Journal
BIOCHIMIE
Volume 88, Issue 1, Pages 69-76Publisher
ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER
DOI: 10.1016/j.biochi.2005.06.015
Keywords
snake venom phospholipase A(2); factor Xa; anticoagulant site; prothrombinase inhibition; surface plasmon resonance; site-directed mutagenesis
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Ammodytoxin A (AtxA) from the venom of Vipera ammodytes ammodytes belongs to group IIA secreted phospholipase A(2) (sPLA(2)), for which the major pathologic activity is presynaptic neurotoxicity. We show here that this toxin also affects hemostasis because it exhibits strong anticoagulant activity. AtxA binds directly to human coagulation factor Xa (FXa) with K-d(app) of 32 nM, thus inhibiting the activity of the prothrombinase complex with an IC50 of 20 nM. To map the FXa-interaction site on AtxA, various mutants of AtxA produced by site-directed mutagenesis and expressed in Escherichia coli were tested in the study. In surface plasmon resonance (SPR) measurements, with FXa covalently attached to the sensor chip, we show that the FXa-binding site on AtxA includes several basic amino acid residues at the C-terminal and beta-wing regions of the molecule. Applying an in vitro biological test for inhibition of prothrombinase activity, we further demonstrate that the same residues are also very important for the anticoagulant activity of AtxA. We conclude that the anticoagulant site of AtxA is located in the C-terminal and beta-wing regions of this phospholipase A(2). Synthetic peptides comprising residues of the deduced anticoagulant site of AtxA provide a basis to synthesize novel anticoagulant drugs. (C) 2005 Elsevier SAS. All rights reserved.
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