Several proteins are attached to the cell membrane by a glycosyl- phosphatidylinositol (GPI) anchor. In this report, we show that during vesiculation of human RBCs in vitro, two of these proteins, acetylcholinesterase and decay accelerating factor, redistribute on the cell surface and become enriched in the released vesicles. As a result, the remnant cells are depleted of these proteins. We suggest that alterations in the architecture of the RBC membrane that precede vesiculation lead to selective polarization of GPI-anchored proteins within the domain of the membrane destined to become a vesicle. Since vesiculation occurs in many cell types, and if the loss of GPI-anchored proteins accompanies this process, it may have important biologic significance.

A deficiency of membrane proteins having a glycosylphosphatidylinositol (GPI) anchor is characteristic of the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) and is currently believed to be the basis for the enhanced susceptibility to lysis by activated complement observed in these cells. Our recent observation that GPI-anchored proteins are preferentially lost into membrane vesicles shed from normal erythrocytes after calcium loading led us to examine the hypothesis that the remnant erythrocytes might also have increased sensitivity to complement-mediated hemolysis. Indeed, red blood cells treated in such a manner became more sensitive to lysis by antibody and complement or to lysis initiated by activated cobra venom factor complexes (CoFBb). As a consequence of membrane vesiculation, the erythrocytes lost up to approximately 50% of their immunoreactive decay- accelerating factor and 25% to 30% of their immunoreactive membrane inhibitor of reactive lysis (MIRL). Closer examination of the defect responsible for the marked increase in sensitivity to CoFBb-initiated hemolysis seen in calcium-loaded erythrocytes showed that a complex combination of factors produced the defect. These included a decrease in both functional and immunoreactive MIRL and depletion of intracellular potassium and adenosine triphosphate (ATP). These results suggest the possibility that loss of DAF and MIRL via membrane vesiculation, as well as decreases in intracellular potassium and/or ATP, might contribute to the phenotype of PNH erythrocytes. Further, normal or pathologic red blood cells might develop a PNH-like defect after membrane vesiculation if sufficient decreases in potassium and ATP also occurred.