GPIb is involved in platelet aggregation induced by mucetin, a snake C-type lectin protein from Chinese habu (Trimeresurus mucrosquamatus) venom

Qiumin Lu; Alexei Navdaev; Jeannine M. Clemetson; Kenneth J. Clemetson

doi:10.1160/TH03-12-0747

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2004; 91(06): 1168-1176
DOI: 10.1160/TH03-12-0747

Platelets and Blood Cells

Schattauer GmbH

GPIb is involved in platelet aggregation induced by mucetin, a snake C-type lectin protein from Chinese habu (Trimeresurus mucrosquamatus) venom

Authors

Qiumin Lu

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

²Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China
Alexei Navdaev

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

¹Theodor Kocher Institute, University of Bern, Bern, Switzerland
Kenneth J. Clemetson

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

Abstract

Summary

Mucetin (Trimeresurus mucrosquamatus venom activator, TMVA) is a potent platelet activator purified from Chinese habu (Trimeresurus mucrosquamatus) venom. It belongs to the snake venom heterodimeric C-type lectin family and exists in several multimeric forms. We now show that binding to platelet glycoprotein (GP) Ib is involved in mucetin-induced platelet aggregation. Antibodies against GPIb as well as the GPIb-blocking C-type lectin echicetin inhibited mucetin-induced platelet aggregation. Binding of GPIb was confirmed by affinity chromatography and Western blotting. Antibodies against GPVI inhibited convulxin- but not mucetin-induced aggregation. Signalling by mucetin involved rapid tyrosine phosphorylation of a number of proteins including Syk, Src, LAT and PLCγ2. Mucetininduced phosphorylation of the Fcγ chain of platelet was greatly promoted by inhibition of α_IIbβ₃ by the peptidomimetic EMD 132338, suggesting that phosphatases downstream of α_IIbβ₃ activation are involved in dephosphorylation of Fcγ. Unlike other multimeric snake C-type lectins that act via GPIb and only agglutinate platelets, mucetin activates α_IIbβ₃. Inhibition of α_IIbβ₃ strongly reduced the aggregation response to mucetin, indicating that activation of α_IIbβ₃ and binding of fibrinogen are involved in mucetin-induced platelet aggregation. Apyrase and aspirin also inhibit platelet aggregation induced by mucetin, suggesting that ADP and thromboxane A₂ are involved in autocrine feedback. Sequence and structural comparison with closely related members of this protein family point to features that may be responsible for the functional differences.

Keywords

Snake venom - C-type lectin - platelets - GPIb - tyrosine phosphorylation

Full Text

References

References
1 Clemetson KJ. Platelet collagen receptors: A new target for inhibition?. Haemostasis 1999; 29: 16-29.
2 Clemetson KJ. Primary haemostasis: Sticky fingers cement the relationship. Curr Biol 1999; 09: R110-2.
3 Du X, Plow EF, Frelinger III AL. et al. Ligands “activate” integrin αIIbβ3 (platelet GPIIb-IIIa). Cell 1991; 65: 409-16.
4 Moroi M, Onisuka I, Imaizumi T. et al. Involvement of activated integrin α2β1 in the firm adhesion of platelets onto a surface of immobilized collagen under flow conditions. Thromb Haemost 2000; 83: 769-76.
5 Clemetson JM, Polgar J, Magnenat E. et al. The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcγR and the natural killer receptors. J Biol Chem 1999; 274: 29019-24.
6 Clemetson KJ, Polar J, Clemetson JM. Snake venom C-type lectins as tools in platelet research. Platelets 1998; 09: 165-9.
7 Clemetson KJ, Navdaev A, Dörmann D. et al. Multifunctional snake C-type lectins affecting platelets. Hamostasis 2000; 31: 148-54.
8 Polgar J, Magnenat EM, Peitsch MC. et al. Amino acid sequence of the alpha subunit and computer modelling of the alpha and beta subunits of echicetin from the venom of Echis carinatus (saw-scaled viper). Biochem J 1997; 323: 533-7.
9 Fujimura Y, Ikeda Y, Miura S. et al. Isolation and characterization of jararaca GPIb-BP, a snake venom antagonist specific to platelet glycoprotein Ib. Thromb Haemost 1995; 74: 743-50.
10 Kawasaki T, Fujimura Y, Usami Y. et al. Complete amino acid sequence and identification of the platelet glycoprotein Ib-binding site of jararaca GPIb-BP, a snake venom protein isolated from Bothrops jararaca. J Biol Chem 1996; 271: 10636-9.
11 Kawasaki T, Taniuchi Y, Hisamichi N. et al. Tokaracetin, a new platelet antagonist that binds to platelet glycoprotein Ib and inhibits von Willebrand factor-dependent shearinduced platelet aggregation. Biochem J 1995; 308: 947-53.
12 Andrews RK, Kroll MH, Ward CM. et al. 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.
13 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.
14 Peng M, Lu W, Kirby EP. Alboaggregin-B: a new platelet agonist that binds to platelet membrane glycoprotein Ib. Biochemstry 1991; 30: 11529-36.
15 Peng M, Lu W, Kirby EP. Characterization of three alboaggregins purified from Trimeresurus albolabris venom. Thromb Haemost 1992; 67: 702-7.
16 Taniuchi Y, Kawasaki T, Fujimura Y. et al. 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.
17 Sakurai Y, Fujimura Y, Kokubo T. et al. The cDNA cloning and molecular characterization of a snake venom platelet glycoprotein Ibbinding protein, mamushigin, from Agkistrodon halys blomhoffii venom. Thromb Haemost 1998; 79: 1199-207.
18 Dörmann D, Clemetson JM, Navdaev A. et al. Alboaggregin A activates platelets by a mechanism involving glycoprotein VI as well as glycoprotein Ib. Blood 2001; 97: 929-36.
19 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.
20 Navdaev A, Dörmann D, Clemetson JM. et al. Echicetin, a GPIb-binding snake C-type lectin from Echis carinatus, also contains a binding site for IgMκ responsible for platelet agglutination in plasma and inducing signal transduction. Blood 2001; 97: 2333-41.
21 Fukuda K, Mizuno H, Atoda H. et al. Crystal structure of flavocetin-A, a platelet glycoprotein Ib-binding protein, reveals a novel cyclic tetramer of C-type lectin heterodimers. Biochemistry 2000; 39: 1915-23.
22 Huang K-F, Ko T-P, Hung C-C. et al. Crystal structure of a platelet agglutinating factor isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus). Biochem J 2004; 378: 399-407.
23 Wei Q, Lu QM, Jin Y. et al. Purification and cloning of a novel C-type lectin protein with platelet aggregation activity from Trimeresurus mucrosquamatus venom. Toxicon 2002; 40: 1331-8.
24 Taniuchi Y, Kawasaki T, Fujimura Y. The high molecular mass, glycoprotein Ib-binding protein flavocetin-A induced only small platelet aggregates in vitro . Thromb Res 2000; 97: 69-75.
25 Wicki AN, Clemetson JM, Kehrel BE. et al. Isolation and characterization of glycoprotein Ib. Methods Enzymol 1992; 215: 276-88.
26 Polar J, Clemetson JM, Kehrel BE. et al. 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.
27 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 1970; 227: 680-5.
28 Morrissey JH. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 1981; 117: 307-10.
29 Shen Y, Romo GM, Dong JF. et al. Requirement of leucine-rich repeats of glycoprotein (GP) Ibα for shear-dependent and static binding of von Willebrand factor to the platelet membrane GP Ib-IX-V complex. Blood 1999; 95: 903-10.
30 Mazurov AV, Vinogradov DV, Vlasik TN. et al. Characterization of an antiglycoprotein Ib monoclonal antibody that specifically inhibits platelet-thrombin interaction. Thromb Res 1991; 62: 673-84.
31 Du XY, Magnenat E, Timothy NCW. et al. Alboluxin, a snake C-type lectin from Trimeresurus albolabris venom is a potent platelet agonist acting via GPIb and GPVI. Thromb Haemost 2002; 87: 692-8.
32 Asazuma N, Ozaki Y, Satoh K. et al. Glycoprotein Ib-von Willebrand factor interactions activate tryrosine kinases in human platelets. Blood 1997; 90: 4789-98.
33 Navdaev A, Clemetson KJ. Glycoprotein Ib cross-linking/ligation on echicetin-coated surfaces or echicetin IgMκ in stirred suspension activates platelets by cytoskeleton modulated calcium release. J Biol Chem 2002; 277: 45928-34.
34 Wu Y, Suzuki-Inoue K, Satoh K. et al. Role of Fc receptor gamma-chain in platelet glycoprotein Ib-mediated signalling. Blood 2001; 97: 3836-45.
35 Rathore V, Stapleton MA, Hillery CA. et al. PECAM-1 negatively regulates GPIb/V/IX signalling in murine platelets. Blood 2003; 102: 3658-64.
36 Zaffran Y, Meyer SC, Negrescu E. et al. Signalling across the platelet adhesion receptor glycoprotein Ib-IX induces α_IIbβ₃ activation both in platelets and a transfected Chinese hamster ovary cell system. J Biol Chem 2000; 275: 16779-87.
37 Navdaev A, Clemetson JM, Polgar J. et al. Aggretin, a heterodimeric C-type lectin from Calloselasma rhodostoma (Malayan pit viper) stimulates platelets by binding to α₂β₁ integrin and glycoprotein Ib, activating Syk and phospholipase Cγ2, but does not involve the glycoprotein VI/Fc receptor γ chain collagen receptor. J Biol Chem 2001; 276: 20882-9.
38 Bergmeier W, Bouvard D, Eble JA. et al. Rhodocytin (aggretin) activates platelets lacking α₂β₁ integrin, glycoprotein VI, and the ligand-binding domain of glycoprotein Ibα. J Biol Chem 2001; 276: 25121-6.