Characterization of Microvesicles Released from Human Red Blood Cells
Cellular Physiology and Biochemistry
Background/Aims: Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process
... rd budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca 2+ uptake and activation of protein kinase C. Methods: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Results: Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca 2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca 2+ , especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from -40 mV to -10 mV depended on the solutions and buffers used. Conclusion: An increase of intracellular Ca 2+ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs. dissolved in absolute ethanol at 1 mM. LPA and PMA were dissolved in DMSO at 1 mM and stored at -20°C. NAC was prepared as 0.5 M stock solution in water placed in a heating block for 1 h. Glutaraldehyde (for electron microscopy), and all other chemicals were at analytical grade and purchased from Sigma-Aldrich. Statistics Data are presented as the mean values ± S.D. of at least 3 experiments of different blood samples. Statistical analysis was performed using Student's t-test when Gaussian distributed. The values were taken as significant difference when p ≤ 0.05 or p ≤ 0.01. Otherwise, a Mann-Whitney test was performed. Fig. 4. Flow cytometry analysis of stimulated RBCs stained with annexin V-FITC. RBCs were stimulated with A23187, LPA or PMA for 2 h. The dot plots display the characteristics of the RBCs and MVs, the SSC vs. FSC (upper row) and the FL1 vs. FSC (lower row). Gates R1: instrument noise; R2: MVs and exosomes; R3: intact RBCs; R4: negative populations of small particles, MVs and exosomes; R5: positive population of MVs and exosomes; R6: positive population of RBCs; R7: negative population of RBCs.