Characterization of isoprostane signaling: Evidence for a unique coordination profile of 8-iso-PGF2α with the thromboxane A2 receptor, and activation of a separate cAMP-dependent inhibitory pathway in human platelets

Since isoprostanes are thought to participate in the pathogenesis of thrombosis, presumably through their interaction with thromboxane receptors (TPRs), we examined the ability of 8-iso-PGF2α to bind/signal through TPRs. Using TPR expressing HEK cells, it was found that 8-iso-PGF2α mobilized calcium and bound TPRs with a dissociation constant (Kd) of 57 nM. Interestingly, site-directed-mutagenesis revealed that 8-iso-PGF2α has a unique coordination profile with TPRs. Thus, while Phe184 and Asp193 are shared by both 8-iso-PGF2α and classical TPR ligands, Phe196 was found to be required only for 8-iso-PGF2α binding. Functional studies also revealed interesting results. Namely, that 8-iso-PGF2α signals in human platelets through both a stimulatory (TPR-dependent) and an inhibitory (cAMP-dependent) pathway. Consistent with the existence of two signaling pathways, platelets were also found to possess two separate binding sites for 8-iso-PGF2α. While the stimulatory site is represented by TPRs, the second cAMP inhibitory site is presently unidentified, but does not involve receptors for PGI2, PGD2 or PGE2. In summary, these studies provide the first documentation that: (1) 8-iso-PGF2α coordinates with Phe184, Asp193 and Phe196 on platelet TPRs; (2) Phe196 serves as a unique TPR binding site for 8-iso-PGF2α; (3) 8-iso-PGF2α signals through both stimulatory and inhibitory pathways in platelets; (4) 8-iso-PGF2α inhibits human platelet activation through a cAMP-dependent mechanism; (5) 8-iso-PGF2α interacts with platelets at two separate binding sites. Collectively, these results provide evidence for a novel isoprostane function in platelets which is mediated through a cAMP-coupled receptor.