Sixma J J, Schiphorst M E, Verweij C L, Pannekoek H
Department of Haematology, University Hospital Utrecht, The Netherlands.
Eur J Biochem. 1991 Mar 14;196(2):369-75. doi: 10.1111/j.1432-1033.1991.tb15826.x.
In order to study the functional importance of the collagen, heparin and glycoprotein-Ib-binding domain, we deleted the A1 domain of von Willebrand factor (vWF), corresponding to residues 478-716, by oligonucleotide-directed mutagenesis. The resulting delta A1-vWF cDNA was expressed in COS-1 monkey kidney cells and compared to wild-type vWF. The higher-molecular-mass multimers were decreased in delta A1 recombinant von Willebrand factor (delta A1-rvWF) compared to plasma vWF and rvWF. The reactivity of delta A1-rvWF and rvWF with monoclonal antibodies directed against the collagen-binding domain (residues 969-992), the vessel-wall-binding domain, and the binding site for glycoprotein IIb-IIIa on platelets was identical. The interaction with vWF of the monoclonal antibody directed against the glycoprotein Ib binding domain was abolished for delta A1-rvWF, and similar to plasma vWF for rvWF. The binding of factor VIII to delta A1-rvWF and rvWF was similar. delta A1-rvWF and rvWF bound similarly to collagen, but the binding of delta A1-rvWF to heparin and to platelets in the presence of ristocetin were abolished. These data indicate that the heparin-binding site in the A1 domain is essential. There is no second binding domain for glycoprotein Ib outside the A1 domain. The collagen-binding domain in the A1 domain is either not active or its action can be compensated by the second collagen-binding domain.
为了研究胶原蛋白、肝素和糖蛋白-Ib结合结构域的功能重要性，我们通过寡核苷酸定向诱变缺失了血管性血友病因子（vWF）的A1结构域，该结构域对应于第478 - 716位氨基酸残基。将所得的ΔA1 - vWF cDNA在COS - 1猴肾细胞中表达，并与野生型vWF进行比较。与血浆vWF和重组vWF（rvWF）相比，ΔA1重组血管性血友病因子（ΔA1 - rvWF）中高分子量多聚体减少。ΔA1 - rvWF和rvWF与针对胶原蛋白结合结构域（第969 - 992位氨基酸残基）、血管壁结合结构域以及血小板上糖蛋白IIb - IIIa结合位点的单克隆抗体的反应性相同。针对糖蛋白Ib结合结构域的单克隆抗体与ΔA1 - rvWF的vWF相互作用被消除，而与rvWF的相互作用与血浆vWF相似。因子VIII与ΔA1 - rvWF和rvWF的结合相似。ΔA1 - rvWF和rvWF与胶原蛋白的结合相似，但在存在瑞斯托霉素的情况下，ΔA1 - rvWF与肝素和血小板的结合被消除。这些数据表明A1结构域中的肝素结合位点至关重要。在A1结构域之外不存在糖蛋白Ib的第二个结合结构域。A1结构域中的胶原蛋白结合结构域要么不活跃，要么其作用可被第二个胶原蛋白结合结构域补偿。
