Binding Site on Human von Willebrand Factor of Bitiscetin, a Snake Venom-Derived Platelet Aggregation Inducer
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- 作者：Taei Matsui ; Jiharu Hamako ; Tadashi Matsushita ; Takayuki Nakayama ; Yoshihiro Fujimura ; and Koiti Titani
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：June 25, 2002
- 年：2002
- 卷：41
- 期：25
- 页码：7939 - 7946
- 全文大小：235K
- 年卷期：v.41,no.25(June 25, 2002)
- ISSN：1520-4995
文摘
Bitiscetin, a C-type lectin-like heterodimeric snake venom protein purified from Bitis arietans,binds to human von Willebrand factor (VWF) and induces the platelet membrane glycoprotein (GP) Ib-dependent platelet agglutination in vitro similar to botrocetin. In contrast with botrocetin which binds tothe A1 domain of VWF, the A3 domain, a major collagen-binding site of VWF, was proposed to be abitiscetin-binding site. In the competitive binding assay, neither bitiscetin nor botrocetin had an inhibitoryeffect on the VWF binding to the immobilized type III collagen on a plastic plate. The anti-VWFmonoclonal antibody NMC-4, which inhibits VWF-induced platelet aggregation by binding to 
4 helixof the A1 domain, also inhibited bitiscetin binding to the VWF. Binding of VWF to the immobilizedbitiscetin was competitively inhibited by a high concentration of botrocetin. A panel of recombinant VWF,in which alanine-scanning mutagenesis was introduced to the charged amino acid residues in the A1domain, showed that the bitiscetin-binding activity was reduced in mutations at Arg632, Lys660, Glu666,and Lys673 of the A1 domain. Those substituted at Arg629, Arg636, and Lys667, which decreased thebotrocetin binding, showed no effect on the bitiscetin binding. These results indicate that bitiscetin bindsto a distinct site in the A1 domain of VWF spanning over 4a, 5 helices and the loop between 5 and
6 but close to the botrocetin- and NMC-4-binding sites. Monoclonal antibodies recognizing the -subunitof bitiscetin specifically inhibited bitiscetin-induced platelet agglutination without affecting the bindingbetween VWF and bitiscetin, suggesting that the -subunit of bitiscetin is located on VWF closer to theGPIb-binding site than the -subunit is. Bitiscetin and botrocetin might modulate VWF by binding to thehomologous region of the A1 domain to induce a conformational change leading to an increased accessibilityto platelet GPIb.
4 helixof the A1 domain, also inhibited bitiscetin binding to the VWF. Binding of VWF to the immobilizedbitiscetin was competitively inhibited by a high concentration of botrocetin. A panel of recombinant VWF,in which alanine-scanning mutagenesis was introduced to the charged amino acid residues in the A1domain, showed that the bitiscetin-binding activity was reduced in mutations at Arg632, Lys660, Glu666,and Lys673 of the A1 domain. Those substituted at Arg629, Arg636, and Lys667, which decreased thebotrocetin binding, showed no effect on the bitiscetin binding. These results indicate that bitiscetin bindsto a distinct site in the A1 domain of VWF spanning over 4a, 5 helices and the loop between 5 and6 but close to the botrocetin- and NMC-4-binding sites. Monoclonal antibodies recognizing the -subunitof bitiscetin specifically inhibited bitiscetin-induced platelet agglutination without affecting the bindingbetween VWF and bitiscetin, suggesting that the -subunit of bitiscetin is located on VWF closer to theGPIb-binding site than the -subunit is. Bitiscetin and botrocetin might modulate VWF by binding to thehomologous region of the A1 domain to induce a conformational change leading to an increased accessibilityto platelet GPIb.