A sensitive assay of human blood platelet cyclic nucleotide phosphodiesterase activity by HPLC using fluorescence derivatization and its application to assessment of cyclic nucleotide phosphodiesterase inhibitors

Abstract

A selective and sensitive HPLC measurement of 3′,5′-cyclic nucleotide phosphodiesterase (PDE) activity in human platelets using (3,4-dimethoxyphenyl)glyoxal (DMPG) as a fluorogenic reagent for guanine and its nucleosides and nucleotides is described. cGMP, a substrate for PDE, and GMP, which was produced by the enzyme reaction, are selectively converted by the reaction with DMPG to the fluorescent derivatives. The derivatives were separated by reversed-phase HPLC. Human platelet PDE activity was measured and the inhibitory effects of several compounds were investigated.