Abstract

In vitro platelet glycoprotein Ib (GPIb) binding of the 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 the VWF A1 domain, which consists of a disulfide loop Cys1272(509)-Cys1458(695) where amino acid residues are numbered from the starting methionine as +1. The previous numbering from the N-terminal Ser of the mature processed VWF is indicated in parentheses. In contrast, several gain-of-function mutations have been found in two regions comprised of the disulfide loop and its N- and C-terminal flanking regions. In this study, Cys1222(459)-Tyr1271(508), Gln1238(475)-Tyr1271(508), Glu1260(497)-Tyr1271(508), and Asp1459(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, the addition of a mutation at Arg1308(545) on each deletion mutant resulted in spontaneous GPIb binding without requiring modulators, suggesting that both regions are important for the inhibition of GPIb binding. Spontaneous binding was completely inhibited by monoclonal antibodies that recognize the GPIb binding sites. Interestingly, mutant proteins with N-terminal but not C-terminal deletions lost binding to monoclonal antibodies B328, B710, and 23C7, which selectively inhibit ristocetin-induced GPI binding. Their epitopes were found at His1268(505) or Asp1269(506). The crystallographic structure of the A1 domain suggests that GPIb binding is influenced by the molecular interface between the two regions and that the antibody binding to the interface inhibits binding.

Highlights

  • In vitro platelet glycoprotein Ib (GPIb) binding of the human von Willebrand factor (VWF) increases markedly by exogenous modulators such as ristocetin or botrocetin, and the binding does not occur in normal circulation

  • Increased GPIb Binding of the Deletion Mutants—Deletion mutants with R545A substitution, ⌬R497–508 and ⌬R696 – 709, resulted in increased ristocetin-induced GPIb binding and in marked spontaneous GPIb binding (Figs. 4 and 5)

  • The observed spontaneous binding appears to depend on the GPIb binding sites of the A1 domain because it is completely inhibited by NMC4 or AvW3, which blocks the GPIb binding sites (Fig. 7)

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Summary

VWF Regulation Sites for GPIb Binding

L697V, A698V) and in a cluster of mutations between Met1303(540) and Arg1341(578) in the disulfide loop of Cys1272(509)– Cys1458(695) (type 2B region, Human Gene Mutation data base, uwcm.ac.uk/uwcm/mg/hgmd0.html). Alanine-scanning mutagenesis had indicated that several mutations in flanking regions resulted in the gain-of-function phenotype for GPIb binding that is similar to type 2B (9). To further characterize the role of these regions in regulation of GPIb binding, we have produced deletion mutants of flanking regions with or without an additional mutation in the type 2B region. These mutants were tested for ristocetin- and botrocetin-induced GPIb binding and for spontaneous GPIb binding. We have identified the epitopes of several monoclonal antibodies for the VWF A1 domain that inhibits VWF activation

EXPERIMENTAL PROCEDURES
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