Lipid rafts are sphingolipid- and cholesterol-rich membrane microdomains that are insoluble in nonionic detergents, have a low buoyant density, and preferentially contain lipid-modified proteins, like glycosyl phosphatidylinositol (GPI)-anchored proteins. The lipid rafts were isolated from human erythrocytes and major protein components were identified. Apart from the GPI-anchored proteins, the most abundant integral proteins were found to be the distantly related membrane proteins stomatin (band 7.2b), flotillin-1, and flotillin-2. Flotillins, already described as lipid raft components in neurons and caveolae-associated proteins in A498 kidney cells, have not been recognized as red cell components yet. In addition, it was shown that the major cytoskeletal proteins, spectrin, actin, band 4.1, and band 4.2, are partly associated with the lipid rafts. Stomatin and the flotillins are present as independently organized high-order oligomers, suggesting that these complexes act as separate scaffolding components at the cytoplasmic face of erythrocyte lipid rafts.

Cytosolic Ca ؉؉ induces the shedding of microvesicles and nanovesicles from erythrocytes. Atomic force microscopy was used to determine the sizes of these vesicles and to resolve the patchy, fine structure of the microvesicle membrane. The vesicles are highly enriched in glycosyl phosphatidylinositol-linked proteins, free of cytoskeletal components, and depleted of the major transmembrane proteins. Both types of vesicles contain 2 as-yet-unrecognized red cell proteins, synexin and sorcin, which translocate from the cytosol to the membrane upon Ca ؉؉ binding. In nanovesicles, synexin and sorcin are the most abundant proteins after hemoglobin. In contrast, the microvesicles are highly enriched in stomatin. The membranes of both microvesicles and nanovesicles contain lipid rafts. Stomatin is the major protein of the microvesicular lipid rafts, whereas synexin and sorcin represent the major proteins of the nanovesicular rafts in the presence of Ca ؉؉ . Interestingly, the raft proteins flotillin-1 and flotillin-2 are not found in the vesicles but remain in the red cell membrane. These data indicate the presence of different types of lipid rafts in the erythrocyte membrane with distinct fates after Ca ؉؉ entry. Synexin, which is known to be vital to the process of membrane fusion, is suggested to be a key component in the process of vesicle release from erythrocytes. ( IntroductionErythroctes are known to respond to physiological stimuli via a diversity of receptors and effectors. 1 In particular, prostaglandin E 2 and lysophosphatidic acid (LPA) induce Ca ϩϩ -dependent processes. 2,3 The rise of cytosolic Ca ϩϩ in erythrocytes triggers a sequence of biochemical and morphologic changes that finally result in the release of hemoglobin-containing exovesicles. Two sorts of vesicles, differing in size, have been described; these have been named microvesicles (150 nm diameter) and nanovesicles (60 nm diameter). 4 Elevated cytosolic Ca ϩϩ levels cause alterations in the membrane protein pattern, notably the aggregation of proteins catalyzed by transaminases and cytoskeletal rearrangements owing to the proteolysis of protein 4.1. 5 Another Ca ϩϩ -mediated effect is the breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with a concomitant increase in 1,2-diacylglycerol and phosphatidic acid. 6 Moreover, the phospholipidscramblase is activated and the aminophospholipid translocase is inhibited, 7 thereby leading to a randomization of the phospholipid asymmetry over the 2 membrane leaflets. The Ca ϩϩ -activated potassium efflux via the Gardos channel causes the loss of cell water and concomitant shrinkage of the erythrocyte. 8 The latter factors are essential for the release of the vesicles from the echinocytic erythrocytes.The physiological importance of the vesiculation process can be seen in a protection strategy of the red cell against the destruction by complement. 9 An erythrocyte being attacked by the complement components C5b through C9 faces a local Ca ϩϩ influx and responds by a r...

These data indicate that a stomatin-specific, raft-based process is involved in storage-associated vesiculation. A model of the vesiculation process in RBCs is proposed considering the raft-stabilizing properties of stomatin, the low storage temperature favoring raft aggregation, and the previously reported storage-associated changes in the cytoskeletal organization.