Studies of the Affinity and Stoichiometry of Convulxin Binding to Glycoprotein VI: Clues into Its Potent Agonism.

The primary cell-surface receptor that mediates platelet activation upon exposure to collagen is glycoprotein VI (GPVI). GPVI can be activated by fibrous collagen, soluble collagen mimics, or several snake venom proteins. Upon binding of these agonists, GPVI signaling activates key integrins and triggers secretion of platelet granule contents, leading in turn to activation of neighboring circulating platelets. One of the most potent GPVI agonists from snake venom is convulxin (CVX), an α₄β₄ C-type lectin-like protein from the South American rattlesnake, Crotalus durissus terrificus. Using analytical ultracentrifugation, we have discovered that CVX exists in solution as a stable dimer of α₄β₄ rings, creating eight potential binding sites for GPVI. Using a combination of kinetic and equilibrium surface plasmon resonance binding experiments, we show that CVX binds GPVI with high picomolar to low nanomolar affinity at each site, and that all eight sites are likely to be available for binding. Analysis of the previously published crystal structures of CVX suggests that its potency could be due to both high-affinity binding and clustering of GPVI on a single platelet as well as potential bridging of adjacent platelets via the dimeric CVX architecture.