Purification, characterization, and cDNA cloning of acidic platelet aggregation inhibiting phospholipases A2 from the snake venom of Vipera lebetina (Levantine viper)

Two novel acidic phospholipase A2s (PLA2) were isolated by size exclusion chromatography and reversed-phase chromatography from the crude Vipera lebetina venom. The molecular masses of VLPLA2-1 (13,704 Da) and VLPLA2-2 (13,683 Da) and their internal tryptic peptides were determined by MALDI-TOF mass-spectrometry. When tested in human platelet-rich plasma, both enzymes showed a potent inhibitory effect on aggregation induced by ADP and collagen. Chemical modification with p-bromophenacylbromide abolished the enzymatic activity of PLA2; its anti-platelet activity was fully inhibited in case of collagen as inducer and partially inhibited in case of ADP as inducer. The complete cDNAs encoding PLA2 were cloned from a single venom gland cDNA library. Complete amino acid sequences of the VLPLA2 were deduced from the cDNA sequences. The full-length cDNA sequences of the VLPLA2 possess 615 bp and encode an open reading frame of 138 amino acids that include signal peptide (16 amino acids) and mature enzyme (122 amino acids). The VLPLA2s have significant sequence similarity to many other phospholipase A2s from snake venoms. The phylogenetic analysis on the basis of the amino acid sequence homology demonstrates that VLPLA2s grouped with other Asp49 PLA2s and they appear to share a close evolutionary relationship with the European vipers.