ASYMMETRIC DISTRIBUTION OF PHOSPHATIDYLSERINE IN ERYTHROCYTE MEMBRANE AT CRYOPRESERVATION WITH GLYCEROL AND POLYETHYLENE GLYCOL

Zemlianskykh N. G., Babiychuk L. A., Migunova R. K.

BIOLOGY

Scentific article

This study was aimed to the examination of disturbances of the transmembrane distribution of phosphatidylserine (PS) in human erythrocytes under freeze-thawing effect in the presence of glycerol and polyethylene glycol (PEG), as well as after removing of cryoprotective agents out of freeze-thawed samples and transferring them to the physiological conditions in vitro. The PS externalization on the outer surface was assessed using annexin V-FITC by the flow cytometry. Freezing with glycerol that penetrates through the plasma membrane led to the appearance of cell population with externalized PS, which was destroyed during the cryoprotectant removal out of the thawed samples. Transfer of the glycerol-free erythrocytes to the physiological conditions in vitro was characterized by the preserved PS asymmetric distribution in the membrane and the cell stability. Freezing of erythrocytes in the PEG presence, which does not penetrate through the plasma membrane, caused more pronounced disturbances of PS asymmetry in erythrocytes in a comparison with glycerol, despite a low level of hemolysis after freeze-thawing. PEG removal is not a mandatory procedure, since it does not penetrate through the membrane, and in the case of transfusion, its concentration could be reduced by gradual dilution in bloodstream. However, to determine the ability of erythrocytes cryopreserved with PEG to control the PS asymmetry distribution in membranes under the physiological conditions, the cryoprotectant was removed and the washed cells were transferred into Ringer’s solution. The incubation of PEG-free erythrocytes under the physiological conditions in vitro was characterized by an increase in the amount of erythrocytes with the externalized PS and a rise of hemolysis with the course of time that indicated the instability of structural and functional parameters of the erythrocytes. Disturbances in cell membranes during freezing with PEG can resulted from an increase in Ca2+ concentration inside the cells, since previously a reduction in Ca2+-ATPase activity and an increase in Ca2+ influx at the presence of this compound have been shown, unlike glycerol, whose effect on the intracellular Ca2+ regulation was not as substantial as in the PEG presence. Increasing in Ca2+ concentration inside the cells stimulates the scramblase activity and the flipase inhibition that leads to the PS externalization in erythrocyte membranes. The instability of the cells frozen with PEG under the physiological conditions in vitro may be related to impaired protein-lipid interactions in the membrane-cytoskeleton complex due to the disturbances in the PS asymmetry distribution. Prevention of the lipid asymmetry distortion in erythrocyte membranes can be used to improve the outcomes of erythrocyte cryopreservation with the use of cryoprotective medium based on non-penetrating substances. Given the most likely mechanism of the development of disorders in membrane structure the inclusion of Ca2+ blockers in the PEG-based cryoprotective medium can help to preserve the PS asymmetry in erythrocyte membrane and increase the cell stability

erythrocyte, membrane, lipid asymmetry, cryopreservation, glycerol, PEG.

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«Bulletin of problems biology and medicine» Issue 3 (152), 2019 year, 50-54 pages, index UDK 577.352:57.053:577.15:576.3:57.086.13

10.29254/2077-4214-2019-3-152-50-54