The present proposal is aimed at an understanding of several aspects of the liberation of arachidonic acid from phospholipid stores in stimulated platelets. Since the enzyme cyclooxygenase and lipoxygenase can only utilize the free arachidonate, the mechanisms of its release, which are as yet incompletely understood, are an important control point in the formation of biologically active oxygenated products, such as the pro-aggregatory platelet-derived thromboxane A2. In addition, liberated arachidonate can be utilized by other cells to form additional, biologically active compounds such as anti-aggregatory prostacyclin by endothelial cells, and hydroxy acids and leukotrienes by leukocytes. I plan to investigate: 1) the source of arachidonate freed for cyclooxygenation from platelet phospholipids, and thereby the mechanisms by which this liberation occurs, 2) the role of albumin (a plasma protein, which avidly binds fatty acids as well as the eicosanoids thromboxane A2, prostacyclin, and leukotriene A4) in the spectrum of both released fatty acids an eicosanoids, 3) the pathway(s) by which platelets remove (lytic) lyso phospholipids formed upon stimulation, 4) whether the time course of changes in platelet content of polyphosphoinositides is consistent with the hypothesis that polyphosphoinositides might represent the site from which membrane-bound calcium is released upon platelet stimulation, and 5) the extent to which platelets might contribute arachidonic acid (and endoperoxides as well as hydro (per) oxides) to monocytes--cells which appear to have a greater capacity than PMN leukocytes to form a variety of oxygenated arachidonate products, including both thromboxane A2 and prostacyclin, as well as leukotrienes B4 and C4. The techniques to be used for most of these studies are operational in the independent laboratory of the Principal Investigator, and consist of TLC, GC and HPLC, as well as radiochemical procedures. The knowledge gained from the proposed investigations should increase our understanding of the role of platelets in the generation of bioactive eicosanoids and cell-cell interactions as related to hemostasis, thrombosis and atherosclerosis.
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