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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 2α to bind/signal through TPRs. Using TPR expressing HEK cells, it was found that 8-iso-PGF 2α mobilized calcium and bound TPRs with a dissociation constant (K d ) of 57 nM. Interestingly, site-directed-mutagenesis revealed that 8-iso-PGF 2α has a unique coordination profile with TPRs. Thus, while Phe 184 and Asp 193 are shared by both 8-iso-PGF 2α and classical TPR ligands, Phe 196 was found to be required only for 8-iso-PGF 2α binding. Functional studies also revealed interesting results. Namely, that 8-iso-PGF 2α 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 2α . 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 2α coordinates with Phe 184 , Asp 193 and Phe 196 on platelet TPRs; (2) Phe 196 serves as a unique TPR binding site for 8-iso-PGF 2α ; (3) 8-iso-PGF 2α signals through both stimulatory and inhibitory pathways in platelets; (4) 8-iso-PGF 2α inhibits human platelet activation through a cAMP-dependent mechanism; (5) 8-iso-PGF 2α 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.

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Gini coefficient, Health inequality, Simulation, Statistical analysis,