Epitope mapping of inhibitory antibodies against platelet glycoprotein Ibα reveals interaction between the leucine-rich repeat N-terminal and C-terminal flanking domains of glycoprotein Ibα

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Abstract
The interaction of von Willebrand factor (vWF) with the platelet receptor glycoprotein Ibα (GPIbα) is important for platelet adhesion at high shear stress. Two functionally important antigenic areas within GPIbα 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 GPIbα, 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 GPIbα, 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 GPIbα 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 GPIbα. However, although 24G10 competed with 6B4 for binding to platelets, it bound to an epitope within aa 1-81 of GPIbα. In conclusion, 2 functionally important areas within GPIbα 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.

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