This renewal application proposes a series of studies to elucidate the molecular basis and regulation of human umbilical vein endothelial cell (HUVEC) ecto-ADPase. The project will clone, sequence and express HUVEC ADPase and characterize it biochemically; study structure/function relationships to identify regions critical for nucleotide binding and enzyme activity; determine the mechanisms by which lysophosphatidylcholine (lysoPC), homocysteine, cytokines and growth factors affect enzyme activity; and assess transcriptional regulation by elements of the 5'-flanking region of the ADPase gene. In summary, research proposed in this application will elucidate molecular mechanisms by which an enzyme on the surface of human endothelial cells immediately metabolizes ADP emerging from activated platelets. To this end we will clone, sequence and express isolated HUVEC ADPase and characterize it biochemically; study structure/function relationships to identify regions critical for enzyme activity; determine mechanisms by which biologic substances such as lysophosphatidylcholine, homocysteine, cytokines and growth factors affect enzyme activity; and assess transcriptional regulation by elements of the 5'-flanking region of the ADPase gene. Experiments proposed in this application will furnish information for future development of rational and effective forms of antithrombotic therapy which will act at the very earliest stages of thrombus formation.
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