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.

Non-enzymatic proteins from snake venoms: A gold mine of pharmacological tools and drug leads

“…It is a heterodimeric tetramer protein of approximately 84,000 Da size, with one subunit of 135 amino acid residues and a second of 125 amino acid residues (Leduc and Bon, 1998), and belongs to the family of the snaclecs (snake C-type lectins; Polgar et al, 1997;Clemetson et al, 2009). Within mammalian prey, convulxin acts by inducing platelet aggregation through binding and clustering of the p62/GPVI collagen receptor (Jandrot-Peruus et al, 1997) and can bind eight GPVI (Horii et al, 2009) (Fig. 5).…”

“…Tetramers are formed by covalent disulfide linkages(Murakami et al, 2003) Horii et al (2009). determined that convulxin exists as the dimer of a4b4 tetramers in solution, resulting in eight potential binding sites for GPVI.…”

Trowaglerix Venom Polypeptides As a Novel Antithrombotic Agent by Targeting Immunoglobulin-Like Domains of Glycoprotein VI in Platelet

“…14,15 It also was suggested that CVX exists in solution as a dimer of α4β4 rings and contains 8 distinct GPVI-binding sites and binds GPVI with high-binding affinities. 16 Also, presence of 2 distinct GPVI-binding surfaces on the (α4β4)2 CVX dimers allows CVX to both cluster GPVI on a single cell and to cross-link target cells via GPVI.…”

The SCHOOL of nature: I. Transmembrane signaling

“…134 Convulxin, a C-type lectin-like protein from the venom of the South American rattlesnake that functions as a potent agonist of GPVI, has been reported recently to form a dimer in solution and bind eight copies of GPVI. 248 Other MIRRs. Human TREM-1 receptor has been shown to exist as a "head-to-tail" dimer in crystal, suggesting that the dimeric TREM-1 most likely contains two distinct ligand-binding sites.…”