Isoprostanes are a family of prostaglandin (PG) isomers formed in an enzyme-independent manner. They circulate in plasma and are excreted in urine. One of them, 8-epi PGF2αis a vasoconstrictor and mitogen, effects which are prevented by thromboxane antagonists. Given that 8-epi PGF2α may be formed by cyclooxygenase (COX) (Corey, E. J., Shih, C., Shig, N-Y., and Shimoji, K. (1984) Tetrahedron Letts. 44, 5013-5016; Hecker, M., Ullrich, V., Fischer, C., and Meese, C. O. (1987) Eur J. Biochem. 169, 113-123) and that this might confound its use as an index of free radical generation, we sought to characterize the mechanism of its formation by human platelets.
Activation of platelets by threshold concentrations of collagen, thrombin, and arachidonic acid resulted in formation of 8-epi PGF2αcoincident with that of the COX product, thromboxane, and the 12 lipoxygenase product, 12-hydroxyeicosatetraenoic acid, as detected by selected ion monitoring assays using gas chromatography-mass spectrometry. The effect appeared selective for 8-epi PGF2α among the F2isoprostanes. Pretreatment of platelets with aspirin or indomethacin abolished 8-epi PGF2α formation. COX-independent activation of platelets by high doses of collagen or thrombin, by the phorbol ester, phorbol 12-myristate 13-acetate, or the prostaglandin endoperoxide analog, U 46619 was not associated with 8-epi PGF2α formation.
Confirmation of the nature of the material formed by platelet COX as 8-epi PGF2α included its cochromatography over three highly resolving high performance liquid chromatography systems, identification by electron impact mass spectrometry, and its formation by partially purified COX. Inhibition of platelet thromboxane formation was associated with augmented 8-epi PGF2α formation.A major component of 8-epi PGF2α formed in serum by healthy volunteers was shown to be sensitive to inhibition by aspirin ex vivo.
In addition to its generation by free radical catalyzed mechanisms, 8-epi PGF2α may be formed as a PG by human platelets. Given that activation of platelet COX characterizes many of the human syndromes which are putatively associated with free radical generation, assessment of the contribution of this pathway is relevant to the use of 8-epi PGF2α as an index of lipid peroxidation in vivo.
