Vitamin E protects against acetone-induced oxidative stress in rat red blood cells

Acetone may induce oxidative stress leading to disturbance of the biochemical and physiological functions of red blood cells (RBCs) thereby affecting membrane integrity. Vitamin E (vit E) is believed to function as an antioxidant in vivo protecting membranes from lipid peroxidation. The aim of the present study was the evaluation of possible protective effects of vit E treatment against acetone-induced oxidative stress in rat RBCs. Thirty healthy male Wistar albino rats, weighing 200–230 g and averaging 12 weeks old were randomly allotted into one of three experimental groups: Control (A), acetone-treated (B) and acetone + vit E-treated groups (C), each containing ten animals. Group A received only drinking water. Acetone, 5% (v/v), was given with drinking water to B and C groups. In addition, C group received vit E dose of 200 mg/kg/day i.m. The experiment continued for 10 days. At the end of the 10th day, the blood samples were obtained for biochemical and morphological investigation. Acetone treatment resulted in RBC membrane destruction and hemolysis, increased thiobarbituric acid reactive substance (TBARS) levels in plasma and RBC, and decreased RBC vit E levels. Vit E treatment decreased elevated TBARS levels in plasma and RBC and also increased reduced RBC vit E levels, and prevented RBC membrane destruction and hemolysis. In conclusion, vit E treatment appears to be beneficial in preventing acetone-induced oxidative RBC damage, and therefore, it can improve RBC rheology.     

Role of membrane thermotropic properties on hypotonic hemolysis and hypertonic cryohemolysis of human red blood cells

The hypothesis of a correlation between the effects of temperature on red blood cells hypotonic hemolysis and hypertonic cryohemolysis and two thermotropic structural transitions evidenced by EPR studies has been tested. Hypertonic cryohemolysis of red blood cells shows critical temperatures at 7 degrees C and 19 degrees C. In hypotonic solution, the osmotic resistance increases near 10 degrees C and levels off above 20 degrees C. EPR studies of red blood cell membrane of a 16-dinyloxyl stearic acid spin label show, in the 0-50 degrees C range, the presence of three thermotropic transitions at 8, 20, and 40 degrees C. Treatments of red blood cells with acidic or alkaline pH, glutaraldehyde, and chlorpromazine abolish hypertonic cryohemolysis and reduce the effect of temperature on hypotonic hemolysis. 16-Dinyloxyl stearic acid spectra of red blood cells treated with glutaraldehyde and chlorpromazine show the disappearance of the 8 degrees C transition. Both the 8 degrees C and the 20 degrees C transitions were abolished by acidic pH treatment. The correlation between the temperature dependence of red blood cell lysis and thermotropic breaks might be indicative of the presence of structural transitions producing areas of mismatching between differently ordered membrane components where the osmotic resistance is decreased.     

Influence of increased membrane cholesterol on membrane fluidity and cell function in human red blood cells

Cholesterol and phospholipid are the two major lipids of the red cell membrane. Cholesterol is insoluble in water but is solubilized by phospholipids both in membranes and in plasma lipoproteins. Morever, cholesterol exchanges between membranes and lipoproteins. An equilibrium partition is established based on the amount of cholesterol relative to phospholipid (C/PL) in these two compartments. Increases in the C/PL of red cell membranes have been studied under three conditions: First, spontaneous increases in vivo have been observed in the spur red cells of patients with severe liver disease; second, similar red cell changes in vivo have been induced by the administration of cholesterol-enriched diets to rodents and dogs; third, increases in membrane cholesterol have been induced in vitro by enriching the C/PL of the lipoprotein environment with cholesterol-phospholipid dispersions (liposomes) having a C/PL of >1.0. In each case, there is a close relationship between the C/PL of the plasma environment and the C/PL of the red cell membrane. In vivo, the C/PL mole ratio of red cell membranes ranges from a normal value of 0.9–1.0 to values which approach but do not reach 2.0. In vitro, this ratio approaches 3.0. Cholesterol enrichment of red cell membranes directly influences membrane lipid fluidity, as assessed by the rotational diffusion of hydrophobic fluorescent probes such as diphenyl hexatriene (DPH). A close correlation exists between increases in red cell membrane C/PL and decreases in membrane fluidity over the range of membrane C/PL from 1.0 to 2.0; however, little further change in fluidity occurs when membrane C/PL is increased to 2.0–3.0. Cholesterol enrichment of red cell membranes is associated with the transformation of cell contour to one which is redundant and folded, and this is associated with a decrease in red cell filterability in vitro. Circulation in vivo in the presence of the slpeen further modifies cell shape to a spiny, irregular (spur) form, and the survival of cholesterol-rich red cells is decreased in the presence of the spleen. Although active Na-K transport is not influenced by cholesterol enrichment of human red cells, several carrier-mediated transport pathways are inhibited. We have demonstrated this effect for the cotransport of Na + K and similar results have been obtained by others in studies of organic acid transport and the transport of small neutral molecules such as erythritol and glycerol. Thus, red cell membrane C/PL is sensitive to the C/PL of the plasma environment. Increasing membrane C/PL causes a decrease in membrane fluidity, and these changes are associated with a reduction in membrane permeability, a distortion of cell contour and filterability and a shortening of the survival of redcells in vivo.     

Membrane bending energy and shape determination of phospholipid vesicles and red blood cells

A procedure is developed to calculate red blood cell and phospholipid vesicle shapes within the bilayer couple model of the membrane. The membrane is assumed to consist of two laterally incompressible leaflets which are in close contact but unconnected. Shapes are determined by minimizing the membrane bending energy at a given volume of a cell (V), given average membrane area (A) and given difference of the areas of two leaflets (A). Different classes of shapes exist in parts of the v/a phase diagram, where v and a are the volume and the leaflet area difference relative to the sphere with area A. The limiting shapes are composed of sections of spheres with only two values allowed for their radii. Two low energy axisymmetrical classes, which include discocyte and stomatocyte shapes are studied and their phase diagrams are analyzed. For v=0.6, the discocyte is the lowest energy shape, which transforms by decreasing a continuously into a stomatocyte. The spontaneous membrane curvature (C ₀) and compressibility of membrane leaflest can be incorporated into the model.A model, where A is free and C ₀ determines the shapes at given V and A, is also studied. In this case, by decreasing C ₀, a discocyte transforms discontinuously into an almost closed stomatocyte.     

Relationship between the shape and the membrane potential of human red blood cells

Summary Microscopic observations of isotonic suspensions of human red blood cells demonstrate that cell shape is unaltered when the transmembrane electrical potential, orE _m , is set in the range –85 to +10 mV with valinomycin at varied external K⁺, or K _o .E _m was measured with the fluorescent potentiometric indicator, diS-C₃(5), as calibrated by a pH method. Repeating Glaser's experiments in which echinocytosis was attributed to hyperpolarization, we found that at low ionic strength the pH-dependent effects of amphotericin B appear to be unrelated toE _m . The effects of increased intracellular Ca²⁺, or Ca _o , on echinocytosis and onE _m are separable. With Ca ionophore A23187 half-maximal echinocytosis occurs at greater Ca _o than that which induces the half-maximal hyperpolarization associated with Ca-induced K⁺ conductance (Gardos effect). Thus, cells hyperpolarized by increased Ca _o remain discoidal when Ca is below the threshold for echinocytosis. With A23187 and higher Ca _o , extensive echinocytosis occurs in cells which are either hyperpolarized or at their resting potential. The Ca-activation curve for echinocytosis is left-shifted by low K _o , a new observation consistent with increased DIDS-sensitive uptake of⁴⁵Ca by hyperpolarized cells. These results support the following conclusions: (1) the shape and membrane potential of human red blood cells are independent under the conditions studied; (2) in cells treated with A23187, the Gardos effect facilitates echinocytosis by increasing Ca.     

人多能干细胞向红细胞的诱导分化

目前,体外生成人红细胞的实验技术较为复杂,为优化诱导步骤,采用两步法将人多能干细胞诱导分化为红细胞。首先,利用人多能干细胞(包括Rh阴性A型的脐带间充质干细胞(hUCMSC~(Rh-A))和人iPS(hiPS)细胞)在BVF培养液中进行诱导分化得到CD31~~+和CD34~+的阳性细胞群。经PCR和流式细胞仪检测CD31和CD34的表达发现,hUCMSC~(Rh-A)细胞诱导得到的CD31和CD34阳性细胞率分别是5.3%和22.7%;hiPS细胞诱导得到的CD31和CD34阳性细胞率分别是31.2%和8.2%。第二步,将获得的CD31~+和CD34~+的阳性细胞群在多种生长因子的作用下经过36 d诱导,分化为成熟红细胞。经吉姆萨染色检测得到的红细胞在形态和大小上与正常人红细胞相近,且存在血细胞去核的现象。荧光定量RT-PCR检测到了globin的表达,其中β-globin的表达量占20%以上。将得到的红细胞收集到离心管中,自然沉降后可见红色的红细胞沉淀。上述研究为大量制备人红细胞提供了新的有效的技术方法。     

Thermodynamic studies on oxygen binding by human red blood cells

Oxygen equilibrium curves have been measured on human normal red blood cells, at the temperatures of 20, 25, 30, 37 and 41 degrees C, and at pHs ranging from 6.8 to 8.2. The thermodynamical parameters have been determined for the four successive steps of oxygenation and for overall oxygenation, according to the Adair and MWC models [Monod J, Wyman J, Changeux JP. On the nature of allosteric transitions: a plausible model. J Mol Biol 1965;12:88-118]. The heat release appears to be nearly equal for the four steps. At the first three steps, the delta H change is counterbalanced by a nearly equivalent change of delta S, resulting in a rather small delta G value. delta G is greater at the fourth step, because of diminution of this enthalpy-entropy compensation phenomenon. The four steps are both enthalpy and entropy driven. According to the MWC model, the T to R transition is endothermic, and allosteric quaternary transition occurs at binding of the third oxygen. The average heat release increases by 2.8 kcal/mol when pH raises from 7.4 to 8.2, but flattens below pH 7.4. After correction for the heat of solution of oxygen and for the heat of proton release (referred to intracellular pH), an intrinsic heat for oxygenation of the heme of approximately--13 kcal/mol is obtained for the successive steps of oxygenation (at pH 7.4, 37 degrees C). These results are compared with those previously obtained for pigeon and trout red blood cells.     

Inhibition of the heme-induced hemolysis of red blood cells by the chlorite-based drug WF10

Excessive release of hemoglobin from red blood cells markedly disturbs the health status of patients due to cytotoxic effects of free hemoglobin and heme. The latter component is able to initiate novel hemolytic events in unperturbed red blood cells. We modeled this process by incubation of ferric protoporphyrin IX with freshly isolated red blood cells from healthy volunteers. The heme-induced hemolysis was inhibited in a concentration-dependent manner by the chlorite-based drug WF10, whereby the hemolysis degree was totally abolished at a molar ratio of 1:2 between chlorite and heme. Upon incubation of heme with WF10, the ultraviolet-visible spectrum changed, whereas the release of iron from heme and the appearance of fluorescent breakdown products of the porphyrin ring were negligible at this ratio, but increased with increasing excess of chlorite over heme. Thus, inhibition of hemolysis by WF10 takes already place at those chlorite concentrations, where no degradation of the porphyrin ring occurs. As WF10 is applied in form of an intravenous infusion to patients with severe inflammatory states, these data support the hypothesis that the beneficial WF10 effects are closely associated with inactivation of free heme.     

Effect of solution environment on the permeability of red blood cells

The cell membrane permeability governs the rate of solute transport into and out of the cell, significantly affecting the cell's metabolic processes, viability, and potential usefulness in both biotechnological applications and physiological systems. Most previous studies of the cell membrane permeability have neglected the possible effects of suspending medium on membrane transport, even though there is extensive experimental evidence that suspending phase composition can significantly affect other properties related to the cell membrane (e.g., cell deformability, fragility, and aggregation rate). This study examined the effects of suspending phase composition (both proteins and electrolytes) on the permeability of human red blood cells to the metabolites creatinine and uric acid. Data were obtained using a stirred ultrafiltration device with direct cell- and proteinfree sampling through a semipermeable membrane. Both the uric acid and creatinine permeabilities were strongly affected by the suspending phase composition, with the permeabilities in different buffer solutions varying by as much as a factor of three. The predominant factors affecting the permeability were the presence (or absence) of chloride, phosphate/adenine, and proteins, although the magnitude and even the direction of these effects were significantly different for creatinine and uric acid transport. The dramatic differences in behavior for uric acid and creatinine reflect the different transport mechanisms for these solutes, with uric acid transported by a carrier-mediated mechanism and creatinine transported by passive diffusion through the lipid bilayer. These results provide important insights into the effects of solution environment on cell membrane transport and other cell membrane-mediated properties. (c) 1994 John Wiley & Sons, Inc.     

Changes of phosphatidylserine distribution in human red blood cells during the process of loading sugars

The plasma membrane of red blood cells permits sugars to be loaded into the cytoplasm simply by incubation in a suitable buffer solution containing the sugar. This may provide some hope for the freeze-drying of human red blood cells. However, the effect of the loading process on red blood cells has not been fully investigated. The exposure of phosphatidylserine (PS) on the surface of the cell can be recognized by macrophages and result in shortened circulation in vivo. This study evaluates the effects of the concentration, the incubation time, and the temperature of exposure of human red blood cells to extracellular trehalose or glucose. Exposure of PS was demonstrated by annexin V labeling. It was shown that the efficiency of loading of glucose was significantly greater than that of trehalose. The loading efficiency of both sugars increased with increase in extracellular sugar concentration, prolongation of incubation time, and increase of incubation temperature. The percentages of cells with exposed PS and of damaged cells were dependent on the extracellular sugar concentration, the incubation time, and the temperature. With an extracellular glucose concentration of 0.8M, the percentage of cells with exposed PS was more than 80% and significantly higher than that of red blood cells loaded with trehalose (approximate 20%, P<0.01). As the incubation time was prolonged, the percentage of PS exposure and of damaged cells also increased. After incubation for 5h, the percentage of red cells with exposed PS following loading with glucose was more than 80% and significantly higher than that of cells loaded with trehalose (40%, P<0.01). In addition, the incubation temperature had a major effect on PS exposure. The percentage of cells with PS exposure and the proportion of damaged cells increased with increase of incubation temperature. At 37 degrees C, the percentage of cells with exposed PS and of damaged cells after loading with glucose was more than 80% and significantly higher than that of cells loaded with trehalose (P<0.01). However, when the temperature was below 25 degrees C, the percentage of cells with exposed PS and of damaged cells after loading with glucose or trehalose were both less than 10%. In conclusion, the loading efficiency for glucose was higher than that for trehalose, but the lesser effect of trehalose on exposure of PS suggests that it can maintain the asymmetrical distribution of membrane phospholipids and the intracellular trehalose can increase the osmotic tolerance of cells.     

Iron-deficiency anaemia enhances red blood cell oxidative stress

Oxidative stress associated with iron deficiency anaemia in a murine model was studied feeding an iron-deficient diet. Anaemia was monitored by a decrease in hematocrit and haemoglobin. For the 9 week study an increase in total iron binding capacity was also demonstrated. Anaemia resulted in an increase in red blood cells (RBC) oxidative stress as indicated by increased levels of fluorescent heme degradation products (1.24-fold after 5 weeks; 2.1-fold after 9 weeks). The increase in oxidative stress was further confirmed by elevated levels of methemoglobin for mice fed an iron-deficient diet. Increased haemoglobin autoxidation and subsequent generation of ROS can account for the shorter RBC lifespan and other pathological changes associated with iron-deficiency anaemia.     

Protoporphyrin IX-induced structural and functional changes in human red blood cells,haemoglobin and myoglobin

Protoporphyrin IX and its derivatives are used as photosensitizers in the photodynamic therapy of cancer. Protoporphyrin IX penetrates into human red blood cells and releases oxygen from them. This leads to a change in the morphology of the cells. Spectrophotometric studies reveal that protoporphyrin IX interacts with haemoglobin and myoglobin forming ground state complexes. For both proteins, the binding affinity constant decreases, while the possible number of binding sites increases, as the aggregation state of the porphyrin is increased. The interactions lead to conformational changes of both haemoglobin and myoglobin as observed in circular dichroism studies. Upon binding with the proteins, protoporphyrin IX releases the heme-bound oxygen from the oxyproteins, which is dependent on the stoichiometric ratios of the porphyrin: protein. The peroxidase activities of haemoglobin and myoglobin are potentiated by the protein-porphyrin complexation. Possible mechanisms underlying the relation between the porphyrin-induced structural modifications of the heme proteins and alterations in their functional properties have been discussed. The findings may have a role in establishing efficacy of therapeutic uses of porphyrins as well as in elucidating their mechanisms of action as therapeutic agents.     

Membrane potentials associated with Ca-induced K conductance in human red blood cells: Studies with a fluorescent oxonol dye,WW 781

Summary A divalent anionic dye, bis-[3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-pentamethine oxonol (WW 781) is a rapidly responding fluorescent indicator of KCl diffusion potentials induced in human red blood cells with valinomycin, gramicidin, and with the Ca ionophore A 23187 in the presence of external Ca. WW 781 has a sensitivity of 0.13% F/mV, a detection limit of 10 mV, a response time of less than 1 sec, and exhibits a decrease in fluorescence intensity upon hyperpolarization without detectable shifts in absorption or emission peaks. This dye does not perturb the normal resting potential, and unlike the slow permeant cyanine dyes, does not inhibit Ca-induced K conductance in human red blood cells. However, WW 781 does stimulate Ca-induced unidirectional Rb efflux. With Ca plus A 23187, the initial rapid change in dye fluorescence is sensitive to [Ca] _o and to [A 23187], is reversible with excess EGTA, and is inhibited by quinine, oligomycin, and by trifluoperazine. A biphasic dependence of hyperpolarization on K _o is evident at pH 6, where the ionic selectivity of activation is K, Rb>Cs>Na and that of conductance is K, Rb>Cs. Conditions were defined which permitted continuous monitoring ofE _m for at least 10 min, and the time dependence of the Ca-induced potentials was characterized. Since the properties of the Ca-induced changes in dye fluorescence correlate well with the known characteristics of Ca-induced K permeability, we conclude that WW 781 is a useful indicator of changes inE _m, provided that sufficient controls are employed to separate direct effects of Ca on dye fluorescence from the effects ofE _m on fluorescence.     

Membrane and cytoplasmic resistivity properties of normal and sickle red blood cells

The cytoplasmic resistivities and membrane breakdown potentials of normal (AA), sickle-cell-trait (AS), and sickle (SS) red blood cells have been measured by the biophysical methodology of resistive pulse spectroscopy over a range of osmolalities. At isotonicity, the average membrane breakdown potentials are virtually identical for the three types of cells occurring at about 1150 V/cm. Average isotonic cytoplasmic resistivities are somewhat higher for the SS cells (166.7±7.49 ohm-cm) compared to the AA (147.6±1.98 ohm-cm) or AS cells (148.7±1.79 ohm-cm). As medium osmolality is varied, the differences in resistive properties become enlarged, especially at very low and very high osmolalities. At high osmolalities, both types of sickle cells show a large increase in internal resistivity compared to the normals; at low osmolality, the SS samples exhibit a distinctly different membrane breakdown characteristic, decreasing in this parameter, whereas the other two groups increase. Of the 15 SS samples tested, three displayed much higher cytoplasmic resistivities at isotonicity: 218.2±5.25 ohm-cm, compared to an average of 153.5±3.46 ohm-cm for the other 12. The relationship between these high resistivities and the subfraction of irreversibly sickled cells in the sample is discussed.     

The immune-stimulation capacity of liposome-treated red blood cells

Our in vivo studies on a rat model established safety of transfusing liposome-treated red blood cells (RBCs) but identified the potential for immune modulation as related to transfusion efficacy of liposome-treated RBCs. The aim of this study was at assessing the impact of liposome-induced membrane changes on the immune profile of liposome-treated RBCs by (a) evaluating their interaction with endothelial cells and monocytes; and (b) the resulting immune response derived from this interaction, in the form of cytokine release, adhesion molecules expression and phagocytosis. Unilamellar liposomes were synthesized to contain unsaturated phospholipids (1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC]:CHOL, 7:3?mol%). The human RBCs immune profile was assessed by incubating control and DOPC-treated RBCs with human umbilical vein endothelial cells (HUVECs) and monocytes. Cytokine release measured by Luminex technology, vascular cell adhesion molecule (VCAM)-1 and E-selectin on HUVECs measured by flow cytometry, and the erythrophagocytic activity of monocytes by monocyte monolayer assay (MMA) were determined. Fibroblast growth factor [FGF]-2 was the only cytokine released by HUVECs that remained increased after incubation with DOPC-treated RBCs compared to control throughout storage. The expression of both VCAM-1 (15.3?±?5.6% versus 6.3?±?0.9%, p?=?0.008) and E-selectin (18.0?±?6.3% versus 6.6?±?0.7%, p?=?0.004) by HUVECs were significantly increased after incubation with DOPC-treated RBCs at day 2 of storage. The MMA resulted in phagocytic indexes of zero for both control and DOPC-treated RBCs at day 2 and 42 of storage. The liposome treatment did not result in significant changes to the immune profile of stored DOPC-treated RBCs. These findings combined with previous in vivo results, make liposome treatment a potential candidate for application in RBC preservation and open the possibility for clinical use with other cell types.     

Effects of mobile phone emissions on human red blood cells

Raman spectroscopy was performed on GSM 900 and 1800 MHz mobile phone signal exposed red blood cells (RBCs). The observed changes in the Raman spectra of mobile signal exposed RBCs compared to unexposed control suggest reduced hemoglobin-oxygen affinity for the exposed cells. The possible mechanism may involve activation of the voltage gated membrane Ca²⁺ channels by the mobile phone emissions resulting in an increase in the levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) in cells via altered metabolic activities. Further studies carried out with fluorescent Ca²⁺ indicator confirmed increased intracellular Ca²⁺ level in the exposed cells. Since intracellular ATP level influences the shape and mechanics of RBCs, exposed cells were studied using diffraction phase microscopy and optical tweezers. Detectable changes in shape and mechanical properties were observed due to mobile signal exposure.     

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

Summary The interaction ofActinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.     

Inhibition of invitro sickling by liposome-mediated transport of amino acids into intact human red blood cells

To investigate the role of phenylalanine and tryptophane as potential antisickling agents in intact human SS-red blood cells a liposomal transport system was employed to transfer phenyl-alanine or tryptophane into intact SS-red blood cells. Aromatic amino acids and short peptides containing phenylalanine have been demonstrated to increase the minimum gelling concentration and solubility of deoxy-hemoglobin S in aqueous solution. However, these compounds do not cross the red blood cell membrane under usual incubation conditions. Incorporation of phenylalanine or tryptophane into intact SS-red blood cells $\text{via}$ liposomal transport system markedly inhibited the $\text{in}\text{vitro}$ sickling of deoxy-hemoglobin S. These findings raise the possibility that a nontoxic liposomal transport system which facilitates incorporation of antisickling agents into intact SS-RBC may have significant therapeutic implications in the treatment of sickle cell disease.     

Immune recognition of exposed xenoantigens on the surface of PEGylated bovine red blood cells

Due to potential problems that can occur during blood transfusion and increasing blood shortages, our group engineered methoxypolyethylene glycol conjugated bovine red blood cells (mPEG-bRBCs) as a potential universal oxygen therapeutic. This current work investigates the immunological properties of mPEG-bRBCs incubated with human plasma (hP) and correlates these properties to exposed Galalpha(1,3)Gal xenoantigens. After mPEG-bRBCs were incubated with hP, the amount of bound IgG and IgM was assessed via flow cytometry. Flow cytometry also assessed the amount of GS-IB4 bound to exposed Galalpha(1,3)Gal xenoantigens. The results of this study demonstrate that most hP samples strongly promote agglutination of mPEG-bRBCs regardless of the extent of mPEG surface coverage or donor blood type. IgG and IgM from hP bound strongly to mPEG-bRBCs. In general, the Galalpha(1,3)Gal xenoantigen remains exposed at all levels of PEG surface coverage. PEGylation did block some of the xenoantigens as the amount of exposed Galalpha (1,3)Gal decreased with increased mPEG surface coverage. However, this was not sufficient to prevent a strong agglutination reaction. Taken together, the results of this study indicate that the current strategy for PEGylating bRBCs is unsatisfactory for the development of immunologically silent oxygen therapeutics.