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Erythrocytes play a fundamental role in oxygen delivery to tissues and binding to inflammatory mediators. Evidences suggest that dysregulated erythrocyte function could contribute to the pathophysiology of several neurodegenerative diseases. We aimed to evaluate changes in morphological, biomechanical, and biophysical properties of erythrocytes from amyotrophic lateral sclerosis (ALS) patients, as new areas of study in this disease. Blood samples were collected from ALS patients, comparing with healthy volunteers. Erythrocytes were assessed using atomic force microscopy (AFM) and zeta potential analysis. The patients' motor and respiratory functions were evaluated using the revised ALS Functional Rating Scale (ALSFRS-R) and percentage of forced vital capacity (%FVC). Patient survival was also assessed. Erythrocyte surface roughness was significantly smoother in ALS patients, and this parameter was a predictor of faster decline in ALSFRS-R scores. ALS patients exhibited higher erythrocyte stiffness, as indicated by reduced AFM tip penetration depth, which predicted a faster ALSFRS-R score and respiratory subscore decay. A lower negative charge on the erythrocyte membrane was predictor of a faster ALSFRS-R and FVC decline. Additionally, a larger erythrocyte surface area was an independent predictor of lower survival. These changes in morphological and biophysical membrane properties of ALS patients' erythrocytes, lead to increased cell stiffness and morphological variations. We speculate that these changes might precipitate motoneurons dysfunction and accelerate disease progression. Further studies should explore the molecular alterations related to these observations. Our findings may contribute to dissect the complex interplay between respiratory function, tissue hypoxia, progression rate, and survival in ALS., (© 2023 The Authors. BioFactors published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Introduction: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease of the motor system. It has been hypothesised that red blood cells (RBCs) may be involved in the disease process by the release of damaging molecules., Objective: The aim of this ex vivo study is to compare RBCs biochemical and hemorheological parameters between ALS patients and healthy donors to identify novel biomarkers of the ALS disease., Methods: We included 82 ALS patients and 40 gender age-matched healthy donors. We performed quantification of erythrocyte aggregation and deformability, nitric oxide (NO) efflux from RBCs, acetylcholinesterase (AChE) enzyme activity and intraerythrocytic concentration of nitrite, nitrate and S-nitrosogluthatione (GSNO)., Results: Erythrocyte deformability and AChE activity were increased in patients with ALS in comparison to healthy donors. NO efflux from RBCs and concentration of intraerythrocytic nitrite were lower in ALS patients. In patients, we found that for higher NO range of values the respiratory function is worse and that for higher AChE range of values the RBCs nitrite content increase., Conclusion: The results of the present study indicate that NO efflux from RBCs and RBCs AChE should be further explored as potential biomarkers for ALS.

Introduction: Acetylcholinesterase (AChE) is located on outer surface of erythrocyte membrane. Gender-related differences in erythrocyte AChE enzyme activity had been verified in young adults. It is also known that binding of acetylcholine (ACh) with AChE on erythrocyte membrane initiates a signal transduction mechanism that stimulates nitric oxide (NO) efflux., Aims: This ex vivo study was done to compare the amount of NO efflux obtained from erythrocytes of healthy donors in males and females., Methods: We included 66 gender age-matched healthy donors (40-60 years old). We performed quantification of erythrocyte NO efflux from erythrocytes and of the membrane AChE enzyme activity., Results: There are no significant differences in NO efflux from erythrocytes between men and women. Regarding AChE enzyme activity values, in this range of age, no differences between genders were obtained. However, the values of AChE enzyme activity in the third quartile of NO efflux values were significantly higher (p < 0.05) in women than in men., Conclusions: The efflux of NO from erythrocyte of healthy humans did not change with gender. For the same range of values of NO efflux from erythrocytes, in both gender, it was verified higher values of AChE enzyme activity in women.

Aims: Oxidized low density lipoprotein (ox-LDL) has been reported as an inhibitor of nitric oxide (NO)-mediated dilatation in microcirculation. Oxidized LDL effect on NO metabolism of erythrocytes is not known. Therefore, this study aims to evaluate the effect of ox-LDL on erythrocytes NO metabolism., Methods: The effect of different concentrations of human purified ox-LDL (25, 50 and 100 μg/mL) on NO metabolism was evaluated on blood of healthy subjects., Results: An inhibitory effect of higher concentrations of ox-LDL on erythrocyte NO efflux levels was verified. Concentrations of NO efflux from erythrocytes were lower as consequence of treatments with 50 μg/mL ox-LDL treatment (1.6±0.27 nM) and 100 μg/mL ox-LDL treatment (1.3±0.22 nM) than control (1.9±0.28 nM). Opposite, ox-LDL incubation has a positive effect on GSNO content of erythrocytes. That effect is proportional to concentrations of ox-LDL treatments (10.8±1.4 nM for 25 μg/mL, 12.9±1.5 nM for 50 μg/mL and 12.1±1.9 nM for 100 μg/mL) and is significant relative to control (8.56±0.76 μM) and ACh (8.9±0.52 μM) aliquots., Conclusions: Presence of oxidized LDL in erythrocyte NO metabolism induces a decrease of NO efflux amount and an increase on intra-erythrocyte GSNO concentrations. These results suggest a role of ox-LDL in mobilization of NO between NO derivatives molecules in dependence of oxidized LDL concentration. An anti - reactive nitrogene role can be attributed to ox-LDL for its contribution in the erythrocyte scavenged ability for nitric oxide.

Background: Glaucoma is an optic neuropathy associated with vascular dysregulation and increased intra-ocular pressure (IOP). Timolol is used as treatment for reducing IOP, by limiting aqueous humour production. Increased NOS expression as well as decreased levels of nitric oxide (NO) metabolites, and high activity of erythrocyte acetylcholinesterase (AChE) were observed in primary open angle glaucoma patients., Objective: This ex vivo study aims to evaluate timolol effect in NO efflux and its derivatives in glaucoma patient's erythrocytes., Methods: Venous blood from 15 glaucoma patients was collected. Erythrocyte suspensions were incubated with the AChE modulators acetylcholine (ACh) and timolol at 10 μM. Erythrocyte NO efflux and S-nitrosoglutathione (GSNO) concentration were measured., Results: No significant differences were obtained in erythrocyte NO efflux and GSNO concentration in response to ACh or timolol when compared with the untreated erythrocytes of glaucoma patients. When comparing the same incubation conditions for erythrocyte suspensions between glaucoma patients and healthy subjects, those from glaucoma patients showed higher NO efflux in presence and absence of timolol, and higher values of GSNO in the presence of timolol., Conclusions: We demonstrated that erythrocytes from glaucoma patients have similar availability to release NO both in absence and presence of timolol, and have higher GSNO values in presence of timolol.

Aims: The objectives of this study were to evaluate the effects of high fibrinogen concentration on erythrocyte deformability on mobilization of nitric oxide (NO) and of its metabolites in the presence of acetylcholine (ACh) in healthy human blood samples., Main Methods: Levels of NO were evaluated by amperometric method. Nitrite, nitrate and S-nitrosoglutathione (GSNO) were measured using the spectrophotometric Griess reaction. Erythrocyte deformability was determined using the Rheodyn SSD laser diffractometer., Key Findings: In the presence of high concentrations of fibrinogen and ACh (10 μM) in the blood samples from healthy humans the erythrocyte nitrites, nitrates and GSNO concentrations increased without significant changes in NO efflux. Mobilization of NO in erythrocytes' presence was enhanced in the presence of ACh and high fibrinogen levels., Significance: These results suggest that during inflammation when both ACh and high levels of fibrinogen are present, NO delivery by erythrocytes might be compromised by their NO scavenging ability that acts as a compensatory mechanism against the overproduced NO by endothelial inducible nitric oxide synthase., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Erythrocyte hyperaggregation, a cardiovascular risk factor, is considered to be caused by an increase in plasma adhesion proteins, particularly fibrinogen. We have recently reported a specific binding between fibrinogen and an erythrocyte integrin receptor with a β(3) or β(3)-like subunit. In this study we evaluate the influence of erythrocyte aging on the fibrinogen binding. By atomic force microscopy-based force spectroscopy measurements we found that increasing erythrocyte age, there is a decrease of the binding to fibrinogen by decreasing the frequency of its occurrence but not its force. This observation is reinforced by zeta-potential and fluorescence spectroscopy measurements. We conclude that upon erythrocyte aging the number of fibrinogen molecules bound to each cell decreases significantly, due to the progressive impairment of the specific fibrinogen-erythrocyte receptor interaction. Knowing that younger erythrocytes bind more to fibrinogen, we could presume that this population is the main contributor to the cardiovascular diseases associated with increased fibrinogen content in blood, which could disturb the blood flow. Our data also show that the sialic acids exposed on the erythrocyte membrane contribute for the interaction with fibrinogen, possibly by facilitating its binding to the erythrocyte membrane receptor.

We assessed the redox thiol status influence on nitric oxide (NO) metabolism and efflux in erythrocytes stimulated with acetylcholinesterase substrate (acetylcholine, ACh) and inhibitor (velnacrine maleate, VM). Erythrocyte suspensions from healthy donors were incubated with increasing concentrations of dithiothreitol (1-50microM), in the presence and absence of acetylcholine/velnacrine (10microM). Levels of NO, nitrite/nitrate, S-nitrosohemoglobin, peroxynitrite and S-nitrosoglutathione were determined by spectrofluorimetric and spectrophotometric methods. Dithiothreitol significantly mobilized NO toward nitrite/nitrate and S-nitrosoglutathione, and decreased the amount of NO efflux. Both ACh/VM induce changes on the levels of erythrocyte nitrite/nitrate dependent on the DTT concentration. Higher levels of peroxynitrite and S-nitrosoglutathione were seen with velnacrine in presence of DTT 1 and 50microM. We concluded that dithiothreitol-induced activation of erythrocyte thiol status decreases NO efflux and allows greater intracellular NO mobilization onto different derivative molecules, both in the absence and presence of acetylcholinesterase substrate and inhibitor.

Background: There is growing knowledge about the association between hemorheological blood disorders and compromised microcirculation in erythrocyte abnormalities. Effects of the non-neuronal cholinergic elements, especially acetylcholinesterase, on the erythrocyte hemorheological parameters were characterized in the past. However, alterations of these parameters have not been studied under the influence of the cellular redox thiol status., Methods: Aliquots of venous blood from ten healthy male subjects were incubated in vitro with increasing concentrations of a thiol reducer agent (dithiothreitol 1, 10, 50 microM final concentrations) in the presence and absence of acetylcholinesterase substrate (acetylcholine) or inhibitor (velnacrine maleate). The following parameters were determined in all blood samples aliquots: erythrocyte aggregation, erythrocyte deformability and lipid membrane fluidity. Blood smears were performed., Results: Dithiothreitol induces no significant changes on the hemorheological behaviour of human red cells. Upon intracellular thiol stimulation, the presence of AChE effectors (either acetylcholine or velnacrine) decreases erythrocyte aggregation and elongation indexes., Conclusion: The addition of DTT to blood samples aliquots, contributing to the redox thiol status, is not directly involved in the modulation of erythrocyte rheological properties. However, upon acetylcholinesterase modulation by its substrate or inhibitor, changes on the hemorheological parameters are triggered by DTT. Associated pharmacological interest is considerable to address the hemorheology-hemostasis-microcirculation triad disorders.