Purification and characterization of kaouthiagin, a von Willebrand factor-binding and -cleaving metalloproteinase from Naja kaouthia cobra venom

Jiharu Hamako, Taei Matsui, Sachiyo Nishida, Shosaku Nomura, Yoshihiro Fujimura, Masayuki Ito, Yasuhiro Ozeki, Koiti Titani

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Abstract

A von Willebrand factor (vWF)-binding and -cleaving metalloproteinase, termed 'kaouthiagin', was purified from the venom of cobra snake Naja kaouthia. Kaouthiagin is a monomer with a molecular mass of about 46 kDa and 51 kDa under non-reducing and reducing conditions, respectively, and the N-terminal amino acid sequence is homologous to high molecular mass snake venom metalloproteinases. Kaouthiagin bound to vWF in a divalent ion-independent manner, but the reduced kaouthiagin failed to interact with vWF, suggesting that the protein conformation maintained by intrachaindisulfide linkages of the molecule is essential for the binding to vWF. Neither botrocetin nor bitiscetin, vWF-binding modulators from another snake venom, interfered with the binding between kaouthiagin and vWF, but a monoclonal antibody VW92-3 specific to the N-terminal region of vWF (residues 1-910) inhibited the binding. Without affecting platelet GPIb/IX and GPIIb/IIIa, kaouthiagin specifically cleaved vWF between residues Pro-708 and Asp-709 in a divalent ion-dependent manner to diminish the multimeric structure of vWF in plasma, resulting in the loss of ristocetin-induced platelet aggregability and the collagen-binding activity of vWF. These results indicate that kaouthiagin is a unique metalloproteinase which specifically binds to and cleaves vWF at a specific site and that it will be a useful tool for functional dissection of vWF.

Original languageEnglish
Pages (from-to)499-505
Number of pages7
JournalThrombosis and Haemostasis
Volume80
Issue number3
Publication statusPublished - 01-09-1998

All Science Journal Classification (ASJC) codes

  • Hematology

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