In general, the studies described in this proposal are designed to further our understanding of platelet and leukocyte function in health and disease. Most of the experiments are designed to translate metabolic, biochemical and biophysical data into mean- ingful morphologic concepts. Thus, we wish to determine what membrane changes occur when platelets adhere, aggregate or degranulate, and how integral membrane proteins/lipids are rearranged. Elucidation of the mechanism(s) of secretion, endocytosis and intracellular transport form part of the objective. Although the emphasis will be on surface membranes, intracellular proteins and organelles which play a role in specific cell functions will also be investigated. Similar studies will be carried out on leukocytes to explain the transduction of stimuli which eventuate in migration or mitosis. The methodologies will include conventional fluorescence and electron microscopy, immunohistochemistry on the light microscopic and ultrastructural level, freeze-fracture, cell fractionation and tissue culture. Three new techniques will be introduced: 1) Low-icryl low temperature embedding, which permits detection of intracellular antigens at higher resolution than previously possible, 2) label- fracture, which permits delineation of surface molecules coincident with proteins anchored in the outer leaflet of the plasma membrane and 3) in situ hybridization, which permits a correlation of subcellular topography with higher order gene expression. Specimens obtained from patients with abnormal platelet or leukocyte function will be compared with those obtained from healthy subjects, and with cells that have been experimentally modified in vitro. The morphologic information derived from such studies seems important because, in the future, it may become possible to selectively alter structure e.g., by insertion of molecules into membranes which may restore normal function to pathologic cells. At all times, the ultimate goal is to help our understanding of aberrant blood cell function, which is a prerequisite for the rational management of patients with hematologic disease.
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