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

体外实验中，人血管性血友病因子(VWF)与血小板膜糖蛋白Ib(GPIB)的结合力在瑞斯托西汀或肉豆蔻素等外源性调节剂作用下显著增加，而在正常循环中这种结合是不存在的。GPIB结合位点已被指定在VWF Al结构域中，该结构域由二硫键Cysl272(5091)-Cys1458(695)组成。相反，在由二硫键环及其N-和CT末端侧翼区域组成的两个区域中发现了几个功能增益突变。在本研究中，依次缺失了Cysl222(459)-Tyr1271(508)、Gin1238(475)Tyr1271(508)、Glu L260(497)-Tyr1271(508)和Asp 1459(696)-Asp1472(709)。两侧的缺失导致正常的GPIB结合，表明侧翼区域不是GPIB结合位点。然而，每个缺失突变体上的Arg1308(545)处的相加突变导致了自发的GPIB结合，而不需要调节子，这表明这两个区域对抑制GPIB结合是重要的。这种自发结合可被识别GPIB结合部位的单抗完全抑制。有趣的是，带有N-末端而不是CT端侧翼区域的突变失去了与单抗B328、B710和23C7的结合，这些单抗选择性地抑制了瑞斯托菌素诱导的GPIB结合，它们的表位位于His 1268(505)或Asp1269(506)。Al结构域的晶体结构表明，GPIB结合受到两个区域之间的分子界面的影响，而与界面结合的抗体抑制了结合。

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.