The physiological role of the interaction of VWF-GPIb : Amino acid residues of the platelet GPIb to bind VWF and the generation of knock-in mice mutated at Lys599 to A1a.

在血管损伤部位，血管性假血友病因子(VWF)通过与血小板膜糖蛋白Ibα链(GPIba)的N-端结构域结合，介导血小板与内皮下结缔组织的黏附。我们在含有GPIBαN端287个氨基酸的可溶性片段中发现了产生的功能缺失突变。Glu128、Glu172和Asp175上的突变特异性地减少了瑞斯托菌素和肉豆素诱导的VWF结合，这表明这些位点对于VWF与血小板GPIb的结合是重要的。单抗6d1抑制了瑞斯托菌素和肉毒菌素诱导的VWF结合，Glu125上的突变特异性地将这种结合减少到6d1。相反，抗体HPL7对VWF的结合没有影响，突变体E121A降低了HPL7的结合。His12和Glu14的突变减少了瑞斯托菌素诱导的VWF结合，而正常的肉毒杆菌素诱导的结合。对VWF-GPIBA络合物的晶体模拟表明，Glu128和Asp175形成VWF结合位点和…此外，6D1与Glu125的结合中断了Glu128的VWF结合，而HPL7与Glu121的结合不影响VWF的结合。此外，His12和Glu14与VWF A1结构域的Glu613和Arg571相接触，这两个突变具有相似的表型。利用靶向基因阻断和Cre-loxP基因切除技术，我们研究了VWF-GPIB在正常止血中的作用。采用RT-PCR方法从C57BL/6J小鼠的mRNA中扩增出小鼠血管性血友病因子的部分基因。以扩增产物为探针，从129SVJ lambda fix II基因组文库中分离出含有小鼠vwf基因片段的基因组克隆。构建了用于胚胎干细胞同源重组的靶向载体。它以pBS/MC1DTpA+loxpGKneW载体和vwf基因片段为基础。制备靶向D3胚胎干细胞(ES细胞)并注射囊胚来实施转基因策略。研究结果表明，VWF与人GPIBα结合所需的新的结合位点

At the site of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion to subendothelial connective tissue through binding to N-terminal domain of the a chain of platelet glycoprotein Ib (GPIba). We have found the loss-of-function mutations generated in soluble fragment containing the N-terminal 287 amino acids of GPIbα. Mutations at Glu128, Glu172 and Asp175 specifically decreased both ristocetin-and botrocetin-induced VWF binding, suggesting that these sites are important for VWF binding of platelet GPIb. Monoclonal antibody 6D1 inhibited ristocetin-and botrocetin-induced VWF binding and a mutation at Glu125 specifically reduced the binding to 6D1. In contrast, antibody HPL7 had no effect for VWF binding and mutant E121A reduced the HPL7 binding. Mutations at His12 and Glu14 decreased the ristocetin-induced VWF binding with normal botrocetin-induced binding. Crystallographic modeling of the VWF-GPIba complex indicated that Glu128 and Asp175 form VWF binding sites and … More the binding of 6D1 to Glu125 interrupts the VWF binding of Glu128 but HPL7 binding to Glu121 has no effect on VWF binding. Moreover, His12 and Glu14 contact with Glu613 and Arg571 of VWF A1 domain whose mutations had shown similar phenotype. Using targeted gene disruption and Cre-loxP gene excision technique, we investigated the role of VWF-GPIb in normal hemostasis. Oligonucleotide primer were used to obtain a partial cDNA of mouse VWF in from mRNA of C57BL/6J mice using RT-PCR. The resulting PCR product was used as a probe to isolate a genomic clone containing a segment of mouse VWF gene from a 129SVJ lambda FIX II genomic library. A targeting vector was constructed for homologous recombination in embryonic stem cells. It was based on a vector pBS/MC1DTpA+loxpGKneoW and the VWF gene fragments. The generation of targeted D3 embryonic stem (ES) cells and blastocyst injection were prepared to perform the knock-in strategies. Obtained findings indicates the novel binding sites required for VWF binding of human GPIbα Less