Red blood cell washing,nitrite therapy,and antiheme therapies prevent stored red blood cell toxicity after trauma–hemorrhage

Transfusion of stored red blood cells (RBCs) is associated with increased morbidity and mortality in trauma patients. Pro-oxidant, pro-inflammatory, and nitric oxide (NO) scavenging properties of stored RBCs are thought to underlie this association. In this study we determined the effects of RBC washing and nitrite and antiheme therapy on stored RBC-dependent toxicity in the setting of trauma-induced hemorrhage. A murine (C57BL/6) model of trauma–hemorrhage and resuscitation with 1 or 3 units of RBCs stored for 0–10 days was used. Tested variables included washing RBCs to remove lower MW components that scavenge NO, NO-repletion therapy using nitrite, or mitigation of free heme toxicity by heme scavenging or preventing TLR4 activation. Stored RBC toxicity was determined by assessment of acute lung injury indices (airway edema and inflammation) and survival. Transfusion with 5 day RBCs increased acute lung injury indexed by BAL protein and neutrophil accumulation. Washing 5 day RBCs prior to transfusion did not decrease this injury, whereas nitrite therapy did. Transfusion with 10 day RBCs elicited a more severe injury resulting in ~90% lethality, compared to <15% with 5 day RBCs. Both washing and nitrite therapy significantly protected against 10 day RBC-induced lethality, suggesting that washing may be protective when the injury stimulus is more severe. Finally, a spectral deconvolution assay was developed to simultaneously measure free heme and hemoglobin in stored RBC supernatants, which demonstrated significant increases of both in stored human and mouse RBCs. Transfusion with free heme partially recapitulated the toxicity mediated by stored RBCs. Furthermore, inhibition of TLR4 signaling, which is stimulated by heme, using TAK-242, or hemopexin-dependent sequestration of free heme significantly protected against both 5 day and 10 day mouse RBC-dependent toxicity. These data suggest that RBC washing, nitrite therapy, and/or antiheme and TLR4 strategies may prevent stored RBC toxicities.     

Red blood cells prevent inhibition of hypoxic pulmonary vasoconstriction by nitrite in isolated, perfused rat lungs

Nitrite reduction to nitric oxide (NO) may be potentiated by a nitrite reductase activity of deoxyHb and contribute to systemic hypoxic vasodilation. The effect of nitrite on the pulmonary circulation has not been well characterized. We explored the effect of nitrite on hypoxic pulmonary vasoconstriction (HPV) and the role of the red blood cell (RBC) in nitrite reduction and nitrite-mediated vasodilation. As to method, isolated rat lungs were perfused with buffer, or buffer with RBCs, and subjected to repeated hypoxic challenges, with or without nitrite. As a result, in buffer-perfused lungs, HPV was reduced at nitrite concentrations of 7 muM and above. Nitrite inhibition of HPV was prevented by excess free Hb and RBCs, suggesting that vasodilation was mediated by free NO. Nitrite-inhibition of HPV was not potentiated by mild acidosis (pH = 7.2) or xanthine oxidase activity. RBCs at 15% but not 1% hematocrit prevented inhibition of HPV by nitrite (maximum nitrite concentration of approximately 35 muM) independent of perfusate Po(2). Degradation of nitrite was accelerated by hypoxia in the presence of RBCs but not during buffer perfusion. In conclusion, low micromolar concentrations of nitrite inhibit HPV in buffer-perfused lungs and when RBC concentration is subphysiological. This effect is lost when RBC concentration approaches physiological levels, despite enhanced nitrite degradation in the presence of RBCs. These data suggest that, although deoxyHb may generate NO from nitrite, insufficient NO escapes the RBC to cause vasodilation in the pulmonary circulation under the dynamic conditions of blood flow through the lungs and that RBCs are net scavengers of NO.     

Nitric oxide red blood cell membrane permeability at high and low oxygen tension.

Red blood cell (RBC) encapsulated hemoglobin in the blood scavenges nitric oxide (NO) much more slowly than cell-free hemoglobin would. Part of this reduced NO scavenging has been attributed to an intrinsic membrane barrier to diffusion of NO through the RBC membrane. Published values for the permeability of RBCs to NO vary over several orders of magnitude. Recently, the rate that RBCs scavenge NO has been shown to depend on the hematocrit (percentage volume of RBCs) and oxygen tension. The difference in rate constants was hypothesized to be due to oxygen modulation of the RBC membrane permeability, but also could have been due to the difference in bimolecular rate constants for the reaction of NO and oxygenated vs deoxygenated hemoglobin. Here, we model NO scavenging by RBCs under previously published experimental conditions. A finite-element based computer program model is constrained by published values for the reaction rates of NO with oxygenated and deoxygenated hemoglobin as well as RBC NO scavenging rates. We find that the permeability of RBCs to NO under oxygenated conditions is between 4400 and 5100 microm s(-1) while the permeability under deoxygenated conditions is greater than 64,000 microm s(-1). The permeability changes by a factor of 10 or more upon oxygenation of anoxic RBCs. These results may have important implications with respect to NO import or export in physiology.     

Stored red blood cells: A changing universe waiting for its map(s)

The availability of stored red blood cells (RBCs) for transfusion remains an important aspect of the treatment of polytrauma, acute anemia or major bleedings. RBCs are prepared by blood banks from whole blood donations and stored in the cold in additive solutions for typically six weeks. These far from physiological storage conditions result in the so-called red cell storage lesion that is of importance both to blood bankers and to clinical practitioners. Here we review the current state of knowledge about the red cell storage lesion from a proteomic perspective. In particular, we describe the current models accounting for RBC aging and response to lethal stresses, review the published proteomic studies carried out to uncover the molecular basis of the RBC storage lesion, and conclude by suggesting a few possible proteomic studies that would provide further knowledge of the molecular alterations carried by RBCs stored in the cold for six weeks.     

Cryopreservation of red blood cells: Effect on rheologic properties and associated metabolic and nitric oxide related parameters

AimHigh glycerol cryopreservation of red blood cells (RBCs) reduces metabolic processes at ultralow temperatures but less is known regarding the effect of cryopreservation on RBC nitric oxide (NO) metabolism, haemorheological properties, structural behaviour and membrane fragility.MethodsBlood from ten healthy participants was sampled, glycerolized and stored at −80 °C (SB). Aliquots were thawed and further processed after 4, 8 and 12 weeks, respectively. At these time points, fresh blood (FB) was additionally sampled from each participant. FB/SB mixtures were prepared corresponding to transfusion of 1–3 blood bags. Additionally, mixtures were exposed to shear stress similar to that found in the circulation and deformability was measured to estimate possible behaviour of cryopreserved RBC in vivo.ResultsAgeing of RBC was reduced during cryopreservation. Markers for RBC metabolism (ATP, 2,3-DPG) were not altered but RBC sodium levels increased and potassium and calcium decreased, respectively. Mean cellular volume was higher and accordingly, mean cellular haemoglobin concentration was lower in SB. Deformability was altered during storage with less shear stress necessary to deform RBCs. Changes were also detectable in blood mixtures. Deformability remained unaltered in shear stress settings in FB and SB. RBC viscosity was reduced in SB. RBC-NOS content and phosphorylation sites as well as nitrite and RxNO levels seem not to be affected by the intervention.ConclusionCryopreservation maintains RBC metabolic function in vitro, but structure and function of cryopreserved RBC seems to be altered. Impact of these alterations in vivo seems to be less but needs further investigation.     

Phospholipid composition of packed red blood cells and that of extracellular vesicles show a high resemblance and stability during storage

Red blood cells (RBCs) are stored up to 35–42 days at 2–6 °C in blood banks. During storage, the RBC membrane is challenged by energy depletion, decreasing pH, altered cation homeostasis, and oxidative stress, leading to several biochemical and morphological changes in RBCs and to shedding of extracellular vesicles (EVs) into the storage medium. These changes are collectively known as RBC storage lesions. EVs accumulate in stored RBC concentrates and are, thus, transfused into patients. The potency of EVs as bioactive effectors is largely acknowledged, and EVs in RBC concentrates are suspected to mediate some adverse effects of transfusion. Several studies have shown accumulation of lipid raft–associated proteins in RBC EVs during storage, whereas a comprehensive phospholipidomic study on RBCs and corresponding EVs during the clinical storage period is lacking. Our mass spectrometric and chromatographic study shows that RBCs maintain their major phospholipid (PL) content well during storage despite abundant vesiculation. The phospholipidomes were largely similar between RBCs and EVs. No accumulation of raft lipids in EVs was seen, suggesting that the primary mechanism of RBC vesiculation during storage might not be raft -based. Nonetheless, a slight tendency of EV PLs for shorter acyl chains was observed.     

Optical tweezers as a new biomedical tool to measure zeta potential of stored red blood cells

During storage, red blood cells (RBCs) for transfusion purposes suffer progressive deterioration. Sialylated glycoproteins of the RBC membrane are responsible for a negatively charged surface which creates a repulsive electrical zeta potential. These charges help prevent the interaction between RBCs and other cells, and especially among each RBCs. Reports in the literature have stated that RBCs sialylated glycoproteins can be sensitive to enzymes released by leukocyte degranulation. Thus, the aim of this study was, by using an optical tweezers as a biomedical tool, to measure the zeta potential in standard RBCs units and in leukocyte reduced RBC units (collected in CPD-SAGM) during storage. Optical tweezers is a sensitive tool that uses light for measuring cell biophysical properties which are important for clinical and research purposes. This is the first study to analyze RBCs membrane charges during storage. In addition, we herein also measured the elasticity of RBCs also collected in CPD-SAGM. In conclusion, the zeta potential decreased 42% and cells were 134% less deformable at the end of storage. The zeta potential from leukodepleted units had a similar profile when compared to units stored without leukoreduction, indicating that leukocyte lyses were not responsible for the zeta potential decay. Flow cytometry measurements of reactive oxygen species suggested that this decay is due to membrane oxidative damages. These results show that measurements of zeta potentials provide new insights about RBCs storage lesion for transfusion purposes.     

The Capacity of Red Blood Cells to Reduce Nitrite Determines Nitric Oxide Generation under Hypoxic Conditions

Nitric oxide (NO) is a key regulator of vascular tone. Endothelial nitric oxide synthase (eNOS) is responsible for NO generation under normoxic conditions. Under hypoxia however, eNOS is inactive and red blood cells (RBC) provide an alternative NO generation pathway from nitrite to regulate hypoxic vasodilation. While nitrite reductase activity of hemoglobin is well acknowledged, little is known about generation of NO by intact RBC with physiological hemoglobin concentrations. We aimed to develop and apply a new approach to provide insights in the ability of RBC to convert nitrite into NO under hypoxic conditions. We established a novel experimental setup to evaluate nitrite uptake and the release of NO from RBC into the gas-phase under different conditions. NO measurements were similar to well-established clinical measurements of exhaled NO. Nitrite uptake was rapid, and after an initial lag phase NO release from RBC was constant in time under hypoxic conditions. The presence of oxygen greatly reduced NO release, whereas inhibition of eNOS and xanthine oxidoreductase (XOR) did not affect NO release. A decreased pH increased NO release under hypoxic conditions. Hypothermia lowered NO release, while hyperthermia increased NO release. Whereas fetal hemoglobin did not alter NO release compared to adult hemoglobin, sickle RBC showed an increased ability to release NO. Under all conditions nitrite uptake by RBC was similar. This study shows that nitrite uptake into RBC is rapid and release of NO into the gas-phase continues for prolonged periods of time under hypoxic conditions. Changes in the RBC environment such as pH, temperature or hemoglobin type, affect NO release.     

Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation

Nitrite anions comprise the largest vascular storage pool of nitric oxide (NO), provided that physiological mechanisms exist to reduce nitrite to NO. We evaluated the vasodilator properties and mechanisms for bioactivation of nitrite in the human forearm. Nitrite infusions of 36 and 0.36 micromol/min into the forearm brachial artery resulted in supra- and near-physiologic intravascular nitrite concentrations, respectively, and increased forearm blood flow before and during exercise, with or without NO synthase inhibition. Nitrite infusions were associated with rapid formation of erythrocyte iron-nitrosylated hemoglobin and, to a lesser extent, S-nitroso-hemoglobin. NO-modified hemoglobin formation was inversely proportional to oxyhemoglobin saturation. Vasodilation of rat aortic rings and formation of both NO gas and NO-modified hemoglobin resulted from the nitrite reductase activity of deoxyhemoglobin and deoxygenated erythrocytes. This finding links tissue hypoxia, hemoglobin allostery and nitrite bioactivation. These results suggest that nitrite represents a major bioavailable pool of NO, and describe a new physiological function for hemoglobin as a nitrite reductase, potentially contributing to hypoxic vasodilation.     

Nitrite-dependent vasodilation is facilitated by hypoxia and is independent of known NO-generating nitrite reductase activities

The reduction of circulating nitrite to nitric oxide (NO) has emerged as an important physiological reaction aimed to increase vasodilation during tissue hypoxia. Although hemoglobin, xanthine oxidase, endothelial NO synthase, and the bc(1) complex of the mitochondria are known to reduce nitrite anaerobically in vitro, their relative contribution to the hypoxic vasodilatory response has remained unsolved. Using a wire myograph, we have investigated how the nitrite-dependent vasodilation in rat aortic rings is controlled by oxygen tension, norepinephrine concentration, soluble guanylate cyclase (the target for vasoactive NO), and known nitrite reductase activities under hypoxia. Vasodilation followed overall first-order dependency on nitrite concentration and, at low oxygenation and norepinephrine levels, was induced by low-nitrite concentrations, comparable to those found in vivo. The vasoactive effect of nitrite during hypoxia was abolished on inhibition of soluble guanylate cyclase and was unaffected by removal of the endothelium or by inhibition of xanthine oxidase and of the mitochondrial bc(1) complex. In the presence of hemoglobin and inositol hexaphosphate (which increases the fraction of deoxygenated heme), the effect of nitrite was not different from that observed with inositol hexaphosphate alone, indicating that under the conditions investigated here deoxygenated hemoglobin did not enhance nitrite vasoactivity. Together, our results indicate that the mechanism for nitrite vasorelaxation is largely intrinsic to the vessel and that under hypoxia physiological nitrite concentrations are sufficient to induce NO-mediated vasodilation independently of the nitrite reductase activities investigated here. Possible reaction mechanisms for nitrite vasoactivity, including formation of S-nitrosothiols within the arterial smooth muscle, are discussed.     

Dose-dependent inhibitory effect of CD47 in macrophage uptake of IgG-opsonized murine erythrocytes

The cell surface glycoprotein CD47 on target cells can bind to the inhibitory receptor SIRPalpha on macrophages to inhibit phagocytosis of antibody sensitized blood cells. The aim of this study was to determine if CD47 dose-dependently can regulate macrophage uptake of IgG-opsonized RBCs. CD47(+/-) RBCs express about 50% of the CD47 level found on CD47(+/+) RBCs. When injected into CD47(+/+) mice, CD47(+/-) RBCs showed a significantly faster antibody-mediated clearance as compared with CD47(+/+) RBCs injected into the same recipient. In vitro phagocytosis experiments confirmed that CD47(+/-) RBCs were taken up significantly more than CD47(+/+) RBCs, but significantly less than CD47(-/-) RBCs. A reduction in RBC CD47 expression just below 50% of that in normal RBCs can significantly accelerate RBC clearance by macrophages in the presence of RBC autoantibodies. This may have relevance for transfusion of stored RBCs, where loss of CD47 is seen over time, and in clearance of these cells by antibody-dependent phagocytosis.     

Intermediates detected by visible spectroscopy during the reaction of nitrite with deoxyhemoglobin: the effect of nitrite concentration and diphosphoglycerate

The reaction of nitrite with deoxyhemoglobin (deoxyHb) results in the reduction of nitrite to NO, which binds unreacted deoxyHb forming Fe(II)-nitrosylhemoglobin (Hb(II)NO). The tight binding of NO to deoxyHb is, however, inconsistent with reports implicating this reaction with hypoxic vasodilation. This dilemma is resolved by the demonstration that metastable intermediates are formed in the course of the reaction of nitrite with deoxyHb. The level of intermediates is quantitated by the excess deoxyHb consumed over the concentrations of the final products formed. The dominant intermediate has a spectrum that does not correspond to that of Hb(III)NO formed when NO reacts with methemoglobin (MetHb), but is similar to metHb resulting in the spectroscopic determinations of elevated levels of metHb. It is a delocalized species involving the heme iron, the NO, and perhaps the beta-93 thiol. The putative role for red cell reacted nitrite on vasodilation is associated with reactions involving the intermediate. (1) The intermediate is less stable with a 10-fold excess of nitrite and is not detected with a 100-fold excess of nitrite. This observation is attributed to the reaction of nitrite with the intermediate producing N2O3. (2) The release of NO quantitated by the formation of Hb(II)NO is regulated by changes in the distal heme pocket as shown by the 4.5-fold decrease in the rate constant in the presence of 2,3-diphosphoglycerate. The regulated release of NO or N2O3 as well as the formation of the S-nitroso derivative of hemoglobin, which has also been reported to be formed from the intermediates generated during nitrite reduction, should be associated with any hypoxic vasodilation attributed to the RBC.     

Vitamin C in mouse and human red blood cells: an HPLC assay

Although vitamin C (ascorbate) is present in whole blood, measurements in red blood cells (RBCs) are problematic because of interference, instability, limited sensitivity, and sample volume requirements. We describe a new technique using HPLC with coulometric electrochemical detection for ascorbate measurement in RBCs of humans, wild-type mice, and mice unable to synthesize ascorbate. Exogenously added ascorbate was fully recovered even when endogenous RBC ascorbate was below the detection threshold (25 nM). Twenty microliters of whole blood or 10 μl of packed RBCs was sufficient for assay. RBC ascorbate was stable for 24h from whole-blood samples at 4°C. Processed, stored samples were stable for >1 month at -80°C. Unlike other tissues, ascorbate concentrations in human and mouse RBCs were linear in relation to plasma concentrations (R=0.8 and 0.9, respectively). In healthy humans, RBC ascorbate concentrations were 9-57 μM, corresponding to ascorbate plasma concentrations of 15-90 μM. Mouse data were similar. In human blood stored as if for transfusion, initial RBC ascorbate concentrations varied approximately sevenfold and decreased 50% after 6 weeks of storage under clinical conditions. With this assay, it becomes possible for the first time to characterize ascorbate function in relation to endogenous concentrations in RBCs.     

Paradoxical hypotension following increased hematocrit and blood viscosity

Hematocrit (Hct) of awake hamsters and CD-1 mice was acutely increased by isovolemic exchange transfusion of packed red blood cells (RBCs) to assess the relation between Hct and blood pressure. Increasing Hct 7-13% of baseline decreased mean arterial blood pressure (MAP) by 13 mmHg. Increasing Hct above 19% reversed this trend and caused MAP to rise above baseline. This relationship is described by a parabolic function (R2 = 0.57 and P < 0.05). Hamsters pretreated with the nitric oxide (NO) synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) and endothelial NOS-deficient mice showed no change in MAP when Hct was increased by <19%. Nitrate/nitrite plasma levels of Hct-augmented hamsters increased relative to control and L-NAME treated animals. The blood pressure effect was stable 2 h after exchange transfusion. These findings suggest that increasing Hct increases blood viscosity, shear stress, and NO production, leading to vasodilation and mild hypotension. This was corroborated by measuring A1 arteriolar diameters (55.0 +/- 21.5 microm) and blood flow in the hamster window chamber preparation, which showed statistically significant increased vessel diameter (1.04 +/- 0.1 relative to baseline) and microcirculatory blood flow (1.39 +/- 0.68 relative to baseline) after exchange transfusion with packed RBCs. Larger increases of Hct (>19% of baseline) led blood viscosity to increase >50%, overwhelming the NO effect through a significant viscosity-dependent increase in vascular resistance, causing MAP to rise above baseline values.     

Hemoglobin mediated nitrite activation of soluble guanylyl cyclase

Nitrite has long been known to be vasoactive when present at large concentrations but it was thought to be inactive under physiological conditions. Surprisingly, we have recently shown that supraphysiological and near physiological concentrations of nitrite cause vasodilation in the human circulation. These effects appeared to result from reduction of nitrite by deoxygenated hemoglobin. Thus, nitrite was proposed to play a role in hypoxic vasodilation. We now discuss these results in the context of nitrite reacting with hemoglobin and effecting vasodilation and present new data modeling the nitric oxide (NO) export from the red blood cell and measurements of soluble guanylate cyclase (sGC) activation. We conclude that NO generated within the interior of the red blood cell is not likely to be effectively exported directly as nitric oxide. Thus, an intermediate species must be formed by the nitrite/deoxyhemoglobin reaction that escapes the red cell and effects vasodilation.     

Red blood cell membrane water permeability increases with length of ex vivo storage

Water transport across the red blood cell (RBC) membrane is an essential cell function that needs to be preserved during ex vivo storage. Progressive biochemical depletion during storage can result in significant conformational and compositional changes to the membrane. Characterizing the changes to RBC water permeability can help in evaluating the quality of stored blood products and aid in the development of improved methods for the cryopreservation of red blood cells. This study aimed to characterize the water permeability (L_p), osmotically inactive fraction (b), and Arrhenius activation energy (E_a) at defined storage time-points throughout storage and to correlate the observed results with other in vitro RBC quality parameters. RBCs were collected from age- and sex-matched blood donors. A stopped flow spectrophotometer was used to determine L_p and b by monitoring changes in hemoglobin autofluorescence when RBCs were exposed to anisotonic solutions. Experimental values of L_p were characterized at three different temperatures (4, 20 and 37 °C) to determine the E_a. Results showed that L_p, b, and E_a of stored RBCs significantly increase by day 21 of storage. Degradation of the RBC membrane with length of storage was seen as an increase in hemolysis and supernatant potassium, and a decrease in deformability, mean corpuscular hemoglobin concentration and supernatant sodium. RBC osmotic characteristics were shown to change with storage and correlate with changes in RBC membrane quality metrics. Monitoring water parameters is a predictor of membrane damage and loss of membrane integrity in ex vivo stored RBCs.     

Glutathione loading prevents free radical injury in red blood cells after storage

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity^[1]. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1-6°C for 0, 42 and 84 days in a conventional additive solution (Adsol®) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4-6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (¹⁴C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (∼ 50 %), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.     

Modulation of endothelial nitric oxide synthase expression by red blood cell aggregation

The effects of enhanced red blood cell (RBC) aggregation on nitric oxide (NO)-dependent vascular control mechanisms have been investigated in a rat exchange transfusion model. RBC aggregation for cells in native plasma was increased via a novel method using RBCs covalently coated with a 13-kDa poloxamer copolymer (Pluronic F-98); control experiments used RBCs coated with a nonaggregating 8.4-kDa poloxamer (Pluronic F-68). Rats exchange transfused with aggregating RBC suspensions demonstrated significantly enhanced RBC aggregation throughout the 5-day follow-up period, with mean arterial blood pressure increasing gradually over this period. Arterial segments ( approximately 300 microm in diameter) were isolated from gracilis muscle on the fifth day and mounted between two glass micropipettes in a special chamber equipped with pressure servo-control system. Dose-dependent dilation by ACh and flow-mediated dilation of arterial segments pressurized to 30 mmHg and preconstricted to 45-55% of the original diameter by phenylephrine were significantly blunted in rats with enhanced RBC aggregation. Both responses were totally abolished by nonspecific NO synthase (NOS) inhibitor (Nomega-nitro-l-arginine methyl ester) treatment of arterial segments, indicating that the responses were NO related. Additionally, expression of endothelial NOS protein was found to be decreased in muscle samples obtained from rats exchanged with aggregating cell suspensions. These results imply that enhanced RBC aggregation results in suppressed expression of NO synthesizing mechanisms, thereby leading to altered vasomotor tonus; the mechanisms involved most likely relate to decreased wall shear stresses due to decreased blood flow and/or increased axial accumulation of RBCs.     

Survival of the fittest?--survival of stored red blood cells after transfusion.

During the last 90 years many developments have taken place in the world of blood transfusion. Several anticoagulants and storage solutions have been developed. Also the blood processing has undergone many changes. At the moment, in The Netherlands, red blood cell (RBC) concentrates (prepared from a whole blood donation and leukocyte-depleted by filtration) are stored for a maximum of 35 days at 4 degrees C in saline adenine glucose mannitol (SAGM). Most relevant studies show that approximately 20% of the RBCs is lost in the first 24 hr after transfusion. Even more remarkable is that the average life span is 94 days after a storage period of 42-49 days. Such observations create the need for a parameter to measure the biological age of RBCs as a possible predictor of the fate of RBCs after transfusion. The binding of IgG to RBCs can lead to recognition and subsequent phagocytosis by macrophages. This occurs during the final stages of the RBC life span in vivo. We determined the quantity of cell-bound IgG during storage, and found considerable variation between RBCs, but no significant storage-related change in the quantity of cell-bound IgG. The significance of this finding for predicting the survival of transfused RBCs in vivo remains to be established. Hereto we developed a flow cytometric determination with a sensitivity of 0.1% for the measurement of survival in vivo based on antigenic differences. This technique has various advantages compared with the 'classical' 51Cr survival method.     

Glutathione loading prevents free radical injury in red blood cells after storage

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity^[1]. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1–6°C for 0, 42 and 84 days in a conventional additive solution (Adsol®) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4–6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (¹⁴C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (～ 50 %), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.