Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 277, Issue 24, Pages 22063-22072Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M201327200
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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 Cys(1272(509))-cys(1458(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 Nand C-terminal flanking regions. In this study, cys(1222(459))-Tyr(1271(508)), Gln(1238(475))-Tyr(1271(508)), Glu(1260(497))-Tyr(1271(508)), and Asp(1459(696))Asp(1472(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 Arg(1308(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 13328, 13710, and 23C7, which selectively inhibit ristocetin-induced GPI binding. Their epitopes were found at His(1268(505)) or Asp(1269(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.
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