Purification, characterization, and amino acid sequence determination of acanthins, potent inhibitors of platelet aggregation from Acanthophis antarcticus (common death adder) venom

Abstract

Venom of Acanthophis antarcticus, a common death adder, exhibits potent antiplatelet effects. By a combination of gel-filtration, cation-exchange, and reversed-phase chromatographic methods, two inhibitors of platelet aggregation, named acanthin I and II, were purified to homogeneity as assessed by capillary electrophoresis and electrospray mass spectrometry. These isoforms exhibit the most potent antiplatelet activity known thus far, with IC50 values of 7 nM for acanthin I and 4 nM for acanthin II in human whole blood when collagen was used as an agonist, whereas with ADP the IC50 values were 10 and 12 nM, respectively. Acanthin I and II are basic proteins with pIs of 10.2 μ 0.1 and 10.4 μ 0.1 and molecular weights of 12,844.58 μ 0.61 and 12,895.63 μ 0.48, respectively, as determined by electrospray mass spectrometry. They exhibit phospholipase enzyme activity, and acanthin I and II hydrolyzed 51.57 ± 1.30 and 46.85 ± 2.90 μmol of phosphatidylcholine/min/mg, respectively. The complete amino acid sequences of acanthin I and II showed that they have a high homology with each other and with other elapids' phospholipase A2 neurotoxin, especially pseudexin A.