Red Blood Cell Membrane Deformability Research Articles

Alterations in plasma and erythrocyte membrane fatty acid composition following exposure to toxic copper level affect membrane deformability and fluidity in female wistar rats

BackgroundDeformability and fluidity function of the red blood cell membrane are properties defined by the lipid composition. Toxic copper level induces membrane lipid peroxidation which could cause membrane instability. This study therefore investigated the effect of exposure to toxic copper level for 30 days on red blood cell membrane deformability and fluidity in female Wistar rats. MethodsTwelve (12) female Wistar rats (160 ± 10 g) were randomly grouped (n = 6) into control (given 0.1 ml distilled water p.o.) and copper-toxic (100 mg/kg Copper Sulphate, p.o.), and treated for 30 days. Plasma obtained and RBC membrane prepared from blood collected over EDTA post-treatment were assayed for total cholesterol (TC), phospholipids and fatty acid profile using spectrophotometry and Gas chromatography while heparinized blood was subjected to fragility test. Data were analyzed using student T-test for statistical significance at p < 0.05. Results and conclusionPlasma TC increased by 4.33% while RBC membrane TC decreased by 20.32% in copper-toxic group compared to control. Compared to control, excess copper significantly increased membrane phospholipids level (0.72 ± 0.01 vs 0.59 ± 0.04 mg/dL) but reduced membrane cholesterol/phospholipid ratio (46.61 ± 4.72 vs 72.66 ± 6.47) and stability (by 23.53%). Number of cis- and saturated fatty acids increased in copper-treated plasma and RBC membrane compared to control. Exposure to toxic copper level alters erythrocyte membrane fluidity and deformability by disrupting membrane lipid composition, saturation, bond configuration in phospholipids and permeability.

Pharmacological Profile of Nigella sativa Seeds in Combating COVID-19 through In-Vitro and Molecular Docking Studies

COVID-19 infection is associated with elevated oxidative stress, systemic hyper-inflammatory responses, endothelial dysfunction, and red blood cell membrane deformability. Nigella sativa extract is widely used in alternative and complementary medicine systems in a large population, due to its highly therapeutic, economic, natural, and safe nature. The aim of this study was to evaluate the effect of N. sativa extract on oxidative stress, hemolysis, proteolysis, and glycation through in vitro studies, as well as to find out its anti-viral potential against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) using in silico studies. N. sativa seed extract (at 600 µg/mL) displayed 67.33% scavenging activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test, and 70.28% hydrogen peroxide reducing activity. N. sativa exhibited anti-proteolytic activity by decreasing heat-induced denaturation of bovine serum albumin (BSA) and egg albumin by 63.14% and 57.95%, respectively, and exhibited anti-proteinase potential of 66.28% at 600 μg/mL. In addition, heat-induced hemolysis and hypersalinity-induced hemolysis were inhibited by 57.86% and 61.7%, respectively, by the N. sativa seeds. N. sativa also inhibited browning intensity by 56.38%, and percent aggregation index by 51.38%, amyloid structure by 48.28%, and AGE-specific fluorescence by 52.18%, thereby protecting the native structure of BSA from glycation. The binding interactions between bioactive molecules of N. sativa seed with SARS-CoV-2 spike glycoprotein were proven by using in silico molecular docking tools. The functional amino acids involved in the interactions are Asp467, Thr108, Thr114, Ile468, Asn234, Gln155, Glu465, Arg466, Gly232, and Ile233, indicating the inhibiting property of N. sativa on SARS-CoV-2. Finally, we may infer that phytoconstituents of N. sativa seeds have the potential to protect against the spike protein of SARS-CoV-2. Studies on N. sativa seeds might act as a path to develop a potent alternative therapy against viral infections, especially COVID-19 infection, in the future. However, the limitations linked with the use of natural products are also needed to be considered in this regard.

Etavopivat, an Allosteric Activator of Pyruvate Kinase-R, Improves Sickle RBC Functional Health and Survival and Reduces Systemic Markers of Inflammation and Hypercoagulability in Patients with Sickle Cell Disease: An Analysis of Exploratory Studies in a Phase 1 Study

Etavopivat, an Allosteric Activator of Pyruvate Kinase-R, Improves Sickle RBC Functional Health and Survival and Reduces Systemic Markers of Inflammation and Hypercoagulability in Patients with Sickle Cell Disease: An Analysis of Exploratory Studies in a Phase 1 Study

Common host variation drives malaria parasite fitness in healthy human red cells.

The replication of Plasmodium falciparum parasites within red blood cells (RBCs) causes severe disease in humans, especially in Africa. Deleterious alleles like hemoglobin S are well-known to confer strong resistance to malaria, but the effects of common RBC variation are largely undetermined. Here, we collected fresh blood samples from 121 healthy donors, most with African ancestry, and performed exome sequencing, detailed RBC phenotyping, and parasite fitness assays. Over one-third of healthy donors unknowingly carried alleles for G6PD deficiency or hemoglobinopathies, which were associated with characteristic RBC phenotypes. Among non-carriers alone, variation in RBC hydration, membrane deformability, and volume was strongly associated with P. falciparum growth rate. Common genetic variants in PIEZO1, SPTA1/SPTB, and several P. falciparum invasion receptors were also associated with parasite growth rate. Interestingly, we observed little or negative evidence for divergent selection on non-pathogenic RBC variation between Africans and Europeans. These findings suggest a model in which globally widespread variation in a moderate number of genes and phenotypes modulates P. falciparum fitness in RBCs.

Evaluation of erythrocyte deformability in patients with hypertension

Background. In the longterm course of hypertension, changes occur not only at the systemic, but also at the membrane level. The predominance of red blood spherocytic cells in hypertension, due to the low deformability of their membrane, can lead to further deterioration of the perfusion of the microcirculatory bed and the development of oxygen starvation of tissues. It is important to assess the extent of gas exchange disorders in organs and tissues in hypertension as early as possible in order to choose the correct and timely treatment strategy for patients with this disease. Currently, in order to assess the ability of red blood cells to deformability in the conditions of clinical units, it is necessary to have special equipment, certain conditions and specialist qualifications.Design and methods. We have developed a method of evaluating deformability of erythrocytes in hypertensive patients. The method includes measuring the patient’s blood pressure, blood sampling, lipidogram, measuring red blood cell parameters on a hematological analyzer with the calculation of the red blood cell sphericity index, and calculating the indicator of the red blood cell membrane deformability by the proposed formula.Conclusions. This method allows of timely diagnostics of the systemic microcirculation abnormality in hypertensive patients. The proposed method is intended for pathophysiologists, physicians, cardiologists, specialists of clinical laboratory diagnostics, it can also be applied for research purposes.

Automated Oxygen Gradient Ektacytometry: A Novel Biomarker in Sickle Cell Anemia

Sickle cell anemia (SCA) is a hereditary hemoglobinopathy with a variable phenotype. There is no single biomarker that adequately predicts disease severity and can be used to monitor treatment response in patients in clinical trials and clinical care. The use of clinical outcomes, such as vaso-occlusive crises (VOC), requires long and expensive studies, sometimes with inconclusive results. To address these limitations, there are several biomarkers under study to improve the ability to predict complications and assess treatment response in both clinical and research settings. Oxygen gradient ektacytometry, also called as oxygenscan, is an assay that measures the effects of deoxygenation and reoxygenation on red blood cell (RBC) deformability and is gaining popularity in SCA research, because it captures the dynamic sickling capacity of a patient’s RBCs as they are subjected to an oxygen gradient under steady shear stress. We describe here the oxygenscan methodology and evaluate the correlation between oxygenscan parameters and more well-known biomarkers of SCA such as fetal hemoglobin (HbF), F-cells, and dense red blood cells (DRBCs). Our data indicate that the oxygenscan curve is affected by all these parameters and the result incorporates the effects of %HbF, %F-cells, RBC hydration, and RBC membrane deformability.

Differential dielectroscopic data on the relation of erythrocyte membrane skeleton to erythrocyte deformability and flicker.

Two dielectric relaxations, βsp (1.5MHz) and γ1sp (7MHz), have been detected on spectrin-based membrane skeleton (MS) of red blood cells (RBCs) using the plot of admittance changes at the spectrin denaturation temperature (Ivanov and Paarvanova in Bioelectrochemistry 110: 59-68, 2016, Electrochim Acta 317: 289-300, 2019a). In this study, we treated RBCs and RBC ghost membranes with agents that make membranes rigid and suppress membrane flicker, and studied the effect on βsp and γ1sp relaxations. Diamide (diazene dicarboxylic acid bis-(N,N-dimethylamide)) (up to 0.85mM), taurine mustard (tris(2-chloroethyl)amine) (up to 2mM), known to specifically cross-link and stiffen spectrin, and glutaraldehyde (up to 0.044%) all inhibited the relaxations in RBC ghost membranes. Similar inhibition was obtained resealing RBC ghost membranes with 2,3-diphosphoglicerate (up to 15mM), binding WGA (wheat germ agglutinin) (up to 0.025mg/ml) to exofacial aspect of RBCs, incubating RBCs in hypotonic (200mOsm) and hypertonic (600-900mOsm) media and depleting RBCs of ATP. By contrast, concanavalin A (1mg/ml) and DIDS (4,4'-diiso-thiocyanato stilbene-2,2'-disulfonic acid) (75μM, pH 8.2), both known to bind specifically band 3 integral protein of RBCs without effect on RBC membrane rigidity, did not affect the relaxations. We conclude there might be a relation between the strength of dielectric relaxations on MS spectrin and the deformability and flicker of RBC membrane.

Oral Administration of FT-4202, an Allosteric Activator of Pyruvate Kinase-R, Has Potent Anti-Sickling Effects in a Sickle Cell Anemia (SCA) Mouse Model, Resulting in Improved RBC Survival and Hemoglobin Levels

Introduction: Sickle cell anemia (SCA) results from a mutant β-globin gene that produces abnormal hemoglobin S (HbS). HbS polymerizes upon deoxygenation, resulting in red blood cell (RBC) sickling and membrane damage, leading to vaso-occlusions and hemolysis. Additionally, sickle RBCs contain less ATP and more 2,3-diphosphoglycerate (2,3-DPG) than normal RBCs; 2,3,DPG allosterically reduces hemoglobin (Hb) oxygen (O2)-affinity [i.e. increases P50], promoting faster unloading of O2, which potentiates HbS polymerization and RBC sickling. FT-4202, a selective and orally bioavailable allosteric activator of RBC pyruvate kinase (PKR), decreases 2,3-DPG and increases ATP in normal human RBCs (Blood, 2019, 134, Supplement 1:616). We hypothesized that oral administration of FT-4202 to SCA mice will increase HbS O2-affinity, and thereby decrease RBC sickling and membrane damage. Methods: Berkeley SCA mice were given 500-1000 mg/kg/day FT-4202 in chow (FT-4202 group) or control chow (control group) in 4 cohorts for 2 weeks (total 17-18 mice/group). In all cohorts, the health status, weight, and average chow consumption of each mouse was determined 3 times/week. Three cohorts were injected with sulfo-NHS-biotin 1 week into treatment (10-11 mice/group), and RBC survival assessed over the next week with serial micro-bleeds while on treatment. The 4th cohort was only bled at 2 week time-point to obtain P50 (Hemox Analyzer) and Hb levels (Hemavet). At experiment termination, all cohorts were terminally bled to determine (a) RBC levels of 2,3-DPG and ATP, (c) plasma levels of FT-4202 by LC-MS/MS, (d) the proportion of irreversibly sickled RBC (ISC) on blood smears (Image-J analysis), (e) the kinetics of experimentally-induced sickling (Lorrca®Oxygenscan) and (f) membrane deformability (Lorrca®Ektacytometry). Results: SCA mice on FT-4202 consumed a similar amount of food, and had similar weights and survival, compared to SCA mice on control chow throughout the 2-week period. As hypothesized, HbS O2 affinity increased, reflected by a decrease in P50 from 29.6 ± 0.62 mmHg (mean ± SEM) in the control group to 27.6 ± 0.58 mmHg in the FT-4202 group (p&amp;lt;0.03). Determinations of 2,3-DPG, ATP and FT-4202 are ongoing and will be presented. As expected, this increased HbS O2-affinity in the FT-4202 group reduced RBC sickling and membrane damage. At 2 weeks, the proportion of ISCs on blood smears was reduced in the FT-4202 group to 2.4 ± 0.3% vs. 5.9 ± 1.4% in the control group (p&amp;lt;0.02). The sickle RBC half-life increased to 1.8 ± 0.07 days in FT-4202 group vs. 1.4 ± 0.1 days in the control group, a 28% increase in RBC survival (p&amp;lt;0.01, Figure 1A). Hence, Hb levels in the FT-4202 group increased from 9.1 ± 0.2 g/dL before treatment, to 10.8 ± 0.3 g/dL 2 weeks after treatment (p&amp;lt;0.001), while Hb levels in the control group remained unchanged (Figure 1B). The reticulocytes remained unchanged in both groups before and after treatment. When sickle RBCs were de-oxygenated from an ambient pO2 of ~150 mmHg to a pO2 of 10-15 mmHg, followed by their re-oxygenation to ambient pO2 at a constant shear stress of 30 Pa (Oxygenscan), the point of sickling (PoS; pO2 level when the EI becomes 95% of the EI at ambient O2) decreased on average from 37% pO2 in the control group, to 30% pO2 in the FT-4202 group (p&amp;lt;0.002, Figure 1C), with a significantly improved Elongation Index at the point of minimum pO2 (EImin), (p&amp;lt;0.05). Next, RBC membrane deformability was measured under ambient pO2 (normoxic conditions), but varying shear stress after the de-oxygenation/re-oxygenation cycle on the Oxygenscan. Sickle RBCs from the FT-4202 group were significantly more deformable [i.e. had a higher Elongation Index (EI)] compared to control sickle RBCs (p&amp;lt;0.01, Figure 1D), as shear stress increased to ≥3 Pa, demonstrating that FT-4202 sickle RBCs sustained significantly less membrane damage following sickling and un-sickling. Conclusion: A 2-week oral FT-4202 administration was well tolerated by SCA mice and demonstrated beneficial biological effects: improved RBC membrane deformability and sickling parameters, with a shift in the PoS to lower pO2, and increased RBC survival and Hb levels. A parallel human phase-I study in healthy subjects and sickle cell disease patients to assess the safety and PK/PD of FT-4202 is ongoing (NCT03815695). Overall, our results suggest that FT-4202 can be a potentially useful orally available agent with significant anti-sickling effect. Disclosures Drake: Forma Therapeutics: Other: Shareholder of Forma Therapeutics. Fulzele:FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics. Guichard:FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics; AstraZeneca: Other: Shareholder. Malik:Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents &amp; Royalties.

Mixing commonly used crystalloid solutions with red blood cells in five common additives does not negatively impact hemolysis, aggregometry, or deformability.

Literature is beginning to challenge the belief that it is unsafe to coinfuse red blood cells (RBCs) with solutions other than isotonic saline. We recently showed that additive-free RBCs tolerated coincubation with Plasma-Lyte or catecholamines dissolved in normal saline (NS), though 5% dextrose in water (D5W) promoted hemolysis. Herein, we evaluate the effect of coincubating crystalloids on additive-preserved RBC hemolysis, aggregation, and membrane deformability. RBCs were coincubated 5 minutes with plasma, NS, Plasma-Lyte, lactated Ringer's (LR) or D5W (1 mL PRBC +131.3 μL solution). Samples were then assessed for hemolysis (free hemoglobin), aggregation (critical shear stress [mPa]), and membrane deformability (elongation index [EI]). Significance (P ≤ .05) by t test or ANOVA with post-hoc Tukey-Kramer test. Additive-prepared RBCs coincubated with crystalloid instead of plasma demonstrated: (a) no increase in hemolysis as indicated by plasma free hemoglobin levels that is likely to be clinically relevant; (b) no increase, but in some cases a decrease, in aggregation as indicated by critical shear stress; and (c) in some combinations, a deterioration in deformability. When present, the deformability decrease was likely clinically insignificant in degree, and always returned to normal when the crystalloid was subsequently diluted out with plasma. Our data suggest that additive-prepared RBCs coincubated for 5 minutes with any of four common crystalloids demonstrate no clinically relevant increased lysis, increased aggregation, or decreased deformability.

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).

Stochastic bond dynamics facilitates alignment of malaria parasite at erythrocyte membrane upon invasion.

Malaria parasites invade healthy red blood cells (RBCs) during the blood stage of the disease. Even though parasites initially adhere to RBCs with a random orientation, they need to align their apex toward the membrane in order to start the invasion process. Using hydrodynamic simulations of a RBC and parasite, where both interact through discrete stochastic bonds, we show that parasite alignment is governed by the combination of RBC membrane deformability and dynamics of adhesion bonds. The stochastic nature of bond-based interactions facilitates a diffusive-like re-orientation of the parasite at the RBC membrane, while RBC deformation aids in the establishment of apex-membrane contact through partial parasite wrapping by the membrane. This bond-based model for parasite adhesion quantitatively captures alignment times measured experimentally and demonstrates that alignment times increase drastically with increasing rigidity of the RBC membrane. Our results suggest that the alignment process is mediated simply by passive parasite adhesion.

A Novel Composition of Endogenous Metabolic Modulators, AXA4010, Impacts Adhesion, Inflammation and RBC Membrane Deformability in Preclinical Models of Sickle Cell Disease

Background: In sickle cell disease (SCD), red blood cell (RBC) sickling propagates hemolysis, inflammation, vascular and endothelial injury with associated disruption in metabolic pathways including nitric oxide depletion, oxidative stress and an increased anabolic and energetic demand due to high RBC turnover. These metabolic functions highlight the role of amino acid biology in blood health and diseases such as SCD, which has motivated numerous individual amino acid (AA) based clinical studies. Here, we measured AA profiles of SCD plasma samples and examined the impact of combinations of AAs and metabolites on preclinical models of vascular adhesion, inflammation, and RBC function. This combined work serves as the basis for investigational product AXA4010, a composition of 10 distinct AAs and metabolites designed to target and support RBC, inflammatory, endothelial and vascular health, with the potential to impact the multifaceted pathology of SCD. Methods: Plasma AA profiles of SCD adults (n=38) and healthy adults (n=40) were measured by LC-MS analysis. Plasma AA profiles from SCD adults informed the design of a defined "SCD medium" used for culturing primary human lung microvascular endothelial cells (HLMVEC) as a preclinical SCD model of adhesion and inflammation. HLMVEC cells cultured in "SCD medium" were co-treated with TNFα and either AXA4010, components of AXA4010, or individual constituent AAs/metabolites, and adhesion, inflammation, and cell migration pathways were characterized using functional assays. Healthy RBCs were treated ex vivo with AXA4010 or individual constituents and stressed using tert-Butyl hydroperoxide (t-BuOOH) treatment, RBC deformability was characterized by ektacytometry (LORRCA, RR Mechatronics). Results: When compared to age-match controls, plasma AA profiles of SCD adults had significantly reduced arginine (-36%, p = 1.8e-4), leucine (-38%, p = 3.6e-6), valine (-36%, p = 2.8e-8), lysine (-38%, p = 6.2e-9), and histidine (-23%, p = 2.8e-4). In "SCD medium" cultured HLMVECs treated with TNFα, we observed AXA4010 reduced markers of adhesion (↓VCAM-1, ↓ICAM-1), inflammation (↓IL-6), and cell migration (↓MCP-1) in a dose dependent manner. In RBCs, we observed AXA4010 protected against t-BuOOH induced loss of RBC deformability. Deconvolution of AXA4010 and subset components revealed unexpected combinatorial interactions and potentiating effects of the constituents within the composition. Conclusion: We combined human plasma AA profiling and combinatorial cell-based experimentation to design AXA4010. In primary human cell systems with AXA4010 we observed improved adhesion, inflammation, and RBC morphology markers, which are associated with blood health and SCD pathogenesis, in a composition specific manner. These results warrant clinical investigation of AXA4010, which we have now initiated in a cohort of SCD subjects to investigate safety, tolerability and impact on blood physiology. Disclosures Hamill: Axcella Health: Employment. Carroll:Axcella Health: Employment. Wani:Axcella Health: Employment. Russell:Axcella Health: Employment. Steuert:Axcella Health: Employment. Nitzel:Axcella Health: Employment. Downey:Axcella Health: Employment. Cokol:Axcella Health: Employment. Afeyan:Axcella Health: Employment. Marukian:Axcella Health: Employment. Tramontin:Axcella Health: Employment.

PS1205 META‐ANALYSIS USING DATA OF RED BLOOD CELL ENZYME ASSAY AND PROTEOME IN PATIENTS WITH DIAMOND‐BLACKFAN ANEMIA

Background:Diamond‐Blackfan anemia (DBA) is a form of congenital bone marrow failure syndrome, which is characterized by RBC aplasia with varied malformation in infants. Elevated enzymatic activity of adenosine deaminase (ADA) is known to be a valuable biomarker of DBA. The ADA assay has been shown to be more sensitive than the gene test of ribosome subunit genes. We recently reported that reduced glutathione (GSH) in red blood cells (RBCs) is another biomarker of DBA, and simultaneous assessment of ADA and GSH enabled us to discriminate DBA patients from normal subjects as well as unaffected family members (Blood Cells Mol Dis 59:31–36, 2016).Aims:This study was performed to investigate the detailed changes in enzymatic activity and enzyme protein mass in DBA erythrocytes. We reanalyzed the enzymatic activity and GSH in 14 DBA patients and compared the results with those obtained from simultaneously assayed 14 normal subjects. Furthermore, we performed a meta‐analysis using proteome data as recently reported by Pesciotta, et al. (PLoS ONE 10:e0140036, 2015).Methods:The gene mutations of the enrolled patients were previously confirmed. We estimated the activity of RBC enzymes and GSH content by a standard method using a spectrophotometer. We calculated the relative expression of both enzymatic activity and protein content. The statistical analyses were performed by Wilcoxon rank‐sum test and a P value of &lt;0.001 was considered statistically significant.Results:The combined analyses of proteomic and enzymatic data revealed that ADA, PGD, and HK‐1 (proteome)/HK (activity) were elevated, while PFKL (proteome)/PFK (activity) and PKLR (proteome)/PK (activity) were decreased. The G6PD enzyme activity, but not its protein level, was characteristically increased.Summary/Conclusion:We speculate that the discrepancy between the activity and protein level of G6PD was attributable to the decreased inhibition of G6PD activity by NADPH. In other words, G6PD activity was supposedly activated by a lower NADPH level in DBA. To maintain the affordable level of GSH in RBCs, reduction of GSSG was necessarily maximized through the reaction catalyzed by glutathione reductase, which used NADPH as a cofactor. As a result, NADPH content was presumably exhausted, resulting in decreased inhibition of G6PD. In this study, we found that HK activity and HK‐1 content were elevated significantly. On the other hand, the activity and protein content of two important rate‐limiting enzymes (PFK/PK and PFKL/PKLR) involved in glycolysis were found to be decreased. Higher demand for NADPH production through the pentose phosphate pathway leads to a reduction in glucose consumption in the glycolytic pathway, and/or activation of p53 leads to inhibition of glycolysis, as previously reported. The present study shows the inhibition of glycolysis in the RBCs in DBA. It needs to be elucidated whether impaired glycolysis partly accounts for premature cell death of erythroblasts in DBA.Abbreviations: DBA: Diamond‐Blackfan anemia; RBC: red blood cell; ADA: adenosine deaminase; GSH: reduced glutathione; PGD: 6‐phosphogluconate dehydrogenase; HK: hexokinase; PFK (L): 6‐phosphofructokinase (liver type); PK (LR): pyruvate kinase (liver and erythrocytes type); G6PD: glucose‐6‐phosphate dehydrogenase; NADPH: reduced nicotinamide adenine dinucleotide phosphate; GSSG: glutathione disulfide.

Computational analysis of nitric oxide biotransport in a microvessel influenced by red blood cells

It is pivotal that endothelium-dependent Nitric Oxide (NO) consumed by hemoglobin (Hb) inside red blood cells (RBCs) membrane, regulates the vascular tone. The whole processes of NO transport in vessel containing flowing RBCs is still not clear, such as NO production in endothelium, diffusion in plasma and consumption inside RBCs. In this work, the motion of RBCs in a microvessel is investigated by using immersed boundary lattice Boltzmann method (IB-LBM) first and the deformability of RBCs is expressed by using spring network model which is based on the minimum energy principle. Furthermore, the interaction between RBCs is considered. Based on the wall shear stress (WSS), NO production rate originated from endothelium was obtained by using a hyperbolic model. NO distribution inside the microvessel with multiple RBCs was computed by using immersed boundary finite difference method (IB-FDM). The result shows that a large (small) WSS exists at locations with a relatively wide(narrow) gap between the wall and cell. In terms of mass transfer, an increase of RBC membrane permeability leads to a decrease of NO concentration in the vessel and the surrounding endothelium significantly. In addition, with the increasing of hematocrit (Hct) value, NO concentration distribution in the whole vessel decreases both in the lumen and vascular wall. Finally, the thickness of RBCs-depleted layer gradually decreases with the weakened deformability of RBCs membrane, and the change degree of cell free layer (CFL) thickness decreases as the bending stiffness is relatively higher. Thus, when bending stiffness is higher, the NO concentration in vascular wall is reduced resulting from the thinner CFL.

Numerical Determination of Red Blood Cell Membrane Deformability from Intravital Microscopy

IntroductionRed cells in microvessels deform extensively in order to flow, thus resulting in high observed apparent viscosities and high stress concentrations localized to the red cell membrane. Flow through microvessels is consequently aided by the high compliance in the red cell membrane. However, changes in the mechanical properties of Red Blood Cells (RBC) as a result of storage lesions, can increase stress concentrations in the membrane, impairing oxygen transport and resulting in increased vesiculation and likely cell lysis. The objective of this study is to determine the mechanical properties and thus wall shear stress experienced by a RBC membrane during capillary flow by applying numerical methods and image analysis to videos obtained using cremaster muscle preparation.MethodsThe RBC membrane is modeled as a two dimensional Hookean Isotropic Solid. Videos of capillary flow of both fresh RBCs and RBCs stored for 2 weeks were taken in the rat cremaster muscle. The mechanical properties, including the elastic modulus, shear stress, and shear strain, of both conditions were compared for both groups. In order to determine strain, each RBC membrane will be parameterized by N (X,Y) points determined by the Moore Neighbor Tracing algorithm of videos of capillary flow obtained by intravital microscopy of the rat cremaster muscle, from which N deformation gradient tensors, [F], can be obtained numerically for each frame of the video. From these deformation gradient tensors, the updated Cauchy Strain Tensor will be determined as a function of position on the Cartesian plane relative to the centroid of the RBC. The strains determined from the updated Langrangian Green Strain are numerically fit to the Navier‐Lame Equations along the boundary of the RBC in order to experimentally fit the two Lame's Constants during capillary flow. These constants will then be used to determine the Cauchy Stress Tensor, using Hooke's Law, along the boundary of the RBC, allowing for the calculation of the Wall Shear Stress.Results and DiscussionThe average Elastic Modulus of fresh cells was found to be 5.4 ×106 ± 3.6 × 106 Pa, while of that of RBCs stored for two weeks was found to be statistically larger and was calculated to be 6.0 ×106 ± 9.7 × 107 Pa. Additionally, stored RBCs experienced higher shear strains and wall shear strains over larger areas of the membrane as compared with stored cells. Additionally, stress concentrators in the membrane were observed at the points of contact with the capillary wall. However, for stored cells, the high stress acted on a larger segment of the membrane. This algorithm for determining wall shear stress experienced by RBC membranes in vivo during capillary flow was thus able to detect statistically significant differences in the both the mechanical properties and stress concentrations between fresh cells and stored cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rho‐kinase Inhibition Improves Hemodynamic Responses and Circulating ATP During Hypoxia and Exercise in Healthy Older Adults

Circulating ATP is a potent vasodilator believed to assist in the matching of tissue oxygen delivery to oxygen demand. Older adults have impaired skeletal muscle hemodynamic responses to hypoxia and exercise and blunted increases in circulating ATP during these stimuli, which may be due to reduced deoxygenation‐mediated red blood cell (RBC) ATP release. We recently demonstrated that treatment of isolated RBCs with a Rho‐kinase inhibitor improves RBC membrane deformability and restores ATP release from RBCs of older adults. Thus, the goal of the present study was to test the hypothesis that in vivo Rho‐kinase inhibition would improve hemodynamic responses and circulating ATP during hypoxia and exercise in older adults. Healthy young (Y; 27 ± 1 years; n = 5) and older (O; 67 ± 2 years; n = 7) adults participated in a double‐blind, randomized, placebo‐controlled, crossover design study on two days (≥ 5 days between visits). A deep venous catheter in the forearm was used to administer the placebo (100 ml saline/60 min) or the Rho‐kinase inhibitor (60 mg fasudil/60 min) and to sample blood for plasma [ATP]. Forearm vascular conductance (FVC; mean arterial pressure from finometry and forearm blood flow (FBF) from Doppler ultrasound) was calculated at rest, during 5 min of isocapnic hypoxia (80% SpO2), and during graded intensity rhythmic handgrip exercise at 5, 15 and 25% of maximum voluntary contraction (MVC; 4 min per workload). Plasma [ATP] was measured at rest and at the end of each condition, and RBCs were isolated to measure ATP release in response to normoxic (PO2 ~120 mmHg) and hypoxic (PO2 ~24 mmHg) stimuli using the luciferin‐luciferase technique. In the placebo trial, the increase in FVC during hypoxia was ~50% lower in O vs Y, and during exercise the greatest age impairment occurred at 25% MVC (266.4 ± 27.6 vs 394.9 ± 35.0 ml/min/100 mmHg; P &lt; 0.05). Plasma [ATP] and ATP effluent (FBF × [ATP]; an index of the total circulating rate of ATP) tended to be lower in O vs Y during hypoxia (75.3 ± 16.6 vs 100.6 ± 18.8 nmol/L and 2.3 ± 0.7 vs 3.1 ± 0.7 nmol/min, respectively) and were lower at 25% MVC (92.8 ± 12.6 vs 168.7 ± 31.5 nmol/L and 28.1 ± 4.9 vs 66.9 ± 9.6 nmol/min, respectively; P &lt; 0.05). The increase in isolated RBC ATP release from normoxia to hypoxia was also impaired in O vs Y (21.4 ± 17.2% vs 77.8 ± 13.3%; P &lt; 0.05). Compared to control, fasudil in O improved FVC during hypoxia (35.2 ± 3.7 ml/min/100 mmHg; P &lt; 0.05) and 25% MVC exercise (324.4 ± 21.7 ml/min/100 mmHg; P &lt; 0.05), and abolished the impairment vs Y. Fasudil tended to improve plasma [ATP] and ATP effluent during hypoxia in O (98.7 ± 29.0 nmol/L and 3.34 ± 0.8 nmol/min), and during 25% MVC exercise (152.2 ± 26.7 nmol/L and 48.3 ± 6.7 nmol/min). Fasudil also tended to improve isolated RBC ATP release (55.6 ± 17.5% vs 21.4 ± 17.2%) and abolished the impairment vs Y. These preliminary data suggest that in vivo Rho‐kinase inhibition improves hemodynamic responses to hypoxia and exercise in older adults, and that this may be related to elevated circulating ATP.Support or Funding InformationHL119337, F31HL126377This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Shelph2, a bacterial-like phosphatase of the malaria parasite Plasmodium falciparum, is dispensable during asexual blood stage.

During the erythrocytic cycle of the malaria parasite Plasmodium falciparum, egress and invasion are essential steps finely controlled by reversible phosphorylation. In contrast to the growing number of kinases identified as key regulators, phosphatases have been poorly studied, and calcineurin is the only one identified so far to play a role in invasion. PfShelph2, a bacterial-like phosphatase, is a promising candidate to participate in the invasion process, as it was reported to be expressed late during the asexual blood stage and to reside within an apical compartment, yet distinct from rhoptry bulb, micronemes, or dense granules. It was also proposed to play a role in the control of the red blood cell membrane deformability at the end of the invasion process. However, genetic studies are still lacking to support this hypothesis. Here, we take advantage of the CRISPR-Cas9 technology to tag shelph2 genomic locus while retaining its endogenous regulatory regions. This new strain allows us to follow the endogenous PfShelph2 protein expression and location during asexual blood stages. We show that PfShelph2 apical location is also distinct from the rhoptry neck or exonemes. We further demonstrate PfShelph2 dispensability during the asexual blood stage by generating PfShelph2-KO parasites using CRISPR-Cas9 machinery. Analyses of the mutant during the course of the erythrocytic development indicate that there are no detectable phenotypic consequences of Pfshelph2 genomic deletion. As this lack of phenotype might be due to functional redundancy, we also explore the likelihood of PfShelph1 (PfShelph2 paralog) being a compensatory phosphatase. We conclude that despite its cyclic expression profile, PfShelph2 is a dispensable phosphatase during the Plasmodium falciparum asexual blood stage, whose function is unlikely substituted by PfShelph1.

A multiscale framework for large deformation modeling of RBC membranes

In the present contribution, a multiscale framework for nonlinear analysis of finite deformation of red blood cell (RBC) membrane is developed. The first-order Cauchy–Born rule is adopted to establish an atomistic enriched hyperelastic constitutive model and to develop macroscale stress–strain relation of the RBC membrane. In order to circumvent the inherent limitations of utilizing mesh-based methods for large deformation analysis, we systematically coupled the 3D multiscale scheme with the element-free IMLS-Ritz method for numerical modeling of RBC deformability by simulating the optical tweezers experiment. This development was partly motivated by the need for a more precise scheme for modeling membrane structures. The effectiveness of the proposed approach is affirmed by the better prediction of RBC membrane deformability in comparison with experimental and numerical results found in literature and a significant reduction in computational cost. Our approach enables precise characterization of the effect of varying microstructure parameters, physiological, and osmolality conditions on the deformability of RBC membrane.

Genotype-phenotype correlations in hereditary elliptocytosis and hereditary pyropoikilocytosis

Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are heterogeneous red blood cell (RBC) membrane disorders that result from mutations in the genes encoding α-spectrin (SPTA1), β-spectrin (SPTB), or protein 4.1R (EPB41). The resulting defects alter the horizontal cytoskeletal associations and affect RBC membrane stability and deformability causing shortened RBC survival. The clinical diagnosis of HE and HPP relies on identifying characteristic RBC morphology on peripheral blood smear and specific membrane biomechanical properties using osmotic gradient ektacytometry. However, this phenotypic diagnosis may not be readily available in patients requiring frequent transfusions, and does not predict disease course or severity. Using Next-Generation sequencing, we identified the causative genetic mutations in fifteen patients with clinically suspected HE or HPP and correlated the identified mutations with the clinical phenotype and ektacytometry profile. In addition to identifying three novel mutations, gene sequencing confirmed and, when the RBC morphology was not evaluable, identified the diagnosis. Moreover, genotypic differences justified the phenotypic differences within families with HE/HPP.

112 Membrane changes in liposome-treated rat red blood cells upon hypothermic storage

Liposomes, which are microscopic synthetic lipid vesicles, have been shown to improve red blood cell (RBC) in vitro quality by minimizing membrane damage occurring during 42 day storage. Considering that the use of animal models is an essential step in preclinical studies for evaluating transfusion efficacy with novel manipulations of blood transfusion products, the aim of this study was to evaluate the effect of four liposome formulations on rat RBC membrane-related quality parameters during hypothermic storage. Unilamellar liposomes were synthesized using an extrusion method to contain lipid bilayer of saturated (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC):cholesterol, 7:3 mol%), unsaturated (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):cholesterol, 7:3 mol%), saturated charged (DPPC:cholesterol: phosphatidylserine (PS), 6:3:1 mol%) and unsaturated charged (DOPC:cholesterol:PS, 6:3:1 mol%) phospholipids. The leukoreduced, packed RBCs from Sprague-Dawley rats ( n = 3) were incubated for 1 h at 37 °C in a mixture of AS-3 solution with either HEPES-NaCl solution (control) or 2 mM lipid from four different liposome compositions. RBC membrane quality was assessed after treatment, one and five weeks of hypothermic storage by % hemolysis (Drabkins), deformability (ektacytometry), hematological indices (Coulter), morphology (microscopy), cholesterol and phospholipid concentration (colorimetry). All four liposome treatments resulted in significant decrease in percent hemolysis, with the effect most prominent with DOPC-treated RBCs (1.6 ± 0.1% vs. 3.1 ± 0.2 %, p = 0.05). No alterations in rat RBC membrane deformability, morphology, or hematological indices were detected in liposome treated RBCs immediately after the treatment. While DOPC liposome treatment resulted in significant increase in rat RBC phospholipid concentration, DPPC liposome treatment seemed to induce loss of phospholipids from the rat RBC membranes (0.30 ± 0.03 mg vs 0.15 ± 0.01 mg, p = 0.05). The cholesterol: phospholipid ratio in DOPC-treated rat RBCs was comparable to control (0.49 ± 0.06 vs 0.51 ± 0.08, p = 0.80). Upon the first week of storage DOPC-treated RBCs demonstrated significantly lower hemolysis (1.4 ± 0.1% vs. 2.5 ± 0.1%, p = 0.05) with values that are comparable to the ones of fresh RBC samples ( p = 0.25). All liposome treated RBCs continued to exhibit decreased hemolysis after 5 weeks of storage compared to the control ( p = 0.05), with uncharged formulations resulting in lower hemolysis than RBCs treated with charged liposomes (3.4 ± 0.2% vs 3.9 ± 0.4%, p = 0.01). The cell rigidity was significantly improved in DOPC-treated RBCs as demonstrated by the decreased KEI values compared to non-treated RBCs (1.01 ± 0.13 vs 1.36 ± 0.16, p = 0.05). The liposome treatment resulted in an improvement in rat RBC in vitro quality upon storage. The stabilizing effect was related to the chemical characteristics of each liposome formulation. The storage induced detrimental changes in the rat RBC membrane were minimized to a greater extent with the unsaturated than saturated liposome treatment. Also, negatively charged liposomes seemed to increase hemolysis upon storage. Therefore, unsaturated-uncharged liposomes will be used in the upcoming in vivo rat studies to further evaluate the pre-clinical effect of transfusing liposome-treated RBCs. Source of funding: Canadian Blood Services. Conflict of interest: None declared. jelena.holovati@ualberta.ca