Convulxin Binds to Native, Human Glycoprotein Ib *

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 GPIb . First, CVX binds to human GPIb expressed on the surface of CHO cells. Second, CVX binds weakly to murine platelet GPIb but more strongly to human platelet GPIb , as evidenced by comparative binding to wild-type, GPVI( / ), FcR ( / ), and human GPIb transgenic mice. Third, the binding of CVX to human GPIb is inhibited by soluble, recombinant human GPVI. Fourth, CVX binding to GPIb is disrupted by phenylalanine substitutions at GPIb tyrosine-276, tyrosine-278, and tyrosine-279, which also disrupts von Willebrand factor and -thrombin binding to GPIb . Fifth, CVX binding to GPIb on Chinese hamster ovary cell transfectants is inhibited by function-blocking murine monoclonal anti-GPIb antibodies. Lastly, CVX fails to bind to denatured GPIb 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 GPIb and GPVI. Furthermore, the binding site on GPIb for CVX may be close to that for von Willebrand factor. Therefore, a contribution of GPIb to CVX-induced platelet responses needs to be carefully re-evaluated.