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

The interaction of von Willebrand factor (vWF) with the platelet receptor g lycoprotein Ib alpha (GPIb alpha) is important for platelet adhesion at hig h shear stress. Two functionally important antigenic areas within GPIb alph a were identified through the characterization of 5 new inhibitory anti-GPI b monoclonal antibodies (mAbs). The binding sites of 3 of these anti-GPIb m Abs, which were intercompeting and potently inhibiting shear stress-induced binding of vWF, were mapped within the N-terminal amino acid (aa) 1-59 are a by the use of canine-human chimeras. These antibodies, however, had littl e 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 GPIb alpha, aa 1-81 and aa 201-268, respe ctively. The epitope for 6B4 was further narrowed by phage display revealin g 2 sets of peptide sequences aligning within aa 259-262 and aa 230-242. In the latter region of GPIb alpha, the gain-of-function platelet-type von Wi llebrand disease (PT-vWD) mutations have been identified. Alignment was par tially confirmed because the binding of 6B4 to recombinant GPIb alpha fragm ents 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 GPIb alpha. However, although 24G10 competed with 6B4 for bi nding to platelets, it bound to an epitope within aa 1-81 of GPIb alpha. In conclusion, 2 functionally important areas within GPlb alpha were identifi ed: one localized within the leucine-rich repeat N-terminal aa 1-59 area an d one composed of residues aa 1-81 In close contact with aa 201-268. Moreov er, further support is provided for the existence of an intramolecular inte raction between the N-terminal flanking (aa 1-81) and C-terminal flanking ( aa 201-268) regions.