Isolation of an acidic phospholipase A2 from the venom of Agkistrodon acutus (five pace snake) and its effect on platelet aggregation.

A phospholipase A2 from the venom of the snake Agkistrodon acutus was purified by immunoaffinity chromatography and fast protein liquid chromatography (FPLC) as a single band by PAGE and SDS-PAGE. The estimated mol.wt was 16,400 by SDS-PAGE and 16,900 by gel filtration and the isoelectric point was 4.9. The ten N-terminal amino acid residues are homologous to those of the acidic phospholipases A2 from other crotalid venoms. The purified enzyme showed a potent inhibitory effect on platelet aggregation induced by ADP, collagen and sodium arachidonate in human platelet-rich plasma. The platelet aggregation by these inducers was completely suppressed when the concentration of the venom phospholipase A2 was 10-100 micrograms/ml. However, at 20 micrograms/ml, platelet aggregation could be elicited in washed human platelet suspension. Aspirin (28 micrograms/ml), an inhibitor of cyclooxygenase, inhibited the aggregating effect of the phospholipase A2. It is proposed that the stimulatory mechanism of the phospholipase A2 might be due to the liberation of arachidonic acid from phospholipids in the membrane of platelets and the formation of thromboxane A2.