Abstract

Convulxin (CVX), a C-type lectin derived from the venom of Crotalus durissus terrificus, is a potent activator of human platelets, binding predominantly to platelet glycoprotein (GP) VI, but also to GP Ibα. Native CVX is an octamer composed of four αβ-heterodimers. It has been speculated that multimer size contributes greatly to CVX function, but proof of this hypothesis has not yet been reported. Additionally, two different native sequences have been reported (GenBank protein accession numbers CAA76182 and AAQ11362), one bearing lysine (K), the other glutamic acid (E), at β chain residue 89, but the physiological relevance of this difference is unknown.

In an earlier report, we have described the production of monomeric recombinant CVX (rCVX) heterodimers using the Drosophila S2 system. We now employ this recombinant protein and site-directed mutagenesis to evaluate the influence of multimer size and the substitution βK89E on CVX function.

By flow cytometry, native CVX (αβ)4 and both recombinant forms of CVX (αβ89K and αβ89E) bind to CHO cells expressing human recombinant GPVI (hrGPVI) and to human platelets in whole blood. Surface plasmon resonance (BIAcore) was employed to assess the kinetics of the interaction between hrGPVI and native or recombinant CVX. The calculated equilibrium dissociation constants (KD) were: for rCVX αβ89K, 11.3 x 10−8 M; for rCVX αβ89E, 9 x 10−8 M; and for native CVX (αβ)4, 2.8 x 10−8 M. These results indicate that the affinities of the two rCVX forms for hrGPVI are essentially the same and that the relative affinity of native CVX is about three-fold higher. Next, aggregation of human PRP was used to assess the relative function of the CVX forms. The minimum concentration of native CVX that induces platelet aggregation (70 picomolar) is roughly 400-fold lower than that of either rCVX αβ89K or rCVX αβ89E (each, 29 nanomolar).

Based on these findings, it is apparent that: 1) There is little, if any difference, in the affinity or activity of the two recombinant forms of CVX; 2) the octameric form of native CVX results in roughly a three-fold increase in affinity for GPVI; and 3) this increase in affinity of the octameric form of native CVX cannot account for the substantial increase in its ability to induce platelet aggregation relative to the recombinant forms. These results are consistent with the hypothesis that the ability of 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 signal transduction and platelet activation. Moreover, smaller forms of CVX, such as the rCVX heterodimers, may prove to be efficient inhibitors of platelet activation by native CVX and other GPVI agonists.

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