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.
Highlights
Convulxin (CVX)1 is a 72-kDa protein from Crotalus durissus terrificus venom that has been shown to bind strongly to the platelet-specific collagen receptor, glycoprotein VI (GPVI) [1, 2]
This conclusion relies on the assumption that the epitopes on glycoprotein Ib (GPIb) that are occupied by von Willebrand factor (VWF) are identical to or sufficiently proximal to epitopes occupied by CVX
CVX.1 is the same batch used in studies published previously, where it was shown to induce platelet aggregation in citrated platelet-rich plasma at concentrations Ն20 pM, stimulate platelet prothrombinase activity, and bind exclusively to GPVI in ligand blot assays of total SDS-denatured platelet proteins [9]
Summary
Purification and Labeling of CVX—CVX was purified from Crotalus durissus terrificus venom (Miami Serpentarium Laboratories, Punta Gorda, FL) as described previously [9]. A stable CHO cell line was established that expresses human GPIb and human GPIX, cloned into the Tet-OFF expression vector pTRE, and the selection plasmid, pTK/Hyg (CHO:(t/IX)). The cell line CHO:(t/IX) was transfected with the plasmid pCDNA3.1Zeo in which were cloned the wild-type human GPIb␣ to generate CHO:(t/IX)␣ or the mutant GPIb␣3F to generate CHO:(t/ IX)␣3F. In the case of the CHO:VI(t/IX)␣ cell, we transfected the cDNA of human wild-type GPIb␣ and GPVI/FCR␥ into the CHO:(t/IX) cell This cell line constitutively expresses GPIb␣ and GPVI/FcR␥, whereas the expression of GPIb and GPIX remains Tet-repressible. A 9-kb HindIII restriction fragment spanning mouse GPVI gene exons I–III was cloned into pBS/KSϪ Within this subcloned fragment a premature stop codon was created immediately 3Ј to the initiation codon by double-stranded, site-directed mutagenesis using Platinum Pfx DNA polymerase (Stratagene). Twenty g of protein were separated by SDSPAGE and transferred electrophoretically onto a Sequi-Blot polyvinyl-
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