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

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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.