Impaired Red Blood Cell Deformability Research Articles

Red Blood Cell Membrane Cholesterol May Be a Key Regulator of Sickle Cell Disease Microvascular Complications.

Cell membrane lipid composition, especially cholesterol, affects many functions of embedded enzymes, transporters and receptors in red blood cells (RBC). High membrane cholesterol content affects the RBCs' main vital function, O2 and CO2 transport and delivery, with consequences on peripheral tissue physiology and pathology. A high degree of deformability of RBCs is required to accommodate the size of micro-vessels with diameters significantly lower than RBCs. The potential therapeutic role of high-density lipoproteins (HDL) in the removal of cholesterol and its activity regarding maintenance of an optimal concentration of RBC membrane cholesterol have not been well investigated. On the contrary, the focus for HDL research has mainly been on the clearance of cholesterol accumulated in atherosclerotic macrophages and plaques. Since all interventions aiming at decreasing cardiovascular diseases by increasing the plasma level of HDL cholesterol have failed so far in large outcome studies, we reviewed the potential role of HDL to remove excess membrane cholesterol from RBC, especially in sickle cell disease (SCD). Indeed, abundant literature supports a consistent decrease in cholesterol transported by all plasma lipoproteins in SCD, in addition to HDL, low- (LDL) and very low-density lipoproteins (VLDL). Unexpectedly, these decreases in plasma were associated with an increase in RBC membrane cholesterol. The concentration and activity of the main enzyme involved in the removal of cholesterol and generation of large HDL particles-lecithin cholesterol ester transferase (LCAT)-are also significantly decreased in SCD. These observations might partially explain the decrease in RBC deformability, diminished gas exchange and tendency of RBCs to aggregate in SCD. We showed that incubation of RBC from SCD patients with human HDL or the HDL-mimetic peptide Fx5A improves the impaired RBC deformability and decreases intracellular reactive oxygen species levels. We propose that the main physiological role of HDL is to regulate the cholesterol/phospholipid ratio (C/PL), which is fundamental to the transport of oxygen and its delivery to peripheral tissues.

The place of glucosylated enteral solution in the correction of hemorheological abnormalities in acute poisoning by psychopharmacological drugs

Introduction. In acute exogenous poisoning, hemorheological abnormalities are observed. Various extracorporeal, physico-chemical methods are used to correct them. There is an opinion that the enteral route of administration of corrective agents may be a more physiological way to restore homeostatic imbalances.Aim. To conduct a comparative assessment of the effect of glucosylated enteral solution and standard infusion therapy on hemorheological abnormalities in acute poisoning by psychopharmacological drugs.Material and methods. Patients with acute poisoning by psychopharmacological drugs who were treated at the N.V. Sklifosovsky Research Institute for Emergency Medicine in 2017–2021 were examined. Of these, 23 people, in whose treatment the enteral correction program was used, made up the study group, and 22 patients (the comparison group) underwent a standard set of therapeutic measures. Indicators of hemorheological status were examined on the 1st, 3rd and 5th days against the background of ongoing therapy. Statistical data analysis was carried out using the Statistica 10 software package (StatSoft, Inc., USA).Results. The use of a glucosylated enteral solution led to a reduction in plasma viscosity under normal hematocrit conditions at all follow-up periods. In patients of both groups, there was a decrease in blood viscoelasticity under conditions of high shear potential at all stages of the study, which indicates impaired red blood cell deformability. This process was more pronounced in individuals of the comparison group. The conducted studies have shown that the use of infusion therapy and glucosylated enteral solution in the early stages of acute poisoning by psychopharmacological drugs generally has a unidirectional positive effect on hemorheological indicators, contributing to the stabilization of blood circulation. At the same time, the effect of glucosylated enteral solution therapy is faster and more pronounced.Conclusions. The use of glucosylated enteral solution and infusion therapy as a supportive treatment in the early period of acute poisoning by psychopharmacological drugs in most cases has a unidirectional effect on hemorheological parameters. In cases of glucosylated enteral solution therapy, there was an outrunning positive dynamics on the part of the majority of the studied hemorheological parameters. Glucosylated enteral solution can be the method of choice as a maintenance therapy after the end of detoxification process for acute poisoning by psychopharmacological drugs.

Flows of healthy and hardened RBC suspensions through a micropillar array.

Red blood cell (RBC) deformability is an important haemorheological factor; it is impaired in many pathologies leading to microvascular complications. Several microfluidic platforms have been utilized to examine the role of deformability in RBC flows but their geometries tend to be simplified. In the present study, we extend our previous work on healthy RBC flows in micropillar arrays [1] to probe the effect of impaired RBC deformability on the velocity and haematocrit distributions in microscale RBC flows. Healthy and artificially hardened RBC suspensions at 25% haematocrit were perfused through the micropillar array at various flow rates and imaged. RBC velocities were determined by Particle Image Velocimetry (PIV) and haematocrit distributions were inferred from the image intensity distributions. The pillars divide the flow into two distinct RBC streams separated by a cell-depleted region along the centreline and in the rear/front stagnation points. RBC deformability was not found to significantly affect the velocity distributions; the shape of the velocity profiles in the interstitial space remained the same for healthy and hardened RBCs. Time-averaged and spatiotemporal intensity distributions, however, reveal differences in the dynamics and local distributions of healthy and hardened cells; hardened cells appear to enter the cell-depleted regions more frequently and their interstitial distributions are more uniform. The study highlights the importance of local RBC distributions and the impact of RBC deformability on cell transport in complex microscale flows.

Even patients with mild COVID‐19 symptoms after SARS‐CoV‐2 infection show prolonged altered red blood cell morphology and rheological parameters

Infection with the novel severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) and the associated coronavirus disease‐19 (COVID‐19) might affect red blood cells (RBC); possibly altering oxygen supply. However, investigations of cell morphology and RBC rheological parameters during a mild disease course are lacking and thus, the aim of the study. Fifty individuals with mild COVID‐19 disease process were tested after the acute phase of SARS‐CoV‐2 infection (37males/13 females), and the data were compared to n = 42 healthy controls (30 males/12 females). Analysis of venous blood samples, taken at rest, revealed a higher percentage of permanently elongated RBC and membrane extensions in COVID‐19 patients. Haematological parameters and haemoglobin concentration, MCH and MCV in particular, were highly altered in COVID‐19. RBC deformability and deformability under an osmotic gradient were significantly reduced in COVID‐19 patients. Higher RBC‐NOS activation was not capable to at least in part counteract these reductions. Impaired RBC deformability might also be related to morphological changes and/or increased oxidative state. RBC aggregation index remained unaffected. However, higher shear rates were necessary to balance the aggregation‐disaggregation in COVID‐19 patients which might be, among others, related to morphological changes. The data suggest prolonged modifications of the RBC system even during a mild COVID‐19 disease course.

Temporal relationship between systemic endothelial dysfunction and alterations in erythrocyte function in a murine model of chronic heart failure.

AimsEndothelial dysfunction (ED) and red blood cell distribution width (RDW) are both prognostic factors in heart failure (HF), but the relationship between them is not clear. In this study, we used a unique mouse model of chronic HF driven by cardiomyocyte-specific overexpression of activated Gαq protein (Tgαq*44 mice) to characterize the relationship between the development of peripheral ED and the occurrence of structural nanomechanical and biochemical changes in red blood cells (RBCs).Methods and resultsSystemic ED was detected in vivo in 8-month-old Tgαq*44 mice, as evidenced by impaired acetylcholine-induced vasodilation in the aorta and increased endothelial permeability in the brachiocephalic artery. ED in the aorta was associated with impaired nitric oxide (NO) production in the aorta and diminished systemic NO bioavailability. ED in the aorta was also characterized by increased superoxide and eicosanoid production. In 4- to 6-month-old Tgαq*44 mice, RBC size and membrane composition displayed alterations that did not result in significant changes in their nanomechanical and functional properties. However, 8-month-old Tgαq*44 mice presented greatly accentuated structural and size changes and increased RBC stiffness. In 12-month-old Tgαq*44 mice, the erythropathy was featured by severely altered RBC shape and elasticity, increased RDW, impaired RBC deformability, and increased oxidative stress (gluthatione (GSH)/glutathione disulfide (GSSG) ratio). Moreover, RBCs taken from 12-month-old Tgαq*44 mice, but not from 12-month-old FVB mice, coincubated with aortic rings from FVB mice, induced impaired endothelium-dependent vasodilation and this effect was partially reversed by an arginase inhibitor [2(S)-amino-6-boronohexanoic acid].ConclusionIn the Tgαq*44 murine model of HF, systemic ED accelerates erythropathy and, conversely, erythropathy may contribute to ED. These results suggest that erythropathy may be regarded as a marker and a mediator of systemic ED in HF. RBC arginase and possibly other RBC-mediated mechanisms may represent novel therapeutic targets for systemic ED in HF.

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.

Hemochromatosis alters the sensitivity of red blood cells to mechanical stress.

Hemochromatosis (HH) is characterized by chronic iron accumulation, leading to deleterious effects to various organ systems. A common approach to managing iron load involves large-volume venesection. Some countries authorize HH venesections to be used in the development of transfusable blood products, although concerns remain regarding suitability. Due to the high oxidative load associated with hyperferritinemia, it has been proposed that HH blood products may be susceptible to mechanical damage. This is particularly relevant given that typical blood product destinations (eg, transfusion, cardiopulmonary bypass) expose blood to supraphysiologic levels of mechanical stress. We sought to explore the mechanical tolerance of red blood cells (RBC) derived from HH venesections to varied magnitudes and durations of sublethal shear stress. Initially, 110 individuals with HH were recruited; to eliminate the effects of comorbidities, only those who were untreated and uncomplicated were included for comparisons with age-matched healthy controls (Con). RBC were exposed to 25 discrete magnitudes (1-64 Pa) and durations (1-64 seconds) of shear stress. Cellular deformability was assessed before, and immediately after, each shear exposure. In the absence of prior shear exposure, RBC deformability of HH was significantly decreased by 11.5%, compared with Con. For both HH and Con, supraphysiologic shear exposure significantly impaired RBC deformability, although the rate and magnitude of deterioration were elevated for HH. Given that blood products are commonly exposed to high-shear environments (eg, during high-volume transfusion), venesections from asymptomatic and untreated individuals with HH appear suboptimal for the development of therapeutic RBCs.

Altered red blood cell deformability-A novel hypothesis for retinal microangiopathy in diabetic retinopathy.

Impaired red blood cell (RBC) deformability impedes tissue perfusion. This study aims to investigate RBC biomechanics in type 2 diabetes mellitus (DM) patients with different grades of diabetic retinopathy (DR) and to correlate RBC deformability with hematological and serum biochemical markers. This cross-sectional study included 86 type 2 DM patients (31 with no DR, 31 with non-proliferative DR [NPDR] and 24 with proliferative DR [PDR]) and 32 control subjects. RBC deformability was measured by a microfluidic cross-slot channel (elongation index, EI). Venous blood samples were taken for assessment of hematological and serum biochemical markers. RBC deformability showed significant reduction in diabetic patients, being lowest in the PDR group, followed by NPDR and DM with no DR groups, and highest in control group (P=.018). RBC deformability was not affected by age or gender but showed significant associations with certain hematological and serum biochemical markers. In the regression analysis controlling for DM status, urea concentration and reticulocyte count were shown to be negatively associated with EI. Impaired RBC deformability measured by a microfluidic cross-slot channel in DM patients with different grades of DR underscores the contribution of RBC rheological properties to the pathogenesis and progression of DM related microangiopathy.

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.

Microfluidic assessment of red blood cell mediated microvascular occlusion.

Abnormal red blood cell (RBC) deformability contributes to hemolysis, thrombophilia, inflammation, and microvascular occlusion in various circulatory diseases. A quantitative and objective assessment of microvascular occlusion mediated by RBCs with abnormal deformability would provide valuable insights into disease pathogenesis and therapeutic strategies. To that end, we present a new functional microfluidic assay, OcclusionChip, which mimics two key architectural features of the capillary bed in the circulatory system. First, the embedded micropillar arrays within the microchannel form gradient microcapillaries, from 20 μm down to 4 μm, which mimic microcapillary networks. These precisely engineered microcapillaries retain RBCs with impaired deformability, such that stiffer RBCs occlude the wider upstream microcapillaries, while less stiff RBCs occlude the finer downstream microcapillaries. Second, the micropillar arrays are coupled with two side passageways, which mimic the arteriovenous anastomoses that act as shunts in the capillary bed. These side microfluidic anastomoses prevent microchannel blockage, and enable versatility and testing of clinical blood samples at near-physiologic hematocrit levels. Further, we define a new generalizable parameter, Occlusion Index (OI), which is an indicative index of RBC deformability and the associated microcapillary occlusion. We demonstrate the promise of OcclusionChip in diverse pathophysiological scenarios that result in impaired RBC deformability, including mercury toxin, storage lesion, end-stage renal disease, malaria, and sickle cell disease (SCD). Hydroxyurea therapy improves RBC deformability and increases fetal hemoglobin (HbF%) in some, but not all, treated patients with SCD. HbF% greater than 8.6% has been shown to improve clinical outcomes in SCD. We show that OI associates with HbF% in 16 subjects with SCD. Subjects with higher HbF levels (HbF > 8.6%) displayed significantly lower OI (0.88% ± 0.10%, N = 6) compared with those with lower HbF levels (HbF ≤ 8.6%) who displayed greater OI (3.18% ± 0.34%, N = 10, p < 0.001). Moreover, hypoxic OcclusionChip assay revealed a significant correlation between hypoxic OI and subject-specific sickle hemoglobin (HbS) level in SCD. OcclusionChip enables versatile in vitro assessment of microvascular occlusion mediated by RBCs in a wide range of clinical conditions. OI may serve as a new parameter to evaluate the efficacy of treatments improving RBC deformability, including hemoglobin modifying drugs, anti-sickling agents, and genetic therapies.

The effect of deformability on the microscale flow behavior of red blood cell suspensions

Red blood cell (RBC) deformability is important for tissue perfusion and a key determinant of blood rheology. Diseases such as diabetes, sickle cell anemia, and malaria, as well as prolonged storage, may affect the mechanical properties of RBCs altering their hemodynamic behavior and leading to microvascular complications. However, the exact role of RBC deformability on microscale blood flow is not fully understood. In the present study, we extend our previous work on healthy RBC flows in bifurcating microchannels [Sherwood et al., “Viscosity and velocity distributions of aggregating and non-aggregating blood in a bifurcating microchannel,” Biomech. Model. Mechanobiol. 13, 259–273 (2014); Sherwood et al., “Spatial distributions of red blood cells significantly alter local hemodynamics,” PLoS One 9, e100473 (2014); and Kaliviotis et al., “Local viscosity distribution in bifurcating microfluidic blood flows,” Phys. Fluids 30, 030706 (2018)] to quantify the effects of impaired RBC deformability on the velocity and hematocrit distributions in microscale blood flows. Suspensions of healthy and glutaraldehyde hardened RBCs perfused through straight microchannels at various hematocrits and flow rates were imaged, and velocity and hematocrit distributions were determined simultaneously using micro-Particle Image Velocimetry and light transmission methods, respectively. At low feed hematocrits, hardened RBCs were more dispersed compared to healthy ones, consistent with decreased migration of stiffer cells. At high hematocrit, the loss of deformability was found to decrease the bluntness of velocity profiles, implying a reduction in shear thinning behavior. The hematocrit bluntness also decreased with hardening of the cells, implying an inversion of the correlation between velocity and hematocrit bluntness with loss of deformability. The study illustrates the complex interplay of various mechanisms affecting confined RBC suspension flows and the impact of both deformability and feed hematocrit on the resulting microstructure.

Shear Stress Ameliorates Superoxide Impairment to Erythrocyte Deformability With Concurrent Nitric Oxide Synthase Activation.

The cellular deformability of red blood cells (RBC) is exceptional among mammalian cells and facilitates nutrient delivery throughout the microcirculation; however, this physical property is negatively impacted by oxidative stress. It remains unresolved whether the molecular determinants of cellular deformability – which in the contemporary model of RBC are increasingly recognized – are sensitive to free radicals. Moreover, given cellular deformability has recently been demonstrated to increase following exposure to specific doses of mechanical stimulation, the potential for using shear “conditioning” as a novel method to reverse free-radical induced impairment of cell mechanics is of interest. We thus designed a series of experiments that explored the effects of intracellular superoxide (O2-) generation on the deformability of RBC and also activation of pivotal molecular pathways known to regulate cell mechanics – i.e., PI3K/Akt kinase and RBC nitric oxide synthase (NOS). In addition, RBC exposed to O2- were conditioned with specific shear stresses, prior to evaluation of cellular deformability and activation of PI3K/Akt kinase and RBC-NOS. Intracellular generation of O2- decreased phosphorylation of RBC-NOS at its primary activation site (Ser1177) (p < 0.001), while phosphorylation of Akt kinase at its active residue (Ser473) was also diminished (p < 0.001). Inactivation of these enzymes following O2- exposure occurred in tandem with decreased RBC deformability. Shear conditioning significantly improved cellular deformability, even in RBC previously exposed to O2-. The improvement in cellular deformability may have been the result of enhanced molecular signaling, given RBC-NOS phosphorylation in RBC exposed to O2- was restored following shear conditioning. Impaired RBC deformability induced by intracellular O2- may be due, in part, to impaired activation of PI3K/Akt, and downstream signaling with RBC-NOS. These findings may shed light on improved circulatory health with targeted promotion of blood flow (e.g., exercise training), and may prove fruitful in future development of blood-contacting devices.

Simple chronic transfusion therapy, a crucial therapeutic option for sickle cell disease, improves but does not normalize blood rheology: What should be our goals for transfusion therapy?

Sickle cell anemia is characterized by a mutation resulting in the formation of an abnormal beta-hemoglobin called hemoglobin S. Hemoglobin S polymerizes upon deoxygenation, causing impaired red blood cell deformability and increased blood viscosity at equivalent hematocrits. Thus, sickle cell disease is a hemorheologic disease that results in various pathologic processes involving multiple organ systems including the lungs, heart, kidneys and brain. Red blood cell mechanics and the perturbations on blood flow-endothelial interaction underlie much of the pathology found in sickle cell disease. Transfusion therapy is one of the few therapeutic options available to patients, acting as both primary and secondary prevention of stroke. Transfusion therapy, both simple and exchange, is also used for unremitting and frequent pain crises and pulmonary hypertension. Therefore, understanding basic rheologic changes following transfusion inform other therapeutic options that aim to mitigate this diffuse pathologic process. This review will aim to highlight transfusion effects on blood rheology.

Microconfined flow behavior of red blood cells

Red blood cells (RBCs) perform essential functions in human body, such as gas exchange between blood and tissues, thanks to their ability to deform and flow in the microvascular network. The high RBC deformability is mainly due to the viscoelastic properties of the cell membrane. Since an impaired RBC deformability could be found in some diseases, such as malaria, sickle cell anemia, diabetes and hereditary disorders, there is the need to provide further insight into measurement of RBC deformability in a physiologically relevant flow field. Here, RBCs deformability has been studied in terms of the minimum apparent plasma-layer thickness by using high-speed video microscopy of RBCs flowing in cylindrical glass capillaries. An in vitro systematic microfluidic investigation of RBCs in micro-confined conditions has been performed, resulting in the determination of the RBCs time recovery constant, RBC volume and surface area and RBC membrane shear elastic modulus and surface viscosity. It has been noticed that the deformability of RBCs induces cells aggregation during flow in microcapillaries, allowing the formation of clusters of cells. Overall, our results provide a novel technique to estimate RBC deformability and also RBCs collective behavior, which can be used for the analysis of pathological RBCs, for which reliable quantitative methods are still lacking.

RBC-NOS-Dependent S-Nitrosylation of Cytoskeletal Proteins Improves RBC Deformability

BackgroundRed blood cells (RBC) possess a nitric oxide synthase (RBC-NOS) whose activation depends on the PI3-kinase/Akt kinase pathway. RBC-NOS-produced NO exhibits important biological functions like maintaining RBC deformability. Until now, the cellular target structure for NO, to exert its influence on RBC deformability, remains unknown. In the present study we analyzed the modification of RBC-NOS activity by pharmacological treatments, the resulting influence on RBC deformability and provide first evidence for possible target proteins of RBC-NOS-produced NO in the RBC cytoskeletal scaffold.Methods/FindingsBlood from fifteen male subjects was incubated with the NOS substrate L-arginine to directly stimulate enzyme activity. Direct inhibition of enzyme activity was induced by L-N5-(1-Iminoethyl)-ornithin (L-NIO). Indirect stimulation and inhibition of RBC-NOS were achieved by applying insulin and wortmannin, respectively, substances known to affect PI3-kinase/Akt kinase pathway. The NO donor sodium nitroprusside (SNP) and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) were additionally applied as NO positive and negative controls, respectively. Immunohistochemical staining was used to determine phosphorylation and thus activation of RBC-NOS. As a marker for NO synthesis nitrite was measured in plasma and RBCs using chemiluminescence detection. S-nitrosylation of erythrocyte proteins was determined by biotin switch assay and modified proteins were identified using LC-MS. RBC deformability was determined by ektacytometry. The data reveal that activated RBC-NOS leads to increased NO production, S-nitrosylation of RBC proteins and RBC deformability, whereas RBC-NOS inhibition resulted in contrary effects.Conclusion/SignificanceThis study first-time provides strong evidence that RBC-NOS-produced NO modifies RBC deformability through direct S-nitrosylation of cytoskeleton proteins, most likely α- and β-spectrins. Our data, therefore, gain novel insights into biological functions of RBC-NOS by connecting impaired RBC deformability abilities to specific posttranslational modifications of RBC proteins. By identifying likely NO-target proteins in RBC, our results will stimulate new therapeutic approaches for patients with microvascular disorders.

Association of increased erythrocyte osmotic resistance with haematological and histopathological findings in dogs with a congenital extrahepatic portosystemic shunt

The aim of the study was to investigate changes in erythrocyte osmotic resistance in relation to haematological and histological changes in dogs with a congenital portosystemic shunt. Osmotic fragility tests with complete blood counts and liver histological examinations were performed in 12 dogs with single extrahepatic portosystemic shunt confirmed by surgical exploration. Laboratory results were compared with those from 30 healthy dogs. Dogs with portosystemic shunt had a significantly increased erythrocyte osmotic resistance (P&lt; 0.01) with 5%, 50% and 90% haemolysis corresponding to 0.45%, 0.35% and 0.30% NaCl solution, respectively. Statistical analyses revealed no correlation between haematological indicators and the osmotic fragility test results. Increased osmotic resistance was significantly associated with hepatic lipogranulomas. Based on these results, dogs with a congenital portosystemic shunt have a significantly increased erythrocyte osmotic resistance suggesting impaired red blood cell deformability. Osmotic resistance test that until now was not studied in canine hepatopathies seems to be independent of routinely performed haematological tests.

Impaired Red Blood Cell Deformability in Patients with Coronary Artery Disease and Diabetes Mellitus

Patients with diabetes mellitus (DM) have an increased cardiovascular morbidity and mortality. There is increasing evidence that diabetes mellitus is associated with pathological hemorheological alterations, which might contribute to impaired coronary blood flow in coronary artery disease (CAD). We hypothesize that red blood cell (RBC) deformability is impaired in diabetic patients with CAD in comparison to nondiabetic patients with CAD. RBC deformability was measured in 21 patients with CAD and type 2 diabetes mellitus (CAD + DM) and 24 patients with CAD (CAD - DM). RBC deformability was measured by the Laser-assisted optical rotational cell analyzer by determining the elongation index (EI). RBC deformability was reduced in patients with CAD + DM in comparison to patients with CAD - DM (EI @ 1.12 Pa 0.236 ± 0.008 vs. 0.260 ± 0.005, p=0.007). Inverse univariate correlations were found between the EI @ 1.12 Pa and plasma glucose concentration (r= - 0.57; p<0.001) and HbA1c (r= - 0.45; p=0.002). Multivariate linear regression analysis identified plasma glucose concentration as the independent predictor of RBC deformability (β= - 0.58; p=0.007) thereby indicating that increased glucose concentrations determine RBC deformability in diabetic patients with CAD. In patients with CAD, diabetes mellitus leads to an impairment of RBC deformability which might contribute to increased morbidity of diabetic patients with CAD.

Assessment of oxidant susceptibility of red blood cells in various species based on cell deformability

The present study was designed to investigate the oxidant susceptibility of red blood cells (RBC) from four species (echidna, human, koala, Tasmanian devil) based on changes in cellular deformability. These species were specifically chosen based on differences in lifestyle and/or biology associated with varied levels of oxidative stress. The major focus was the influence of superoxide radicals generated within the cell (phenazine methosulfate, PMS, 50 μM) or in the extracellular medium (xanthine oxidase-hypoxanthine, XO-HX, 0.1 U/ml XO) on RBC deformability at various shear stresses (SS). RBC deformability was assessed by laser-diffraction analysis using a "slit-flow ektacytometer". Both superoxide-generating treatments resulted in significant increases of methemoglobin for all species (p < 0.01), with Tasmanian devil RBC demonstrating the most sensitivity to either treatment. PMS caused impaired RBC deformability for all species, but vast interspecies variations were observed: human and koala cells exhibited a similar sigmoid-like response to SS, short-beaked echidna values were markedly lower and only increased slightly with SS, while Tasmanian devil RBC were extremely rigid. The effect of XO-HX on RBC deformability was less when compared with PMS (i.e., smaller increase in rigidity) with the exception of Tasmanian devil RBC which exhibited essentially no deformation even at the highest SS; Tasmanian devil RBC response to XO-HX was thus comparable to that observed with PMS. Our findings indicate that ektacytometry can be used to determine the oxidant susceptibility of RBC from different species which varies significantly among mammals representing diverse lifestyles and evolutionary histories. These differences in susceptibility are consistent with species-specific discrepancies between observed and allometrically-predicted life spans and are compatible with the oxidant theory of aging.

Impaired Blood Rheology and Elevated Remnant-like Lipoprotein Particle Cholesterol in Hypercholesterolaemic Subjects

Blood rheology, fasting serum concentrations of remnant-like lipoprotein particle cholesterol (RLP-C) and concentrations of other lipids were compared in 23 hypercholesterolaemic and 69 normocholesterolaemic subjects, and the relationship between red blood cell (RBC) deformability and RLP-C concentrations were studied in a different set of six hypercholesterolaemic and six normocholesterolaemic subjects. Passage time of whole blood and concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and RLP-C were significantly higher in hypercholesterolaemic than in normocholesterolaemic subjects. Passage time of whole blood correlated positively with TC, TG, LDL-C and RLP-C and negatively with high-density lipoprotein cholesterol. Furthermore, the passage time of 10% haematocrit-adjusted RBCs in phosphate-buffered saline, which reflects RBC deformability, correlated positively with the passage time of whole blood and RLP-C. Thus, hypercholesterolaemic subjects had impaired blood rheology and elevated RLP-C concentrations, which may be associated with the pathophysiology of atherosclerosis in hypercholesterolaemic subjects. Impaired RBC deformability may contribute to impaired blood rheology associated with elevated RLP-C in hypercholesterolaemic subjects.

Counterpoint: Sickle cell trait should not be considered asymptomatic and as a benign condition during physical activity

Counterpoint: Sickle cell trait should not be considered asymptomatic and as a benign condition during physical activity