72] Purification of cAMP phosphodiesterase from platelets

Abstract

In human platelets, cAMP plays an important role in the regulation of platelet activation and thus platelet participation in hemostasis and thrombosis. An increase in the intracellular concentration of cAMP is closely associated with inhibition of platelet shape change, aggregation, adhesion, and secretion of granule contents. Three forms of cyclic nucleotide phosphodiesterase activity have been separated by DEAE-Cellulose chromatography from the cytosol of human platelets. One form was reported to be specific for cGMP, another relatively nonspecific, and the third form relatively specific for cAMP. The cytosolic fraction contains at least 75 to 80% of the cAMP hydrolytic activity in human platelets. This chapter describes the purification of the cAMP-specific form, or low-Kin cAMP form, of cyclic nucleotide phosphodiesterase from the cytosolic fraction of human platelets. The purification of this phosphodiesterase consists basically of three steps: preparation of a 150,000 g supernatant fraction from disrupted platelets, DEAE-Cellulose chromatography, and chromatography on blue dextran-Sepharose.