Convulxin binds to native, human glycoprotein Ibα

Convulxin (CVX), a C-type snake protein from Crotalus durissus terrificus venom, is the quintessential agonist for studies of the collagen receptor, glycoprotein VI ( GPVI) and its role in platelet adhesion to collagens. In this study, CVX, purified from venom, behaves as expected, i.e. it binds to platelet GPVI and recombinant human GPVI, induces platelet aggregation and platelet prothrombinase activity, and binds uniquely to GPVI in ligand blots of SDS-denatured proteins. Nonetheless, we find that CVX has a dual specificity for both GPVI and native but not denatured human GPIbalpha. First, CVX binds to human GPIbalpha expressed on the surface of CHO cells. Second, CVX binds weakly to murine platelet GPIbalpha but more strongly to human platelet GPIbalpha, as evidenced by comparative binding to wild-type, GPVI(-/-), FcRgamma (-/-), and human GPIb transgenic mice. Third, the binding of CVX to human GPIbalpha is inhibited by soluble, recombinant human GPVI. Fourth, CVX binding to GPIbalpha is disrupted by phenylalanine substitutions at GPIbalpha tyrosine-276, tyrosine-278, and tyrosine-279, which also disrupts von Willebrand factor and alpha-thrombin binding to GPIbalpha. Fifth, CVX binding to GPIbalpha on Chinese hamster ovary cell transfectants is inhibited by function-blocking murine monoclonal anti-GPIbalpha antibodies. Lastly, CVX fails to bind to denatured GPIbalpha in detergent extracts of platelets. Three separate preparations of CVX (two purified by the authors; one obtained commercially) produced equivalent results. These results indicate that CVX exhibits dual specificity for both native GPIbalpha and GPVI. Furthermore, the binding site on GPIbalpha for CVX may be close to that for von Willebrand factor. Therefore, a contribution of GPIbalpha to CVX-induced platelet responses needs to be carefully re-evaluated.