title: Convulxin binds to native, human glycoprotein Ib alpha.
abstract: 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 alpha. First, CVX binds to human GPIb alpha expressed on the surface of CHO cells. Second, CVX binds weakly to murine platelet GPIb alpha but more strongly to human platelet GPIb alpha, as evidenced by comparative binding to wild-type, GPVI(-/-), FcR gamma (-/-), and human GPIb transgenic mice. Third, the binding of CVX to human GPIb alpha is inhibited by soluble, recombinant human GPVI. Fourth, CVX binding to GPIb alpha is disrupted by phenylalanine substitutions at GPIb alpha tyrosine-276, tyrosine-278, and tyrosine-279, which also disrupts von Willebrand factor and alpha-thrombin binding to GPIb alpha. Fifth, CVX binding to GPIb alpha on Chinese hamster ovary cell transfectants is inhibited by function-blocking murine monoclonal anti-GPIb alpha antibodies. Lastly, CVX fails to bind to denatured GPIb alpha 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 alpha and GPVI. Furthermore, the binding site on GPIb alpha for CVX may be close to that for von Willebrand factor. Therefore, a contribution of GPIb alpha to CVX-induced platelet responses needs to be carefully re-evaluated.
authors.affiliation: Roon Research Center for Arteriosclerosis and Thrombosis, Division of Experimental Hemostasis and Thrombosis, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Kanaji
authors.forename: Taisuke
authors.initials: T
authors.lastname: Kanaji
authors.forename: Kenichi
authors.initials: K
authors.lastname: Furihata
authors.forename: Kazunobu
authors.initials: K
authors.lastname: Kato
authors.forename: Jerry L
authors.initials: JL
authors.lastname: Ware
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Kunicki
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003435:Crotalid Venoms / Q000302:isolation & purification / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D037181:Lectins, C-Type / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017452:Receptors, IgG / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D003435:Crotalid Venoms; D018076:DNA, Complementary; D037181:Lectins, C-Type; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D017452:Receptors, IgG; D011994:Recombinant Proteins; C098341:platelet membrane glycoprotein VI; C028890:convulxin