L-amino acid oxidase from Naja atra venom activates and binds to human platelets

Authors


  • This work was supported by the grants from the Natural Science Foundation of China (30770431), the Natural Science Foundation of Yunnan Science and Technology Committee, Yunnan Province (2007C102M) and the Western Light Project of Chinese Academy of Sciences

*Corresponding author: Tel/ Fax, 86-871-5192476; E-mail, lvqm@mail.kiz.ac.cn

Abstract

An L-amino acid oxidase (LAAO), NA-LAAO, was purified from the venom of Naja atra. Its N-terminal sequence shows great similarity with LAAOs from other snake venoms. NA-LAAO dose-dependently induced aggregation of washed human platelets. However, it had no activity on platelets in platelet-rich plasma. A low concentration of NA-LAAO greatly promoted the effect of hydrogen peroxide, whereas hydrogen per oxide itself had little activation effect on plate lets. NA-LAAO induced tyrosine phosphorylation of a number of platelet proteins including Src kinase, spleen tyrosine kinase, and phospholipase C γ2. Unlike convulxin, Fc receptor γ chain and T lymphocyte adapter protein are not phosphorylated in NA-LAAO-activated platelets, suggesting an activation mechanism different from the glycoprotein VI pathway. Catalase inhibited the platelet aggregation and platelet protein phosphorylation induced by NA-LAAO. NA-LAAO bound to fixed platelets as well as to platelet lysates of Western blots. Furthermore, affinity chromatography of platelet proteins on an NA-LAAO-Sepharose 4B column isolated a few platelet membrane proteins, suggesting that binding of NA-LAAO to the platelet membrane might play a role in its action on platelets.

Abbreviations:
BCIP

5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt

biotin-NHS

biotinamidocaproate N-hydroxysuccinimide ester

BSA

bovine serum albumin

Fcγ

Fc receptor γ chain

FITC

fluorescein-isothiocyanate

GPVI

glycoprotein VI

LAAO

L-amino acid oxidase

LAT

T lymphocyte adapter protein

NA-LAAO

L-amino acid oxidase from Naja atra venom

NBT

p-nitro blue tetrazolium chloride

PBS

phosphate-buffered saline

PLC

phospholipase C

PVDF

polyvinylidene difluoride

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Src

Src kinase

Syk

spleen tyrosine kinase

TBS

Tris-buffered saline

Ancillary