Epitope mapping of inhibitory antibodies against platelet glycoprotein Ibalpha reveals interaction between the leucine-rich repeat N-terminal and C-terminal flanking domains of glycoprotein Ibalpha.
The interaction of von Willebrand factor (vWF) with the platelet receptor glycoprotein Ibalpha (GPIbalpha) is important for platelet adhesion at high shear stress. Two functionally important antigenic areas within GPIbalpha were identified through the characterization of 5 new inhibitory anti-GPIb monoclonal antibodies (mAbs). The binding sites of 3 of these anti-GPIb mAbs, which were intercompeting and potently inhibiting shear stress-induced binding of vWF, were mapped within the N-terminal amino acid (aa) 1-59 area by the use of canine-human chimeras. These antibodies, however, had little or no effect (approximately 40% inhibition) on the binding of vWF induced by either botrocetin or ristocetin. On the other hand, the anti-GPIb mAbs 24G10 and 6B4, which blocked GPIb-vWF binding under all conditions examined, bound to 2 different regions of GPIbalpha, aa 1-81 and aa 201-268, respectively. The epitope for 6B4 was further narrowed by phage display revealing 2 sets of peptide sequences aligning within aa 259-262 and aa 230-242. In the latter region of GPIbalpha, the gain-of-function platelet-type von Willebrand disease (PT-vWD) mutations have been identified. Alignment was partially confirmed because the binding of 6B4 to recombinant GPIbalpha fragments carrying either one of the PT-vWD mutations was considerably impaired but not completely abolished. In contrast, mAb 24G10 bound more strongly to mutant PT-vWD GPIbalpha. However, although 24G10 competed with 6B4 for binding to platelets, it bound to an epitope within aa 1-81 of GPIbalpha. In conclusion, 2 functionally important areas within GPIbalpha were identified: one localized within the leucine-rich repeat N-terminal aa 1-59 area and one composed of residues aa 1-81 in close contact with aa 201-268. Moreover, further support is provided for the existence of an intramolecular interaction between the N-terminal flanking (aa 1-81) and C-terminal flanking (aa 201-268) regions. (Blood. 2001;98:652-660)
Cauwenberghs
N
N
Laboratory for Thrombosis Research, IRC, K U Leuven Campus Kortrijk, E Sabbelaan 53, B-8500 Kortrijk, Belgium.
Vanhoorelbeke
K
K
Vauterin
S
S
Westra
D F
DF
Romo
G
G
Huizinga
E G
EG
Lopez
J A
JA
Berndt
M C
MC
Harsfalvi
J
J
Deckmyn
H
H
D000818:Animals; D019138:Antibodies, Blocking / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D003435:Crotalid Venoms / Q000494:pharmacology; D004285:Dogs; D018604:Epitope Mapping / Q000379:methods*; D006801:Humans; D019151:Peptide Library; D010973:Platelet Adhesiveness / Q000187:drug effects / Q000276:immunology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D012310:Ristocetin / Q000494:pharmacology; D013314:Stress, Mechanical; D014841:von Willebrand Factor / Q000378:metabolism
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D003435:Crotalid Venoms; D019151:Peptide Library; D019038:Platelet Glycoprotein GPIb-IX Complex; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D012310:Ristocetin; C030961:botrocetin