MEMBRANE PROTEIN DEFICIENCY OF PNH ERYTHROCYTES

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Complement is a circulating system composed of many different proteins which is known for its ability to lyse animal cells and bacteria. This application seeks support for a series of investigations on the effects of complement on that do not lead to lysis of the target cell: the sublytic events. This proposal stems from our observation that when complement is activated at sublytical concentrations, influx of Na and L-glucose into the unlysed erthrocytes increases dramatically, albeit transiently, and yet the affected cells do not lyse or swell, as would be expected. We found that this non-lethal permeability increase is produced by the membrane attack complex, which is a macromolecular complex of the terminal complement components. Further we determined that C8, but not C9, is necessary to produce the sublytic change in permeability and that in response to the sublytic complement attack, there is activation of a Ca-dependent K permeability pathway. This promotes net K efflux and prevents the colloidosmotic swelling and lysis that occurs when the membrane damage is permanent and permits the survival of the affected cell. In PNH, a disease characterized by a dramatic increase in the sensitivity of erythrocytes to complement lysis, there is apparently a lack of sublytic changes for reasons not yet completely understood. Our specific aims include: 1) continue our studies on the complement regulatory proteins DAF and C8bp, particularly in relation to their role in the sublytic events; 2) characterize further the transient permeability changes and the volume regulatory transport mechanism through studies on ion fluxes during exposure to sublytic complement; and 3) determine the biological consequences of the sublytic events to the cells that survive a complement attack. We will perform studies in normal and PNH erthrocytes, as well as in cells carrying abnormal hemoglobin, such as SS, SC, or CC which are known to have volume regulatory pathways that we predict would decrease their sensitivity to complement lysis. We expect that our investigations will help better understanding complement mediated diseases, such as lupus erythematous and immune hemolytic anemia, and also provide important information on the physiological functions of the complement system and its regulatory proteins.