Contribution of ecto-5'-nucleotidase to the inhibition of platelet aggregation by human endothelial cells

We studied the role of adenosine (Ado), which is generated from adenine nucleotides via the activity of ecto-5'-nucleotidase (ecto-5'-NT), in the inhibition of platelet aggregation by endothelial cells (ECs). The enzymatic activity of nucleotidases on human umbilical vein endothelial cells (HUVECs) was examined with regard to (1) the inhibition of adenosine diphosphate (ADP)-induced platelet aggregation and (2) the liberation of inorganic phosphate from adenine nucleotides. Adenosine 5'-monophosphate (AMP) preincubated with HUVECs significantly inhibited ADP-induced platelet aggregation. This was completely blocked by the treatment of HUVECs with a specific inhibitor of ecto-5'-NT, 5'-[alphabeta-methylene] diphosphate (APCP), or by the addition of an A(2a) receptor antagonist. Neither nitric oxide nor prostacyclin was involved in this inhibitory activity, suggesting that Ado generated in the incubation medium by the activity of 5'-NT on HUVECs inhibited platelet aggregation. When ADP was incubated on HUVECs, it lost most of its agonistic activity for platelets. Pretreatment of HUVECs with APCP at a concentration that abolished ecto-5'-NT activity partially restored ADP-induced platelet aggregation. Ecto-5'-NT contributes to EC function by inhibiting platelet aggregation in cooperation with ATP diphosphohydrolase, which degrades ADP to AMP.