title: Functional analysis of the C-terminal flanking sequence of platelet glycoprotein Ib alpha using canine-human chimeras.
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 GPIb alpha, consisting of an N-terminal flank (His1-Ile35), 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 GPIb alpha on Chinese hamster ovary cells, binding sites for functional anti-GPIb alpha 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-GPIb alpha 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 Val226-Gly268, surrounding Cys248, which forms a disulfide-bond with Cys209; (3) vWF binding to chimeric GPIb alpha 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 GPIb alpha 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.
authors.affiliation: Hazel and Pip Appel Vascular Biology Laboratory, Baker Medical Research Institute, Melbourne, Australia.
authors.forename: Yang
authors.initials: Y
authors.lastname: Shen
authors.forename: Jing-fei
authors.initials: JF
authors.lastname: Dong Jf
authors.forename: Gabriel M
authors.initials: GM
authors.lastname: Romo
authors.forename: Wendy
authors.initials: W
authors.lastname: Arceneaux
authors.forename: Andrea
authors.initials: A
authors.lastname: Aprico
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Gardiner
authors.forename: José A
authors.initials: JA
authors.lastname: López
authors.forename: Michael C
authors.initials: MC
authors.lastname: Berndt
authors.forename: Robert K
authors.initials: RK
authors.lastname: Andrews
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D016466:CHO Cells / Q000378:metabolism; D002448:Cell Adhesion; D006224:Cricetinae; D004220:Disulfides / Q000737:chemistry; D004285:Dogs; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D015870:Gene Expression; D006801:Humans; D007930:Leucine; D010446:Peptide Fragments / Q000737:chemistry / Q000502:physiology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D013329:Structure-Activity Relationship; D014162:Transfection; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D004220:Disulfides; D000939:Epitopes; D010446:Peptide Fragments; D019038:Platelet Glycoprotein GPIb-IX Complex; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D007930:Leucine