The influence of membrane physical properties on microvesicle release in human erythrocytes

Exposure of human erythrocytes to elevated intracellular calcium causes fragments of the cell membrane to be shed as microvesicles. This study tested the hypothesis that microvesicle release depends on microscopic membrane physical properties such as lipid order, fluidity, and composition. Membrane properties were manipulated by varying the experimental temperature, membrane cholesterol content, and the activity of the trans-membrane phospholipid transporter, scramblase. Microvesicle release was enhanced by increasing the experimental temperature. Reduction in membrane cholesterol content by treatment with methyl-β-cyclodextrin also facilitated vesicle shedding. Inhibition of scramblase with R5421 impaired vesicle release. These data were interpreted in the context of membrane characteristics assessed previously by fluorescence spectroscopy with environment-sensitive probes such as laurdan, diphenylhexatriene, and merocyanine 540. The observations supported the following conclusions: 1) calcium-induced microvesicle shedding in erythrocytes relates more to membrane properties detected by diphenylhexatriene than by the other probes; 2) loss of trans-membrane phospholipid asymmetry is required for microvesicle release

Effects of Cholesterol on Physical Properties of Human Erythrocyte Membranes: Impact on Susceptibility to Hydrolysis by Secretory Phospholipase A2

The ability of secretory phospholipase A2 (sPLA2) to hydrolyze cell membranes is highly dependent on the physical properties of the membrane. The effects of cholesterol on these properties have been characterized in artificial bilayers and found to alter sPLA2 activity significantly. It is hypothesized that the natural difference in cholesterol content between erythrocytes and leukocytes is in part responsible for their differing susceptibility to hydrolysis by sPLA2. To test this hypothesis, defined amounts of cholesterol were removed from erythrocyte membranes using methyl-β-cyclodextrin. Treatment of cells with methyl-β-cyclodextrin increased the hydrolysis rate and total substrate hydrolyzed by sPLA2. In general, this effect of cholesterol removal was more pronounced at higher temperatures. Comparison of the level of membrane order (assessed with the fluorescent probe laurdan) with hydrolysis rate revealed that sPLA2 activity was greatly enhanced upon significant reductions in lipid order. Additional treatment of the cells with calcium ionophore further enhanced the hydrolysis rate and altered the relationship with membrane order. These data demonstrated that interactions with sPLA2 observed in artificial bilayers apply to biological membranes. It is also proposed that the high level of cholesterol in erythrocyte membranes is a protective mechanism to guard against hydrolytic enzymes