ELECTROKINETIC PHENOMENA. III : THE "ISOELECTRIC POINT" OF NORMAL AND SENSITIZED MAMMALIAN ERYTHROCYTES

A survey of the published electrophoretic mobilities of certain mammalian red cells reveals that the isoelectric points accorded to these cells are the result of equilibria incidental to red cell destruction. The electrophoretic mobilities of normal washed sheep and human cells have now been studied in 0.85 per cent NaCl solutions from about pH 3.6 to 7.4. All measurements were made within 2 minutes of the preparation of the suspension of red cells. In no case was reversal of sign of charge observed under these conditions. Reversal of sign of charge occurred only after sufficient time had elapsed to permit sufficient adsorption of the products of red cell destruction. There is little change in mobility as the pH of the medium is decreased. Reversal of sign of charge does occur in the presence of normal and immune (anti-sheep) rabbit sera. The isoelectric point determined under these conditions does not appear to be connected specifically with the immune body but is perhaps associated with phenomena incidental to red cell destruction and the presence of serum. The characteristic lowering of mobility by amboceptor occurs, however, from pH 4.0 to pH 7.4. The curves of mobility plotted against pH for normal and for immune sera support the viewpoint that the identity of the isoelectric points for normal and sensitized sheep cells is not primarily concerned with the immune reaction. It is most unlikely that an "albumin" or a "globulin" surface covers red cells with a complete protein film. Although serum protein reacts with red cells in acid solutions, this is not demonstrable for gelatin. The lowering of mobility usually ascribed to anti-sheep rabbit serum may also occur, but to a lesser degree, in normal rabbit serum. This diminution of mobility is not, in the first place, associated with sensitization to hemolysis induced by complement. This supports the view that only a very small part of the red cell surface need be changed in order to obtain complete hemolysis in the presence of complement.     

Membrane solubility of ethanol in chronic alcoholism. The effect of ethanol feeding and its withdrawal on the protection by alcohol of rat red blood cells from hypotonic hemolysis

Membranes from ethanol-fed rats are resistant to the in vitro effects of ethanol on membrane structure and function. We have proposed that the resistance arises from adaptive changes in membrane composition which lower the solubility (partition coefficient) of ethanol in these membranes. The partition of ethanol (and other alcohols and anesthetics) into red blood cells protects the cells from hypotonic hemolysis. Here, we show that the protection by alcohols and anesthetics of red blood cells from ethanol-fed rats is greatly attenuated. This finding indicates that the membrane solubility of these agents is lowered in chronic alcoholism and thus explains the resistance to the acute effects of ethanol. The protection from hemolysis decreases over 2 weeks of ethanol-feeding and returns to normal values within 1 day after ethanol withdrawal. These changes are associated with a parallel increase in total and free serum cholesterol during ethanol feeding and a return to normal values within a day after withdrawal. However, we find only a slight increase in the cholesterol/phospholipid ratio of the red blood cell membranes during the development of ethanol tolerance. In rats fed a cholesterol and saturated fat diet, the increase in serum cholesterol is also associated with an attenuation of the protection from hypotonic hemolysis.     

Assessment of the safety of the cationic arginine-rich peptides (CARPs) poly-arginine-18 (R18 and R18D) in ex vivo models of mast cell degranulation and red blood cell hemolysis

Our laboratory focuses on the development of novel neuroprotective cationic peptides, such poly-arginine-18 (R18: 18-mer of l-arginine; net charge +18) and its d-enantiomer R18D in stroke and other brain injuries. In the clinical development of R18/R18D, their cationic property raises potential safety concerns on their non-specific effects to induce mast cell degranulation and hemolysis. To address this, we first utilised primary human cultured mast cells (HCMCs) to examine anaphylactoid effects. We also included as controls, the well-characterised neuroprotective TAT-NR2B9c peptide and the widely used heparin reversal peptide, protamine. Degranulation assay based on β-hexosaminidase release demonstrated that R18 and R18D did not induce significant mast cell degranulation in both untreated (naïve) and IgE-sensitised HCMCs in a dose-response study to a maximum peptide concentration of 16 μM. Similarly, TAT-NR2B9c and protamine did not induce significant mast cell degranulation. To examine hemolytic effects, red blood cells (RBCs), were incubated with the peptides at a concentration range of 1–16 μM in the absence or presence of 2% plasma. Measurement of hemoglobin absorbance revealed that only R18 induced a modest, but significant degree of hemolysis at the 16 μM concentration, and only in the absence of plasma. This study addressed the potential safety concern of the application of the cationic neuroprotective peptides, especially, R18D, on anaphylactoid responses and hemolysis. The findings indicate that R18, R18D, TAT-NR2B9c and protamine are unlikely to induce histamine mediated anaphylactoid reactions or RBC hemolysis when administered intravenously to patients.     

Possible generation of hydrogen peroxide and lipid peroxidation of erythrocyte membrane by asbestos: cytotoxic mechanism of asbestos

We studied a mechanism of hemolysis induced by asbestos particles or silicic acid. This hemolysis was instantly initiated by mixing red blood cells with asbestos particles or silicic acid, and reached a plateau within 10 min. The hemolysis was suppressed by catalase, radical quenchers, deoxygenation, or phospholipids. The degree of the hemolysis was proportional to either the amount of asbestos added into red blood cell suspension or the amount of thiobarbituric acid-reacting substances formed. These findings suggest that in vitro hemolysis induced by asbestos particles (or silicic acid) is ascribed to membrane lipid peroxidation initiated by hydrogen peroxide which was generated by the interaction of the mineral particles with biological membranes.     

Interaction of cationic carbosilane dendrimers and their complexes with siRNA with erythrocytes and red blood cell ghosts

We have investigated the interactions between cationic NN16 and BDBR0011 carbosilane dendrimers with red blood cells or their cell membranes. The carbosilane dendrimers used possess 16 cationic functional groups. Both the dendrimers are made of water-stable carbon–silicon bonds, but NN16 possesses some oxygen–silicon bonds that are unstable in water. The nucleic acid used in the experiments was targeted against GAG-1 gene from the human immunodeficiency virus, HIV-1.By binding to the outer leaflet of the membrane, carbosilane dendrimers decreased the fluidity of the hydrophilic part of the membrane but increased the fluidity of the hydrophobic interior. They induced hemolysis, but did not change the morphology of the cells. Increasing concentrations of dendrimers induced erythrocyte aggregation. Binding of short interfering ribonucleic acid (siRNA) to a dendrimer molecule decreased the availability of cationic groups and diminished their cytotoxicity. siRNA–dendrimer complexes changed neither the fluidity of biological membranes nor caused cell hemolysis. Addition of dendriplexes to red blood cell suspension induced echinocyte formation.     

Polymerization of protamine sulphate by carbodiimide and interaction of isolated protamine polymers with human red blood cells.

A method using a water-soluble carbodiimide to polymerize protamine sulphate is described. The behaviour of polymerized protamine in Sephadex chromatography and in polyacrylamide gel electrophoresis indicates that protamine has been polymerized into aggregates with defined molecular weights. Turbidimetrical titrations of the isolated protamine polymers with dextran sulphate show that the cationic charge density has been conserved after polymerization. The binding characteristics of the protamine polymers to human red blood cells as measured by cell electrophoresis indicate increased affinity with increased molecular weight of the polymer.     

Footstrike is the major cause of hemolysis during running.

There is a wide body of literature reporting red cell hemolysis as occurring after various forms of exercise. Whereas the trauma associated with footstrike is thought to be the major cause of hemolysis after running, its significance compared with hemolysis that results from other circulatory stresses on the red blood cell has not been thoroughly addressed. To investigate the significance of footstrike, we measured the degree of hemolysis after 1 h of running. To control for the potential effects of oxidative and circulatory stresses on the red blood cell, the same subjects cycled for 1 h at equivalent oxygen uptake. Our subjects were 10 male triathletes, who each completed two separate 1-h sessions of running and cycling at 75% peak oxygen uptake, which were performed in random order 1 wk apart. Plasma free hemoglobin and serum haptoglobin concentrations were measured as indicators of hemolysis. We also measured methemoglobin as a percentage of total hemoglobin immediately postexercise as an indicator of red cell oxidative stress. Plasma free hemoglobin increased after both running (P < 0.01) and cycling (P < 0.01), but the increase was fourfold greater after running (P < 0.01). This was reflected by a significant fall in haptoglobin 1 h after the running trials, whereas no significant changes occurred after cycling at any sample point. Methemoglobin increased twofold after both running and cycling (P < 0.01), with no significant differences between modes of exercise. The present data indicate that, whereas general circulatory trauma to the red blood cells associated with 1 h of exercise at 75% maximal oxygen uptake may result in some exercise-induced hemolysis, footstrike is the major contributor to hemolysis during running.     

Studies of the antigenic properties of fowl erythrocytes

Summary While in all the hemagglutination reactions with viruses the properties showed by complete fowl erythrocytes are exclusively situated in the cell walls, the authors have studied the antigenic properties of both elements of fowl red cells obtained after hemolysis. Cell walls provoke in the blood serum of rabbits formation of hemolysin and agglutinin just as entire normal red cells do. Probably hemolysate contains a protein fraction identic with one in the blood serum. All the antigenic properties of fresh cell walls are preserved after lyophilization.     

Isolation and characterization of transferrin receptor from embryonic chicken red cell

Transferrin receptor is isolated from the plasma membrane of chicken embryo red cell by affinity chromatography on transferrin-Sepharose 4B matrix. The molecular weight of the protein is approximately 58,000. The purified antibody to this protein is capable of agglutinating chicken embryo red cells, and the purified Fab fragments derived from this antibody are capable of inhibiting the antibody-induced agglutination, as well as the complement-induced hemolysis of chicken embryo red cells. The Fab fragments also inhibit the transferrin-mediated uptake of iron by chicken embryo red cells.     

Investigation of photosensitizing dyes for pathogen reduction in red cell suspensions

Despite recent advances in blood safety by careful donor selection and implementation of infectious disease testing, transmission of viruses, bacteria and parasites by transfusion can still rarely occur. One approach to reduce the residual risk from currently tested pathogens and to protect against the emergence of new ones is to investigate methods for pathogen inactivation. The use of photosensitizing dyes for pathogen inactivation has been studied in both red cell and platelet blood components. Optimal properties of sensitizing dyes for use in red cell suspensions include selection of dyes that traverse cell and viral membranes, bind to nucleic acids, absorb light in the red region of the spectrum, inactivate a wide range of pathogens, produce little red cell photodamage from dye not bound to nucleic acid and do not hemolyze red cells in the dark. Early research at the American Red Cross focused on the use of a class of dyes with rigid structures, such as the phenothiazine dyes, beginning with the prototypical sensitizer methylene blue. Results revealed that methylene blue phototreatment could inactivate extracellular virus, but resulted in undesirable defects in the red cell membrane that resulted in enhanced hemolysis that became evident during extended refrigerated blood storage. In addition, methylene blue phototreatment could neither inactivate intracellular viruses nor appreciably inactivate bacteria under conditions of extracellualar viral killing. Attempts to improve intracellular viral inactivation led to the investigations of more hydrophobic phenothiazines, such as methylene violet or dimethylmethylene blue. Although these dyes could inactivate intracellular virus, problems with increased red cell membrane damage and hemolysis persisted or increased. Further studies using red cell additive storage solutions containing high levels of the impermeable ion, citrate, to protect against colloidal osmotic hemolysis as well as competitive inhibitors to limit sensitizer binding to red cell membranes revealed that photoinduced hemolysis stemmed from dye bound to the red cell membrane as well as dye free in solution. Use of red cell additive solutions to prevent colloidal-osmotic hemolysis and use of novel flexible dyes that only act as sensitizers when bound to their targets are two techniques that currently are under investigation for reducing red cell damage. Ultimately, the decision to implement a photodynamic method for pathogen reduction will be determined by weighing the risks of unintended adverse consequences of the procedure itself, such as the potential for genotoxicity and allergic reactions, against the cost and benefits of its implementation.     

Investigation of photosensitizing dyes for pathogen reduction in red cell suspensions

Despite recent advances in blood safety by careful donor selection and implementation of infectious disease testing, transmission of viruses, bacteria and parasites by transfusion can still rarely occur. One approach to reduce the residual risk from currently tested pathogens and to protect against the emergence of new ones is to investigate methods for pathogen inactivation. The use of photosensitizing dyes for pathogen inactivation has been studied in both red cell and platelet blood components. Optimal properties of sensitizing dyes for use in red cell suspensions include selection of dyes that traverse cell and viral membranes, bind to nucleic acids, absorb light in the red region of the spectrum, inactivate a wide range of pathogens, produce little red cell photodamage from dye not bound to nucleic acid and do not hemolyze red cells in the dark. Early research at the American Red Cross focused on the use of a class of dyes with rigid structures, such as the phenothiazine dyes, beginning with the prototypical sensitizer methylene blue. Results revealed that methylene blue phototreatment could inactivate extracellular virus, but resulted in undesirable defects in the red cell membrane that resulted in enhanced hemolysis that became evident during extended refrigerated blood storage. In addition, methylene blue phototreatment could neither inactivate intracellular viruses nor appreciably inactivate bacteria under conditions of extracellualar viral killing. Attempts to improve intracellular viral inactivation led to the investigations of more hydrophobic phenothiazines, such as methylene violet or dimethylmethylene blue. Although these dyes could inactivate intracellular virus, problems with increased red cell membrane damage and hemolysis persisted or increased. Further studies using red cell additive storage solutions containing high levels of the impermeable ion, citrate, to protect against colloidal osmotic hemolysis as well as competitive inhibitors to limit sensitizer binding to red cell membranes revealed that photoinduced hemolysis stemmed from dye bound to the red cell membrane as well as dye free in solution. Use of red cell additive solutions to prevent colloidal-osmotic hemolysis and use of novel flexible dyes that only act as sensitizers when bound to their targets are two techniques that currently are under investigation for reducing red cell damage. Ultimately, the decision to implement a photodynamic method for pathogen reduction will be determined by weighing the risks of unintended adverse consequences of the procedure itself, such as the potential for genotoxicity and allergic reactions, against the cost and benefits of its implementation.     

Investigation of photosensitizing dyes for pathogen reduction in red cell suspensions.

Despite recent advances in blood safety by careful donor selection and implementation of infectious disease testing, transmission of viruses, bacteria and parasites by transfusion can still rarely occur. One approach to reduce the residual risk from currently tested pathogens and to protect against the emergence of new ones is to investigate methods for pathogen inactivation. The use of photosensitizing dyes for pathogen inactivation has been studied in both red cell and platelet blood components. Optimal properties of sensitizing dyes for use in red cell suspensions include selection of dyes that traverse cell and viral membranes, bind to nucleic acids, absorb light in the red region of the spectrum, inactivate a wide range of pathogens, produce little red cell photodamage from dye not bound to nucleic acid and do not hemolyze red cells in the dark. Early research at the American Red Cross focused on the use of a class of dyes with rigid structures, such as the phenothiazine dyes, beginning with the prototypical sensitizer methylene blue. Results revealed that methylene blue phototreatment could inactivate extracellular virus, but resulted in undesirable defects in the red cell membrane that resulted in enhanced hemolysis that became evident during extended refrigerated blood storage. In addition, methylene blue phototreatment could neither inactivate intracellular viruses nor appreciably inactivate bacteria under conditions of extracellualar viral killing. Attempts to improve intracellular viral inactivation led to the investigations of more hydrophobic phenothiazines, such as methylene violet or dimethylmethylene blue. Although these dyes could inactivate intracellular virus, problems with increased red cell membrane damage and hemolysis persisted or increased. Further studies using red cell additive storage solutions containing high levels of the impermeable ion, citrate, to protect against colloidal osmotic hemolysis as well as competitive inhibitors to limit sensitizer binding to red cell membranes revealed that photoinduced hemolysis stemmed from dye bound to the red cell membrane as well as dye free in solution. Use of red cell additive solutions to prevent colloidal-osmotic hemolysis and use of novel flexible dyes that only act as sensitizers when bound to their targets are two techniques that currently are under investigation for reducing red cell damage. Ultimately, the decision to implement a photodynamic method for pathogen reduction will be determined by weighing the risks of unintended adverse consequences of the procedure itself, such as the potential for genotoxicity and allergic reactions, against the cost and benefits of its implementation.     

Uptake of proteins by red blood cells

During hypotonic hemolysis red cells can take up ¹²⁵I-myoglobin and ¹²⁵I-immunoglobulin G. Cells which contain these proteins have distinctive cell morphology and are called gray ghosts. The association of protein with gray ghosts is fairly stable: these cells retain half of the proteins after 3 days. Passive diffusion of protein into the internal cell volume is the most plausible mechanism for uptake, and several lines of evidence indicate that the loaded proteins are freely diffusable within the red cells. Bacteriophage T4 is not taken up during hemolysis so uptake through large gaps in the red cell membrane with subsequent resealing seems unlikely. If an efficient procedure for fusing loaded gray ghosts to culture cells can be devised, it will be possible to introduce selected macromolecules into the cytoplasm of culture cells quite easily.     

Effect of cholesterol on the stability of human erythrocyte membranes to electric breakdown

Electrical stability of human erythrocyte membranes with different cholesterol content was studied. Breakdown in the cell membranes was generated by application of electric pulses with field strengths of 1.4-3.2 kV/cm. The share of perforated cells was registered by measuring hemolysis level. The red blood cells from patients with psoriasis and normal erythrocytes after incubation in the presence of liposomes were used as a model of cells with cholesterol-rich membranes. It was discovered that an increase of cholesterol content in the membranes moved the field-dependent curves to a higher field range. The obtained effect is attributed to the increase of the breakdown membrane potential. Application of high-pulse-electric-field technique for investigating the properties of cell membranes is discussed.     

Red cell ghost-mediated microinjection of RNA into HeLa cells: I. A comparison of two techniques for the entrapment and microinjection of tRNA and mRNA

We have compared two techniques for introducing RNA into red blood cell ghosts. In the pre-swell technique, RNA is introduced into red cells without prior removal of endogenous contents. In the multiple lysis technique, the red cells are subjected to two or three cycles of reversible lysis, prior to introducing the RNA, in order to first remove the normal red cell constituents. The pre-swell technique offers much greater entrapment of both tRNA and protamine messenger RNA (mRNA), but the RNA appears to be degraded during the procedure. This may be due either to nuoleolytic degradation or oxidation by the high concentration of endogenous hemoglobin. The multiple lysis technique offers much lower entrapment but also results in diminished degradation of the entrapped RNA. Although some degradation is apparent, a significant portion of the biological activity of the entrapped protamine mRNA is retained. We have also fused red cells loaded with protamine mRNA by the multiple lysis technique to HeLa cells using polyethylene glycol 6000. The recipient HeLa cells are capable of translating this heterologous message into protamine, a trout testis chromosomal protein.     

Electrical hemolysis of human and bovine red blood cells.

The external electric field strength required for electrical hemolysis of human red blood cells depends sensitively on the composition of the external medium. In isotonic NaCl und KCl solutions the onset of electrical hemolysis is observed at 4 kV per cm and 50 per cent hemolysis at 6 kV per cm, whereas increasing concentrations of phosphate, sulphate, sucrose, inulin and EDTA shift the onset and the 50 per cent hemolysis-value to higher field strengths. The most pronounced effect is observed for inulin and EDTA. In the presence of these substances the threshold value of the electric field strength is shifted to 14 kV per cm. This is in contrast to the dielectric breakdown voltage of human red blood cells which is unaltered by these substances and was measured to be approximately 1 V corresponding in the electrolytical discharge chamber to an external electric field strength of 2 to 3 kV per cm. On the other hand, dielectric breakdown of bovine red blood cell membranes occurs in NaCl solution at 4 to 5 kV per cm and is coupled directly with hemoglobin release. The electrical hemolysis of cells of this species is unaffected by the above substances with exception of inulin. Inulin suppressed the electrical hemolysis up to 15 kV per cm. The data can be explained by the assumption that the reflection coefficients of the membranes of these two species to bivalent anions and uncharged molecules are field-dependent to a different extent. This explanation implies that electrical hemolysis is a secondary process of osmotic nature induced by the reversible permeability change of the membrane (dielectric breakdown) in response to an electric field. This view is supported by the observation that the mean volumes of ghost cells obtained by electrical hemolysis can be changed by changing the external phosphate concentration during hemolysis and resealing, or by subjecting the cells to a transient osmotic stress immediately after the electrical hemolysis step. An interesting finding is that the breakdown voltage, although constant throughout each normally distributed ghost size distribution, increases with increasing mean volume of the ghost populations.     

Role of hydrogen bonding in red cell aggregation.

The role of hydrogen bonding in red cell aggregation induced by dextran was studied with the use of urea, an inhibitor for hydrogen bonding. In order to avoid hemolysis of red cells by the high concentration of urea, the studies were performed on human red cells hardened in glutaraldehyde. The degree of red cell aggregation at Hct = 45% was estimated by the use of a coaxial cylinder viscometer. The viscometric aggregation index (VAI) was calculated from viscosity values at shear rates of 52 sec-1 (eta H) and 0.05 sec-1 (eta L); VAI = (eta L - eta H)/eta H. Red cells with surface charge intact and with charge removal by neuraminidase treatment were studied. Urea at high concentrations, e.g., 6 M, significantly inhibited red cell aggregation induced by dextran. These findings indicate that hydrogen bonding plays an important role in dextran-induced red cell aggregation. An understanding of the nature of the forces involved in red cell aggregation serves to establish the physicochemical principles of cell-to-cell interactions induced by macromolecules.     

Hemolysis of human red blood cells by freezing and thawing in solutions containing sucrose: relationship with posthypertonic hemolysis and solute movements

Human red blood cells were frozen at temperatures down to ?9 °C in solutions containing sucrose, and the hemolysis on thawing was measured. This was compared with the hemolysis caused by exposing the cells to high concentrations of sucrose and then resuspending them in more dilute solutions at 4 °C. The effects of the hypertonic solutions of sucrose on potassium, sodium, and sucrose movements were also investigated. It was found that sucrose does not prevent damage to the cells by very hypertonic solutions (whether during freezing and thawing or at 4 °C) but it does reduce hemolysis of cells previously exposed to these solutions if present in the resuspension (or thawing) solution. Evidence is presented that the damaging effects of the hypertonic solutions of sucrose occurring during freezing are associated with changes in cell membrane permeability but that posthypertonic hemolysis is not primarily associated with a “loading” of the cells with extracellular solutes in the hypertonic phase. It is concluded that sucrose may reduce hemolysis of red blood cells by slow freezing and thawing by reducing colloid osmotic swelling of cells with abnormally permeable membranes.     

Mechanism of polymer-induced hemolysis: nanosized pore formation and osmotic lysis

Hemolysis induced by antimicrobial polymers was examined to gain an understanding of the mechanism of polymer toxicity to human cells. A series of cationic amphiphilic methacrylate random copolymers containing primary ammonium groups as the cationic functionality and either butyl or methyl groups as hydrophobic side chains have been prepared by radical copolymerization. Polymers with 0-47 mol % methyl groups in the side chains, relative to the total number of monomeric units, showed antimicrobial activity but no hemolysis. The polymers with 65 mol % methyl groups or 27 mol % butyl groups displayed both antimicrobial and hemolytic activity. These polymers induced leakage of the fluorescent dye calcein trapped in human red blood cells (RBCs), exhibiting the same dose-response curves as for hemoglobin leakage. The percentage of disappeared RBCs after hemolysis increased in direct proportion to the hemolysis percentage, indicating complete release of hemoglobin from fractions of RBCs (all-or-none leakage) rather than partial release from all cells (graded leakage). An osmoprotection assay using poly(ethylene glycol)s (PEGs) as osmolytes indicated that the PEGs with MW > 600 provided protection against hemolysis while low molecular weight PEGs and sucrose had no significant effect on the hemolytic activity of polymers. Accordingly, we propose the mechanism of polymer-induced hemolysis is that the polymers produce nanosized pores in the cell membranes of RBCs, causing an influx of small solutes into the cells and leading to colloid-osmotic lysis.     

Accumulation and drainage of hemin in the red cell membrane

The subject of hemin intercalation in red cell membranes and the correlation of the accumulated hemin level with the membrane pathology was studied. Methods which made use of dioxan and octan-2-ol mixtures to quantitate small amounts of hemin in membranes were developed. Applying these methods, hemin levels were measured in the cytoskeleton and the remaining lipid core of various red cell membranes. The amount of hemin, in both membrane fractions, was higher in pathological cells of sickle cell anemia and beta-thalassemia as compared to normal circulating cells. Correlation exists between the amount of the membrane-accumulated hemin and the severity of the disease. The level of hemin in the membrane was found to be age dependent, old cells in circulation accumulating more hemin than young cells. The level of hemin in all cells tested was much lower than the amount found previously to cause immediate hemolysis when applied externally (Kirschner-Zilber, I., Rabizadeh, E. and Shaklai, N. (1982) Biochim. Biophys. Acta 690, 20-30). This was explained by the differences between the process leading to immediate lysis and membrane changes recognized as pathological by the in-vivo sequestration mechanism. In search of a physiological mechanism which may drain the cell membrane from the hazardeous hemin, albumin, the main serum protein, was found capable of serving as an efficient agent for extracting hemin trapped in red cell membranes. It is suggested that under normal conditions albumin extracts enough hemin to leave the erythrocyte with unharmful hemin amounts, however, under pathological conditions greater amounts accumulate leading to a shorter cell life span.