Direct treatment of mouse or human blood with soluble 5′-nucleotidase inhibits platelet aggregation

Objective - Adenosine signaling is known to inhibit platelet aggregation. Extracellular adenosine mainly stems from enzymatic phosphohydrolysis of precursor nucleotides via ecto-5'-nucleotidase. Previous studies suggest that soluble 5'-nucleotidase (5'-NT) derived from Crotalus atrox venom may be clinically beneficial in vascular leakage, myocardial, renal, and intestinal ischemia, or acute lung injury. However, the effects of 5'-NT treatment on platelet aggregation remain unknown. We examined the direct effects of 5'-NT treatment on platelet aggregation in vivo and ex vivo using a whole blood aggregation method. Methods and Results - Platelet aggregation in whole human blood was completely inhibited by 5'-NT. When 5'-[alpha beta-methylene] diphosphate (APCP), a specific 5'-ecto-nucleotidase inhibitor, was added together with 5'-NT, APCP fully restored collagen- or ADP-induced aggregation. Adenosine levels in whole blood were significantly increased after 5'-NT treatment compared to controls and inhibition of platelet aggregation by 5'-NT was completely reversed by pretreatment with the nonspecific adenosine receptor antagonist 8-(p-sulfophenyl) theophylline hydrate (8-SPT), suggesting that 5'-NT inhibits aggregation via increased adenosine signaling. Administration of 5'-NT to mice in vivo abolished ADP- and collagen-induced platelet aggregation and increased adenosine concentrations and tail bleeding time. Conclusions - 5'-NT treatment inhibits platelet aggregation via generation of increased levels of extracellular adenosine and subsequent adenosine receptor signaling.