Bothrojaracin, a Proexosite I Ligand, Inhibits Factor Va-Accelerated Prothrombin Activation

Robson Q. Monteiro; Russolina B. Zingali

doi:10.1055/s-0037-1612987

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2002; 87(02): 288-293
DOI: 10.1055/s-0037-1612987

Letters to the Editor

Schattauer GmbH

Bothrojaracin, a Proexosite I Ligand, Inhibits Factor Va-Accelerated Prothrombin Activation

Robson Q. Monteiro

¹Departamento de Bioquímica Médica, ICB/CCS, Universidade Federal do Rio de Janeiro, Brazil

,

Russolina B. Zingali

¹Departamento de Bioquímica Médica, ICB/CCS, Universidade Federal do Rio de Janeiro, Brazil

› Institutsangaben

Abstract

Summary

Bothrojaracin (BJC) is a 27 kDa snake venom protein from Bothrops jararaca that has been characterized as a potent ligand (K _D = 75 nM) of human prothrombin (Monteiro RQ, Bock PE, Bianconi ML, Zingali RB, Protein Sci 2001; 10: 1897-904). BJC binds to the partially exposed anion-binding exosite I (proexosite I) forming a stable 1:1, non-covalent complex with the zymogen whereas no interaction with fragment 1 or 2 domains is observed. In addition, BJC interacts with thrombin through exosites I and II (K _D = 0.7 nM), and influences but does not block the proteinase catalytic site. In the present work we studied the effect of BJC on human prothrombin activation by factor Xa in the absence or in the presence of its cofactors, factor Va and phospholipids. In the absence of phospholipids, BJC strongly inhibited (80%) the zymogen activation by factor Xa in the presence but not in the absence of factor Va, suggesting a specific interference in the cofactor activity. In the presence of phospholipid vesicles (75% phosphatidylcholine, 25% phosphatidylserine), BJC also inhibited (35%) prothrombin activation by factor Xa in the presence but not in the absence of factor Va. BJC showed a higher inhibitory effect (70%) towards thrombin formation by prothrombinase complex assembled on phospholipid vesicles composed by 95% phosphatidylcholine, 5% phosphatidylserine. Activation of prothrombin by platelet-assembled prothrombinase complex (factor Xa, factor Va and thrombin-activated platelets) showed that hirudin^54-65(SO₃-) and BJC efficiently inhibit the thrombin formation (43% and 84%, respectively). Taken together, our results suggest that proexosite I blockage decreases the productive recognition of prothrombin as substrate by factor Xa-factor Va complex and prothrombinase complex. Furthermore, data obtained with human platelets suggest that proexosite I may play an important role in the physiological activation of prothrombin.

Keywords

Bothrojaracin - prothrombin - proexosite I - factor Va - prothrombinase

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Referenzen

References
1 Mann KG, Elion J, Butkowski RJ, Downing M, Nesheim ME. Prothrombin. Meth Enzymol 1981; 80: 286-302.
2 Kalafatis M, Swords NA, Rand MD, Mann KG. Membrane-dependent reactions in blood coagulation: role of vitamin K-dependent enzyme complexes. Biochim Biophys Acta 1994; 1227: 113-29.
3 Rosing J, Tans G, Govers-Riemslag JW, Zwaal RF, Hemker HC. The role of phospholipids and factor Va in the prothrombinase complex. J Biol Chem 1980; 255: 274-83.
4 Liu LW, Ye J, Johnson AE, Esmon CT. Proteolytic formation of either of the two prothrombin activation intermediates results in formation of a hirugen-binding site. J Biol Chem 1991; 266: 23632-6.
5 Wu Q, Picard V, Aiach M, Sadler JE. Activation-induced exposure of the thrombin anion-binding exosite. J Biol Chem 1994; 269: 3725-30.
6 Naski MC, Fenton II JW, Maraganore JM, Olson ST, Shafer JA. The COOH-terminal domain of hirudin. An exosite-directed competitive inhibitor of the action of alpha-thrombin on fibrinogen. J Biol Chem 1990; 265: 13484-9.
7 Liu LW, Vu TKH, Esmon CT, Coughlin SR. The region of the thrombin receptor resembling hirudin binds to thrombin and alters enzyme specificity. J Biol Chem 1991; 266: 16977-80.
8 Ye J, Liu LW, Esmon CT, Johnson AE. The fifth and sixth growth factorlike domains of thrombomodulin bind to the anion-binding exosite of thrombin and alter its specificity. J Biol Chem 1992; 267: 11023-8.
9 Hortin GL, Tollefsen DM, Benutto BM. Antithrombin activity of a peptide corresponding to residues 54-75 of heparin cofactor II. J Biol Chem 1989; 264: 13979-82.
10 Dharmawardana KR, Bock PE. Demonstration of exosite I-dependent interactions of thrombin with human factor V and factor Va involving the factor Va heavy chain: Analysis by affinity chromatography employing a novel method for active-site-selective immobilization of serine proteinases. Biochemistry 1998; 37: 13143-52.
11 Dharmawardana KR, Olson ST, Bock PE. Role of regulatory exosite I in binding of thrombin to human factor V, factor Va, factor Va subunits, and activation fragments. J Biol Chem 1999; 274: 18635-43.
12 Krstenansky JL, Mao SJT. Antithrombin properties of C-terminus of hirudin using synthetic unsulfated N-alpha-acetyl-hirudin45-65. FEBS lett 1987; 211: 10-6.
13 Stubbs MT, Bode W. A player of many parts: the spotlight falls on thrombin’s structure. Thromb Res 1993; 69: 1-58.
14 Guillin MC, Bezeaud A, Bouton MC, Jandrot-Perrus M. Thrombin specificity. Thromb Haemost 1995; 74: 129-33.
15 Sheehan JP, Sadler JE. Molecular mapping of the heparin-binding exosite of thrombin. Proc Natl Acad Sci USA 1994; 91: 5518-22.
16 Anderson PJ, Nesset A, Dharmawardana KR, Bock PE. Characterization of proexosite I on prothrombin. J Biol Chem 2000; 275: 16428-34.
17 Esmon CT, Jackson CM. The conversion of prothrombin to thrombin. IV. The function of the fragment 2 region during activation in the presence of factor V. J Biol Chem 1974; 249: 7791-7.
18 Kotkow KJ, Deitcher SR, Furie B, Furie BC. The second kringle domain of prothrombin promotes factor Va-mediated prothrombin activation by prothrombinase. J Biol Chem 1995; 270: 4551-7.
19 Krishnaswamy S, Walker RK. Contribution of the prothrombin fragment 2 domain to the function of factor Va in the prothrombinase complex. Biochemistry 1997; 36: 3319-30.
20 Anderson PJ, Nesset A, Dharmawardana KR, Bock PE. Role of proexosite I in factor Va-dependent substrate interactions of prothrombin activation. J Biol Chem 2000; 275: 16435-42.
21 Zingali RB, Jandrot-Perrus M, Guillin MC, Bon C. Bothrojaracin, a new thrombin inhibitor isolated from Bothrops jararaca venom: Characterization and mechanism of thrombin inhibition. Biochemistry 1993; 32: 10794-802.
22 Arocas V, Zingali RB, Guillin MC, Bon C, Jandrot-Perrus M. Bothrojaracin: a potent two-site-directed thrombin inhibitor. Biochemistry 1996; 35: 9083-9.
23 Arocas V, Lemaire C, Bouton MC, Bezeaud A, Bon C, Guillin MC, Jandrot-Perrus M. Inhibition of thrombin-catalyzed factor V activation by bothrojaracin. Thromb Haemost 1998; 79: 1157-61.
24 Monteiro RQ, Rapôso JG, Wisner A, Guimarães JA, Bon C, Zingali RB. Allosteric changes of thrombin catalytic site induced by interaction of bothrojaracin with anion-binding exosites I and II. Biochem Biophys Res Commun 1999; 262: 819-22.
25 Monteiro RQ, Bock PE, Bianconi ML, Zingali RB. Characterization of bothrojaracin interaction with human prothrombin. Protein Sci 2001; 10: 1897-904.
26 Monteiro RQ, Zingali RB. Inhibition of prothrombin activation by bothrojaracin, a C-type lectin from Bothrops jararaca venom. Arch Biochem Biophys 2000; 382: 123-8.
27 Ngai PK, Chang JY. A novel one-step purification of human C-thrombin after direct activation of crude prothrombin enriched from plasma. Biochem J 1991; 280: 805-8.
28 Bezeaud A, Denninger MH, Guillin MC. Interaction of human alphathrombin and gamma-thrombin with antithrombin III, protein C and thrombomodulin. Eur J Biochem 1985; 153: 491-6.
29 Fenton II JW, Fasco MJ, Stackrow AB, Aronson DL, Young AM, Finlayson JS. Human thrombins. Production, evaluation, and properties of alpha-thrombin. J Biol Chem 1977; 252: 3587-98.
30 Patscheke H, Worner P. Platelet activation detected by turbidometric shapechange analysis: Differential influence of cytochalasin B and prostaglandin E1. Thromb Res 1978; 12: 485-96.
31 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
32 Arocas V, Castro HC, Zingali RB, Guillin MC, Jandrot-Perrus M, Bon C, Wisner A. Molecular cloning and expression of bothrojaracin, a potent thrombin inhibitor from snake venom. Eur J Biochem 1997; 248: 550-7.
33 Atoda H, Morita T. A novel blood coagulation factor IX/factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake): isolation and characterization. J Biochem (Tokyo) 1989; 106: 808-13.
34 Atoda H, Yoshida N, Ishikawa M, Morita T. Binding properties of the coagulation factor IX/factor X-binding protein isolated from the venom of Trimeresurus flavoviridis . Eur J Biochem 1994; 224: 703-8.
35 Boskovic DS, Giles AR, Nesheim ME. Studies of the role of factor Va in the factor Xa-catalyzed activation of prothrombin, fragment 1·2, prethrombin-2, and dansyl-L-glutamyl-glycyl-L-arginine-meizothrombin in the absence of phospholipid. J Biol Chem 1990; 265: 10497-505.
36 Solum NO. Procoagulant expression in platelets and defects leading to clinical disorders. Arterioscler Thromb Vasc Biol 1999; 19: 2841-6.
37 Bevers EM, Comfurius P, Zwaal RFA. Changes in membrane phospholipid distribution during platelet activation. Biochim Biophys Acta 1983; 736: 57-66.
38 Tracy PB, Nesheim ME, Mann KG. Platelet factor Xa receptor. Meth Enzymol 1992; 215: 329-60.
39 Bouchard BA, Catcher CS, Thrash BR, Adida C, Tracy PB. Effector cell protease receptor-1, a platelet activation-dependent membrane protein, regulates prothrombinase-catalyzed thrombin generation. J Biol Chem 1997; 272: 9244-51.
40 Bevers EM, Comfurius P, Reutelingsperger CPM, Zwaal RFA. Platelet procoagulant activity and its measurement. In: Platelets, a practical approach. SP Watson, KS Authi. New York: Oxford University Press Inc; 1996: 319-39.
41 Jandrot-Perrus M, Huisse MG, Krstenansky JL, Bezeaud A, Guillin MC. Effect of hirudin carboxy-terminal peptide 54-65 on the interaction of thrombin with platelets. Thromb Haemost 1991; 66: 300-5.
42 Krishnaswamy S, Jones KC, Mann KG. Prothrombinase complex assembly. J Biol Chem 1988; 263: 3823-34.
43 Ofosu FA, Sie P, Modi GJ, Fernandez F, Buchanan MR, Blajchman MA, Boneu B, Hirsh J. Inhibition of thrombin-dependent feedback reactions is critical to the expression of the anticoagulant effects of heparin. Biochem J 1987; 243: 579-88.
44 Gallist S, Muntean W, Leis HJ. Effects of heparin and hirudin on thrombin generation and platelet aggregation after intrinsic activation of platelet-rich plasma. Thromb Haemost 1995; 74: 1163-8.