Recent research in our laboratory has led to the concept of endothelial cell Thromboregulation. Platelet activation as a consequence of vascular injury causes endothelial cells (EC) to respond in a manner directed toward limitation or reversal of the occlusive consequences of platelet accumulation. Such limitation or reversal is achieved by EC via 3 separate Thromboregulatory systems: Eicosanoids, endothelium-derived relaxing factor (EDRF/NO), and, most importantly, an endothelial ecto- nucleotidase. The latter rapidly metabolizes platelet-released ADP thereby restoring platelets to the resting state. Research proposed in this project will focus on control systems and mechanisms responsible for modulation of vascular Thromboregulation. Major emphasis will be placed on endothelial ecto-ADPase activity since our previous research has highlighted this enzyme system. To this end we will determine mechanisms by which cytokines, such as IL-1b, TNF-a, IFN-g, and IL-13, regulate ADPase activity and gene expression. In addition, we will assess transcriptional regulation of EC ecto-ADPase by elements of the 5'- regulatory region, including cytokine response elements. Our collaborations with Project II will be crucial to successful completion of these aims, since Drs. K. Hajjar and D. Hajjar have expertise in this area of research. Since preliminary studies in EC indicated that IL-13 induces phosphorylation of the Janus kinase Jak-2, we will investigate whether IL- 13 upregulation of EC ADPase activity is related to activation of the Jak- STAT pathway of signal transduction. Our collaboration with Project IV will be essential for achieving these objectives, since Dr. Hempstead has considerable expertise in this developing discipline. Studies of the antithrombotic function of the double lipoxygenase product, lipoxin A4, will be extended to evaluate its capacity for upregulation of EC ADPase activity. Furthermore, it will be important to determine whether LXA4 can prevent or reverse the prothrombotic downregulation of EC ADPase activity resulting from cytokine exposure. To further elucidate the function of LXA4 as an antithrombotic eicosanoid, we will isolate the EC-specific LXA4 receptor cDNA, using the full open reading frame (ORF) of our recently obtained myeloid LXA4 receptor as a probe. The EC-specific LXA4 receptor will then be biochemically and functionally characterized in mammalian expression system(s). The expertise of Drs. Pomerantz and D. Hajjar in eicosanoid molecular biology will be essential for achieving these aims. An understanding of mechanisms controlling EC ADPase activity may lead to development of rational effective forms of antithrombotic therapy which can be implemented at the very earliest stages of thrombus formation.
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