Red Blood Cell Membrane Structure Research Articles

Molecular diagnosis of hereditary hemolytic anemias: Recent updates

AbstractHereditary hemolytic anemia (HHA) is a heterogeneous group of disorders due to genetically caused defects in red blood cell membrane structure, enzymes, heme and globin synthesis, erythroid proliferation, and differentiation. Traditionally, the diagnostic process is complex and includes a plethora of tests from routine to highly specialized ones. The inclusion of molecular testing has significantly improved the diagnostic yield. The value of molecular testing is broader than just rendering the correct diagnosis, as it may also guide therapeutic decisions. As more molecular modalities become available for clinical use, it is imperative to understand their benefits and disadvantages pertaining to the HHA diagnostics. Re‐evaluation of the traditional diagnostic workflow may also bring forth additional benefits. This review focuses on the current state of molecular testing for HHA.

Impact of the Hydrolysis and Methanolysis of Bidesmosidic Chenopodium quinoa Saponins on Their Hemolytic Activity.

Saponins are specific metabolites abundantly present in plants and several marine animals. Their high cytotoxicity is associated with their membranolytic properties, i.e., their propensity to disrupt cell membranes upon incorporation. As such, saponins are highly attractive for numerous applications, provided the relation between their molecular structures and their biological activities is understood at the molecular level. In the present investigation, we focused on the bidesmosidic saponins extracted from the quinoa husk, whose saccharidic chains are appended on the aglycone via two different linkages, a glycosidic bond, and an ester function. The later position is sensitive to chemical modifications, such as hydrolysis and methanolysis. We prepared and characterized three sets of saponins using mass spectrometry: (i) bidesmosidic saponins directly extracted from the ground husk, (ii) monodesmosidic saponins with a carboxylic acid group, and (iii) monodesmosidic saponins with a methyl ester function. The impact of the structural modifications on the membranolytic activity of the saponins was assayed based on the determination of their hemolytic activity. The natural bidesmosidic saponins do not present any hemolytic activity even at the highest tested concentration (500 µg·mL−1). Hydrolyzed saponins already degrade erythrocytes at 20 µg·mL−1, whereas 100 µg·mL−1 of transesterified saponins is needed to induce detectable activity. The observation that monodesmosidic saponins, hydrolyzed or transesterified, are much more active against erythrocytes than the bidesmosidic ones confirms that bidesmosidic saponins are likely to be the dormant form of saponins in plants. Additionally, the observation that negatively charged saponins, i.e., the hydrolyzed ones, are more hemolytic than the neutral ones could be related to the red blood cell membrane structure.

Erythrocyte membrane-camouflaged gefitinib/albumin nanoparticles for tumor imaging and targeted therapy against lung cancer

Conventional chemotherapeutic drugs may cause serious side effects such as hepatotoxicity and renal toxicity due to lack of targeting, which affects therapy outcome and the prognosis of patients. Therefore, biomimetic nanoparticles with long blood circulation and active targeting have attracted increasing attention. In this work, we fabricated a biomimetic R-RBC@GEF-NPs nano-system by encapsulating gefitinib-loaded albumin nanoparticles (GEF-NPs) inside cRGD-modified red blood cell (RBC) membranes. The complete RBC membrane structure and membrane proteins enabled the NPs to escape phagocytosis by macrophages. In addition, the cRGD moiety significantly improved tumor cell targeting and uptake. R-RBC@GEF-NPs inhibited the growth of A549 cells in vitro in a dose- and time-dependent manner by inducing apoptosis and cell cycle arrest at the G1 phase. Likewise, the R-RBC@GEF-NPs also decreased tumor weight and volume in the mice injected with A549 cells and prolonged survival time. In addition, the 99Tc-labeled R-RBC@GEF-NPs selectively accumulated in the tumor tissues in vivo, and enabled real time tumor imaging. Finally, blood and histological analyses showed that R-RBC@GEF-NPs did not cause any obvious systemic toxicity. Taken together, the biomimetic R-RBC@GEF-NPs is a promising therapeutic formulation for the treatment of lung cancer.

The Role of RBC Oxidative Stress in Sickle Cell Disease: From the Molecular Basis to Pathologic Implications.

Sickle cell disease (SCD) is an inherited monogenic disorder and the most common severe hemoglobinopathy in the world. SCD is characterized by a point mutation in the β-globin gene, which results in hemoglobin (Hb) S production, leading to a variety of mechanistic and phenotypic changes within the sickle red blood cell (RBC). In SCD, the sickle RBCs are the root cause of the disease and they are a primary source of oxidative stress since sickle RBC redox state is compromised due to an imbalance between prooxidants and antioxidants. This imbalance in redox state is a result of a continuous production of reactive oxygen species (ROS) within the sickle RBC caused by the constant endogenous Hb autoxidation and NADPH oxidase activation, as well as by a deficiency in the antioxidant defense system. Accumulation of non-neutralized ROS within the sickle RBCs affects RBC membrane structure and function, leading to membrane integrity deficiency, low deformability, phosphatidylserine exposure, and release of micro-vesicles. These oxidative stress-associated RBC phenotypic modifications consequently evoke a myriad of physiological changes involved in multi-system manifestations. Thus, RBC oxidative stress in SCD can ultimately instigate major processes involved in organ damage. The critical role of the sickle RBC ROS production and its regulation in SCD pathophysiology are discussed here.

Oxidative Stress and Thrombosis during Aging: The Roles of Oxidative Stress in RBCs in Venous Thrombosis.

Mid-life stage adults are at higher risk of developing venous thrombosis (VT)/thromboembolism (VT/E). Aging is characterized by an overproduction of reactive oxygen species (ROS), which could evoke a series of physiological changes involved in thrombosis. Here, we focus on the critical role of ROS within the red blood cell (RBC) in initiating venous thrombosis during aging. Growing evidence has shifted our interest in the role of unjustifiably unvalued RBCs in blood coagulation. RBCs can be a major source of oxidative stress during aging, since RBC redox homeostasis is generally compromised due to the discrepancy between prooxidants and antioxidants. As a result, ROS accumulate within the RBC due to the constant endogenous hemoglobin (Hb) autoxidation and NADPH oxidase activation, and the uptake of extracellular ROS released by other cells in the circulation. The elevated RBC ROS level affects the RBC membrane structure and function, causing loss of membrane integrity, and decreased deformability. These changes impair RBC function in hemostasis and thrombosis, favoring a hypercoagulable state through enhanced RBC aggregation, RBC binding to endothelial cells affecting nitric oxide availability, RBC-induced platelet activation consequently modulating their activity, RBC interaction with and activation of coagulation factors, increased RBC phosphatidylserine exposure and release of microvesicles, accelerated aging and hemolysis. Thus, RBC oxidative stress during aging typifies an ultimate mechanism in system failure, which can affect major processes involved in the development of venous thrombosis in a variety of ways. The reevaluated concept of the critical role of RBC ROS in the activation of thrombotic events during aging will help identify potential targets for novel strategies to prevent/reduce the risk for VT/E or VT/E recurrences in mid-life stage adults.

The stabilizing effect of an oligomeric proanthocyanidin on red blood cell membrane structure of poorly controlled Type II diabetes

Type II diabetes (T2D) is a pandemic characterized by pathological circulating inflammatory markers, high-glucose levels and oxidative stress. The hematological system is especially vulnerable to these aberrant circulating molecules, and erythrocytes (RBCs) show aberrant rheology properties, owing to the direct contact with these molecules. Pathological levels of circulating inflammatory markers in T2D therefore have a direct effect on the molecular and cellular structure of RBCs. Previous research has suggested that antioxidants may reduce oxidative stress that results from the pathological inflammatory markers. Particularly, polyphenol antioxidants like oligomeric proanthocyanidins (OPCs) may act as a hydroxyl mopping agent, and may have a positive effect on the deformability and membrane protein structure of RBCs from T2D. In this paper, we look at the effect of one such agent, Pinus massoniana bark extract (standardized to 95% oligomeric proanthicyanidins), on the RBC membrane structures and RBC shape changes of T2D, after laboratory exposure at physiological levels. Our methods of choice were atomic force microscopy and scanning electron microscopy to study RBC elasticity and ultrastructure. Results showed that in our hands, this OPC could change both the eryptotic nature of the RBCs, as viewed with scanning electron microscopy, as well as the elasticity. We found a significant difference in variation between the elasticity measurement values between the RBCs before and after OPC exposure (P-value <0.0001). In conclusion, the data from both these techniques therefore suggest that OPC usage might contribute to the improvement of RBC functioning.

Hemin_Induced Changes in the Red Blood Cell Membrane Structure

Objective: to determine whether the supplementation with hemin affects the nanostructure of red blood cell memranes. Subjects and methods. Human whole blood was supplemented or not with hemin and nanostructure of red blood cell membranes was studied using atomic force microscopy. Conclusion. Experiments demonstrated that hemin exerted a specific effect on the nanostructure of red blood cell membranes to form domains upon their surface. The size of granular structures in the domains was 100—200 nm, which coincides with that of a spectrin matrix. The effect of domain generation possesed threshold character starting from hemine concentrations of 1.3—1.7 mM. These results demonstrate the potential of the use of nanostructural changes within the erythrocyte membrane as surrogate biomarkers of erythrocyte response to hemoglobin derivatives in future studies. Key words: red blood cell membranes, hemin, nanostructure, atomic force microscopy.

Laser Tweezers Raman Spectroscopy As a Novel Red Blood Cell Functional Assay for Sickle Cell Disease

Abstract 4847The number of individuals with sickle cell disease (SCD) in the U.S. is estimated to be over 70,000, and the average life span of a SCD patient is 39 years. Despite the high incidence of SCD in the U.S. and its significant negative effects on patient health, there are currently only limited treatment options for this disease. Also, although a single mutation in the globin gene is known to cause SCD, there is significant clinical diversity. As technology advances, many studies, including genome-wide analyses and proteomics, have been conducted to understand the disease mechanism and find better treatments. However, none has yet led to the development of novel therapies. We need a practical approach to study red blood cells (RBCs) to increase our knowledge of this heterogeneous disease.Laser tweezers Raman spectroscopy (LTRS) is a label-free, laser-based method for the biochemical analysis of single living cells. Based on the phenomenon of inelastic light scattering of photons by molecular bonds, Raman spectroscopy can provide detailed information, in the form of a Raman spectrum, about the molecular structure and conformations inside single living cells. Moreover, the integration of laser tweezers enables 1) simple, convenient analysis of single suspension cells such as RBCs and 2) a method to impart a mechanical force on a cell (via the optical forces) and simultaneously monitor the biochemical response of that cell to the applied force. Previous studies using optical tweezers have shown different RBC elasticity between samples from SCD patients treated with hydroxyurea and those that were not treated with hydroxyurea. Raman spectroscopy has been applied to both normal and pathological RBCs in previous studies, but the results were limited to the identification of characteristic signatures. Our integration of the two methods, Raman spectroscopy and laser tweezers, is a new biophotonic approach for studying the intricate relationship between the biochemical and mechanical properties of a single cell that was not previously possible and is not easily carried out with existing methods. As such, we believe that this recent emerging single cell-based technology can be developed and used as an RBC functional assay.We have previously shown that LTRS can detect a force dependent oxygenation state transition in individual RBCs trapped at different laser powers, presumably due to the optical forces stretching the cell and inducing changes in the hemoglobin-hemoglobin and hemoglobin-membrane interactions that change the oxygen content of the cell. We have also shown characteristic Raman signatures indicating different oxygen content in different RBC samples at specific applied forces. Signatures were different among RBCs from cord, normal adult, and SCD patient blood, suggesting laser tweezers Raman spectroscopy can be used to measure the different force dependent oxygen content in a single RBC. We are currently validating our system to determine whether Raman spectroscopy can be used to identify and distinguish gene-corrected RBCs from normal and sickle RBCs. Our preliminary results showed distinct Raman fingerprints associated with the membrane structure of RBCs. Spectral fingerprints were observed to be different between normal and sickle RBCs, and gene-corrected RBCs were observed to have Raman profiles similar to those of normal RBCs. We speculate that the signature overlap between normal and gene-corrected samples is due to partial gene correction. These results are encouraging and strongly suggest that Raman spectroscopy can not only identify molecular structures but also be used as a functional assay. Disclosures:No relevant conflicts of interest to declare.

Red blood cell plasmalogens and docosahexaenoic acid are independently reduced in primary open-angle glaucoma

Among several theories involved in the pathogenesis of primary open-angle glaucoma (POAG), the vascular theory considers the disease to be a consequence of reduced ocular blood flow associated with red blood cell abnormalities. Red blood cell membrane structure and function are influenced by their phospholipid composition. We investigated whether specific lipid entities that may affect the membrane physiology, namely, polyunsaturated fatty acids (PUFAs) and plasmalogens, are modified in POAG and whether these potential variations are related to the stage of glaucoma. Blood samples were collected from 31 POAG patients and 10 healthy individuals. The stage of glaucoma was determined according to the Hodapp and Parrish classification. Lipids were extracted from red blood cell membranes and individual phospholipid species were quantified by liquid chromatography combined with mass spectrometry using triple quadrupole technology. POAG patients had reduced erythrocyte levels of phosphatidyl-choline (PC) carrying docosahexaenoic acid (DHA). POAG patients also displayed lower levels of choline plasmalogens (PlsC) carrying PUFAs other than DHA. These differences were greater as the severity of the disease increased. Linear regressions predicted that red blood cell PlsC levels would decrease years before clinical symptoms, whereas the levels of PC carrying DHA were linearly correlated to visual field loss. Our data demonstrate the selective loss of some individual phospholipid species in red blood cell membranes, which may partly explain their loss of flexibility in POAG.

Hsp70 protein is membrane bound in red blood cells of human hereditary haemolytic anaemias

We have demonstrated that red blood cell (RBC) membranes in membranopathies are targets for inducible Hsp70 family members. A similar phenomenon was observed in several types of hereditary haemolytic anaemias. One common feature of the inherited haemolytic anaemias is that the mutations are manifested in the form of an altered RBC membrane structure resulting in premature cell destruction. Hence, we propose that malfolded (and mutated) RBC membrane proteins are recognised by Hsp70 protein. Although the function of Hsp70 family members as a ‘quality control system’ is well known, this is the first report of their presence in human anaemias.

A Markedly Disrupted Skeletal Network With Abnormally Distributed Intramembrane Particles in Complete Protein 4.1-Deficient Red Blood Cells (Allele 4.1 Madrid): Implications Regarding a Critical Role of Protein 4.1 in Maintenance of the Integrity of the Red Blood Cell Membrane

Electron microscopic (EM) studies were performed to clarify the interactions of membrane proteins in the red blood cell membrane structure in situ of a homozygous patient with total deficiency of protein 4.1 who carried a point mutation of the downstream translation initiation codon (AUG → AGG) of the protein 4.1 gene [the 4.1 (−) Madrid; Dalla Venezia et al, J Clin Invest 90:1713, 1992]. Immunologically, as expected, protein 4.1 was completely missing in the red blood cell membrane structure in situ. A markedly disrupted skeletal network was observed by EM using the quick-freeze deep-etching method and the surface replica method, although the number of spectrin molecules was only minimally reduced (395 ± 63/μm2; normal, 504 ± 36/μm2). The number of basic units in the skeletal network was strikingly reduced (131 ± 21/μm2; normal, 548 ± 39/μm2), with decreased small-sized units (17 ± 4/μm2; normal, 384 ± 52/μm2) and increased large-sized units (64% ± 14%; normal, 5% ± 1%). Concomitantly, immuno-EM disclosed striking clustering of spectrin molecules with aggregated ankyrin molecules in the red blood cell membrane structure in situ. Although no quantitative abnormalities in the number and size distribution of the intramembrane particles were observed, there was a disappearance of regular distribution, with many clusters of various sizes, probably reflecting the distorted skeletal network. Therefore, protein 4.1 suggests by EM to play a crucial role in maintenance of the normal integrity of the membrane structure in situ not only of the skeletal network but also of the integral proteins.

A Markedly Disrupted Skeletal Network With Abnormally Distributed Intramembrane Particles in Complete Protein 4.1-Deficient Red Blood Cells (Allele 4.1 Madrid): Implications Regarding a Critical Role of Protein 4.1 in Maintenance of the Integrity of the Red Blood Cell Membrane

AbstractElectron microscopic (EM) studies were performed to clarify the interactions of membrane proteins in the red blood cell membrane structure in situ of a homozygous patient with total deficiency of protein 4.1 who carried a point mutation of the downstream translation initiation codon (AUG → AGG) of the protein 4.1 gene [the 4.1 (−) Madrid; Dalla Venezia et al, J Clin Invest 90:1713, 1992]. Immunologically, as expected, protein 4.1 was completely missing in the red blood cell membrane structure in situ. A markedly disrupted skeletal network was observed by EM using the quick-freeze deep-etching method and the surface replica method, although the number of spectrin molecules was only minimally reduced (395 ± 63/μm2; normal, 504 ± 36/μm2). The number of basic units in the skeletal network was strikingly reduced (131 ± 21/μm2; normal, 548 ± 39/μm2), with decreased small-sized units (17 ± 4/μm2; normal, 384 ± 52/μm2) and increased large-sized units (64% ± 14%; normal, 5% ± 1%). Concomitantly, immuno-EM disclosed striking clustering of spectrin molecules with aggregated ankyrin molecules in the red blood cell membrane structure in situ. Although no quantitative abnormalities in the number and size distribution of the intramembrane particles were observed, there was a disappearance of regular distribution, with many clusters of various sizes, probably reflecting the distorted skeletal network. Therefore, protein 4.1 suggests by EM to play a crucial role in maintenance of the normal integrity of the membrane structure in situ not only of the skeletal network but also of the integral proteins.

Changes in red blood cell membrane structure in patients with chronic renal failure.

The properties of red blood cell membranes in patients with chronic renal failure were investigated using electron paramagnetic resonance spectroscopy. Using spin traps, 5,5-dimethylpirroline-1 oxide and N-tert-butyl-alpha-phenylnitrone, we found generation of hydroxyl radicals in the blood of patients with chronic renal failure after 20 min of regular hemodialysis. The physical state of membrane proteins and membrane osmotic fragility and reductive properties of red blood cells were studied. The increase in the relative correlation time of 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1 oxyl indicates the immobilization of membrane protein molecules in erythrocytes of chronic renal failure patients. The decrease in membrane protein mobility was observed in whole blood incubated with tert-butylhydroperoxide, regardless of the presence of iron. We found that the addition of ferrous ions did not aggravate profound changes in membrane proteins induced with tert-butylhydroperoxide. We also demonstrated higher osmotic fragility of erythrocytes in the patients with renal failure as compared to normal subjects. These alterations in membrane structure of red blood cells in hemodialysed patients suggest that hydroxyl radicals generated during hemodialysis can play an important role in the oxidative mechanism of erythrocyte damage.

The Effect of Copper Overload on the Sheep Erythrocyte Membrane.

The effect of copper overload on the red blood cell (RBC) membrane from experimentally copper-poisoned sheep was evaluated by studying the lipid peroxidation, composition of fatty acids, and protein organization of RBC membranes. Ten ewes were dosed orally with CuSO4 in order to induce copper toxicity. The level of lipid peroxidation products was examined in RBC ghosts by measuring their thiobarbituric acid reactivity. The measurement of fatty acids was accomplished by gas-liquid chromatography; and the study of membrane proteins, by SDS-PAGE. The results showed increased lipid peroxidation levels and reduced content of 18:1, 18:2, and 18:3 fatty acids in RBC from copper-poisoned sheep. Despite the RBC copper-overload, treated sheep showed a qualitatively normal protein pattern, but the amount of band 3 of membrane proteins increased. We established a relationship between copper overload and alterations in the RBC membrane structure.