Metabolism of prostacyclin by 9-hydroxyprostaglandin dehydrogenase in human platelets. Formation of a potent inhibitor of platelet aggregation and enzyme purification.
We have reported the identification of a metabolite of prostacyclin (PGI2) in the liver, 6-keto-PGE1, a substance having similar potency to PGI2 in its vascular and antiaggregative actions but differing in its greater stability. Either PGI2 or its inactive hydrolysis product, 6-keto-PGF1 alpha, can be enzymically transformed via the 9-hydroxyprostaglandin dehydrogenase pathway to 6-keto-PGE1. In this study, we demonstrated 9-OH prostaglandin dehydrogenase activity in the cytoplasmic fraction of human platelets by measuring the release of 3H from positin 9 using [9-3H]PGI2 and [9-3H]6-keto-PGF1 alpha as substrates. The enzyme was further purified by DEAE-cellulose, followed by Sephadex G-200, and finally by isoelectric focusing. The enzyme was found to have a pH optimum of 8.5 to 9.0 and an isoelectric point of 5.0. The molecular weight was estimated to be 60,000 by sodium dodecyl sulfate-gel electrophoresis. Enzymic activity was time- and concentration-dependent and required NAD+ as a cofactor. The activity of the purified enzyme was further confirmed by using a more stable form of PGI2, the methyl ester. Incubation of [11-3H]PGI2 methyl ester with the purified enzyme resulted in formation of [11-3H]6-keto-PGE1 methyl ester, which also inhibited platelet aggregation. Thus, 9-hydroxyprostaglandin dehydrogenase in platelets could be a major enzymic pathway for the transformation of PGI2, and perhaps 6-keto-PGF1 alpha, to 6-keto-PGE1. The possibility that the effects of prostacyclin on platelet aggregation are related to conversion to the biologically active metabolite, 6-keto-PGE1, should be considered.
D000527:Alprostadil / Q000031:analogs & derivatives*; D001792:Blood Platelets / Q000201:enzymology*; D011464:Epoprostenol / Q000097:blood*; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000097:blood* / Q000302:isolation & purification; D007700:Kinetics; D008970:Molecular Weight; D010974:Platelet Aggregation / Q000187:drug effects*; D011453:Prostaglandins / Q000097:blood*; D011458:Prostaglandins E / Q000494:pharmacology
D011453:Prostaglandins; D011458:Prostaglandins E; C022950:6-ketoprostaglandin E1; D011464:Epoprostenol; D006911:Hydroxyprostaglandin Dehydrogenases; D000527:Alprostadil