The broad, long-term objectives are to study the pathogenesis of thrombosis, which is now established as an integrated group of multicellular events. Contact and varying degrees of pathologic interactions between vascular and blood cells govern development and reversibility of occlusive disorders. Several in vitro model systems are described herein to study components of these cell-cell interactions. This includes transcellular metabolism between different cells, resulting in metabolites with novel biological activities. The research proposed will define biochemical and functional roles of mediators and intrinsic cell activities which enhance or prevent thrombotic events. Specifically, interactions of platelets with: I) human endothelial cells (EC), II) neutrophils, and III) erythrocytes will be investigated. In I) preliminary studies indicating that EC ecto-ADPase(s) block platelet reactivity will be extended. EC ecto-ADPase(s) will be isolated and characterized. Monoclonal antibodies will be developed and regulation of ADPase(s) determined. The second component of I) involves endothelium-derived relaxing factor (EDRF/NO), an autacoid which inhibits blood cell and vascular responsiveness. Formation of EDRF/NO by EC and neutrophils will be characterized. EC/NO synthase system(s) will be isolated. Effects of the 3 main antithrombotic defense mechanisms (EDRF/NO, eicosanoids, and ADPases) will be differentiated and correlated. In II) inhibition of platelet activation and recruitment by neutrophils will be separated into EDRF/NO related and unrelated components. Effects of neutrophil activation and priming on the generation of antithrombotic activities will be determined. Stability of the neutrophil inhibitory activitie(s) allow for localization, isolation, and, possibly, transfer. In III) mechanisms underlying the metabolic promotion of platelet activation and recruitment by erythrocytes will be elucidated, including the influence of nucleotide removal, protease inhibition, and aspirin treatment. Established experimental methods include: Separate measurement of platelet activation and recruitment, tissue culture, TLC, HPLC, GC/MS, aggregometry and radioimmunoassay.
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