Scrambling of Phospholipids Activates Red Cell Membrane Cholesterol^†

Cholesterol is predicted to associate more strongly with the outer than the inner leaflet of plasma membrane bilayers based on the relative in vitro affinities of their phospholipids. Complex formation with the high-affinity species (especially saturated sphingomyelins) is said to reduce the chemical activity (escape potential or fugacity) of the sterol. We therefore tested the hypothesis that scrambling the sidedness of plasma membrane phospholipids of intact cells will increase the chemical activity of outer surface cholesterol. Upon activating the plasma membrane scramblase in intact human red cells by introducing ionomycin to raise cytoplasmic Ca⁺⁺, phosphatidylserine became exposed and, concomitantly, the chemical activity of exofacial cholesterol was increased. (This was gauged by its susceptibility to cholesterol oxidase and its rate of transfer to cyclodextrin.) Similar behavior was observed in human fibroblasts. Two other treatments known to activate cell surface cholesterol (namely, exposure to glutaraldehyde and to low-ionic-strength buffer) also brought phosphatidylserine to the cell surface but by a Ca⁺⁺-independent mechanism. Given that phospholipid scrambling is important in blood coagulation and apoptosis, the concomitant activation of cell surface cholesterol could contribute to these and other pathophysiological signaling processes.