RSS-Feed abonnieren
DOI: 10.1055/s-0037-1616536
A Novel Tetrameric Venom Protein, Agglucetin from Agkistrodon acutus , Acts as a Glycoprotein Ib Agonist
Publikationsverlauf
Received
04. Januar 2001
Accepted after resubmission
18. Juni 2001
Publikationsdatum:
09. Dezember 2017 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/200104/lookinside/thumbnails/10-1055s-0037-1616536-1.jpg)
Summary
A novel platelet agglutination inducer, agglucetin, was purified from the Formosan Agkistrodon acutus snake venom. It migrated as a single band with an apparent molecular mass of 58.8 kDa and two distinct bands of 16.2/14.5 kDa under non-reducing and reducing conditions by SDS-PAGE, respectively. Further confirmed by FPLC, electrospray ionization mass spectrometry and 2D-PAGE, native agglucetin exists as a tetramer composed of disulfide-linked α1, α2, β1 and β2 subunits. Partial N-terminal sequence of agglucetin subunit showed a high degree of homology to those of C-type lectin-like glycoprotein (GP) Ib binding proteins. Functional studies showed that agglucetin, in the absence of von Willebrand factor (vWF), dose-dependently induced platelet agglutination and caused a negligible elevation of intracellular Ca+2 mobilization and thromboxane B2 formation in human platelet suspensions. Anti-GP Ib monoclonal antibodies (mAbs), AP1 or LJ-Ib1, specifically inhibited agglucetin-induced platelet agglutination in a dose-dependent manner. However, EDTA, arietin (a long chain RGD-containing disintegrin), 7E3 (an anti-GP IIb/IIIa mAb), heparin, hirudin, PGE1, or indomethacin exhibited no inhibitory effect on agglucetin-induced platelet agglutination. Furthermore, flow cytometric analysis revealed that FITC-agglucetin dose-dependently bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by anti-GP Ib mAb. It is concluded that agglucetin, acts specifically on an epitope of platelet membrane GP Ib overlapping with that of AP1, causing platelet agglutination in a Ca+2- and GP IIb/IIIa-independent manner.
-
References
- 1 Andrews RK, Berndt MC. Snake venom modulators of platelet adhesion receptors and their ligands. Toxicon 2000; 38: 775-91.
- 2 Markland FS. Snake venoms and the hemostatic system. Toxicon 1998; 36: 1749-800.
- 3 Ouyang C, Teng CM, Huang TF. Characterization of snake venom components acting on blood coagulation and platelet function. Toxicon 1992; 30: 945-66.
- 4 Pirkle H, Stocker K. Thrombin-like enzymes from snake venoms: an inventory. Thromb Haemost 1991; 65: 444-50.
- 5 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.
- 6 Latallo ZS. Retrospective study on complications and adverse effects of treatment with thrombin-like enzymes – a multicentre trial. Thromb Haemost 1983; 50: 604-9.
- 7 Coller BS. Monitoring platelet GP IIb/IIIa antagonist therapy. Circulation 1998; 97: 5-9.
- 8 Madan M, Berkowitz SD, Tcheng JE. Glycoprotein IIb/IIIa integrin blockade. Circulation 1998; 98: 2629-35.
- 9 Topol EJ, Byzova TV, Plow EF. Platelet GPIIb-IIIa blockers. Lancet 1999; 353: 227-31.
- 10 Clemetson KJ, Clemetson JM. Platelet GPIb-V-IX complex – structure, function, physiology, and pathology. Semin Thromb Haemost 1995; 21: 130-6.
- 11 Clemetson KJ. Platelet activation: signal transduction via membrane receptors. Thromb Haemost 1995; 74: 111-6.
- 12 Clemetson KJ. Platelet GpIb-V-IX complex. Thromb Haemost 1997; 78: 266-70.
- 13 Ware J. Molecular analyses of the platelet glycoprotein Ib-IX-V receptor. Thromb Haemost 1998; 79: 466-78.
- 14 Andrews RK, Berndt MC. Adhesion-dependent signalling and the initiation of haemostasis and thrombosis. Histol Histopathol 1998; 13: 837-44.
- 15 Simon DI, Chen Z, Xu H, Li CQ, Dong J, McIntire LV, Ballantyne CM, Zhang L, Furman MI, Berndt MC, Lopez JA. Platelet glycoprotein Ib is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18). J Exp Med 2000; 192: 193-204.
- 16 Usami Y, Fujimura Y, Suzuki M, Ozeki Y, Nishio K, Fukui H, Titani K. Primary structure of two-chain botrocetin, a von Willebrand factor modulator purified from the venom of Bothrops jararaca . Proc Natl Acad Sci 1993; 90: 928-32.
- 17 Berndt MC, Ward CM, Booth WJ, Castaldi PA, Mazurov AV, Andrews RK. Identification of aspartic acid 514 through glutamic acid 542 as a glycoprotein Ib-IX complex receptor recognition sequence in von Wille-brand factor. Mechanism of modulation of von Willebrand factor by ristocetin and botrocetin. Biochemistry 1992; 31: 11144-51.
- 18 Peng M, Lu W, Kirby EP. Alboaggregin-B: a new platelet agonist that binds to platelet membrane glycoprotein Ib. Biochemistry 1991; 30: 11529-36.
- 19 Peng M, Lu W, Kirby EP. Characterization of three alboaggregins purified from Trimeresurus albolabris venom. Thromb Haemost 1992; 67: 702-7.
- 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 Usami Y, Suzuki M, Yoshida E, Sukurai Y, Hirano K, Kawsaki 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.
- 22 Sakurai Y, Fujimura Y, Kokubo T, Imamura K, Kawasaki T, Honda 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.
- 23 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.
- 24 Liu CZ, Wang YW, Shen MC, Huang TF. Analysis of human platelet glycoprotein IIb-IIIa by fluorescein isothiocyanate-conjugated disintegrins with flow cytometry. Thromb Haemost 1994; 72: 919-25.
- 25 Huang TF, Wang WJ, Teng CM, Liu CS, Ouyang C. Purification and characterization of an antiplatelet peptide, arietin, from Bitis arietans venom. Biochim Biophys Acta 1991; 1074: 136-43.
- 26 Mustard JF, Perry DW, Ardlie NG, Packham MA. Preparation of suspension of washed platelet from humans. Br J Haematol 1972; l (Suppl. 22) 193-204.
- 27 Kirby EP, Mills DCB. The interaction of bovine factor VIII with human platelets. J Clin Invest 1975; 56: 491-502.
- 28 Pollock WK, Rink TJ. Thrombin and ionomycin can raise platelet cytosolic ca+2 to micromolar levels by discharge of internal Ca+2 stores: studies using fura-2. Biochem Biophys Res Commun 1986; 139: 308-14.
- 29 Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca+2 indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440-50.
- 30 Vlot AJ, Koppelman SJ, Bouma BN, Sixma JJ. Factor VIII and von Wille-brand factor. Thromb Haemost 1998; 79: 456-65.
- 31 Andrews RK, Lopez JA, Berndt MC. Molecular mechanisms of platelet adhesion and activation. Int J Biochem Cell Biol 1997; 29: 91-105.
- 32 Peng M, Lu W, Beviglia L, Niewiarowski S, Kirby EP. Echicetin: a snake venom protein that inhibits binding of von Willebrand factor and alboaggregins to platelet glycoprotein Ib. Blood 1993; 81: 2321-8.
- 33 Peng M, Holt JC, Niewiarowski S. Isolation, characterization and amino acid sequence of echicetinsubunit, a specific inhibitor of von Willebrand factor and thrombin interaction with glycoprotein Ib. Biochem Biophys Res Commun 1994; 205: 68-72.
- 34 Fujimura Y, Ikeda Y, Miura S, Yoshida E, Shima H, Nishida S, Suzuki M, Titani K, Taniuchi Y, Kawasaki T. Isolation and characterization of Jararaca GPIb-BP, a snake venom antagonist specific to platelet glycoprotein Ib. Thromb Haemost 1995; 74: 743-50.
- 35 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.
- 36 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.
- 37 Kawasaki T, Taniuchi Y, Hisamichi N, Fujimura Y, Suzuki M, Titani K, Sakai Y, Kaku S, Satoh N, Takenaka T, Handa M, Sawai Y. 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.
- 38 Taniuchi Y, Kawasaki T, Fujimura Y, Suzuki M, Titani K, Sakai Y, Kaku S, Hisamichi N, Satoh N, Takenaka T, Handa M, Sawai Y. Flavocein-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.
- 39 Yeh CH, Chang MC, Peng HC, Huang TF. Pharmacological characterization and antithrombotic effect of agkistin, a platelet glycoprotein Ib antagonist. Br J Pharm 2001; 132: 843-850.
- 40 Okita JR, Pidard D, Newman PJ, Montgomery RR, Kunicki TJ. On the association of glycoprotein Ib and actin-binding protein in human platelets. J Cell Biol 1985; 100: 317-21.
- 41 Handa M, Titani K, Holland LZ, Roberts JR, Ruggeri ZM. The von Wille-brand factor-binding domain of platelet membrane glycoprotein Ib – characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments. J Biol Chem 1986; 261: 12579-85.
- 42 Kawasaki T, Fujimura Y, Usami Y, Suzuki M, Miura S, Sakurai Y, Makita K, Taniuchi Y, Hirano K, Titani K. Complete amino acid sequence and identification of the platelet glycoprotein Ib-binding site of jararaca GPIbBP, a snake venom protein isolated from Bothrops jararaca . J Biol Chem 1996; 271: 10635-9.
- 43 Polgar J, Magnenat EM, Peitsch MC, Wells TNC, Saqi MSA, Clemetson KJ. Amino acid sequence of thesubunit and computer modelling of theandsubunits of echicetin from the Echis carinatus . Biochem J 1997; 323: 533-7.
- 44 Ware J, Russel SR, Marchese P, Murata M, Mazzucato M, Marco LD, Ruggeri ZM. Point mutation in a leucine-rich repeat of platelet glycoprotein Ib resulting in the Bernard-Soulier syndrome. J Clin Invest 1993; 92: 1213-20.