Immunological identification of the major platelet low-Km cAMP phosphodiesterase: probable target for anti-thrombotic agents.

Abstract

Immunoblot and enzyme-activity analyses, using specific immunological probes, indicated that more than 80% of the total low-Km cAMP phosphodiesterase activity present in bovine and human platelets resided in a single phosphodiesterase isozyme. In the presence of protease inhibitors, the platelet enzyme has an apparent subunit size of 110 kDa and appears immunologically and structurally indistinquishable from a recently purified bovine heart isozyme. When protease inhibitors were absent during homogenization and centrifugation, this platelet phosphodiesterase was susceptible to sequential proteolysis forming 80-kDa and 60-kDa peptides. As a previous report on the purification of the platelet low-Km cAMP phosphodiesterase described a 61-kDa protein, our data would suggest that this was a proteolytic fragment. Moreover, in our study a 40-70% increase in catalytic activity was associated with proteolysis. Further similarities between the platelet and heart phosphodiesterases were demonstrated by pharmacological studies that showed identical inhibitor profiles for both enzymes. Several known phosphodiesterase inhibitor compounds that have been found useful in inhibiting platelet aggregation also inhibited the platelet low-Km cAMP phosphodiesterase with potencies very similar to their antithrombotic effects. Cilostamide, Ro 15-2041, milrinone, papaverine, isobutylmethylxanthine, and theophylline inhibited the 110-kDa platelet enzyme with IC50 values of 0.04, 0.13, 0.46, 1.4, 2.6. and 110 .mu.M, respectively.