Characterization of the quinine-dependent antibody binding domain on GPIbα. (A) GPIbα amino acid sequence residues 269 to 297. The specific cleavage sites for mocarhagin (a novel cobra venom metalloproteinase) and trypsin are shown. The MoAbs AK2, SZ2, and AK3 are specific for GPIbα. AK2 binds distal to amino acid 275, SZ2 between amino acids 276 to 282, and AK3 proximal to amino acid 294. The quinine-dependent antibody is interacting with GPIbα between amino acids 283 to 293. (B) After labeling the surface proteins of the CHO αβIX cells with biotin, the GPIbIX complex was immunoprecipitated using the MoAb panel. The presence of the GPIbα and GPIX native proteins was shown on the surface of the CHO αβIX cells. Lane 1 shows the components immunoprecipitated using AK3, lane 2 with AK2, and lane 3 with SZ2. After cleavage with mocarhagin, AK3 precipitated a 70-kD GPIbα component and the intact GPIX protein from the surface of the cells (lane 4). AK2 (lane 5) and SZ2 (lane 6) were able to precipitate a 40-kD GPIbα component from the supernatant collected after the cleavage step. (C) Sequential cleavage of GPIbα and checking the ability of the quinine-dependent antibody to continue binding enabled the definition of the domain to which the Ibα-specific antibody was binding. All MoAbs bound to the surface of the untreated L αβ cells. AK2 and SZ2 binding were inhibited after cleavage with mocarhagin. AK3 binding was still present, although at a lower level after cleavage with trypsin. The quinine-dependent antibody binding remained after cleavage with mocarhagin but was inhibited after cleavage with trypsin, indicating that the GPIbα-specific quinine-dependent antibody binding site is located between amino acids 283 and 293.