Neutrophils have been implicated as the major cellular mediator of acute vascular injury associated with bacterial endocarditis, immune vasculitis, ischemic heart disease and the adult respiratory distress syndrome (ARDS) by virtue of their ability to become sequestered in the vasculature and cause subsequent damage to the endothelium by release of toxic oxygen radicals and lysosomal enzymes. The close association of platelets with neutrophils in these disorders suggests potential interactions during inflammation. Yet, little is known about the role of platelet-derived factors in regulating neutrophil-mediated injury to endothelium. We have recently described the ability of platelet-derived proteins as well as adenine-containing nucleotides to regulate neutrophil inflammatory functions. We now seek support to purify and biochemically characterize the protein inhibitor(s) and to determine mechanisms of how the protein inhibitor(s) and adenine nucleotides regulate neutrophil-mediated injury to endothelial cells. The objective of the proposed research is to test the HYPOTHESIS that platelet-derived factors inhibit neutrophil-mediated endothelial cell injury resulting in increased permeability edema.
The SPECIFIC AIMS of the project are: 1) to purify to homogeneity the platelet-derived neutrophil inhibitory factors and to examine the effect of these inhibitors on neutrophil functions in vitro; 2) to determine the effect of these platelet-derived factors on neutrophil-mediated injury and functional alterations of cultured endothelial cells, and 3) to examine the cellular mechanisms by which these platelet-derived factors inhibit neutrophil-mediated injury to endothelial cells. We will employ C5a and F-Met-Leu-Phe (FMLP), stimuli that induce rapid release of cellular products, and phorbol myristate acetate (PMA) that induces a more prolonged release of products such as reactive oxygen species. We will establish dose response and kinetic curves for each of the platelet-derived factors to assess their effect on stimulated neutrophil functions and injury of endothelial cell barrier function. The mechanisms of neutrophil regulation by each of the platelet-derived factors will be studied by measurement of their effect on distinct steps involved in neutrophil activation. Results from these studies should provide useful new information on the role of platelet-derived factors on the initiation and resolution of neutrophil-mediated vascular disorders. Results of these studies should also provide new possibilities for prophylactic and therapeutic measures for inflammatory disorders such as ARDS.
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