Effect of multimer size and a natural dimorphism on the binding of convulxin to platelet glycoprotein (GP)VI.
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 alphabeta-heterodimers [(alphabeta)(4)]. Two different native sequences have been reported, one bearing lysine (K), the other glutamic acid (E), at beta 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 (alphabeta) and site-directed mutagenesis to evaluate the influence of multimer size and the substitution betaK89E 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 (K(D)) were: rCVX alphabeta89K, 11.3 x 10(-8) m; rCVX alphabeta89E, 9 x 10(-8) m; and native CVX, 2.8 x 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.
Kato
K
K
Department of Molecular and Experimental Medicine, Division of Experimental Hemostasis and Thrombosis, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Furihata
K
K
Cheli
Y
Y
Radis-Baptista
G
G
Kunicki
T J
TJ
D000818:Animals; D001483:Base Sequence; D003435:Crotalid Venoms / Q000378:metabolism; D017931:DNA Primers; D004330:Drosophila; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D037181:Lectins, C-Type / Q000378:metabolism*; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding
D003435:Crotalid Venoms; D017931:DNA Primers; D037181:Lectins, C-Type; D010980:Platelet Membrane Glycoproteins; C028890:convulxin