Bilinexin, a Snake C-type Lectin from Agkistrodon bilineatus Venom Agglutinates Platelets via GPIb and α2β1

Xiao-Yan Du; Alexey Navdaev; Jeannine M. Clemetson; Edith Magnenat; Timothy N. C. Wells; Kenneth J. Clemetson

doi:10.1055/s-0037-1616062

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2001; 86(05): 1277-1283
DOI: 10.1055/s-0037-1616062

Review Article

Schattauer GmbH

Bilinexin, a Snake C-type Lectin from Agkistrodon bilineatus Venom Agglutinates Platelets via GPIb and α₂β₁

Authors

Xiao-Yan Du

¹Theodor Kocher Institute, University of Berne, Berne, Switzerland
Alexey Navdaev

¹Theodor Kocher Institute, University of Berne, Berne, Switzerland
Jeannine M. Clemetson

¹Theodor Kocher Institute, University of Berne, Berne, Switzerland
Edith Magnenat

²Serono Pharmaceutical Research Institute SA, Geneva, Switzerland
Timothy N. C. Wells

²Serono Pharmaceutical Research Institute SA, Geneva, Switzerland
Kenneth J. Clemetson

¹Theodor Kocher Institute, University of Berne, Berne, Switzerland

Abstract

Summary

A new snake protein, named bilinexin, has been purified from Agkistrodon bilineatus venom by ion-exchange chromatography and gel filtration chromatography. Under non-reducing conditions it has a mass of 110 kDa protein on SDS-PAGE. On reduction, it can be separated into five subunits with masses in the range 13-25 kDa. The N-terminal sequences of these subunits are very similar to those of convulxin or the alboaggregins, identifying bilinexin as a new member of the snake C-type lectin family, unusual in having multiple subunits. Bilinexin agglutinates fixed platelets, washed platelets and platelet rich plasma (PRP) without obvious activation (shape change) as confirmed by light microscope examination. Both inhibitory and binding studies indicate that antibodies against α₂β₁ inhibit not only platelet agglutination induced by bilinexin, but also bilinexin binding to platelets. VM16d, a monoclonal anti-GPIbα antibody, completely inhibits platelet agglutination induced by bilinexin, and polyclonal antibodies against GPIbα prevent its binding to platelets. However, neither convulxin, polyclonal anti-GPVI antibodies, nor GPIIb/IIIa inhibitors affect its binding to and agglutination of platelets. Bilinexin neither activates GPIIb/IIIa integrin on platelets nor induces tyrosine phosphorylation of platelet proteins, nor increases intracellular Ca²⁺ in platelets. Like alboaggregin B, bilinexin agglutinates platelets, which makes it a good tool to investigate the differences in mechanism between snake C-type lectins causing platelet agglutination and those that induce full activation.

Keywords

Bilinexin - platelet agglutination - GPIb - α₂β₁

Full Text

References

References
1 Chen YL, Tsai IH. Functional and sequence characterization of agkicetin, a new glycoprotein Ib antagonist isolated from Agkistrodon acutus venom. Biochem Biophys Res Commun 1995; 210: 472-7.
2 Kawasaki T, Taniuchi Y, Hisamichi N, Fujimura Y, Suzuki M, Titani K, Sakai Y, Kaku S, Satoh N, Takenaka T. Tokaracetin, a new platelet antagonist that binds to platelet glycoprotein Ib and inhibits von Willebrand factor-dependent shear-induced platelet aggregation. Biochem J 1995; 308: 947-53.
3 Sakurai Y, Fujimura Y, Kokubo T, Imamura K, Kawasaki T, Handa M, Suzuki M, Matsui T, Titani K, Yoshioka A. The cDNA cloning and molecular characterization of a snake venom platelet glycoprotein Ib-binding protein, mamushigin, from Agkistrodon halys blomhoffii venom. Thromb Haemost 1998; 79: 1199-207.
4 Navdaev A, Dormann D, Clemetson JM, Clemetson KJ. Echicetin, a GPIb-binding snake C-type lectin from Echis carinatus, also contains a binding site for IgMkappa responsible for platelet agglutination in plasma and inducing signal transduction. Blood 2001; 97: 2333-41.
5 Dormann D, Clemetson JM, Navdaev A, Kehrel BE, Clemetson KJ. Alboaggregin A activates platelets by a mechanism involving glycoprotein VI as well as glycoprotein Ib. Blood 2001; 97: 929-36.
6 Huang TF, Liu CZ, Yang SH. Aggretin, a novel platelet-aggregation inducer from snake (Calloselasma rhodostoma) venom, activates phospholipase C by acting as a glycoprotein Ia/IIa agonist. Biochem J 1995; 309: 1021-7.
7 Navdaev A, Clemetson JM, Polgar J, Kehrel BE, Glauner M, Magnenat E, Wells TN, Clemetson KJ. Aggretin, a heterodimeric C-type lectin from Calloselasma rhodostoma (Malayan pit viper) stimulates platelets by binding to α₂β₁ integrin and GPIb, activating Syk and PLCγ2, but does not involve the GPVI/Fc collagen receptor. J Biol Chem. 2001; 13. April, e-print in press.
8 Polar J, Clemetson JM, Kehrel BE, Wiedemann M, Magnenat EM, Wells TNC, Clemetson KJ. Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor. J Biol Chem 1997; 272: 13576-83.
9 Peng M, Lu W, Kirby PE. Alboaggregin-B: A new platelet agonist that binds to platelet membrane glycoprotein Ib. Biochemistry 1991; 30: 11529-36.
10 Nikai T, Ohara A, Komori Y, Fox JW, Sugihara H. Primary structure of a coagulant enzyme, bilineobin, from Agkistrodon bilineatus venom. Arch Biochem Biophys 1995; 318: 89-96.
11 Nikai T, Taniguchi K, Komori Y, Masuda K, Fox JW, Sugihara H. Primary structure and functional characterization of bilitoxin-1, a novel dimeric P-II snake venom metalloproteinase from Agkistrodon bilineatus venom. Arch Biochem Biophys 2000; 378: 6-15.
12 Nakagaki T, Kazim AL, Kisiel W. Isolation and characterization of a protein C activator from tropical moccasin venom. Thromb Res 1990; 58: 593-602.
13 Clemetson JM, Polgar J, Magnenat E, Wells TN, Clemetson KJ. The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to Fc R and the natural killer receptors. J Biol Chem 1999; 274: 29019-24.
14 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 1970; 227: 680-5.
15 Morrissey JH. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 1981; 117: 307-10.
16 Kirby EP, Mills DCB. The interaction of bovine factor VIII with human platelets. J Clin Invest 1975; 56: 491-502.
17 Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440-50.
18 Fujimura Y, Kawasaki T, Titani K. Snake venom proteins modulating the interaction between von Willebrand factor and platelet glycoprotein Ib. Thromb Haemost 1996; 76: 633-9.
19 Fukuda K, Mizuno H, Atoda H, Morita T. Crystal structure of flavocetin-A, a platelet glycoprotein Ib-binding protein, reveals a novel cyclic tetramer of C-type lectin-like heterodimers. Biochemistry 2000; 39: 1915-23.
20 Kowalska MA, Tan L, Holt JC, Peng M, Karczewski J, Calvete JJ, Niewiarowski S. Alboaggregins A and B. Structure and interaction with human platelets. Thromb Haemost 1998; 79: 609-13.
21 Taniuchi Y, Kawasaki T, Fujimura Y, Suzuki M, Titani K, Sakai Y, Kaku S, Hisamichi N, Satoh N, Takenaka T. Flavocetin-A and -B, two high molecular mass glycoprotein Ib binding proteins with high affinity purified from Trimeresurus flavoviridis venom, inhibit platelet aggregation at high shear stress. Biochim Biophys Acta 1995; 1244: 331-8.
22 Andrews RK, Kroll MH, Ward CM, Rose JW, Scarborough RM, Smith AI, Lopez JA, Berndt MC. Binding of a novel 50-kilodalton alboaggregin from Trimeresurus albolabris and related viper venom proteins to the platelet membrane glycoprotein Ib-IX-V complex. Effect on platelet aggregation and glycoprotein Ib-mediated platelet activation. Biochemistry 1996; 35: 12629-39.
23 Usami Y, Suzuki M, Yoshida E, Sakurai Y, Hirano K, Kawasaki T, Fujimura Y, Titani K. Primary structure of alboaggregin-B purified from the venom of Trimeresurus albolabris . Biochem Biophys Res Commun 1996; 219: 727-33.
24 Leduc M, Bon C. Cloning of subunits of convulxin, a collagen-like platelet-aggregating protein from Crotalus durissus terrificus venom. Biochem J 1998; 333: 389-93.
25 Inoue K, Ozaki Y, Satoh K, Wu Y, Yatomi Y, Shin Y, Morita T. Signal transduction pathways mediated by glycoprotein Ia/IIa in human platelets: comparison with those of glycoprotein VI. Biochem Biophys Res Commun 1999; 256: 114-20.