Molecular characterization of L-amino acid oxidase from Agkistrodon halys blomhoffii with special reference to platelet aggregation

L-Amino acid oxidase (LAO, EC 1.4.3.2) is widely distributed in snake venom , and induces apoptosis in vascular endothelial cells, causing prolonged bl eeding from vessel walls at bite sites. The effect of snake venom LAOs on p latelet function is controversial. Further, we have little information on t heir structural characterization. We purified M (mamushi)LAO, a single-chai n glycoprotein with a molecular mass of 60 kDa and a pI of 4.9, from Agkist rodon halys blomhoffii (Japanese mamushi) venom, and determined the N-termi nal and several internal amino acid sequences of this enzyme. Molecular clo ning based on these data was conducted to elucidate its full-length cDNA st ructure (2192 nucleotides), which includes a putative 18 amino acid residue signal peptide and a 504 residue mature subunit. The predicted M-LAO trans lation product shares 87.35% identity with that of Crotalus adamanteus (Sou theastern diamondback rattlesnake) LAG. M-LAO, up to a final concentration of 2.6 muM, inhibited both agonist- and shear stress-induced platelet aggre gation (SIPA) dose-dependently. In agonist-induced platelet aggregation, M- LAO predominantly inhibited the second aggregation, but with a marginal inh ibition of the first. In SIPA, the inhibition was more dramatic under low-s hear stress than high-shear stress, and was enhanced by the presence of L-l eucine, a substrate of this enzyme. Catalase, a H2O2 scavenger, totally que nched such enhancement. These results suggest that M-LAO inhibits the inter action between activated platelet integrin alpha IIb/beta3 and fibrinogen t hrough the continuous generation of H2O2, and may contribute to prolonged b leeding from the vessels at snake bite sites. (C) 2001 Elsevier Science B.V . All rights reserved.