Eryptosis: a driver of anemia in chronic kidney disease.

PURPOSE OF REVIEW: Anemia, characterized by a reduction in red blood cell (RBC) count or hemoglobin concentration, commonly accompanies chronic kidney disease (CKD), significantly impacting patients' quality of life. This review delves into the multifaceted nature of anemia in CKD, with a focus on novel mechanisms, particularly the dysregulation of eryptosis or programmed cell death of RBCs, leading to shortened RBC lifespan. RECENT FINDINGS: Recent studies in CKD patients and mouse models revealed that eryptosis, driven by factors such as uremic toxins, inflammation, and imbalances in calcium homeostasis, plays a pivotal role in the development of renal anemia. Dysregulated eryptosis results in premature RBC destruction, exacerbating the hypoproliferative character of anemia in CKD. SUMMARY: Recognizing the intricate relationship between eryptosis and anemia in CKD opens promising prospects for improving patient outcomes and enhancing our understanding of this complex condition. Future research and therapeutic development in this area hold the potential to improve anemia treatment of CKD patients.

Pathophysiology of Red Blood Cell Dysfunction in Diabetes and Its Complications.

Diabetes Mellitus (DM) is a complex metabolic disorder associated with multiple microvascular complications leading to nephropathy, retinopathy, and neuropathy. Mounting evidence suggests that red blood cell (RBC) alterations are both a cause and consequence of disturbances related to DM-associated complications. Importantly, a significant proportion of DM patients develop varying degrees of anemia of confounding etiology, leading to increased morbidity. In chronic hyperglycemia, RBCs display morphological, enzymatic, and biophysical changes, which in turn prime them for swift phagocytic clearance from circulation. A multitude of endogenous factors, such as oxidative and dicarbonyl stress, uremic toxins, extracellular hypertonicity, sorbitol accumulation, and deranged nitric oxide metabolism, have been implicated in pathological RBC changes in DM. This review collates clinical laboratory findings of changes in hematology indices in DM patients and discusses recent reports on the putative mechanisms underpinning shortened RBC survival and disturbed cell membrane architecture within the diabetic milieu. Specifically, RBC cell death signaling, RBC metabolism, procoagulant RBC phenotype, RBC-triggered endothelial cell dysfunction, and changes in RBC deformability and aggregation in the context of DM are discussed. Understanding the mechanisms of RBC alterations in DM provides valuable insights into the clinical significance of the crosstalk between RBCs and microangiopathy in DM.

A positive blood culture is associated with a lower haemoglobin increment in hospitalized patients after red blood cell transfusion.

BACKGROUND AND OBJECTIVES: Abundant clinical evidence supports the safety of red blood cell (RBC) concentrates for transfusion irrespective of storage age, but still, less is known about how recipient characteristics may affect post-transfusion RBC recovery and function. Septic patients are frequently transfused. We hypothesized that the recipient environment in patients with septicaemia would blunt the increase in post-transfusion blood haemoglobin (Hb). The main objective was to compare the post-transfusion Hb increment in hospitalized patients with or without a positive blood culture. MATERIALS AND METHODS: A retrospective cohort study using data from the Transfusion Research, Utilization, Surveillance, and Tracking database (TRUST) was performed. All adult non-trauma in-patients transfused between 2010 and 2017 with ≥1 RBC unit, and for whom both pre- and post-transfusion complete blood count and pre-transfusion blood culture data were available were included. A general linear model with binary blood culture positivity was fit for continuous Hb increment after transfusion and was adjusted for patient demographic parameters and transfusion-related covariates. RESULTS: Among 210,263 admitted patients, 6252 were transfused: 596 had positive cultures, and 5656 had negative blood cultures. A modelled Hb deficit of 1.50 g/L in blood culture-positive patients was found. All covariates had a significant effect on Hb increment, except for the age of the transfused RBC. CONCLUSION: Recipient blood culture positivity was associated with a statistically significant but modestly lower post-transfusion Hb increment in hospitalized patients. In isolation, the effect is unlikely to be clinically significant, but it could become so in combination with other recipient characteristics.

Examination of Bovine Red Blood Cell Death in Vitro in Response to Pathophysiologic Proapoptotic Stimuli.

BACKGROUND: Interspecies variations in mammalian red blood cells (RBCs) are observed in circulating RBC lifespan, cell size, fluidity, aggregation, water permeability, metabolism, lipid composition, and the overall proteome. Bovine RBC cell membrane is deficient in phosphatidylcholine and exhibits anomalies in the arrangement of phosphatidylethanolamine within the lipid bilayer. However, like human RBCs, virtually all the aminophospholipid phosphatidylserine (PS) is found within the cytoplasmic side of the cell membrane of intact circulating bovine RBCs. During apoptotic cell death of human and murine RBCs, PS translocates to the outer leaflet of the cell membrane via Ca2+-dependent and -independent signaling mechanisms. However, little is known about this process in bovine RBCs. METHODS: Using cytofluorometry analyses, we characterized and compared the cell death responses in bovine and human RBCs in vitro exposed to various pathophysiologic cell stressors. RESULTS: Ionic stress, by ionophore treatment, and oxidative stress enhanced cytoplasmic Ca2+ levels and cell membrane PS expression in both bovine and human RBCs. Fever-grade hyperthermia and energy starvation promoted Ca2+ influx and elevated reactive oxygen species levels in both human and bovine RBCs. However, bovine RBCs displayed minimal increases in PS expression elicited by hyperthermia, energy starvation, and extracellular hypertonicity as compared to human RBCs. In response to decreased extracellular osmolality, bovine RBCs exhibited significantly enhanced fragility as compared to human RBCs. CONCLUSIONS: Bovine RBCs display differential cell death patterns as compared to human RBCs, only partly explained by increased Ca2+ influx and oxidative stress. Premature removal of circulating RBCs could potentially contribute to the pathogenesis of anemia in cattle caused by a wide range of factors such as systemic diseases, parasitic infections, and nutritional deficiencies.

The bending rigidity of the red blood cell cytoplasmic membrane.

An important mechanical property of cells is the membrane bending modulus, κ. In the case of red blood cells (RBCs) there is a composite membrane consisting of a cytoplasmic membrane and an underlying spectrin network. Literature values of κ are puzzling, as they are reported over a wide range, from 5 kBT to 230 kBT. To disentangle the contribution of the cytoplasmic membrane from the spectrin network, we investigated the bending of red blood cell cytoplasmic membranes (RBCcm) in the absence of spectrin and adenosine triphosphate (ATP). We used a combination of X-ray diffuse scattering (XDS), neutron spin-echo (NSE) spectrometry and Molecular Dynamics (MD) simulations. Our results indicate values of κ of order 4 kBT to 6 kBT, relatively small compared to literature values for most single component lipid bilayers. We suggest two ways this relative softness might confer biological advantage.

Inductively-Coupled Plasma Mass Spectrometry-Novel Insights From an Old Technology Into Stressed Red Blood Cell Physiology.

BACKGROUND: Ion and metal homeostasis are critical to red blood cell physiology and Inductively Coupled Plasma (ICP) is a decades old approach to pursue elemental analysis. Recent evolution of ICP has resulted in its coupling to mass spectrometry (MS) instead of atomic absorption/emission. METHODS: Here we performed Inductively-coupled plasma mass spectrometry (ICP-MS) measurements of intra- and extra-cellular Na, K, Ca, Mg, Fe, and Cu in red blood cells undergoing ionic, heat, or starvation stress. Results were correlated with Ca measurements from other common platforms (e.g., fluorescence-based approaches) and extensive measurements of red blood cell metabolism. RESULTS: All stresses induced significant intra- and extracellular alterations of all measured elements. In particular, ionomycin treatment or hypertonic stress significantly impacted intracellular sodium and extracellular potassium and magnesium levels. Iron efflux was observed as a function of temperatures, with ionic and heat stress at 40°C causing the maximum decrease in intracellular iron pools and increases in the supernatants. Strong positive correlation was observed between calcium measurements via ICP-MS and fluorescence-based approaches. Correlation analyses with metabolomics data showed a strong positive association between extracellular calcium and intracellular sodium or magnesium levels and intracellular glycolysis. Extracellular potassium or iron were positively correlated with free fatty acids (especially mono-, poly-, and highly-unsaturated or odd-chain fatty acid products of lipid peroxidation). Intracellular iron was instead positively correlated with saturated fatty acids (palmitate, stearate) and negatively with methionine metabolism (methionine, S-adenosylmethionine), phosphatidylserine exposure and glycolysis. CONCLUSION: In the era of omics approaches, ICP-MS affords a comprehensive characterization of intracellular elements that provide direct insights on red blood cell physiology and represent meaningful covariates for data generated via other omics platforms such as metabolomics.

Comparative evaluation of serological tests used for the diagnosis of rickettsial diseases prevalent in the temperate region of North India.

PURPOSE: The clinical manifestations of rickettsial diseases mimic other endemic infections with similar presentations thus posing a serious challenge to clinicians for their diagnosis. For the diagnosis of rickettsial disease serological tests like Weil Felix, ELISA and IFA are used. There are limited studies that have evaluated different serological tests for the diagnosis of rickettsial diseases. Therefore, the present study was undertaken to evaluate the ELISA and Weil Felix test for the diagnosis of rickettsial diseases prevalent in this region. METHODS: Samples from 281 patients clinically suspected of rickettsial diseases were tested for spotted fever group (SFG), typhus group (TG) and scrub typhus group (STG) by Weil Felix, ELISA and IFA was taken as the gold standard. Baseline titers and cut-off ODs were calculated by taking samples from healthy blood donors. RESULTS: The sensitivity, specificity, positive and negative predictive value of Weil Felix test ranged from 30% to 44%, 83.46%-97.86%, 9%-77%, 92-96% respectively. The sensitivity and specificity, positive and negative predictive value of ELISA ranged from 80.77% to 96.15%, 96.33%-98.43%, 70.21%-88.64%, 92.89%-99.60% respectively. Maximum cross-reactions were observed between SFG and STG by the Weil Felix test and between STG and TG by ELISA. CONCLUSIONS: ELISA was found to be sensitive and specific for the diagnosis of rickettsial diseases. It is easy to perform, does not require a technical expert for result interpretation and a large number of samples can be processed at a time.

Blood bank storage of red blood cells increases RBC cytoplasmic membrane order and bending rigidity.

Blood banks around the world store blood components for several weeks ensuring its availability for transfusion medicine. Red blood cells (RBCs) are known to undergo compositional changes during storage, which may impact the cells' function and eventually the recipients' health. We extracted the RBC's cytoplasmic membrane (RBCcm) to study the effect of storage on the membranes' molecular structure and bending rigidity by a combination of X-ray diffraction (XRD), X-ray diffuse scattering (XDS) and coarse grained Molecular Dynamics (MD) simulations. Blood was stored in commercial blood bags for 2 and 5 weeks, respectively and compared to freshly drawn blood. Using mass spectrometry, we measured an increase of fatty acids together with a slight shift towards shorter tail lengths. We observe an increased fraction (6%) of liquid ordered (lo) domains in the RBCcms with storage time, and an increased lipid packing in these domains, leading to an increased membrane thickness and membrane order. The size of both, lo and liquid disordered (ld) lipid domains was found to decrease with increased storage time by up to 25%. XDS experiments reveal a storage dependent increase in the RBCcm's bending modulus κ by a factor of 2.8, from 1.9 kBT to 5.3 kBT. MD simulations were conducted in the absence of proteins. The results show that the membrane composition has a small contribution to the increased bending rigidity and suggests additional protein-driven mechanisms.

Proteinuric chronic kidney disease is associated with altered red blood cell lifespan, deformability and metabolism.

Anemia is a common complication of chronic kidney disease, affecting the quality of life of patients. Among various factors, such as iron and erythropoietin deficiency, reduced red blood cell (RBC) lifespan has been implicated in the pathogenesis of anemia. However, mechanistic data on in vivo RBC dysfunction in kidney disease are lacking. Herein, we describe the development of chronic kidney disease-associated anemia in mice with proteinuric kidney disease resulting from either administration of doxorubicin or an inducible podocin deficiency. In both experimental models, anemia manifested at day 10 and progressed at day 30 despite increased circulating erythropoietin levels and erythropoiesis in the bone marrow and spleen. Circulating RBCs in both mouse models displayed altered morphology and diminished osmotic-sensitive deformability together with increased phosphatidylserine externalization on the outer plasma membrane, a hallmark of RBC death. Fluorescence-labelling of RBCs at day 20 of mice with doxorubicin-induced kidney disease revealed premature clearance from the circulation. Metabolomic analyses of RBCs from both mouse models demonstrated temporal changes in redox recycling pathways and Lands' cycle, a membrane lipid remodeling process. Anemic patients with proteinuric kidney disease had an increased proportion of circulating phosphatidylserine-positive RBCs. Thus, our observations suggest that reduced RBC lifespan, mediated by altered RBC metabolism, reduced RBC deformability, and enhanced cell death contribute to the development of anemia in proteinuric kidney disease.

Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance, survival, and deformability of human red blood cells.

BACKGROUND: Thrombospondin-1 (TSP-1), a Ca2+-binding trimeric glycoprotein secreted by multiple cell types, has been implicated in the pathophysiology of several clinical conditions. Signaling involving TSP-1, through its cognate receptor CD47, orchestrates a wide array of cellular functions including cytoskeletal organization, migration, cell-cell interaction, cell proliferation, autophagy, and apoptosis. In the present study, we investigated the impact of TSP-1/CD47 signaling on Ca2+ dynamics, survival, and deformability of human red blood cells (RBCs). METHODS: Whole-cell patch-clamp was employed to examine transmembrane cation conductance. RBC intracellular Ca2+ levels and multiple indices of RBC cell death were determined using cytofluorometry analysis. RBC morphology and microvesiculation were examined using imaging flow cytometry. RBC deformability was measured using laser-assisted optical rotational cell analyzer. RESULTS: Exposure of RBCs to recombinant human TSP-1 significantly increased RBC intracellular Ca2+ levels. As judged by electrophysiology experiments, TSP-1 treatment elicited an amiloride-sensitive inward current alluding to a possible Ca2+ influx via non-selective cation channels. Exogenous TSP-1 promoted microparticle shedding as well as enhancing Ca2+- and nitric oxide-mediated RBC cell death. Monoclonal (mouse IgG1) antibody-mediated CD47 ligation using 1F7 recapitulated the cell death-inducing effects of TSP-1. Furthermore, TSP-1 treatment altered RBC cell shape and stiffness (maximum elongation index). CONCLUSIONS: Taken together, our data unravel a new role for TSP-1/CD47 signaling in mediating Ca2+ influx into RBCs, a mechanism potentially contributing to their dysfunction in a variety of systemic diseases. Video abstract.

Stabilization of Lipid Membranes through Partitioning of the Blood Bag Plasticizer Di-2-ethylhexyl phthalate (DEHP).

The safe storage of blood is of fundamental importance to health care systems all over the world. Currently, plastic bags are used for the collection and storage of donated blood and are typically made of poly(vinyl chloride) (PVC) plasticized with di-2-ethylhexyl phthalate (DEHP). DEHP is known to migrate into packed red blood cells (RBC) and has been found to extend their shelf life. It has been speculated that DEHP incorporates itself into the RBC membrane and alters membrane properties, thereby reducing susceptibility to hemolysis and morphological deterioration. Here, we used high-resolution X-ray diffraction and molecular dynamics (MD) simulations to study the interaction between DEHP and model POPC lipid membranes at high (9 mol %) and low (1 mol %) concentrations of DEHP. At both concentrations, DEHP was found to spontaneously partition into the bilayer. At high concentrations, DEHP molecules were found to aggregate in the aqueous phase before inserting as clusters into the membrane. The presence of DEHP in the bilayers resulted in subtle, yet statistically significant, alterations in several membrane properties in both the X-ray diffraction experiments and MD simulations. DEHP led to (1) an increase of membrane width and (2) an increase in the area per lipid. It was also found to (3) increase the deuterium order parameter, however, (4) decrease membrane orientation, indicating the formation of thicker, stiffer membranes with increased local curvature. The observed effects of DEHP on lipid bilayers may help to better understand its effect on RBC membranes in increasing the longevity of stored blood by improving membrane stability.

Decoding the metabolic landscape of pathophysiological stress-induced cell death in anucleate red blood cells.

BACKGROUND: In response to stress, anucleate red blood cells (RBCs) can undergo a process of atypical cell death characterised by intracellular Ca2+ accumulation and phosphatidylserine (PS) externalisation. Here we studied alterations in RBC metabolism, a critical contributor to their capacity to survive environmental challenges, during this process. MATERIALS AND METHODS: Metabolomics analyses of RBCs and supernatants, using ultra-high-pressure liquid chromatography coupled to mass spectrometry, were performed after in vitro exposure of RBCs to different pathophysiological cell stressors, including starvation, extracellular hypertonicity, hyperthermia, and supraphysiological ionic stress. Cell death was examined by flow cytometry. RESULTS: Our data show that artificially enhancing RBC cytosolic Ca2+ influx significantly enhanced purine oxidation and strongly affected cellular bioenergetics by reducing glycolysis. Depleting extracellular Ca2+ curtailed starvation-induced cell death, an effect paralleled by the activation of compensatory pathways such as the pentose phosphate pathway, carboxylic acid metabolism, increased pyruvate to lactate ratios (methemoglobin reductase activation), one-carbon metabolism (protein-damage repair) and glutathione synthesis; RBCs exposed to hypertonic shock displayed a similar metabolic profile. Furthermore, cell stress promoted lipid remodelling as reflected by the levels of free fatty acids, acyl-carnitines and CoA precursors. Notably, RBC PS exposure, independently of the stressor, showed significant correlation with the levels of free fatty acids, glutamate, cystine, spermidine, tryptophan, 5-oxoproline, lactate, and hypoxanthine. DISCUSSION: In conclusion, different cell death-inducing pathophysiological stressors, encountered in various clinical conditions, result in differential RBC metabolic phenotypes, only partly explained by intracellular Ca2+ levels and ATP availability.

Prothrombin, alone or in complex concentrates or plasma, reduces bleeding in a mouse model of blood exchange-induced coagulopathy.

Prothrombin complex concentrates (PCC) are fractionated plasma protein drugs that reverse warfarin anticoagulation. PCC may control more general bleeding. We sought to identify the dominant procoagulant factor in PCC in vivo. We tested PCC or coagulation factor (F) treatment in CD1 mice made coagulopathic by exchange of whole blood for washed red cells. Anesthetized mice were transfused with murine fresh-frozen plasma (mFFP), PCC, mixtures of human vitamin K-dependent proteins (VKDP) (prothrombin, FVII, FIX, or FX), or purified single human VKDP, immediately prior to tail transection (TT), liver laceration (LL), or intravascular laser injury (ILI). Plasma donor mice were treated with vehicle or control antisense oligonucleotide (ASO-CON) or ASO specific for prothrombin (FII) (ASO-FII) to yield mFFP or ASO-CON mFFP or ASO-FII mFFP. Blood losses were determined spectrophotometrically (TT) or gravimetrically (LL). Thrombus formation was quantified by intravital microscopy of laser-injured arterioles. PCC or four factor- (4F-) VKDP or prothrombin significantly reduced bleeding from TT or LL. Omission of prothrombin from 4F-VKDP significantly reduced its ability to limit bleeding. Mice transfused with ASO-FII mFFP demonstrated inferior haemostasis versus those transfused with ASO-FII following TT, LL, or ILI. Prothrombin is the dominant procoagulant component of PCC and could limit bleeding in trauma.

Oxidative stress, eryptosis and anemia: a pivotal mechanistic nexus in systemic diseases.

The average lifespan of circulating erythrocytes usually exceeds hundred days. Prior to that, however, erythrocytes may be exposed to oxidative stress in the circulation which could cause injury and trigger their suicidal death or eryptosis. Oxidative stress activates Ca2+ -permeable nonselective cation channels in the cell membrane, thus, stimulating Ca2+ entry and subsequent cell membrane scrambling resulting in phosphatidylserine exposure and activation of Ca2+ -sensitive K+ channels leading to K+ exit, hyperpolarization, Cl- exit, and ultimately cell shrinkage due to loss of KCl and osmotically driven water. While the mechanistic link between oxidative stress and anemia remains ill-defined, several diseases such as diabetes, hepatic failure, malignancy, chronic kidney disease and inflammation have been identified to display both increased oxidative stress as well as eryptosis. Recent compelling evidence suggests that oxidative stress is an important perpetrator in accelerating erythrocyte loss in different systemic conditions and an underlying mechanism for anemia associated with these pathological states. In the present review, we discuss the role of oxidative stress in reducing erythrocyte survival and provide novel insights into the possible use of antioxidants as putative antieryptotic and antianemic agents in a variety of systemic diseases.

Genetic deficiency of the tumor suppressor protein p53 influences erythrocyte survival.

The transcription factor p53 suppresses tumor growth by inducing nucleated cell apoptosis and cycle arrest. Because of its influence on primitive erythroid cell differentiation and survival, p53 is an important determinant of erythropoiesis. However, the impact of p53 on the fate of erythrocytes, cells lacking nucleus and mitochondria, during their post-maturation phase in the circulation remained elusive. Erythrocyte survival may be compromised by suicidal erythrocyte death or eryptosis, which is hallmarked by phosphatidylserine translocation and stimulated by increase of cytosolic Ca2+ concentration. Here, we comparatively examined erythrocyte homeostasis in p53-mutant mice (Trp53tm1Tyj/J) and in corresponding WT mice (C57BL/6J) by analyzing eryptosis and erythropoiesis. To this end, spontaneous cell membrane phosphatidylserine exposure and cytosolic Ca2+ concentration were higher in erythrocytes drawn from Trp53tm1Tyj/J mice than from WT mice. Eryptosis induced by glucose deprivation, a pathophysiological cell stressor, was slightly, but significantly more prominent in erythrocytes drawn from Trp53tm1Tyj/J mice as compared to WT mice. The loss of erythrocytes by eryptosis was fully compensated by enhanced erythropoiesis in Trp53tm1Tyj/J mice, as reflected by increased reticulocytosis and abundance of erythroid precursor cells in the bone marrow. Accordingly, erythrocyte number, packed cell volume and hemoglobin were similar in Trp53tm1Tyj/J and WT mice. Taken together, functional p53 deficiency enhances the turnover of circulating erythrocytes by parallel increase of eryptosis and stimulated compensatory erythropoiesis.

Heterotrimeric G-protein subunit Gαi2 contributes to agonist-sensitive apoptosis and degranulation in murine platelets.

Gαi2 , a heterotrimeric G-protein subunit, regulates various cell functions including ion channel activity, cell differentiation, proliferation and apoptosis. Platelet-expressed Gαi2 is decisive for the extent of tissue injury following ischemia/reperfusion. However, it is not known whether Gαi2 plays a role in the regulation of platelet apoptosis, which is characterized by caspase activation, cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) translocation to the platelet surface. Stimulators of platelet apoptosis include thrombin and collagen-related peptide (CoRP), which are further known to enhance degranulation and activation of αIIb ß3-integrin and caspases. Using FACS analysis, we examined the impact of agonist treatment on activation and apoptosis in platelets drawn from mice lacking Gαi2 and their wild-type (WT) littermates. As a result, treatment with either thrombin (0.01 U/mL) or CoRP (2 µg/mL or 5 µg/mL) significantly upregulated PS-exposure and significantly decreased forward scatter, reflecting cell size, in both genotypes. Exposure to CoRP triggered a significant increase in active caspase 3, ceramide formation, surface P-selectin, and αIIb ß3-integrin activation. These molecular alterations were significantly less pronounced in Gαi2 -deficient platelets as compared to WT platelets. In conclusion, our data highlight a previously unreported role of Gαi2 signaling in governing platelet activation and apoptosis.

Red blood cells, still vital after all these years: Commentary on Canadian Blood Services' International Symposium 2017.

Canadian Blood Services (CBS), Canada's national blood transfusion service, has for many years sponsored an annual conference, for the education and awareness of interested participants, showcasing the latest evidence-based understanding of both basic science and clinical issues in transfusion medicine and science. The 15th iteration of this symposium took place September 9, 2017 and focused on some of the vital aspects of red blood cells (RBC), in line with the" 3Rs" concept, namely the provision of the Right red blood cell (RBC) product to the Right patient at the Right time. Presentations touched upon: the evolution of blood banking in North America; the monocyte monolayer assay as a predictor of post-transfusion hemolysis; hemoglobin-based oxygen carriers; RBC alloimmunization; serological approaches to complex RBC antibody problems; randomized clinical trials related to the age of stored RBC; RBC genotyping; pathophysiology, prevention and treatment of hemolytic disease of the fetus and newborn (HDFN); and testing and timing in perinatal serology. This commentary provides summaries of all speakers' presentations annotated with relevant references. Special thanks are due to all contributors for their praiseworthy approaches in sharing their experiences and knowledge on this interesting scientific/clinical and management theme.

Bacterial neuraminidase-mediated erythrocyte desialylation provokes cell surface aminophospholipid exposure.

BACKGROUND: Surface desialylation is associated with erythrocyte aging and mediates phagocytic recognition and clearance of senescent erythrocytes. Neuraminidases, a family of glycohydrolytic enzymes, cleave the glycosidic linkages between sialic acid and mucopolysaccharides and have previously been implicated in erythrocyte dysfunction associated with sepsis. Erythrocytes in septic patients further display a phenotype of accelerated eryptosis characterized by membrane phospholipid scrambling resulting in phosphatidylserine (PS) externalization. Herein, we examined the impact of artificial erythrocyte desialylation on eryptosis. METHODS: Using flow cytometry and/or fluorescence microscopy, we analyzed desialylation patterns and eryptotic alterations in erythrocytes exposed to Clostridium perfringens-derived neuraminidase. RESULTS: Exogenous bacterial neuraminidase significantly augmented membrane PS exposure and cytosolic Ca2+ levels in a dose- and time-dependent manner. Neuraminidase treatment significantly reduced fluorescence-tagged agglutinin binding, an effect temporally preceding the increase in PS externalization. Neuraminidase-induced PS exposure was significantly curtailed by pretreatment with the pan-sialidase inhibitor N-acetyl-2,3-dehydro-2-deoxyneuraminic acid. Neuraminidase treatment further induced hemolysis but did not significantly impact erythrocyte volume, ceramide abundance, or the generation of reactive oxygen species. CONCLUSION: Collectively, our data reveal that alteration of erythrocyte sialylation status by bacterial neuraminidase favors eryptotic cell death, an effect potentially contributing to reduced erythrocyte lifespan and anemia in sepsis.