ADP is a potent inhibitor of human platelet plasma membrane adenylate cyclase

Abstract

The potent inducer of platelet aggregation, ADP, inhibits both basal and stimulated cyclic AMP production in intact platelets^1,2. However, several attempts have failed to detect an effect of the nucleotide on adenylate cyclase activity in broken cell preparations^2,3. This paradox has led to suggestions that ADP may act on unidentified sites before the adenylate cyclase step, such as a nucleoside diphosphate kinase⁴ and a Ca²⁺ transport system^5,6. It is difficult to demonstrate the effect of physiologically effective concentrations (0.1–5 µM) of ADP on a crude membrane preparation of adenylate cyclase because of the presence of nucleoside phosphohydrolase activities which may degrade either the substrate ATP or the putative effector ADP to products (such as ADP, AMP and adenosine) displaying diverse effects. A substrate regenerating system which converts ADP to ATP obviously provides no solution. The contamination by adenine nucleotides and catecholamines in widely used crude lysate preparations aggravates the problem^7–9. Using a purified human platelet plasma membrane preparation, and the non-hydrolysable ATP analogue APP(NH)P as substrate, we have now demonstrated direct inhibition of adenylate cyclase by ADP in the physiologically effective concentration range. The inhibitory effect of ADP is immediate in onset and is equal to that produced by adrenaline; it is shared by ADP-βS (and 2′-deoxy ADP to some extent) but not by AP(CH₂)P or other adenine nucleotides. The K_i, for ADP is ∼ 0.3 µM and maximal inhibition is observed at 2 µM