Effect of blood storage on erythrocyte/wall interactions: implications for surface charge and rigidity

In this report, we study, under flow conditions, the interactions of stored erythrocytes with an artificial surface: a microelectrode whose charge density ranges from –15 to +27 μC/cm². Interactions consist of red cells slowly circulating on the microelectrode and exerting a real contact with the electrode. Interaction is detected and measured by transient fluctuations of the electrolyte resistance obtained by impedance measurement of the microelectrode. Effects of aging induced by storage of whole blood at 4 °C show that the surface charge of erythrocytes rapidly decreases when blood is stored for more than 6 days under our experimental conditions. In comparison with trypsin-treated erythrocytes, an eight day storage induces a 60% decrease in the surface charge of red cells. After two weeks of storage, red cells are no longer negatively charged, presumably be cause of removal of sialic acid. Cells rigidity is significant after 6 days of storage and influences the electrical contact. Membrane rigidity increase could arise from the surface charge decrease. Finally, the surface charge decrease could be of importance in the use of stored blood. Received: 30 October 1996 / Accepted: 12 November 1996     

Effect of intracellular ATP on La(3+)-induced aggregation and fusion of human erythrocytes

The influence of intracellular ATP level on the aggregation and fusion of human erythrocytes, induced by La3+ in the concentration range 20-330 microM was studied. The aggregation of intact red blood cells differs from that of cells with increased and decreased contents of ATP. Incubation of erythrocyte aggregates at 37 degrees C did not lead to cell fusion. At the same time, incubation of erythrocyte aggregates with decreased and increased ATP contents in the presence of La3+ induced a pronounced cell fusion.     

ATP-content and shape of erythrocytes during preservation with adenine and nucleosides]

ACD blood with additions of adenine (A, 0.5 mM in blood), ademine + guanosine ((AG, 0.5 mM each) and adenine + guanosine + inosine (IAG, 0.5: 0.5: 18 mM) was stored for 6 weeks at 4 degrees C and the morphological changes in connection with the ATP content were observed. After a storage of 6 weeks 2--3% of the cells were present as diskocytes, 60% as echinocytes, and 40% as spherocytes. The delayed morphological alterations in the ACD-AG blood in comparison with ACD-A blood were also reflected by a higher ATP content of the ACD-AG blood during its storage. The alterations in the form of erythrocytes recorded in the morphological index Im (a subdivision was made according to 6 different stages of form) correlated with the ATP content. The coefficient of correlation amounted to r = 0.85. Thus, Im is a reliable criterium for evaluating possible storage damages of stored erythrocytes.     

Ex vivo expansion and clonality of CD34+ selected cells from bone marrow and cord blood in a serum-free media

Although umbilical cord blood is increasingly being used in allogeneic marrow transplantation, delayed platelet engraftment is often a concern for cord blood transplant recipients. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from a bone marrow source, and cord blood, in a serum-free Media. The CD34+ cells, selected from bone marrow (BM) and umbilical cord blood (CB), were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at days 0, 4, 7, and 14 under the combination of growth factors, with cell counts. The cytokines included the recombinant human megakaryocyte growth and development (100 ng/ml), interleukin-3 (10 ng/ml), stem cell factor (100 ng/ml), flt-3 ligand (50 ng/ml) and interleukin-11 (200 ng/ml). The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the BM at day 7 (3.0 fold increase than BM), day 14 (2.4 fold), and day 17 (2.6 fold). The colony count of the BFU-E/CFU-E per CD34+ cell at day 0 was 0.14 +/- 0.023 in the CB, which was significantly higher than 0.071 +/- 0.015 in the BM. The CB-selected CD34+ cells produced more BFU-E colonies than the BM on culture days 4, 7, and 14. The BFU-E colonies from the CB cells increased markedly on culture days 4 and 7, with a 4-fold increase at day 14. The colony count of the CFU-Mk per CD34+ cell at day 0 was 0.047 +/- 0.011 in the CB-selected CD34+ cells cultures, which was higher than the 0.026 +/- 0.014 in the BM. The CB-selected CD34+ cells produced more CFU-Mk colonies than the BM on culture days 4, 7 and 14. In conclusion, the ex vivo expansion of the CB cells may be very promising in producing total cellular expansion, CFU-Mk and BFU-E compared with BM, especially at day 7. The ex vivo expansion of the CB may have rationale in making an ex vivo culture for 7 to 14 d.     

Improved stability of 2,3-bisphosphoglycerate during storage of hexokinase-overloaded erythrocytes

Human red blood cells were overloaded with homogeneous human hexokinase using a procedure of encapsulation based on hypotonic hemolysis and isotonic resealing and reannealing to achieve a final activity that was 15 times higher than that in control cells. Storage for 5 weeks at 4 degrees C of hexokinase-overloaded erythrocytes shows that these cells undergo small K+ leakage and mean cell volume increase compared with control cells. Furthermore, after these 5 weeks of storage the 2,3-bisphosphoglycerate content was normal while the ATP concentration was slightly reduced. These results and other properties suggest that encapsulation of key glycolytic enzymes in erythrocytes can provide a new way to maintain in vitro functionally active red blood cells for at least 5 weeks.     

Trypanosoma cruzi: Involvement of proteolytic activity during cell fusion induced by epimastigote form

Human erythrocytes were fused by Trypanosoma cruzi from 7 and 14 day old culture (stationary and declination phases, respectively) while only lysis was induced by "4 day old culture parasite (exponential phase). Lysis and erythrocyte fusion were studied by phase contrast microscopy, measuring of hemolysis and gel electrophoresis. The fusogenicity is Ca²⁺-dependent while lysis is delayed in the absence of exogenous Ca²⁺. The proteolysis of erythrocyte protein bands 1, 2, 2.1, 2.3 and 3 are common features of both fusion and lysis processes. Nevertheless the breakdown rate of ankyrin (band 2.1) and band 3 are different in fused or in lysed cells. The lysis process is associated with a faster degradation of band 2.1 and increase of band 2.3 than in the case of the fusion process. By contrast, degradation of band 3 occurs faster in the fusion than in the lytic event. Treatment of fusogenic parasites but not erythrocytes with TPCK, soybean trypsin inhibitor or FCS inhibited to some extent the fusion process and the decrease of bands 1, 2, 2.1, 2.3 and 3. The results suggest that proteases from fusogenic parasites may be directly or indirectly involved in the proteolysis of band 2.1 in a way related to induction of fusion.Abbreviations TPCK Tos-Phe-CH₂Cl,1-chloro-4 phenyl-3-L-tosylamidobutan-2-one - SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis - FCS Fetal Calf Serum     

The calcium-phosphate-induced fusion of human erythrocytes

The calcium-phosphate-induced fusion of normal human erythrocytes with those having increased (above physiological) levels of intracellular ATP was studied. Fusion of erythrocytes was markedly enhanced in the case of increased content of intracellular ATP. Fusion of such erythrocytes results in formation of giant cells up to 130 microm in diameter. Dithiothreitol practically completely inhibited fusion of erythrocytes with normal ATP content and markedly lowered it between those with increased ATP content.     

The role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes.

To elucidate the role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes, we examined the interaction of erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion of human erythrocytes was monitored by light microscopy as well as spectrophotometrically by the octadecylrhodamine dequenching assay. Phospholipid translocation and distribution between the inner and the outer leaflet of intact red blood cells were determined with spin-labeled phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Significant fusion of lipid-asymmetric red blood cells where PS and PE are predominantly oriented to the inner leaflet was only observed at Ca2+ concentrations greater than or equal to 10 mM (in the presence of 10 mM phosphate buffer) while fusion of lipid-symmetric erythrocyte membranes was established at greater than or equal to 1.5 mM Ca2+. The Ca2+ threshold of fusion of lipid-asymmetric red blood cells was significantly reduced (i) after exposure of PS to the outer layer but not after redistribution of PE alone, and (ii) upon incorporation of spin-labeled PS into the outer leaflet of red blood cells. Spin-labeled PE or PC did not affect fusion, suggesting that the serine headgroup is an important factor in calcium-phosphate-induced fusion.     

The oxygen transport function of the blood and of the erythrocyte concentrate during storage and its importance for blood transfusion

The property of oxygen transport function was investigated in blood which had been stored in solutions of "glugizir" and "zitroglucophosphate" and in erythrocyte concentrates gained from it on the noughth, 7th, 14th und 21st day of storage at -4 +/- 2 degrees C. The parameters of the oxygen binding function (oxygen content of erythrocytes, half time of haemoglobin saturation with oxygen, concentration of organic phosphate [2.3 diphosphoglycerate and adenosine triphosphate] and those of inorganic phosphorus were determined in erythrocytes. During storage for more than 21 days no significant differences could be detected in the property of oxygen transfer between erythrocytes of stored blood and erythrocyte concentrate, with the values for storing in zitroglucophosphate being somewhat higher. Problems of applying all components of donor blood efficiently are discussed. In performing an adequate haemotherapy with blood components the importance of a functional condition of erythrocytes and oxygen balance in the organism of the receiver should be considered. The necessity of transfusing erythrocyte concentrate in the therapy of anaemias of different genesis is emphasized and the differences in applying concentrates in different plasma solutions are referred to. By transfusing concentrates the effectiveness of hemotherapy are elevated and the rate of complications and side-effects of whole blood are diminished.     

Evidence that electrofusion yield is controlled by biologically relevant membrane factors

Rabbit + rabbit and human + human combinations of erythrocyte ghost membranes were fused under the same conditions with an electric pulse. Storage at 4 degrees C of ghost membranes from both rabbit and human erythrocytes showed no change with time but storage of the erythrocytes for various periods before ghost preparation showed consistent storage-dependent changes in fusion yield.     

Proteinase-resistant factors in human erythrocyte membranes mediate CD4-dependent fusion with cells expressing human immunodeficiency virus type 1 envelope glycoproteins.

Murine CD4+ cells are resistant to human immunodeficiency virus type 1 (HIV-1) entry and to fusion with cells expressing HIV-1 envelope glycoproteins (Env). The role of human-specific factors in Env/CD4-mediated fusion is shown by the ability of transient cell hybrids formed between CD4+ murine cells and human HeLa cells to fuse with Env+ cells. Fusion events were observed when other human cells, including erythrocytes, were substituted for HeLa cells in the hybrids. Experiments with erythrocyte ghosts showed that the factors allowing Env/CD4-mediated fusion are located in the plasma membrane. These factors were fully active after extensive digestion of erythrocytes with proteinase K or pronase. Nonprotein components of human plasma membranes, possibly glycolipids, could therefore be required for Env/CD4-mediated fusion and virus entry.     

Involvement of ADAM9 in multinucleated giant cell formation of blood monocytes

Monocytes-macrophages are converted to multinucleated giant cells by stimulation with various cytokines, and osteoclasts are the multinucleated giant cells derived from a monocyte-macrophage lineage. However, at present, the fusion peptides have not been clearly identified in monocytes-macrophages. The ADAM are a family of transmembrane glycoproteins that have a role in various biological functions. Interestingly, fertilin-alpha, ADAM9, and ADAM11 have potential fusion peptides. In this study, which ADAM was specifically expressed in monocytes stimulated with anti-CD98 antibody or RANKL and which factor(s) was functioning in monocytes as a fusion protein were investigated. ADAM1, 8, 10, 12, 15, 17, 20, and 21 mRNAs are expressed in blood monocytes incubated with control antibody, anti-FRP-1/CD98 antibody, or RANKL + M-CSF, while ADAM2, 7, 11, 13, 19, 23, 29, and 30 mRNAs could not be detected in these blood monocytes. Expression of ADAM9 and ADAM10 mRNAs are enhanced by either RANKL + M-CSF or anti-CD98 antibody. The expression of ADAM9 and ADAM10 is also induced in blood monocytes by anti-CD98 mAb. An anti-ADAM9 antibody enhances CD98-mediated cell aggregation, while it blocks CD98-mediated and RANKL-mediated multinucleated giant cell formation. A hydroxamate-based metalloprotease inhibitor, SI-27, which is found to suppress ADAM9 activity, suppresses multinucleated giant cell formation. New protein synthesis is necessary for the expression of ADAM9 mRNA and genistein suppresses induction of ADAM9 mRNA. This is the first report that ADAM9 is involved in monocyte fusion, such as CD98-mediated and RANKL-mediated cell fusion of blood monocytes. Furthermore, AMAM9 is one candidate for a fusion peptide in blood monocytes.     

Magneto-electro-fusion of human erythrocytes

In inhomogeneous (static) magnetic fields close contact between ‘magnetic’ human erythrocytes was established. The cells were made magnetic by incubating them in a medium containing small Fe₃O₄-particles which adsorbed to the outer membrane surface. Fusion was induced by applying two electric field pulses (field strength: 8.5 kV · cm^?1; duration: 60 μs) to the magnetically collected cells. This procedure allowed the use of electrically conductive media (3 · 10^?1 Ω^?1 · cm^?1). Fusion of red blood cells occured very often. If cell suspensions of high density were used fusion resulted in the formation of giant red blood cells with osmotically intact membranes.     

Calpain activation is essential for membrane fusion of erythrocytes in the presence of exogenous Ca2+.

The membrane mobility agent, 2-(methoxyethoxy)ethyl-cis-8-(2-octylcyclopropyl)octanoate (A2C) promotes the fusion of rat, rabbit, and human erythrocytes in the presence of exogenous Ca2+. Under these conditions, the high sensitivity form of calcium-activated neutral protease (mu-calpain) in erythrocytes is activated autolytically. mu-Calpain is activated in accordance with fusion; that is, both erythrocyte fusion and autolytic activation of mu-calpain are induced in rat erythrocytes at 30 min, in rabbit erythrocytes at 150 min, and in human erythrocytes at 240 min after the addition of A2C and Ca2+. When erythrocytes are preincubated with the Ca2+ ionophore A23187, both fusion and autolytic activation start earlier. A leupeptin analogue, Cbz-Leu-Leu-Leu-aldehyde (ZLLLal), inhibits both the autolytic activation of mu-calpain and fusion induced by A2C and Ca2+. These results indicate that treatment of erythrocytes with A2C and Ca2+, results in first an influx of Ca2+ into the cells, followed by autolytic activation of mu-calpain, proteolysis of membrane proteins, exposure of fusion-sites, and, finally, fusion of erythrocytes.     

The immediate induction of extensive cell fusion by Ca2+ addition in Dictyostelium discoideum

In mated cultures (NC4 X V12) of Dictyostelium discoideum containing 1.0 mM CaCl2, cell fusion generates large numbers of binucleate cells which develop into zygote giant cells. In the absence of Ca2+, binucleate formation does not occur. When 1.0 mM CaCl2 is added to Ca2+-deficient cultures at 18 h, 50% of the cells fuse within 45 min producing large multinucleate syncytia. Small, presumptive gametes appear in Ca2+-deficient cultures and reach a peak of about 20% of the cell population by 10 h, but they maintain this plateau and do not fuse. Upon the addition of CaCl2, the presumptive gametes immediately fuse, producing binucleate cells which develop rapidly into morphologically distinct giant cells. Cell fusion continues, resulting in the formation of extremely large (40-80 microns diameter) multinucleate syncytia by 45 min. The induction of this extensive, synchronous cell fusion does not occur in the presence of other chloride salts and EGTA inhibits it, revealing that Ca+ is the regulatory ion.     

Osmotic forces in artificially induced cell fusion

The importance of cell swelling in the fusion of erythrocytes by three different chemical treatments has been investigated with cells that were cytoplasmically labelled with 6-carboxyfluorescein. Hen erythrocytes, which had been pre-incubated with ionophore A23187 and 5 mM Ca2+ to cause a proteolytic breakdown of the membrane skeleton, were induced to fuse by applying an osmotic shock. Human erythrocytes that had been incubated in an isotonic salt/buffer solution, which was progressively diluted and which contained 0.5 mM La3+ to minimise cell lysis, were also fused. In addition, the fusion of human erythrocytes by 40% poly(ethylene glycol) began only when the poly(ethylene glycol) was diluted, and it mostly occurred when the diluted polymer solution was subsequently replaced by isotonic buffer. In related experiments, the effect of an osmotic gradient on electrically induced cell fusion has been studied. Human erythrocytes in 150 mM erythritol fused more readily than less swollen cells in 200-400 mM erythritol when subjected to a 20 microseconds pulse of 3.5 kV X cm-1, indicating that the extent of cell fusion induced by the breakdown pulse is governed by the combined electrical-compressive and osmotic forces. Since osmotic phenomena are already known to be important in exocytosis, we suggest that these observations on cell fusion indicate that osmotic forces may provide the driving force for many membrane fusion reactions in biological systems.     

In vivo study of the influence of adenine and guanosine on the erythrocytes of ACD (pH 5.0 and 5.6) preserved blood

Data are presented for percentage of recovery, survival time (T1/2) and mode of sequestration of erythrocytes from ACD [disodium citrate 95 mmol/l and glucose (C6H12O6 X H2O)] 152 mmol/l or ACD--adenine or adenine + guanosine (pH ranging from 5.0 to 5.6) preserved blood for 35 days at 4-8 degrees C (277-281 K). With the availability of guanosine in 0.25 mmol/l or 0.5 mmol/l final concentration in ACD + 0.25 or 0.5 mmol/l adenine preserved blood a positive effect can be exerted on erythrocyte 24 hrs recovery and survival time (T1/2). This effect is particularly evident when pH of the preservative solution is raised to 5.6. Final concentrations of 0.25 mmol/l adenine and guanosine in ACD preserved blood (whole or packed erythrocytes, pH 5.6, Hct. 0.73 or 0.61) are sufficient to ensure 35 days of storage at 4-8 degrees C (277-281 K).     

Cell age-related monovalent cations content and density changes in stored human erythrocytes

Conversion of erythrocyte membrane protein 4.1b to 4.1a occurs through a non-enzymatic deamidation reaction in most mammalian erythrocytes, with an in vivo half-life of approximately 41 days, making the 4.1a/4.1b ratio a useful index of red cell age [Inaba and Maede, Biochim. Biophys. Acta 944 (1988) 256-264]. Normal human erythrocytes distribute into subpopulations of increasing cell density and cell age when centrifuged in polyarabinogalactan density gradients. We have observed that, when erythrocytes were stored at 4 degrees C under standard blood bank conditions, the deamidation was virtually undetectable, as cells maintained the 4.1a/4.1b ratio they displayed at the onset of storage. By measuring the 4.1a/4.1b values in subpopulations of cells of different density at various time points during storage, a modification of the normal 'cell age/cell density' relationship was observed, as erythrocytes were affected by changes in cell volume in an age-dependent manner. This may stem from a different impact of storage on the imbalance of monovalent cations, Na(+) and K(+), in young and old erythrocytes, related to their different complement of cation transporters.     

Inhibition and stimulation of K+ transport across the frog erythrocyte membrane by furosemide, DIOA, DIDS and quinine

Frog erythrocytes were incubated in iso- or hypotonic media containing 10 mmol/l Rb+ and 0.1 mmol/l ouabain and both Rb+ uptake and K+ loss were measured simultaneously. Rb+ uptake by frog red cells in iso- and hypotonic media was reduced by 30-60% in the presence of 0.01-0.1 mmol/l [(dihydroindenyl)oxy] alkanoic acid (DIOA) or 0.5-1.0 mmol/l furosemide. Furosemide inhibited K+ loss from frog erythrocytes incubated in hypotonic media but did not affect it in isotonic media. DIOA at a concentration of 0.05 mmol/l inhibited of K+ loss from frog erythrocytes in both iso- and hypotonic media. At the concentrations of 0.01 and 0.02 mmol/l DIOA significantly suppressed K+ loss in a K+-free chloride medium but not in a K+-free nitrate medium. The Cl(-)-dependent K+ loss was completely blocked at a concentration of 0.1 mmol/l DIOA and the concentration required for 50% inhibition of K-Cl cotransport was approximately 0.015 mmol/l. However, the inhibitory effect of DIOA on K-Cl cotransport was masked by an opposite stimulatory effect on K+ transport which was also observed in nitrate medium. Quinine in a concentration of 0.2-1.0 mmol/l was able to inhibit Rb+ uptake and K+ loss only in hypotonic media. In isotonic media, quinine produced a stimulation of Rb+ uptake and K+ loss. A three to five-fold activation of Rb+ uptake and K+ loss was consistently observed in frog erythrocytes treated with 0.05-0.2 mmol/l 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). In contrast, another stilbene derivative 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) had no effect on K+ transport in the cells. Thus, of these drugs tested in the present study only DIOA at low concentrations may be considered as a selective blocker of the K-Cl cotransporter in the frog red blood cells.     

The cellular composition of the blood and haematopoietic organs of turbot Scophthalmus maximus L.

The cellular composition of the blood, anterior kidney, spleen and thymus of turbot Scophrhalmus maximus L., aged 1 + was determined. Ninety-four per cent of blood cells belonged to the erythrocyte lineage of which 82% were mature erythrocytes. The leucocytes, which represented 4.5% of the blood cells, were mainly lymphocytes (50%). The presence of crythroblasts in the anterior kidney and the spleen demonstrated an erythropoietic activity in both organs. However, this activity appeared to be prevalent in the spleen which also appeared to act as a storage zone for erythrocytes and as the centre point for thrombopoiesis. Although 96% of the anterior kidney cells were leucocytes, the number of white cells per gram of organ was higher in the spleen.