Journal of Thrombosis and Haemostasis, 4: 1107–1113
ORIGINAL ARTICLE Effect of multimer size and a natural dimorphism on the binding of convulxin to platelet glycoprotein (GP)VI
K. KATO,* K. FURIHATA, Y. CHELI,* G. RADIS-BAPTISTAà and T . J . K U N I C K I * *Department of Molecular and Experimental Medicine, Division of Experimental Hemostasis and Thrombosis, The Scripps Research Institute, La Jolla, CA; Department of Experimental Medicine, Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA, USA; and àDepartment of Biochemistry, Federal University of Pernambuco, Recife, PE, Brazil
To cite this article: Kato K, Furihata K, Cheli Y, Radis-Baptista G, Kunicki TJ. Effect of multimer size and a natural dimorphism on the binding of convulxin to platelet glycoprotein (GP)VI. J Thromb Haemost 2006; 4: 1107–13.
Keywords: convulxin, glycoprotein VI, GPVI, platelet. Summary. Background: Convulxin (CVX), a C-type lectin from the venom of Crotalus durissus terrificus, is a potent Introduction activator of human platelets, binding predominantly to glyco- protein (GP)VI. Native CVX is an octamer composed of four Convulxin (CVX), a glycosylated C-type lectin, was first isolated from the venom of Crotalus durissus cascavella [1] as a ab-heterodimers [(ab)4]. Two different native sequences have been reported, one bearing lysine (K), the other glutamic acid potent inducer of platelet aggregation. It was later shown that it (E), at b chain residue 89, but the physiological relevance of this is also produced in the venom of another related subspecies, difference is unknown. Objective: We used the Drosophila S2 C. durissus terrificus [2]. Because of its high affinity for system to express recombinant CVX (rCVX) heterodimers (ab) glycoprotein (GP)VI, CVX has been used as a powerful tool to and site-directed mutagenesis to evaluate the influence of investigate the function of this important collagen receptor that multimer size and the substitution bK89E on CVX function. is instrumental in the initiation of platelet activation and Methods: By flow cytometry, native CVX and both recombin- thrombus formation. ant forms bind to human platelets in whole blood. By surface Previous findings are consistent with the notion that plasmon resonance (BIAcore, Piscataway, NJ, USA), the clustering of GPVI is necessary to induce platelet aggregation. For example, polypeptides composed of repeat Glycine– calculated equilibrium dissociation constants (KD) were: rCVX )8 )8 ab89K, 11.3 · 10 M;rCVXab89E, 9 · 10 M; and native Proline–Hydroxyproline sequences, one of the fundamental )8 CVX, 2.8 · 10 M. Results: Thus, the affinities of the two collagen-binding motifs, do not activate human platelets unless rCVX forms for human, recombinant GPVI are essentially the they are cross-linked [3]. Moreover, purified F(ab¢)2 fragments same, but the relative affinity of native CVX is about 3-fold of anti-GPVI antibodies can induce platelet aggregation, higher. The minimum concentration of native CVX that whereas Fab fragments of the same do not [4,5]. Likewise, induces maximal human platelet aggregation (70 pM) is roughly rat anti-GPVI antibody (JAQ1) induces mouse platelet aggre- 400-fold lower than that of either rCVX (29 nM). Conclusions: gation only when the antibodies are cross-linked by polyclonal These results are consistent with the hypothesis that the ability rabbit antirat immunoglobulin G (IgG) antibodies [6]. Thus, of the native CVX octamer to cluster mobile GPVI molecules it is reasonable to argue that GPVI-specific ligands, whether within the platelet membrane may be the single most important antibodies, collagens or CVX, will initiate signal transduction factor that contributes to the efficiency with which CVX is able and platelet activation most efficiently when they are able to to induce platelet activation. cluster GPVI on the membrane surface. Studies of the structure of CVX have a direct bearing on this Correspondence: Thomas J. Kunicki, Department of Molecular and hypothesis. The initial reports, based on hydrodynamic studies,
Experimental Medicine, The Scripps Research Institute, 10550 N. suggested that CVX has a hexameric structure (ab)3, involving Torrey Pines Road, MEM-150, La Jolla, CA 92037, USA. two distinct subunits, as is the case with alboluxin [2,7]. Tel.: +1 858 784 2668; fax: +1 858 784 2174; e-mail: tomk@ However, two independent studies employing crystallographic scripps.edu analysis have since shown conclusively that native CVX exists Present address: Kenichi Furihata, SRL Inc., Hachioji, Tokyo, Japan. as a tetra-heterodimer composed of four ab-heterodimers (ab) [8–10]. The ab heterodimers are linked by interchain Present address: Kazunobu Kato, Department of Molecular 4 Biosciences, School of Veterinary Medicine, UC Davis, Davis, CA disulfide bonds between Cys79 of the a-subunit and Cys76 of 95616, USA. the b-subunit. The heterodimers are then linked one to another by intersubunit disulfide bonds between Cys135 of the Received 8 December 2005, accepted 12 January 2006 a-subunit and Cys3 of the b-subunit. This distinctive, cyclic