Anticoagulant effect of Naja naja venom 5′nucleotidase: Demonstration through the use of novel specific inhibitor, vanillic acid

The snake venom proteins affect hemostasis by either advancing/delaying blood coagulation. Apart from proteases and phospholipase A2s (PLA2s), 5′nucleotidase is known to affect hemostasis by inhibiting platelet aggregation. In this study, the possible involvement of Naja naja venom 5′nucleotidase in mediating anticoagulant affect is evaluated. Vanillic acid selectively and specifically inhibited 5′nucleotidase activity among other enzymes present in N. naja venom. It is a competitive inhibitor as evident of inhibition relieving upon increased substrate concentration. Vanillic acid dose dependently inhibited the anticoagulant effect of N. naja venom up to 40%. This partial involvement of 5′nucleotidase in mediating anticoagulant effect is substantiated by concanavalin-A (Con-A) inhibition studies. Con-A, competitively inhibited in vitro protease and 5′nucleotidase activity up to 100%. However, it did not exhibit inhibitory activity on PLA2. The complete inhibition of anticoagulant effect by Con-A upon recalcification time suggests the participation of both 5′nucleotidase and protease in mediating anticoagulant effect of N. naja venom. Vanillic acid and Con-A inhibition studies together suggest that probably 5′nucleotidase interacts with one or more factors of intrinsic pathway of blood coagulation to bring about anticoagulant effect. Thus, this study for the first time demonstrates the involvement of 5′nucleotidase in mediating N. naja venom anticoagulant effect.