Identification of the regulatory elements of the human von Willebrand factor for binding to platelet GPIb. Importance of structural integrity of the regions flanked by the CYS1272-CYS1458 disulfide bond.
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.
Nakayama
Takayuki
T
First Department of Internal Medicine, Nagoya University School of Medicine, Japan.
Matsushita
Tadashi
T
Dong
Zhengyu
Z
Sadler
J Evan
JE
Jorieux
Sylvie
S
Mazurier
Claudine
C
Meyer
Dominique
D
Kojima
Tetsuhito
T
Saito
Hidehiko
H
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D019921:Biotinylation; D001792:Blood Platelets / Q000378:metabolism; D002460:Cell Line; D003435:Crotalid Venoms / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D018604:Epitope Mapping; D017353:Gene Deletion; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D008958:Models, Molecular; D010957:Plasmids / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012310:Ristocetin / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D003435:Crotalid Venoms; D004220:Disulfides; D007074:Immunoglobulin G; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D012310:Ristocetin; D014443:Tyrosine; C030961:botrocetin; D003545:Cysteine