This research proposal represents a continued effort of our laboratory to investigate membrane phospholipid organization (MPO) in the human red blood cell (RBC). The proposal describes several unique methods to define the pathophysiologic consequences of disorders in MPO including the generation of thrombin by RBC as measured with a chromogenic substrate, the adherence of RBC to vascular endothelial cells, the detection of RBC by macrophages based upon surface aminophospholipid content, and the binding of liposomes to RBC as an indication of abnormal cell-cell interaction. We will investigate a variety of RBC disorders in which defects in the membrane have been identified and will attempt to delineate the relative importance of MPO to the abnormal membrane properties noted in these cells. Studies are included to define the role of MPO in RBC vesiculation and the effects of storage on red cell MPO. Enzymatic (phospholipase A2, sphingomyelinase) as well as chemical (fluorescamine binding) methods to study MPO will be used. Both phospholipid exchange protein (PLEP) and liposome technology will be used to introduce phospholipids into RBC to study transbilayer movements of phospholipids in pathologic cells. PLEP will also be used to insert unique phospholipids into RBC to study the effect of phospholipid species on membrane structure and function. Monospecific antibodies to phosphatidylserine, electron microscopic methods to identify outer membrane leaflet distribution of anionic phospholipids, and cation spin probes to study anionic phospholipid domains on the RBC surface represent new methods we have recently developed to study MPO in pathologic RBC. We will continue our investigations of factors which regulate MPO and will measure the effects of pH transmembrane potential, and heat treatment on MPO. We will also study interactions between red cell membrane proteins (normal and abnormal) and phospholipids using a monolayer system. Finally, we will study the biogenesis of MPO in erythroid cells by isolating red cell precursors and correlating the development of red cell membrane proteins with that of MPO. Studies will be performed on bone marrow erythroid cells as well as on precursors isolated during culture in solid or liquid systems. In summary, this project will (1) provide important information concerning the pathophysiologic consequences of MPO, (2) screen for MPO in a variety of pathologic RBC, (3) investigate specific factors which regulate MPO, and (4) investigate the biogenesis of MPO.
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