Hemolytic assay for venom phospholipase A2

A rapid and sensitive spectrophotometric assay for venom phospholipase A₂ based on the hemolysis of guinea pig erythrocytes in the presence of decomplemented serum and cardiotoxin (direct lytic factor) is described. This assay is particularly useful for rapid multisample analyses, such as those used in monitoring chromatography fractions, and is specific for phospholipase A₂ in she presence of other potentially hemolytic venom components. The hemolytic mechanism is shown to be a combination of the action of lysophospholipids liberated from lipoproteins in the serum and the synergistic action of phospholipase A₂ and cardiotoxin on the erythrocyte membrane.     

Differential resistance to calcium-induced bilirubin-dependent hemolysis in mammalian erythrocytes

Washed erythrocytes from human, buffalo, sheep and goat preincubated with different concentrations of calcium chloride (16.7–1830 μM) showed significantly different rates of hemolysis (up to 62%) after addition of bilirubin (72 μM). Goat erythrocytes displayed marked resistance to hemolysis with only 11% hemolysis observed at the highest calcium concentration. Similar trend in hemolysis was also observed when the concentration of CaCl₂ was fixed (330 μM) and bilirubin concentration varied (0–72 μM). (Ca²⁺–Mg²⁺)-ATPase levels were found significantly lower in goat and sheep erythrocyte membranes compared to human and buffalo erythrocyte membranes. This was correlated well with the observed hemolysis in various mammalian erythrocytes.     

Further aspects on the hemolytic activity of the antibiotic lipopeptide iturin A

The bacterial lipopeptide iturin A is able to cause hemolysis of human erythrocytes in a dose-dependent manner. Hemolysis takes place at iturin concentrations below its critical micellar concentration. Relative kinetics determinations clearly show that K⁺ leakage occurs prior to hemoglobin release. Furthermore, hemolysis can be prevented by addition to the outer solution of osmotic protectants of appropriate size. Altogether these results indicate that iturin A-induced hemolysis follows a colloid-osmotic mechanism, with the formation of a membrane pore of average diameter 32 Å. Iturin A is capable of inducing leakage of an aqueous fluorescent probe trapped in human erythrocyte ghosts, but not in large unilamellar liposomes made of various lipid compositions. The different permeabilizing effects of iturin A on model and biological membranes are discussed on the light of the presented results.     

Temperature‐dependent hemolytic activity of membrane pore‐forming peptide toxin,tolaasin

Tolaasin, a pore‐forming peptide toxin produced by Pseudomonas tolaasii, causes brown blotch disease on cultivated mushrooms. Hemolysis using red blood cells was measured to evaluate the cytotoxicity of tolaasin. To investigate the mechanism of tolaasin‐induced cell disruption, we studied the effect of temperature on the hemolytic process. At 4 °C, poor binding of the tolaasin molecules to the erythrocyte membrane was observed and most of the tolaasin molecules stayed in the solution. However, once tolaasin bound to erythrocytes at 37 °C and the temperature was decreased, complete hemolysis was observed even at 4 °C. These results indicate that tolaasin binding to cell membrane is temperature‐sensitive while tolaasin‐induced membrane disruption is less sensitive to temperature change. The effect of erythrocyte concentration was measured to understand the membrane binding and pore‐forming properties of tolaasin. The percentage of hemolysis measured by both hemoglobin release and cell lysis decreased as erythrocyte concentration increased in the presence of a fixed amount of tolaasin. The result shows that hemolysis is dependent on the amount of tolaasin and multiple binding of tolaasin is required for the hemolysis of a single cell. In analysis of dose‐dependence, the hemolysis was proportional to the tenth power of the amount of tolaasin, implying that tolaasin‐induced hemolysis can be explained by a multi‐hit model. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Hemolytic activity of Serratia marcescens

A cell-bound hemolytic activity was found in several strains of Serratia marcescens. One Serratia cell per ten erythrocytes was sufficient to cause complete lysis of human erythrocytes within 2 h in the liquid assay. The hemolytic activity resided in the membrane fraction and could be inactivated by incubating cells with proteases. The hemolytic activity was greatly enhanced in actively metabolizing Serratia cells and was partially controlled by the iron supply. Hemolysis was accompanied by degradation of erythrocyte membrane proteins (band 3 and 6, glycophorin) and was independent of the blood group. The exoprotease secreted by S. marcescens in large amounts was not involved in hemolysis. Comparison with various hemolytic strains of Escherichia coli showed that hemolysis of erythrocytes was more pronounced with S. marcescens than with E. coli. In contrast to hemolysis by E. coli, lysis of erythrocytes by S. marcescens was not enhanced by Ca²⁺ ions.Dedicated to Professor Dr. Gerhart Drews on the occasion of his 60th birthday     

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.     

Role of membrane sialic acid and glycophorin protein in thorium induced aggregation and hemolysis of human erythrocytes

Thorium-232 (²³²Th), a natural radionuclide from the actinide family, is abundantly present in monazite and other ores. It is used as one of the prime fuel materials in nuclear industry and may pose an exposure risk to nuclear workers and members of the public. Human erythrocytes, as a classical cellular membrane model, were coincubated with ²³²Th in order to elucidate whether this naturally occurring important radionuclide produced perturbations to cell membrane. Present study revealed that erythrocytes underwent aggregation or lysis depending on the ratio of ²³²Th to cell. Scanning electron micrographs showed that erythrocytes transformed into equinocytes and/or spherocytes after ²³²Th treatment. Further examination of erythrocyte by atomic force microscopy suggested significant increase in surface roughness after ²³²Th treatment. Experiments on neuraminidase treated and/or anti-GpA antibody blocked erythrocytes suggested significant role of membrane sialic acid and glycophorin A (GpA) protein in aggregation or hemolytic effects of ²³²Th. Further results showed that ²³²Th caused hemolysis by colloid osmotic mechanism, as evidenced by potassium efflux, osmotic protection and osmotic fragility studies. Osmoprotection experiments indicated that hemolysis get elicited through the formation of membrane pores of ∼2.0 nm in size. Hemolysis studies in presence of inhibitors (TEA, bumetanide, DIDS and amiloride) revealed the role of K⁺ channel, Na⁺/K⁺/2Cl⁻ channel, Cl⁻/HCO₃⁻ anion exchanger and Na⁺/H⁺ antiporter in ²³²Th induced erythrolysis. Presence of non-diffusible cation (N-methyl d-glucasamine) or anion (gluconate) in erythrocyte suspending medium further confirm the role of Na⁺ and Cl⁻ influx in hemolytic effect of ²³²Th. These findings provide significant insight in structural, biochemical and osmotic toxic effects of ²³²Th on human erythrocytes.     

Signaling Pathway in the Osmotic Resistance Induced by Angiotensin II AT2 Receptor Activation in Human Erythrocytes

Background:Angiotensin II regulates blood volume via AT1 (AT1R) and AT2 (AT2R) receptors. As cell integrity is an important feature of mature erythrocyte, we sought to evaluate, in vitro, whether angiotensin II modulates resistance to hemolysis and the signaling pathway involved.Methods:Human blood samples were collected and hemolysis assay and angiotensin II signaling pathway profiling in erythrocytes were done.Results:Hemolysis assay created a hemolysis curve in presence of Ang II in several concentrations (10^-6 M, 10^-8 M, 10^-10 M, 10^-12 M). Angiotensin II demonstrated protective effect, both in osmotic stressed and physiological situations, by reducing hemolysis in NaCl 0.4% and 0.9%. By adding receptors antagonists (losartan, AT1R antagonist and PD 123319, AT2R antagonist) and/or signaling modulators for AMPK, Akt/PI3K, p38 and PKC we showed the protective effect was enhanced with losartan and abolished with PD 123319. Also, we showed activation of p38 as well as PI3K/Akt pathways in this system.Conclusion:Ang II protects human erythrocytes from hypo-osmotic conditions-induced hemolysis by activating AT2 receptors and triggering intracellular pathways.Key Words: Angiotensin II, Erythrocyte, Osmotic fragility, Signaling pathway     

Enzymatic oxidation of membrane cholesterol in relation to lysis of sheep erythrocytes by corynebacterial enzymes

Synergistic hemolysis of sheep erythrocytes brought about by the combined action of Corynebacterium ovis (C. pseudotuberculosis) and Corynebacterium equi depends upon the extracellular sphingomyelin-specific phospholipase D of the former species and a partially characterized agent(s) of the latter. Fractionation of the culture supernatant of C. equi revealed a cholesterol oxidase which was purified to near homogeneity by gel filtration and isoelectric focusing. The enzyme was isoelectric at pH 9–10 and had a molecular weight of 61,000. Sheep erythrocytes pretreated with purified sphingomyelinase D of C. ovis were hemolyzed by incubation with C. equi cholesterol oxidase or by the same enzyme from Brevibacterium sp. Lipid analysis revealed complete conversion of membrane cholesterol to cholest-4-en-3-one, the product of cholesterol oxidase action. Cells not pretreated with sphingomyelinase D did not undergo cholesterol oxidation or hemolysis when treated with cholesterol oxidase. Studies with crude culture supernatant of C. equi confirmed the presence of a phospholipase active in hydrolyzing ceramide phosphate generated in the erythrocyte membrane by C. ovis sphingomyelinase. Ceramide thus produced in the membrane is known to make the cells labile to hemolysis. There are, therefore, at least two mechanisms underlying synergistic hemolysis by these coryne-bacteria.     

Interactions Between Sendai Virus and Human Erythrocytes

Concentrated Sendai virus, when adsorbed to erythrocytes at 4 C, caused invaginations in the plasma membrane. Following elevation of the temperature to 37 C, the plasma membrane became fused with the viral envelope before dissolution of the virions and rupture of the cells. Cell lysis was accompanied by rapid and total loss of hemoglobin to the extracellular space. Following aqueous pyridine extraction, the hemoglobin-free ghosts remaining were found to be devoid of N-acetylneuraminic acid and to have solubility properties different from those of normal erythrocyte ghosts. By the action of viral neuraminidase, bound N-acetylneuraminic acid was also liberated from purified virus receptor substance whose electrophoretic mobility was thereby substantially reduced. Cu⁺⁺ selectively inhibited hemolysis and neuraminidase without interfering with hemagglutination and attachment. Neuraminidase appeared to be essential for Sendai virus hemolysis; viral particle size may also be a critical factor in this process.     

On the interaction of cobra venom protein cardiotoxins with erythrocytes

The principally active hemolytic toxin (cardiotoxin) previously purified from the venom of the Thailand cobra, Naja naja siamensis, was shown to produce spontaneous twitching, contractures and membrane depolarization in sartorius muscles from the frog, Rana pipiens. Spontaneous twitching, observed at concentrations greater than 0.1 uM was completely abolished by addition of tetrodotoxin and not affected by d-tubocurarine. Dose and time dependent membrane depolarization of muscle fibers was observed to occur within 10-30 min at 0.2 to 1.0 uM concentrations of the toxin. These observations, taken together with an amino acid analysis characteristic of previously described cobra venom cardiotoxins, characterized this hemolytic toxin as a cardiotoxin. In the absence of EDTA the initial velocities of erythrocyte hemolysis for this toxin showed a sigmoidal concentration dependence which became hyperbolic in the presence of EDTA. The largest increases in hemolysis rates on addition of 1 mM EDTA were observed at low toxin concentrations. In the presence of EDTA extracellular and membrane associated divalent cations are complexed, thus alleviating their competition with toxin for binding to the membrane, a key and apparently rate-determining initial step which leads to hemolysis. In the presence of EDTA hemolysis rates increased linearly at low toxin concentration and reached an extrapolated maximum value at toxin concentrations at which, given its molecular dimensions, there are just sufficient toxin molecules to cover the entire membrane surface area provided by the erythrocytes.     

Hemolysis induced by Prymnesium parvum toxin effect of primaquine treatment

The effect of primaquine (1 mM) incubation on rabbit erythrocytes was studied at 25° using the hemolytic toxin (prymnesin) of the chrysomonad Prymnesium parvum. One notable effect is the alteration of rate of prymnesin-induced hemolysis. The hemolysis rate constant, k_ψ, showed a biphasic dependence on length of primaquine incubation: a gradual increase in k_ψ was observed (0–4 h), followed by a more pronounced increase (4–6 h). Incubation with primaquine (an antimalarial) is known to cause invaginations and loss of cell surface by subsequent internalization. No biphasic effect of primaquine incubation was noted in the tendency of prymnesin to be bound (using ³H-labeled toxin). Median cell volume, however, does show a biphasic relationship, and it appears that the biphasic effect depends upon changes in one out of two or more populations of cells.     

Oxidative stress,inflammation, and glycometabolism disorder-induced erythrocyte hemolysis in selenium-deficient exudative diathesis broilers

Selenium (Se) deficiency causes injury of diversified tissues and cells, including livers, hearts, skeletal muscles, and erythrocytes. The aim of the present study is to explore the molecular mechanism of erythrocyte hemolysis due to Se deficiency in broilers. One hundred and eighty broilers (male/female, 1 day old) were randomly divided into two groups and fed with either a normal Se content diet (C group, 0.2 mg Se/kg) or a Se-deficient diet (ED group, 0.008 mg Se/kg) for 45 days. During the trial period of 15–30 days, biological properties such as osmotic fragility, fluidity, phospholipid components of cell membrane, adenosine triphosphatase activities, and antioxidant function of erythrocytes in broilers were examined. Moreover, the messenger RNA (mRNA) expressions of genes associated with inflammation, glycometabolism, and avian uncoupling protein (avUCP) were detected. We found that compared with the C group, hemolysis rate, degree of polarization, and microviscosity of erythrocytes were increased in broilers of the ED group. The composition of erythrocyte membrane lipids was changed. Meanwhile, the antioxidant function of erythrocytes was weakened and mRNA levels of inflammatory genes were stimulated by Se deficiency (p < 0.05). In addition, mRNA expressions of rate-limiting enzymes in glycometabolism were effected and avUCP mRNA level was downregulated (p < 0.05) in the ED group. It has been concluded from the results that oxidative stress, inflammatory response, and glycometabolism disorder lead to erythrocyte hemolysis by changing the structure and function of erythrocyte membrane in ED broilers suffered from Se deficiency.     

Functional significance of glutamate–cysteine ligase modifier for erythrocyte survival in vitro and in vivo

Erythrocytes endure constant exposure to oxidative stress. The major oxidative stress scavenger in erythrocytes is glutathione. The rate-limiting enzyme for glutathione synthesis is glutamate–cysteine ligase, which consists of a catalytic subunit (GCLC) and a modifier subunit (GCLM). Here, we examined erythrocyte survival in GCLM-deficient (gclm^−/−) mice. Erythrocytes from gclm^−/− mice showed greatly reduced intracellular glutathione. Prolonged incubation resulted in complete lysis of gclm^−/− erythrocytes, which could be reversed by exogenous delivery of the antioxidant Trolox. To test the importance of GCLM in vivo, mice were treated with phenylhydrazine (PHZ; 0.07 mg/g b.w.) to induce oxidative stress. Gclm^−/− mice showed dramatically increased hemolysis compared with gclm^+/+ controls. In addition, PHZ-treated gclm^−/− mice displayed markedly larger accumulations of injured erythrocytes in the spleen than gclm^+/+ mice within 24 h of treatment. Iron staining indicated precipitations of the erythrocyte-derived pigment hemosiderin in kidney tubules of gclm^−/− mice and none in gclm^+/+ controls. In fact, 24 h after treatment, kidney function began to diminish in gclm^−/− mice as evident from increased serum creatinine and urea. Consequently, while all PHZ-treated gclm^+/+ mice survived, 90% of PHZ-treated gclm^−/− mice died within 5 days of treatment. In vitro, upon incubation in the absence or presence of additional oxidative stress, gclm^−/− erythrocytes exposed significantly more phosphatidylserine, a cell death marker, than gclm^+/+ erythrocytes, an effect at least partially due to increased cytosolic Ca²⁺ concentration. Under resting conditions, gclm^−/− mice exhibited reticulocytosis, indicating that the enhanced erythrocyte death was offset by accelerated erythrocyte generation. GCLM is thus indispensable for erythrocyte survival, in vitro and in vivo, during oxidative stress.     

The relationship of membrane lipids to species specific hemolysis by hemolytic factors from Stomoxys calcitrans (L.) (Diptera: Muscidae)

Posterior-midgut homogenate from female stable flies prepared at 12 h after feeding hemolyzed erythrocytes from 6 different mammalian species more readily than homogenate prepared at 22 h. A significant correlation was obtained between the per cent sphingomyelin content of the erythrocyte membrane and the time required for lysis by the 12 h homogenate. Erythrocytes with low sphingomyelin content were more sensitive to lysis than cells with high sphingomyelin. No such correlation exists for hemolysis by 22 h homogenate. Mean corpuscular volume and osmotic fragilities of erythrocytes were not related to hemolysis either by 12 or 22 h homogenate. Determination of phospholipase C and sphingomyelinase activities showed that the hydrolysis rate of phospholipase C in homogenates prepared at 12–14 h was almost twice as much as sphingomyelinase activity. Whereas hydrolysis rates in 22–24 h homogenate were not different and markedly reduced compared to the 12–14 h homogenate. The times required for erythrocyte hemolysis related to the phospholipase C and sphingomyelinase activity profiles suggests that these enzyme activities participate in the in vitro hemolysis of red blood cells. Bovine and human erythrocytes change their biconcave contour into a spiculated spherical shape when they are exposed to midgut homogenate. This shape change is interpreted as a detergent induced modification of the red cell membrane which renders the erythrocytes more vulnerable to hemolysis.     

Role of the interaction between puerarin and the erythrocyte membrane in puerarin-induced hemolysis

Adverse drug reactions (ADR), especially intravenous hemolysis, have largely limited the application of puerarin injections in clinics. This study investigated the underlying mechanisms of puerarin-induced hemolysis. Our results show that puerarin induced concentration-dependent and time-dependent hemolysis when human erythrocytes were incubated in saline solution with more than 2 mM puerarin for over 2 h. However, incubation in PBS or addition of 1 mM of lidocaine to the saline solution completely abolished the hemolysis. Providing materials that could start ATP synthesis did not reverse the hemolysis, and puerarin did not affect Na⁺–K⁺–ATPase activity. In addition, puerarin (0.1–2 mM) did not cause calcium influx or exhibited pro-oxidant activity in erythrocytes. Puerarin exhibited different influences on the membrane microviscosity of erythrocytes in saline and PBS. Moreover, 1 mM lidocaine inhibited 8 mM puerarin-induced reduction of membrane microviscosity in saline solution. SDS–PAGE analysis of membrane proteins revealed that 2 mM puerarin treatment induced the appearance of several new protein bands but attenuated the expression of protein bands 2.1, 3, 4.1, 4.2 and 5. These results suggest that high concentrations of puerarin-induced hemolysis were associated with the changes of membrane lipids and of the composition of erythrocytes membrane proteins but not with ATP depletion, pro-oxidation and calcium influx. These changes could be related to the intercalation of amphiphilic puerarin at high concentration into the erythrocyte membrane in certain media, resulting in membrane disorganization and, eventually, cytolysis. Hence, in clinics, determining the optimal dose of puerarin is critical to avoid overdosing and ADR.     

Effect of hypervitaminosis A on hemolysis and lipid peroxidation in the rat

Erythrocytes from rats fed large doses of Vitamin A alone, or large doses of vitamin A and vitamin E or diphenyl-p-phenylene diamine (DPPD) were studied for H2O2-induced hemolysis. The vitamin A-dosed rats were more susceptible than normal rats to H2O2-induced hemolysis. Hemolysis was not accompanied by lipid peroxidation. Nevertheless, the antioxidants vitamin E and DPPD inhibited hemolysis in erythrocytes from vitamin A-dosed rats. These antioxidants had the same inhibitory effect when they were included in the diet or added to erythrocyte suspensions in vitro. Erythrocytes from vitamin A-dosed rats with or without added vitamin E or DPPD were less susceptible than the erythrocytes from normal rats to osmotic challenge, showing that vitamin A was present in levels sufficient to alter the structure of the erythrocyte membrane. These studies show that oxidative hemolysis occurs when the erythrocyte membrane is modified. Furthermore, this oxidative hemolysis is unrelated to lipid peroxidation.     

Hemolysis and clearance of erythrocytes in Scylla serrata are related to the agglutination by the native sialic acid-specific lectin

The hemolymph of the crab Scylla serrata contains a lectin specific for N-glycolylneuraminic acid. The role of the sialic acid-specific lectin on natural immunity of the crab is studied by using several kinds of mammalian erythrocytes as a pathogen model. A significant correlation is observed between in vivo clearance of exogenous erythrocytes with the extent of erythrocyte agglutination by the lectin. Similarly, another correlation is noticed between the susceptibility of erythrocytes to lectin-dependent hemocytc-mediated hemolysis and the extent of lectin-mediated erythrocyte agglutination. Two hours after administration of the erythrocytes into the hemocoel, induced augmentation of hemagglutinating activity was observed against all erythrocytes, whether agglutinated highly or least by the lectin, suggesting an increase in the circulating lectin. This study documents that “opsonization” of foreign pathogen by the native lectin is an important step in hemocyte recognition, hemolysis and clearance of the pathogen.     

Mechanical lysis of human erythrocytes. Membrane stabilization by plasma proteins]

Mechanical properties of erythrocyte membranes play an important role in red cell functions. Stability of human erythrocytes under deforming mechanical tensions which occur in the rapidly moving fluid is studied. The activation energy of the mechanical hemolysis determined by the temperature dependence of the hemolysis rate is 55 + 7 kJ/mol. The fragility of erythrocytes rises sharply as the salt concentrations increase. Glutaric dialdehyde forms a certain number of interprotein bonds which increase the fragility of erythrocytes. The mechanical stability of the erythrocyte membrane falls at high (0.5 M) ethanol concentrations. Blood plasma proteins, particularly human serum albumin, have a pronounced stabilizing effect. The hemolysis occurring during the rapid mixing is not probably associated with an osmotic mechanism since high sucrose concentrations do not prevent this process. The mechanical hemolysis depends both on the deforming tension arising in the membrane and on the state of the erythrocyte membrane.