Inhibition of platelet aggregation by 5'-nucleotidase purified from Trimeresurus gramineus snake venom.

By means of DEAE-Sephadex A-50 column chromatography and gel filtrations on Sephadex G-75, Sephacryl S-300 and Sephadex G-100, successively, a potent 5'-nucleotidase was purified from Trimeresurus gramineus venom. The venom 5'-nucleotidase is a single polypeptide chain and homogeneous as judged by SDS-polyacrylamide gel electrophoresis. It is a thermostable glycoprotein consisting of 589 amino acid residues. Its molecular weight was estimated to be 74,000 by SDS-polyacrylamide gel electrophoresis. It possessed nucleotidase activities toward adenosine monophosphate and adenosine diphosphate. The specific activities toward AMP and ADP were 504 +/- 28 and 101 +/- 8 micrograms Pi/min per mg, respectively. Pre-incubation of this venom's 5'-nucleotidase with ADP resulted in the cleavage of ADP and formation of adenosine. The 5'-nucleotidase activity was inhibited by EDTA. Both Zn2+ and Co2+/- reversed the inhibitory effect of EDTA. In rabbit platelet-rich plasma, it inhibited completely the ADP (2 x 10(-5) g/ml)-induced platelet aggregation. It also inhibited the platelet aggregations induced by sodium arachidonate (100 microM), collagen (20 micrograms/ml) and ionophore A-23187 (5 microM)-induced platelet aggregations were not affected significantly by this venom 5'-nucleotidase. In ADP-refractory platelet-rich plasma, the venom 5'-nucleotidase inhibited the platelet aggregations induced by collagen (20 micrograms/ml) or sodium arachidonate (100 microM). The venom 5'-nucleotidase showed a more pronounced inhibitory effect on sodium arachidonate-induced platelet aggregation than creatine phosphate/creatine phosphokinase and apyrase did. No lactate dehydrogenase was released by this venom 5'-nucleotidase, indicating that no platelet lysis occurred. It is concluded that removal of ADP, which is released by these platelet aggregation inducers, and the subsequent accumulation of adenosine are responsible for the inhibitory effect of the venom 5'-nucleotidase on platelet aggregations.