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.

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 ofκare puzzling, as they are reported over a wide range, from 5 k_BT to 230 k_BT. To disentangle the contribution of the cytoplasmic membrane from the spectrin network, we investigated the bending of red blood cell cytoplasmic membranes (RBC_cm) 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 ofκof order 4 k_BT to 6 k_BT, relatively small compared to literature values for most single component lipid bilayers. We suggest two ways this relative softness might confer biological advantage.

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 (RBC_cm) 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 (l_o) domains in the RBC_cms with storage time, and an increased lipid packing in these domains, leading to an increased membrane thickness and membrane order. The size of both,l_oand liquid disordered (l_d) lipid domains was found to decrease with increased storage time by up to 25%. XDS experiments reveal a storage dependent increase in the RBC_cm’s bending modulusκby a factor of 2.8, from 1.9 k_BT to 5.3 k_BT. 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.

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.

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.