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′-[αβ-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.