Effects of Inhibitors of ADP-induced Aggregation on Platelet Nucleo-sidediphosphate Kinase (NDK) Activity

Abstract

We showed previously (Am. J. Physiol. 227, 1143, 1974) that ADP reacts with NDK on the surface of platelets; when 14C-ADP is added to suspensions of washed rabbit platelets, it is partially converted to 14C-ATP in the suspending fluid. This is more difficult to show with human platelets because of their greater ATPase activity. With both species, -external ATP (10 μM), and other NTP’s at higher concentrations, enhance formation of “C-ATP form “C-ADP. All the NTP’s inhibit ADP-induced aggregation, possibly by acting as external donors of a phosphoryl group which, in their absence, is normally donated to ADP by platelets. Studies of other inhibitors have been done with suspensions -of washed rabbit platelets. The other NDP’s and AMP inhibit, ADP-induced shape change, aggregation, and conversion of 14C-ADP to 14C-ATP, probably by competition for the same site on NDK. AMP inhibits NDK both in the presence and absence of external ATP. Parachloromercuribenzene sulfonic acid has a similar effect. In contrast, prostaglandin E1 and hydroxylamine inhibit ADP-induced shape change and aggregation, but inhibit NDK only in the absence of external ATP, not in its presence. ADP-induced shape change without aggregation occurs in the presence of EGTA or EDTA but only half as much 14C-ADP is converted to 14C-ATP in the first minute. EGTA does not inhibit NDK in the presence of external ATP but EDTA does. Inhibition of NDK by adenosine was observed in some experiments but the adenosine derivatives 2-n-amylthioadenosino, 2-cyclopentylthioadenosine and 2-cyclophexylthioadenosine (Kohjin Co., Tokyo) inhibited strongly both in the presence and absence of external ATP; these compounds also inhibit ADP-induced aggregation. Thus all compounds tested which inhibit NDK also inhibit ADP-induced aggregation, indicating that NDK may be the ADP receptor on the platelet surface. Donation of a phosphoryl group from the platelets to ADP, catalyzed by this enzyme, may be responsible for initiating ADP-induced platelet aggregation.