Mechanical Properties Of Red Blood Cells Research Articles

High-throughput single-cell assay for precise measurement of the intrinsic mechanical properties and shape characteristics of red blood cells.

The intrinsic physical and mechanical properties of red blood cells (RBCs), including their geometric and rheological characteristics, can undergo changes in various circulatory and metabolic diseases. However, clinical diagnosis using RBC biophysical phenotypes remains impractical due to the unique biconcave shape, remarkable deformability, and high heterogeneity within different subpopulations. Here, we combine the hydrodynamic mechanisms of fluid-cell interactions in micro circular tubes with a machine learning method to develop a relatively high-throughput microfluidic technology that can accurately measure the shear modulus of the membrane, viscosity, surface area, and volume of individual RBCs. The present method can detect the subtle changes of mechanical properties in various RBC components at continuum scales in response to different doses of cytoskeletal drugs. We also investigate the correlation between glycosylated hemoglobin and RBC mechanical properties. Our study develops a methodology that combines microfluidic technology and machine learning to explore the material properties of cells based on fluid-cell interactions. This approach holds promise in offering novel label-free single-cell-assay-based biophysical markers for RBCs, thereby enhancing the potential for more robust disease diagnosis.

MODELING A RED BLOOD CELL CYTOSKELETON: INSIGHTS AND TIPS

The aim of this work is to provide a clear and ready-to-implement mathematical model to describe the red blood cell (RBC) cytoskeleton. The motivation for this methodo- logy work lies in the lack of available sources facilitating the implementation of computational (in silico) modelling of RBC mechanical properties. Our approach adopts a worm-like chain force model to mimic an individual spectrin deformation and the virtual work principle to extrapolate the spectrin network dynamics. We report modelling tips using coarse-grained scaling techniques and optical tweezers experiments. We validate the model in virtual expe- riments gaining insights on single spectrin equilibrium, the impact of cytoskeletal symmetry defects on system behaviour, and stress transmission and distribution within the network. By performing these virtual experiments, we elucidate the role that the cytoskeleton plays in de- termining the remarkable RBC mechanical behaviour. These findings emphasise the need to consider the cytoskeleton component in the latest and most advanced single-cell mathematical models.

Red blood cell rheology during a complete blood count: A proof of concept.

Counting and sizing blood cells in hematological analyzers is achieved using the Coulter principle. The cells flow in a micro-aperture in which a strong electrical field is imposed, so that an electrical perturbation, called pulse, is measured each time a cell crosses the orifice. The pulses are expected to contain information on the shape and deformability of Red Blood Cells (RBCs), since recent studies state that RBCs rotate and deform in the micro-orifice. By implementing a dedicated numerical model, the present study sheds light on a variety of cells dynamics, which leads to different associated pulse signatures. Furthermore, simulations provide new insights on how RBCs shapes and mechanical properties affect the measured signals. Those numerical observations are confirmed by experimental assays. Finally, specific features are introduced for assessing the most relevant characteristics from the various pulse signatures and shown to highlight RBCs alterations induced by drugs. In summary, this study paves the way to a characterization of RBC rheology by routine hematological instruments.

Vitamin E-coated dialyzer alleviates erythrocyte deformability dysfunction in patients with end-stage renal disease undergoing hemodialysis.

Patients with end-stage renal disease (ESRD) are characterized by augmented oxidative stress (OS) due to the imbalance between the generation of increased concentrations of oxidative molecules and decreased antioxidant capacity. Vitamin E-coated dialyzer membranes (VEMs) have previously been reported to alleviate the imbalance of redox metabolism in patients with ESRD undergoing hemodialysis (HD); however, their effect on the deformability of red blood cells (RBCs) remains unknown. In the present study, 48 patients with ESRD undergoing HD were enrolled and randomly assigned into two groups: HD with VEMs (VEM group; n=24) and HD with polysulfone dialyzer membranes (PM group; n=24), and another 24 healthy volunteers served as the control group. The present study investigated the morphological changes and deformability of RBCs in patients with ESRD and healthy volunteers. The concentration of serum vitamin E, the parameters of antioxidant stress and OS, and the degree of oxidative phosphorylation and clustering of anion exchanger 1 (Band 3) in RBCs were measured. The results obtained suggested that VEM treatment markedly ameliorated the abnormalities of RBC morphology and deformability in patients with ESRD undergoing HD. Mechanistic studies showed that VEM treatment led to a marked improvement in the concentration of serum vitamin E, which was positively associated with the restored antioxidant capacity, and decreased oxidative phosphorylation and clustering of Band 3 in RBCs of patients with ESRD undergoing HD. Taken together, the results of the present study have demonstrated that VEM treatment effectively restored the imbalance of redox metabolism, and improved the oxidative phosphorylation and clustering of Band 3 in RBCs of patients with ESRD undergoing HD via delivering vitamin E, which may alleviate the abnormal morphological and mechanical properties of RBCs. These findings are anticipated to be useful with respect to improving the nursing care and cure rate of patients with ESRD.

Dynamics of Individual Red Blood Cells Under Shear Flow: A Way to Discriminate Deformability Alterations.

In this work, we compared the dynamics of motion in a linear shear flow of individual red blood cells (RBCs) from healthy and pathological donors (Sickle Cell Disease (SCD) or Sickle Cell-β-thalassemia) and of low and high densities, in a suspending medium of higher viscosity. In these conditions, at lower shear rates, biconcave discocyte-shaped RBCs present an unsteady flip-flopping motion, where the cell axis of symmetry rotates in the shear plane, rocking to and fro between an orbital angle ±ϕ observed when the cell is on its edge. We show that the evolution of ϕ depends solely on RBC density for healthy RBCs, with denser RBCs displaying lower ϕ values than the lighter ones. Typically, at a shear stress of 0.08 Pa, ϕ has values of 82 and 72° for RBCs with average densities of 1.097 and 1.115, respectively. Surprisingly, we show that SCD RBCs display the same ϕ-evolution as healthy RBCs of same density, showing that the flip-flopping behavior is unaffected by the SCD pathology. When the shear stress is increased further (above 0.1 Pa), healthy RBCs start going through a transition to a fluid-like motion, called tank-treading, where the RBC has a quasi-constant orientation relatively to the flow and the membrane rotates around the center of mass of the cell. This transition occurs at higher shear stresses (above 0.2 Pa) for denser cells. This shift toward higher stresses is even more remarkable in the case of SCD RBCs, showing that the transition to the tank-treading regime is highly dependent on the SCD pathology. Indeed, at a shear stress of 0.2 Pa, for RBCs with a density of 1.097, 100% of healthy RBCs have transited to the tank-treading regime vs. less than 50% SCD RBCs. We correlate the observed differences in dynamics to the alterations of RBC mechanical properties with regard to density and SCD pathology reported in the literature. Our results suggest that it might be possible to develop simple non-invasive assays for diagnosis purpose based on the RBC motion in shear flow and relying on this millifluidic approach.

Impact of Liposomal Drug Formulations on the RBCs Shape, Transmembrane Potential, and Mechanical Properties.

Liposomal technologies are used in order to improve the effectiveness of current therapies or to reduce their negative side effects. However, the liposome–erythrocyte interaction during the intravenous administration of liposomal drug formulations may result in changes within the red blood cells (RBCs). In this study, it was shown that phosphatidylcholine-composed liposomal formulations of Photolon, used as a drug model, significantly influences the transmembrane potential, stiffness, as well as the shape of RBCs. These changes caused decreasing the number of stomatocytes and irregular shapes proportion within the cells exposed to liposomes. Thus, the reduction of anisocytosis was observed. Therefore, some nanodrugs in phosphatidylcholine liposomal formulation may have a beneficial effect on the survival time of erythrocytes.

Calcium/protein kinase C signaling mechanisms in shear-induced mechanical responses of red blood cells

Red blood cell (RBC) deformability has vital importance for microcirculation in the body, as RBCs travel in narrow capillaries under shear stress. Deformability can be defined as a remarkable cell ability to change shape in response to an external force which allows the cell to pass through the narrowest blood capillaries. Previous studies showed that RBC deformability could be regulated by Ca2+/protein kinase C (PKC) signaling mechanisms due to the phosphorylative changes in RBC membrane proteins by kinases and phosphatases. We investigated the roles of Ca2+/PKC signaling pathway on RBC mechanical responses and impaired RBC deformability under continuous shear stress (SS). A protein kinase C inhibitor Chelerythrine, a tyrosine phosphatase inhibitor Calpeptin, and a calcium channel blocker Verapamil were applied into human blood samples in 1 micromolar concentration. Samples with drugs were treated with or without 3 mM Ca2+. A shear stress at 5 Pa level was applied to each sample continuously for 300 s. RBC deformability was measured by a laser-assisted optical rotational cell analyzer (LORRCA) and was calculated as the change in elongation index (EI) of RBC upon a range of shear stress (SS, 0.3–50 Pa). RBC mechanical stress responses were evaluated before and after continuous SS through the parameterization of EI-SS curves. The drug administrations did not produce any significant alterations in RBC mechanical responses when they were applied alone. However, the application of the drugs together with Ca2+ substantially increased RBC deformability compared to calcium alone. Verapamil significantly improved Ca2+-induced impairments of deformability both before and after 5 Pa SS exposure (p < 0.0001). Calpeptin and Chelerythrine significantly ameliorated impaired deformability only after continuous SS (p < 0.05). Shear-induced improvements of deformability were conserved by the drug administrations although shear-induced deformability was impaired when the drugs were applied with calcium. The blocking of Ca2+ channel by Verapamil improved impaired RBC mechanical responses independent of the SS effect. The inhibition of tyrosine phosphatase and protein kinase C by Calpeptin and Chelerythrine, respectively, exhibited ameliorating effects on calcium-impaired deformability with the contribution of shear stress. The modulation of Ca2+/PKC signaling pathway could regulate the mechanical stress responses of RBCs when cells are under continuous SS exposure. Shear-induced improvements in the mechanical properties of RBCs by this signaling mechanism could facilitate RBC flow in the microcirculation of pathophysiological disorders, wherein Ca2+ homeostasis is disturbed and RBC deformability is reduced.

Targeting spectrin redox switches to regulate the mechanoproperties of red blood cells.

The mechanical properties of red blood cells (RBCs) are fundamental for their physiological role as gas transporters. RBC flexibility and elasticity allow them to survive the hemodynamic changes in the different regions of the vascular tree, to dynamically contribute to the flow thereby decreasing vascular resistance, and to deform during the passage through narrower vessels. RBC mechanoproperties are conferred mainly by the structural characteristics of their cytoskeleton, which consists predominantly of a spectrin scaffold connected to the membrane via nodes of actin, ankyrin and adducin. Changes in redox state and treatment with thiol-targeting molecules decrease the deformability of RBCs and affect the structure and stability of the spectrin cytoskeleton, indicating that the spectrin cytoskeleton may contain redox switches. In this perspective review, we revise current knowledge about the structural and functional characterization of spectrin cysteine redox switches and discuss the current lines of research aiming to understand the role of redox regulation on RBC mechanical properties. These studies may provide novel functional targets to modulate RBC function, blood viscosity and flow, and tissue perfusion in disease conditions.

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.

What determines blood viscosity at the highest city in the world?

What determines blood viscosity at the highest city in the world?

Calcium dynamically alters erythrocyte mechanical response to shear

Red blood cells (RBC) are constantly exposed to varying mechanical forces while traversing the cardiovascular system. Upon exposure to mechanical stimuli (e.g., shear stress), calcium enters the cell and prompts potassium-efflux. Efflux of potassium is accompanied by a loss of intracellular fluid; thus, the volume of RBC decreases proportionately (i.e., ‘Gárdos effect’). The mechanical properties of the cell are subsequently impacted due to complex interactions between cytosolic viscosity (dependent on cell hydration), the surface-area-to-volume ratio, and other molecular processes. The dynamic effects of calcium on RBC mechanics are yet to be elucidated, although accumulating evidence suggests a vital role. The present study thus examined the effects of calcium on contemporary biomechanical properties of RBC in conjunction with high-precision geometrical analyses with exposure to shear. Mechanical stimulation of RBC was performed using a co-axial Couette shearing system to deform the cell membrane; intracellular signaling events were observed via fluorescent imaging. Calcium was introduced into RBC using ionophore A23187. Increased intracellular calcium significantly impaired RBC deformability; these impairments were mediated by a calcium-induced reduction of cell volume through the Gárdos channel. Extracellular calcium in the absence of the ionophore only had an effect under shear, not at stasis. Under low shear, the presence of extracellular calcium induced progressive lysis of a sub-population of RBC; all remaining RBC exhibited exceptional capacity to deform, implying preferential removal of potentially aged cells. Collectively, we provide evidence of the mechanism by which calcium acutely regulates RBC mechanical properties.

Mechanical Signature of Red Blood Cells Flowing Out of a Microfluidic Constriction Is Impacted by Membrane Elasticity, Cell Surface-to-Volume Ratio and Diseases.

Despite the fact that Red Blood Cells (RBCs) have been intensively studied in the past 50 years to characterize mechanical phenotypes associated with both healthy and pathological states, only ektacytometry (i.e., laser diffractometry) is currently used by hematologists to screen for RBC membrane disorders. Therefore, the development of new diagnostic tools able to perform analysis at the scale of a single cell, over a statistically relevant population, would provide important complementary information. But these new diagnostic tools would have to be able to discriminate between different disorders causing a change in RBCs mechanical properties. We evaluated the mechanical response of artificially rigidified RBCs flowing through a microfluidic constriction. The geometry consists in a 50 μm wide channel with a succession of 14 tooth-like patterns, each composed of a 5 μm wide and 10 μm long constriction, associated with a 25 μm wide and 10 μm long enlargement. RBCs deformability was altered using two chemical treatments, known to affect RBCs membrane surface area and membrane deformability, lysolecithine (LPC) and diamide, respectively. Differences between samples were highlighted by the representation of the inverse of the shape recovery time (1/τr), versus the extension at the exit of the constriction, Dout. The results demonstrate that our approach is able to provide a direct signature of RBCs membrane composition and architecture, as it allows discriminating the effect of changes in RBCs membrane surface area from changes in RBCs membrane deformability. Finally, in order to evaluate the potential of our microsystem to detect pathological cells, we have performed preliminary experiments on patients with Hereditary Spherocytosis (HS) or Sickle Cell Anemia (SCA).

High-Throughput Microfluidic Characterization of Erythrocyte Shapes and Mechanical Variability

The motion of red blood cells (RBCs) in microchannels is important for microvascular blood flow and biomedical applications such as blood analysis in microfluidics. The current understanding of the complexity of RBC shapes and dynamics in microchannels is mainly based on several simulation studies, but there are a few systematic experimental investigations. Here, we present a combined study that systematically characterizes RBC behavior for a wide range of flow rates and channel sizes. Even though simulations and experiments generally show good agreement, experimental observations demonstrate that there is no single well-defined RBC state for fixed flow conditions but rather a broad distribution of states. This result can be attributed to the inherent variability in RBC mechanical properties, which is confirmed by a model that takes the variation in RBC shear elasticity into account. This represents a significant step toward a quantitative connection between RBC behavior in microfluidic devices and their mechanical properties, which is essential for a high-throughput characterization of diseased cells.

Deformation of a Red Blood Cell in a Narrow Rectangular Microchannel.

The deformability of a red blood cell (RBC) is one of the most important biological parameters affecting blood flow, both in large arteries and in the microcirculation, and hence it can be used to quantify the cell state. Despite numerous studies on the mechanical properties of RBCs, including cell rigidity, much is still unknown about the relationship between deformability and the configuration of flowing cells, especially in a confined rectangular channel. Recent computer simulation techniques have successfully been used to investigate the detailed behavior of RBCs in a channel, but the dynamics of a translating RBC in a narrow rectangular microchannel have not yet been fully understood. In this study, we numerically investigated the behavior of RBCs flowing at different velocities in a narrow rectangular microchannel that mimicked a microfluidic device. The problem is characterized by the capillary number , which is the ratio between the fluid viscous force and the membrane elastic force. We found that confined RBCs in a narrow rectangular microchannel maintained a nearly unchanged biconcave shape at low , then assumed an asymmetrical slipper shape at moderate , and finally attained a symmetrical parachute shape at high . Once a RBC deformed into one of these shapes, it was maintained as the final stable configurations. Since the slipper shape was only found at moderate , measuring configurations of flowing cells will be helpful to quantify the cell state.

Mechanical properties of RBCs under oxidative stress measured by optical tweezers

Mechanical characterization of human red blood cells (RBC) is crucial to the study of the pathology, drug screening, clinical diagnosis, and treatment of Parkinson’s disease. Oxidative stress has been found to be a dominant factor among the causes of Parkinson’s disease. This paper proposes a method for measuring cell mechanical properties using optical tweezers. By measuring the membrane shear modulus of RBCs treated with hydrogen peroxide (H2O2) at different concentrations, mechanical properties of RBCs under oxidative stress were analyzed. The experimental results reveal that increased membrane shear modulus and decreased cell deformability are commensurate with increasing concentrations of H2O2. This proposal will benefit both basic research and clinical applications in the field of Parkinson’s disease research. The results will also provide a guideline for measuring the mechanical properties of other cells.

ATP Release by Red Blood Cells under Flow: Model and Simulations

ATP is a major player as a signaling molecule in blood microcirculation. It is released by red blood cells (RBCs) when they are subjected to shear stresses large enough to induce a sufficient shape deformation. This prominent feature of chemical response to shear stress and RBC deformation constitutes an important link between vessel geometry, flow conditions, and the mechanical properties of RBCs, which are all contributing factors affecting the chemical signals in the process of vasomotor modulation of the precapillary vessel networks. Several in vitro experiments have reported on ATP release by RBCs due to mechanical stress. These studies have considered both intact RBCs as well as cells within which suspected pathways of ATP release have been inhibited. This has provided profound insights to help elucidate the basic governing key elements, yet how the ATP release process takes place in the (intermediate) microcirculation zone is not well understood. We propose here an analytical model of ATP release. The ATP concentration is coupled in a consistent way to RBC dynamics. The release of ATP, or the lack thereof, is assumed to depend on both the local shear stress and the shape change of the membrane. The full chemo-mechanical coupling problem is written in a lattice-Boltzmann formulation and solved numerically in different geometries (straight channels and bifurcations mimicking vessel networks) and under two kinds of imposed flows (shear and Poiseuille flows). Our model remarkably reproduces existing experimental results. It also pinpoints the major contribution of ATP release when cells traverse network bifurcations. This study may aid in further identifying the interplay between mechanical properties and chemical signaling processes involved in blood microcirculation.

Numerical Simulation of Tank-Treading and Tumbling Motion of Red Blood Cell in the Poiseuille Flow in a Microchannel With and Without Obstacle

In the present study, the dynamics of a red blood cell (RBC) in a simple microchannel and in a microchannel with obstacle is simulated using combined lattice Boltzmann-immersed boundary method. The fluid flow field is solved for using LBM and the interaction between the fluid and the RBC is simulated using the IBM. The RBC is considered as a deformable boundary immersed in the fluid flow. When the RBC is stiffer, the flow passage is further blocked due to the lack of flexibility of the RBC causing the flow velocity to decrease and greater drag force (resistance force against the motion of cell) from the fluid to be exerted on it. As a result, the pressure around the RBC increases and becomes even higher than the inlet pressure of microchannel. This increased pressure is thought to be the reason of many serious diseases including cardiovascular diseases. If the number of RBCs used in the simulation increases, the pressure increase would be more appreciable. In addition, the high-flexibility RBC experienced tank-treading motion due to its low elastic and bending coefficients whereas for the low-flexibility RBC the tumbling motion took place. The mechanical properties of RBCs are the determining factor in the distribution of hematocrit in microcirculation. The current results showed good agreement with the available results.

Investigation of red blood cell mechanical properties using AFM indentation and coarse-grained particle method

BackgroundRed blood cells (RBCs) deform significantly and repeatedly when passing through narrow capillaries and delivering dioxygen throughout the body. Deformability of RBCs is a key characteristic, largely governed by the mechanical properties of the cell membrane. This study investigated RBC mechanical properties using atomic force microscopy (AFM) with the aim to develop a coarse-grained particle method model to study for the first time RBC indentation in both 2D and 3D. This new model has the potential to be applied to further investigate the local deformability of RBCs, with accurate control over adhesion, probe geometry and position of applied force.ResultsThe model considers the linear stretch capacity of the cytoskeleton, bending resistance and areal incompressibility of the bilayer, and volumetric incompressibility of the internal fluid. The model’s performance was validated against force–deformation experiments performed on RBCs under spherical AFM indentation. The model was then used to investigate the mechanisms which absorbed energy through the indentation stroke, and the impact of varying stiffness coefficients on the measured deformability. This study found the membrane’s bending stiffness was most influential in controlling RBC physical behaviour for indentations of up to 200 nm.ConclusionsAs the bilayer provides bending resistance, this infers that structural changes within the bilayer are responsible for the deformability changes experienced by deteriorating RBCs. The numerical model presented here established a foundation for future investigations into changes within the membrane that cause differences in stiffness between healthy and deteriorating RBCs, which have already been measured experimentally with AFM.

Blood Quality Diagnostic Device Detects Storage Differences Between Donors.

In 2013, nearly 15 million units of banked blood were transfused in the United States of America alone. Blood shortages are expected to increase globally. Donated blood is not equal due to differences in quality and deterioration rate. There are no methods to detect time-dependent biochemical and biophysical changes of red blood cells (RBCs) or the deterioration rate of donated RBCs. Nine randomly selected RBC units collected by the San Diego Blood Bank were examined for interdonor variability over six weeks of storage. In vitro RBC quality was assessed weekly by conventional biochemical tests including free Hb, K+, ATP, P50, 2,3 DPG, lactate, and pH. Deformability was measured via cell filtration. Briefly, the RBC suspension (10% Hct), was forced through a 5.0-μm pore membrane (106 mm2) at various flow rates. No interdonor variability in biochemical or mechanical parameters was observed at baseline. Interdonor variability in biochemical properties (free Hb, K+, ATP, P50, 2,3 DPG, lactate, and pH) was observed after 14 days of storage. However, significant differences from baseline in RBC mechanical properties (i.e., filterability) were observed as early as 7 days into storage at the lowest flow rates and after 28 days of storage at all flow rates. There was a net decrease in filterability over time for all donors, but the rate at which filterability decreased (i.e., deterioration rates) was different when comparing individual donors. Changes in all biochemical parameters were significant different between donors. These data suggest that filterability is more sensitive to changes in blood quality than conventional biochemical parameters.

Stabilization of F-actin by tropomyosin isoforms regulates the morphology and mechanical behavior of red blood cells.

The short F-actins in the red blood cell (RBC) membrane skeleton are coated along their lengths by an equimolar combination of two tropomyosin isoforms, Tpm1.9 and Tpm3.1. We hypothesized that tropomyosin's ability to stabilize F-actin regulates RBC morphology and mechanical properties. To test this, we examined mice with a targeted deletion in alternatively spliced exon 9d of Tpm3 (Tpm3/9d-/- ), which leads to absence of Tpm3.1 in RBCs along with a compensatory increase in Tpm1.9 of sufficient magnitude to maintain normal total tropomyosin content. The isoform switch from Tpm1.9/Tpm3.1 to exclusively Tpm1.9 does not affect membrane skeleton composition but causes RBC F-actins to become hyperstable, based on decreased vulnerability to latrunculin-A-induced depolymerization. Unexpectedly, this isoform switch also leads to decreased association of Band 3 and glycophorin A with the membrane skeleton, suggesting that tropomyosin isoforms regulate the strength of F-actin-to-membrane linkages. Tpm3/9d-/- mice display a mild compensated anemia, in which RBCs have spherocytic morphology with increased osmotic fragility, reduced membrane deformability, and increased membrane stability. We conclude that RBC tropomyosin isoforms directly influence RBC physiology by regulating 1) the stability of the short F-actins in the membrane skeleton and 2) the strength of linkages between the membrane skeleton and transmembrane glycoproteins.