Purification and Characterization of Kaouthiagin, a von Willebrand Factor-Binding and -Cleaving Metalloproteinase from Naja kaouthia Cobra Venom

Abstract

SummaryA 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.

Antihemostatic Activity of Human Granzyme B Mediated by Cleavage of von Willebrand Factor

“…It is also likely that GrB cleavage disrupts interactions of the A1 domain with molecules such as collagen, heparin, and sulfatides (40,44,45). Interestingly, the snake venom metalloproteinase kaouthiagin cleaves VWF at a single site adjacent to the GrB site 1, between 708 P2D 709 and inhibits VWF-mediated platelet aggregation (46). Asp 709 is also the site of a single nucleotide polymorphism that alters residue 709 to His (47).…”

The Cysteine-rich Domain of Snake Venom Metalloproteinases Is a Ligand for von Willebrand Factor A Domains

“…Three PIII SVMPs, jararhagin from B. jararaca venom, atrolysin A from C. atrox venom, and kaouthiagin from Naja kaouthia venom, have been demonstrated to bind and/or proteolytically cleave VWF (9,10,32). In the case of kaouthiagin, the site of cleavage in VWF was identified at Pro 708 -Asp 709 in the C-terminal hinge region of the VWA1 domain (32). This cleavage yields two dimeric VWF fragments of 350 and 220 kDa that are incapable of promoting platelet aggregation (32).…”

“…In the case of kaouthiagin, the site of cleavage in VWF was identified at Pro 708 -Asp 709 in the C-terminal hinge region of the VWA1 domain (32). This cleavage yields two dimeric VWF fragments of 350 and 220 kDa that are incapable of promoting platelet aggregation (32). As for which of the PIII domain(s) is involved in VWF binding, less is known compared with ADAMTS13.…”

Análise dos elementos estruturais de metaloproteinases das classes P-I e P-III do veneno de Bothrops jararaca importantes para suas interações com proteínas plasmáticas e da matriz extracelular.

“…Essas enzimas também são capazes de digerir proteínas da cascata da coagulação sangüínea, tais como, fibrinogênio, fibrina, e fator von Willebrand (KAMIGUTI et al, 1994;HAMAKO et al, 1998;LAING e MOURA-DA-SILVA, 2005;SERRANO et al, 2007).…”

“…Da mesma forma, a crovidisina, também da classe P-III, isolada do veneno de C. viridis, foi capaz de inibir a interação plaqueta-colágeno (LIU e HUANG, 1997). Paralelamente, o fato de outras metaloproteinases P-III, tais como a jararhagina e a kaouthiagina, serem capazes de clivar o vWF e assim causar a ruptura de sua estrutura, pode também contribuir para a geração da hemorragia (KAMIGUTI et al, 1996;HAMAKO et al, 1998;SERRANO et al, 2007).…”