Effect of multimer size and a natural dimorphism on the binding of convulxin to platelet glycoprotein (GP)VI

Abstract

Background: Convulxin (CVX), a C-type lectin from the venom of Crotalus durissus terrificus, is a potent activator of human platelets, binding predominantly to glycoprotein (GP)VI. Native CVX is an octamer composed of four ab-heterodimers [(ab)4]. Two different native sequences have been reported, one bearing lysine (K), the other glutamic acid (E), at b chain residue 89, but the physiological relevance of this difference is unknown. Objective: We used the Drosophila S2 system to express recombinant CVX (rCVX) heterodimers (ab) and site-directed mutagenesis to evaluate the influence of multimer size and the substitution bK89E on CVX function. Methods: By flow cytometry, native CVX and both recombinant forms bind to human platelets in whole blood. By surface plasmon resonance (BIAcore, Piscataway, NJ, USA), the calculated equilibrium dissociation constants (KD) were: rCVX ab89K, 11.3 · 10)8 M; rCVX ab89E, 9 · 10)8 M; and native CVX, 2.8 · 10)8 M. Results: Thus, the affinities of the two rCVX forms for human, recombinant GPVI are essentially the same, but the relative affinity of native CVX is about 3-fold higher. The minimum concentration of native CVX that induces maximal human platelet aggregation (70 pM) is roughly 400-fold lower than that of either rCVX (29 nM). Conclusions: These results are consistent with the hypothesis that the ability of the native CVX octamer to cluster mobile GPVI molecules within the platelet membrane may be the single most important factor that contributes to the efficiency with which CVX is able to induce platelet activation.