Thromboxane A2 (TXA2) is an effective constrictor of vascular smooth muscle and aggregator of platelets. These effects are mediated via membrane receptors that belong to the super family of G protein coupled receptors. The synthesis of TXA2, particularly by platelets, is increased in a variety of cardiovascular and renal diseases. Aspirin inhibits the platelet synthesis of TXA2 and because aspirin is effective in the treatment and prevention of acute coronary artery syndromes, it has been postulated that TXA2 plays an important pathophysiologic role in these syndromes. The long range objectives of this laboratory are to determine the relationship of structure to function for human platelet TXA2 receptors. To meet these objectives the PI has proposed the following specific aims: 1) determine the role of conserved histidines, serines and arginine-295 in ligand binding to the platelet TXA2 receptor, 2) determine the sites of attachment for affinity probes to TXA2 receptors and 3) synthesize affinity and photoaffinity probes that will be used to elucidate the relationship of structure to function for TXA2 receptors. These studies will use a combination of molecular biology to clone and mutate specific amino acids of the cloned, carboxy terminal hexahistidine tagged TXA2 receptors, rapid receptor purification techniques, cell culture, radioligand equilibrium and competition binding assays, pharmacologic and biochemical assays, and affinity labeling of TXA2 receptors coupled with proteolysis and sequencing using state of the art mass spectrometry. The results of these experiments will be used to begin to develop a computer generated model of the ligand binding domain of TXA2 receptors. These studies should provide a better fundamental understanding of the relationship of structure to function for this important class of receptors and may ultimately lead to the development of specific antagonists of TXA2 receptors for the treatment of a variety of cardiovascular and renal diseases.
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