title: Convulxin forms a dimer in solution and can bind eight copies of glycoprotein VI: implications for platelet activation.
abstract: Convulxin (CVX) is a C-type lectin-like protein from the venom of the South American rattlesnake that functions as a potent agonist of the platelet collagen receptor glycoprotein VI (GPVI). Although CVX is widely used as a platelet agonist, the molecular basis for its extremely high potency is not clear. In order to delineate possible mechanisms for CVX-induced GPVI activation, we used analytical ultracentrifugation to determine the assembly state of CVX in solution and surface plasmon resonance in order to understand the affinity, kinetics, and stoichiometry of GPVI binding to CVX. We show here that CVX exists in solution as a dimer of alpha4beta4 rings, yielding eight potential binding sites for GPVI. Binding studies confirm that all eight sites are able to bind GPVI tightly, each with high picomolar or low nanomolar affinity. Reanalysis of previously determined crystal structures of CVX revealed the dimer in both structures. The dimeric nature of CVX and its ability to bind eight GPVI molecules suggest that it might be capable of binding to GPVI expressed on two opposing surfaces. Agglutination assays using GPVI-coated beads confirm that CVX is able to bridge distinct GPVI-coated surfaces and indicate that CVX agglutination of platelets is dependent on GPVI binding. Thus, in addition to clustering up to eight GPVI receptors, CVX may facilitate platelet activation by bridging platelets directly.
authors.affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524, USA.
authors.forename: Katsunori
authors.initials: K
authors.lastname: Horii
authors.forename: Monica T
authors.initials: MT
authors.lastname: Brooks
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Herr
mesh_terms: D000818:Animals; D001665:Binding Sites; D003435:Crotalid Venoms / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D037181:Lectins, C-Type / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D015539:Platelet Activation; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D051381:Rats; D012996:Solutions; D055672:Static Electricity; D020349:Surface Plasmon Resonance; D013499:Surface Properties
chemical_list: D003435:Crotalid Venoms; D037181:Lectins, C-Type; D010980:Platelet Membrane Glycoproteins; D012996:Solutions; C098341:platelet membrane glycoprotein VI; C028890:convulxin