title: Characterization and structural analysis of a potent anticoagulant phospholipase A2 from Pseudechis australis snake venom.
abstract: Pseudechis australis is one of the most venomous and lethal snakes in Australia. Numerous phospholipase A2 (PLA2) isoforms constitute a major portion of its venom, some of which have previously been shown to exhibit not only enzymatic, but also haemolytic, neurotoxic and anticoagulant activities. Here, we have purified a potent anticoagulant PLA2 (identified as PA11) from P. australis venom to investigate its phospholipase, anticoagulant, haemolytic and cytotoxic activities and shown that addition of 11 nM PA11 resulted in a doubling of the clotting time of recalcified whole blood. We have also demonstrated that PA11 has high PLA2 enzymatic activity (10.9 × 10(4) Units/mg), but low haemolytic activity (0.6% of red blood cells hydrolysed in the presence of 1 nM PA11). PA11 at a concentration lower than 600 nM is not cytotoxic towards human cultured cells. Chemical modification experiments using p-bromophenacyl bromide have provided evidence that the catalytic histidine of PA11 is critical for the anticoagulant activity of this PLA2. PA11 that was subjected to trypsin digestion without previous reduction and alkylation of the disulfide bonds maintained enzymatic and anticoagulant activity, suggesting that proteolysis alone cannot abolish these properties. Consistent with these results, administration of PA11 by gavage in a rabbit stasis thrombosis model increased the clotting time of recalcified citrated whole blood by a factor of four. These data suggest that PA11 has potential to be developed as an anticoagulant in a clinical setting.
authors.affiliation: School of Chemistry and Molecular Bioscience, The University of Queensland, QLD, 4072, Australia; Centre for Kidney Disease-Venomics Research, School of Medicine, The University of Queensland, Level 5, 37 Kent Street, Woolloongabba, QLD, 4102, Australia.
authors.forename: Qianyun Sharon
authors.initials: QS
authors.lastname: Du
authors.affiliation: School of Chemistry and Molecular Bioscience, The University of Queensland, QLD, 4072, Australia.
authors.forename: Manuela
authors.initials: M
authors.lastname: Trabi
authors.affiliation: The University of Queensland, UQ Centre for Clinical Research, Brisbane, QLD, 4029, Australia.
authors.forename: Renée Stirling
authors.initials: RS
authors.lastname: Richards
authors.affiliation: Venom Science Pty Ltd, Tanunda South Australia, 5352, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Mirtschin
authors.affiliation: Venom Science Pty Ltd, Tanunda South Australia, 5352, Australia.
authors.forename: Frank
authors.initials: F
authors.lastname: Madaras
authors.affiliation: School of Chemistry and Molecular Bioscience, The University of Queensland, QLD, 4072, Australia.
authors.forename: Amanda
authors.initials: A
authors.lastname: Nouwens
authors.affiliation: Centre for Kidney Disease-Venomics Research, School of Medicine, The University of Queensland, Level 5, 37 Kent Street, Woolloongabba, QLD, 4102, Australia.
authors.forename: Kong-Nan
authors.initials: KN
authors.lastname: Zhao
authors.affiliation: School of Chemistry and Molecular Bioscience, The University of Queensland, QLD, 4072, Australia.
authors.forename: John
authors.initials: J
authors.lastname: de Jersey
authors.affiliation: The University of Queensland, UQ Centre for Clinical Research, Brisbane, QLD, 4029, Australia.
authors.forename: Martin F
authors.initials: MF
authors.lastname: Lavin
authors.affiliation: School of Chemistry and Molecular Bioscience, The University of Queensland, QLD, 4072, Australia. Electronic address: luke.guddat@uq.edu.au.
authors.forename: Luke W
authors.initials: LW
authors.lastname: Guddat
authors.affiliation: Centre for Kidney Disease-Venomics Research, School of Medicine, The University of Queensland, Level 5, 37 Kent Street, Woolloongabba, QLD, 4102, Australia; The University of Queensland, UQ Centre for Clinical Research, Brisbane, QLD, 4029, Australia. Electronic address: p.masci@uq.edu.au.
authors.forename: Paul P
authors.initials: PP
authors.lastname: Masci
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry / Q000494:pharmacology*; D004546:Elapid Venoms / Q000737:chemistry*; D017815:Elapidae / Q000502:physiology*; D006801:Humans; D008958:Models, Molecular; D015840:Oculomotor Nerve Diseases; D054467:Phospholipases A2 / Q000378:metabolism*; D011487:Protein Conformation; D011517:Prothrombin Time; D011817:Rabbits; D013916:Thrombelastography
chemical_list: D001798:Blood Proteins; D004546:Elapid Venoms; C400481:PLIalpha; D054467:Phospholipases A2
keywords: Anticoagulant; Oligomer; Phospholipase A(2); Protein purification; Snake venom protein