The C-terminal and beta-wing regions of ammodytoxin A, a neurotoxic phospholipase A2 from Vipera ammodytes ammodytes, are critical for binding to factor Xa and for anticoagulant effect.
Ammodytoxin A (AtxA) from the venom of Vipera ammodytes ammodytes belongs to group IIA secreted phospholipase A2 (sPLA2), 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 Kdapp 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 A2. Synthetic peptides comprising residues of the deduced anticoagulant site of AtxA provide a basis to synthesize novel anticoagulant drugs.
Prijatelj
Petra
P
Unité des Venins, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris cedex 15, France.
Charnay
Marlène
M
Ivanovski
Gabriela
G
Jenko
Zala
Z
Pungercar
Joze
J
Krizaj
Igor
I
Faure
Grazyna
G
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry* / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance; D014757:Viper Venoms / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*
D000925:Anticoagulants; D014757:Viper Venoms; C068117:ammodytoxin A; D010741:Phospholipases A; D054467:Phospholipases A2; D015951:Factor Xa