Characterization of isoprostane signaling: evidence for a unique coordination profile of 8-iso-PGF(2alpha) with the thromboxane A(2) 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-PGF(2alpha) to bind/signal through TPRs. Using TPR expressing HEK cells, it was found that 8-iso-PGF(2alpha) mobilized calcium and bound TPRs with a dissociation constant (K(d)) of 57 nM. Interestingly, site-directed-mutagenesis revealed that 8-iso-PGF(2alpha) has a unique coordination profile with TPRs. Thus, while Phe184 and Asp193 are shared by both 8-iso-PGF(2alpha) and classical TPR ligands, Phe196 was found to be required only for 8-iso-PGF(2alpha) binding. Functional studies also revealed interesting results. Namely, that 8-iso-PGF(2alpha) 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-PGF(2alpha). While the stimulatory site is represented by TPRs, the second cAMP inhibitory site is presently unidentified, but does not involve receptors for PGI(2), PGD(2) or PGE(2). In summary, these studies provide the first documentation that: (1) 8-iso-PGF(2alpha) coordinates with Phe184, Asp193 and Phe196 on platelet TPRs; (2) Phe196 serves as a unique TPR binding site for 8-iso-PGF(2alpha); (3) 8-iso-PGF(2alpha) signals through both stimulatory and inhibitory pathways in platelets; (4) 8-iso-PGF(2alpha) inhibits human platelet activation through a cAMP-dependent mechanism; (5) 8-iso-PGF(2alpha) 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.