL-C-lowering therapy with high dose atorvastatin reduced major cardiovascular events as compared with standard-dose pravastatin [1]. Based on the available clinical trial evidence, an LDL-C goal of <70 mg/dl is a reasonable clinical strategy when risk is very high [2]. Since cholesterol is one of the basic elements of the cell membranes, Uyuklu et al. addressed the question, whether lowering of cholesterol beyond a certain level – with supra-pharmacological dose of atorvastatin – may cause significant alterations in red blood cell (RBC) deformability. In their study they found no adverse rheological change, despite significant structural and functional alterations of the RBC membrane.

Structural and mechanical characteristics of erythrocyte membranes in patients with type 2 diabetes mellitus

The morphology and structure of erythrocyte cytoskeleton and local mechanical characteristics in patients with type 2 diabetes mellitus and donors were studied by atomic force microscopy. Poikilocytosis and anisocytosis, spatial reorganization of the cytoskeleton (loosening and condensation of the actin-spectrin network), and modification of local mechanical properties of erythrocytes were characteristic of diabetics. These results indicate significant heterogeneity of erythrocyte population in the patients, most likely due to the presence of erythrocytes of different age groups, which can promote the development of diabetes complications (angiopathy).

The Deformation of an Erythrocyte Under the Radiation Pressure by Optical Stretch

In this paper, the mechanical properties of erythrocytes were studied numerically based upon the mechanical model originally developed by Pamplona and Calladine (ASME J. Biomech. Eng., 115, p. 149, 1993) for liposomes. The case under study is the erythrocyte stretched by a pair of laser beams in opposite directions within buffer solutions. The study aims to elucidate the effect of radiation pressure from the optical laser because up to now little is known about its influence on the cell deformation. Following an earlier study by Guck et al. (Phys. Rev. Lett., 84, p. 5451, 2000; Biophys. J., 81, p. 767, 2001), the empirical results of the radiation pressure were introduced and imposed on the cell surface to simulate the real experimental situation. In addition, an algorithm is specially designed to implement the simulation. For better understanding of the radiation pressure on the cell deformation, a large number of simulations were conducted for different properties of cell membrane. Results are first discussed parametrically and then evaluated by comparing with the experimental data reported by Guck et al. An optimization approach through minimizing the errors between experimental and numerical data is used to determine the optimal values of membrane properties. The results showed that an average shear stiffness around 4.611x10-6 Nm(-1), when the nondimensional ratio of shear modulus to bending modulus ranges from 10 to 300. These values are in a good agreement with those reported in literature.

Headpiece domain of dematin is required for the stability of the erythrocyte membrane.

Dematin is an actin-binding and bundling protein of the erythrocyte membrane skeleton. Dematin is localized to the spectrin-actin junctions, and its actin-bundling activity is regulated by phosphorylation of cAMP-dependent protein kinase. The carboxyl terminus of dematin is homologous to the "headpiece" domain of villin, an actin-bundling protein of the microvillus cytoskeleton. The headpiece domain contains an actin-binding site, a cAMP-kinase phosphorylation site, plays an essential role in dematin self-assembly, and bundles F-actin in vitro. By using homologous recombination in mouse embryonic stem cells, the headpiece domain of dematin was deleted to evaluate its function in vivo. Dematin headpiece null mice were viable and born at the expected Mendelian ratio. Hematological evaluation revealed evidence of compensated anemia and spherocytosis in the dematin headpiece null mice. The headpiece null erythrocytes were osmotically fragile, and ektacytometry/micropore filtration measurements demonstrated reduced deformability and filterability. In vitro membrane stability measurements indicated significantly greater membrane fragmentation of the dematin headpiece null erythrocytes. Finally, biochemical characterization, including the vesicle/cytoskeleton dissociation, spectrin self-association, and chemical crosslinking measurements, revealed a weakened membrane skeleton evidenced by reduced association of spectrin and actin to the plasma membrane. Together, these results provide evidence for the physiological significance of dematin and demonstrate a role for the headpiece domain in the maintenance of structural integrity and mechanical properties of erythrocytes in vivo.

Physiological activity of some organophosphorous compounds and their influence on mechanical properties of erythrocytes.

Hemolysis and fluidization of erythrocytes (RBC) membranes by some newly synthesized aminophosphonates as well as their potency to induce electrolyte efflux from cucumber (Cucumis sativus cv "Wisconsin") cotyledons were studied. Also, the chlorophyll content in aminophosphonate-treated cotyledons was affected. The compounds studied differed mainly in hydrophobicity of their substituents at the carbon, nitrogen and phosphorus atoms. It was found that aminophosphonate potency to fluidize RBC membranes depended on the combination of its overall lipophilicity and/or the kind of substituent at the P atom. Especially, iso-propyl groups enhanced that potency. The sequence of aminophosphonates that exhibited the strongest fluidization activity was paralelled by their physiological and hemolytic activities; in the latter case for these compounds that hemolyzed RBC under used concentrations. A general conclusion is that both the stereochemistry and lipophilicity determine the efficiency of the aminophosphonates studied. This efficiency is most probably related to the interaction of aminophosphonates with the lipid phase of biological objects.

Mild spherocytic hereditary elliptocytosis and altered levels of α- and γ-adducins in β-adducin-deficient mice

AbstractThe membrane skeleton, a dynamic network of proteins associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Deficiencies or defects in membrane skeletal proteins are associated with inherited disorders of erythrocyte morphology and function. Adducin is one of the proteins localized at the spectrin-actin junction of the membrane skeleton. In this work we show that deficiency of β-adducin produces an 80% decrease of α-adducin and a fourfold up-regulation of γ-adducin in erythrocytes. β-Adducin or any other isoform generated by translation of abnormally spliced messenger RNAs could not be detected by our antibodies either in ghosts or in cytoplasm of −/− erythrocytes. Actin levels were diminished in mutant mice, suggesting alterations in the actin-spectrin junctional complexes due to the absence of adducin. Elliptocytes, ovalocytes, and occasionally spherocytes were found in the blood film of −/− mice. Hematological values showed an increase in reticulocyte counts and mean corpuscular hemoglobin concentration, decreased mean corpuscular volume and hematocrit, and normal erythrocyte counts that, associated to splenomegaly, indicate that the mice suffer from mild anemia with compensated hemolysis. These modifications are due to a loss of membrane surface and dehydration that result in an increase in the osmotic fragility of red blood cells. The marked alteration in osmotic fragility together with the predominant presence of elliptocytes is reminiscent of the human disorder called spherocytic hereditary elliptocytosis. Our results suggest that the amount of adducin remaining in the mutant animals (presumably αγ adducin) could be functional and might account for the mild phenotype.

Mild spherocytic hereditary elliptocytosis and altered levels of α- and γ-adducins in β-adducin-deficient mice

The membrane skeleton, a dynamic network of proteins associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Deficiencies or defects in membrane skeletal proteins are associated with inherited disorders of erythrocyte morphology and function. Adducin is one of the proteins localized at the spectrin-actin junction of the membrane skeleton. In this work we show that deficiency of β-adducin produces an 80% decrease of -adducin and a fourfold up-regulation of γ-adducin in erythrocytes. β-Adducin or any other isoform generated by translation of abnormally spliced messenger RNAs could not be detected by our antibodies either in ghosts or in cytoplasm of −/− erythrocytes. Actin levels were diminished in mutant mice, suggesting alterations in the actin-spectrin junctional complexes due to the absence of adducin. Elliptocytes, ovalocytes, and occasionally spherocytes were found in the blood film of −/− mice. Hematological values showed an increase in reticulocyte counts and mean corpuscular hemoglobin concentration, decreased mean corpuscular volume and hematocrit, and normal erythrocyte counts that, associated to splenomegaly, indicate that the mice suffer from mild anemia with compensated hemolysis. These modifications are due to a loss of membrane surface and dehydration that result in an increase in the osmotic fragility of red blood cells. The marked alteration in osmotic fragility together with the predominant presence of elliptocytes is reminiscent of the human disorder called spherocytic hereditary elliptocytosis. Our results suggest that the amount of adducin remaining in the mutant animals (presumably γ adducin) could be functional and might account for the mild phenotype.