Identification of the regulatory elements of human von Willebrand factor for binding to platelet GPlb.

在体外血小板糖蛋白Ib（GPIb）的人血管性血友病因子（VWF）的结合显着增加外源性调节剂，如利托那肽或botrocetin和结合不发生在正常循环。GPIb结合位点已被分配在由二硫环Cys 1272（5091）-Cys 1458（695）组成的VWF Al结构域中。相反，在由二硫环及其N端和C端侧翼区组成的两个区域中发现了几个功能获得性突变。在该研究中，Cysl 222（459）-Tyrl 271（508）、Glnl 238（475）Tyrl 271（508）、Glul 260（497）-Tyrl 271（508）和Aspl 459（696）-Aspl 472（709）依次缺失全长多聚体重组VWF。两侧的缺失导致正常的GPIB结合，这表明侧翼区域不是GPIB结合位点。然而，在Arg 1308（545）的添加突变到每个缺失突变体中导致自发GPIb结合而不需要调节剂，这表明这两个区域对于抑制GPIb结合是重要的。识别GPIb结合位点的单克隆抗体完全抑制自发结合。有趣的是，缺失N-末端但不缺失C-末端侧翼区的突变失去了与选择性抑制瑞斯托菌素诱导的GPIb结合的单克隆抗体B328、B710和23 C7的结合，并且在His 1268（505）或Asp 1269（506）处发现了它们的表位。Al结构域的晶体学结构表明GPIb结合受两个区域之间的分子界面的影响，并且与界面结合的抗体抑制结合。

In vitro platelet glycoprotein Ib (GPIb) binding of human von Willebrand factor (VWF) increases markedly by exogenous modulators such as ristocetin or botrocetin and the binding does not occur in normal circulation. GPIb binding sites have been assigned in VWF Al domain that consists of a disulfide loop Cysl272 (509l)-Cys1458(695). In contrast, several gain-of-function mutations have been found in two regions comprised of the disulfide loop and its N- and Cterminal flanking regions. In this study, Cysl222(459)-Tyr1271(508), Gin1238(475)Tyrl271 (508), Glu l260(497)-Tyrl27 1(508), and Asp 1459(696)-Asp1472(709) were sequentially deleted of full-length multimeric recombinant VWF. Deletions at either side resulted in normal GPIb binding, indicating that the flanking regions are not GPIb binding sites. However, an additive mutation at Arg1308(545) onto each deletion mutant resulted in the spontaneous GPIb binding without requirements of modulators, suggesting both regions are important for inhibition of GPIb binding. The spontaneous binding was completely inhibited by monoclonal antibodies that recognize the GPIb binding sites. Interestingly, mutations deleted with N-terminal, but not Cterminal flanking regions lost the binding to monoclonal antibodies B328, B710, and 23C7 that selectively inhibit ristocetin-induced GPIb binding and their epitopes were found at His 1268(505) or Asp1269(506). The crystalographic structure of the Al domain suggest that GPIb binding is influenced by the molecular interface between two regions, and the antibody binding to the interface inhibit the binding.