Properties of Soluble His-Tagged rGPIbα_1–483 Wild-Type and Increase-of-Function Mutants.

The extracellular domain of human platelet GPIbα contains the binding site for von Willebrand factor (vWF) within the amino-terminal 45 kDa region comprising approximately 300 amino acids. In the present studies, we have used CHO cells to synthesize a longer fragment of His₁-Phe₄₈₃, representing nearly the complete extracellular domain of GPIbα, with an additional 8 histidine residues added at the carboxyl end to provide recognition by his-tag reagents. Both wild-type and the increase-of-function mutants Gly₂₃₃→Val₂₃₃ and/or Met₂₃₉→Val₂₃₉ were employed. For studies of antibody binding, modulator-induced vWF binding, or antibody inhibition of this vWF binding, capture of the recombinant GPIbα could be accomplished by nickel chelate binding of the his-tag. Both botrocetin and ristocetin induced saturable binding of vWF by ELISA, as demonstrated by HRP-conjugated anti-vWF polyclonal antibody. In the case of the different increase-of-function mutants, significant vWF binding was seen even in the absence of added modulator. Inhibition of botrocetin-induced vWF binding was greater for wild-type than for the Met₂₃₉→Val₂₃₉ GPIbα mutant with some mabs (e.g., AS-2), whereas the degree of inhibition was essentially equivalent with other mabs (e.g., C-34). When a rabbit polyclonal anti-his antibody was used to immobilize the recombinant proteins instead of the nickel chelate system, binding of vWF and of conformation-dependent mabs was impaired. However, when this same antibody was biotinylated and then captured on a streptavidin plate, excellent capture and subsequent binding characteristics of the recombinant GPIbα were restored. With both this latter system and with the nickel chelate system, a highly sensitive functional vWF activity assay could be established, using botrocetin-induced binding of vWF from human plasma. Linearity of binding was observed between 0.1–0.6% normal plasma, with saturation occurring around 2%. Whereas higher concentrations of plasma actually appeared to displace bound GPIbα to the nickel chelate, the streptavidin-biotin-anti-his attachment method appeared more resistant to such effects. The ELISA-based assay systems developed in this study accordingly provide an and effective approach to study functional interactions of both wild-type and mutant forms of GPIbα with its ligand vWF and also with antibodies directed against different epitopes within the entire extracellular domain of this receptor molecule. The high sensitivity, reproducibility, and rapidity of the assay system also offer the possibility of a useful test for functional assay of plasma vWF.