Functional analysis of the C-terminal flanking sequence of platelet glycoprotein Ibα using canine-human chimeras

Yang Shen, Jing Fei Dong, Gabriel M. Romo, Wendy Arceneaux, Andrea Aprico, Elizabeth E. Gardiner, José A. López, Michael C. Berndt, Robert K. Andrews

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25 Citations (Scopus)

Abstract

Platelet glycoprotein Ib-IX-V (GPIb-IX-V) mediates adhesion to von Willebrand factor (vWF) in (patho)physiological thrombus formation. vWF binds the N-terminal 282 residues of GPIbα, consisting of an N-terminal flank (His1-IIe35), 7 leucine-rich repeats (Leu36-Ala200), a C-terminal flank (Phe201-Gly268), and a sulfated tyrosine sequence (Asp269-Glu282). By expressing canine-human chimeras of GPIbα on Chinese hamster ovary cells, binding sites for functional anti-GPIbα antibodies to individual domains were previously mapped, and it was shown that leucine-rich repeats 2 to 4 were required for optimal vWF recognition under static or flow conditions. Using novel canine-human chimeras dissecting the C-terminal flank, it is now demonstrated that (1) Phe201-Glu225 contains the epitope for AP1, an anti-GPIbα monoclonal antibody that inhibits both ristocetin- and botrocetin-dependent vWF binding; (2) VM16d, an antibody that preferentially inhibits botrocetin-dependent vWF binding, recognizes the sequence Va1226-Gly268, surrounding Cys248, which forms a disulfide-bond with Cys209; (3) vWF binding to chimeric GPIbα is comparable to wild-type in 2 chimeras in which the sixth leucine-rich repeat was of the same species as the first disulfide loop (Phe201-Cys248) of the C-terminal flank, suggesting an interaction between these domains may be important for optimal vWF binding; and (4) replacing the C-terminal flank second disulfide loop (Asp249-Gly268) in human GPIbα with the corresponding canine sequence enhanced vWF binding under static and flow conditions, providing the first evidence for a gain-of-function phenotype associated with the second loop of the C-terminal flank.

Original languageEnglish
Pages (from-to)145-150
Number of pages6
JournalBlood
Volume99
Issue number1
DOIs
Publication statusPublished - 1 Jan 2002
Externally publishedYes

Cite this

Shen, Y., Dong, J. F., Romo, G. M., Arceneaux, W., Aprico, A., Gardiner, E. E., López, J. A., Berndt, M. C., & Andrews, R. K. (2002). Functional analysis of the C-terminal flanking sequence of platelet glycoprotein Ibα using canine-human chimeras. Blood, 99(1), 145-150. https://doi.org/10.1182/blood.V99.1.145