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Thromb Haemost 2014; 112(04): 700-715
DOI: 10.1160/TH13-12-1063
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Identification and characterisation of novel inhibitors on extrinsic tenase complex from Bungarus fasciatus (banded krait) venom

Wan Chen   #
1   Department of Pharmacy, National University of Singapore, Singapore, Singapore
,
Leng Chuan Goh   #
1   Department of Pharmacy, National University of Singapore, Singapore, Singapore
,
Tse Siang Kang
1   Department of Pharmacy, National University of Singapore, Singapore, Singapore
,
Manjunatha R. Kini
2   Department of Biological Sciences, National University of Singapore, Singapore, Singapore
3   Department of Biochemistry and Molecular Biology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia, USA
4   School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia, Australia
› Author Affiliations
Further Information

Publication History

Received: 31 December 2014

Accepted after major revision: 06 May 2014

Publication Date:
04 December 2017 (online)

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Summary

Snake venoms are excellent sources of pharmacologically active proteins and peptides, and hence are potential sources of leads for drug developments. It has been previously established that krait (Bungarus genus) venoms contain mainly neurotoxins. A screening for anticoagulants showed that Bungarus fasciatus venom exhibits potent anticoagulant effect in standard clotting assays. Through sequential fractionation of the venom by size exclusion and high performance liquid chromatographies, coupled with functional screening for anticoagulant activities, we have isolated and purified two anticoagulant proteins, termed BF-AC1 ( Bungarus fasciatus anticoagulant 1) and BFAC2. They have potent inhibitory activities (IC50 of 10 nM) on the extrinsic tenase complex. Structurally, these proteins each has two subunits covalently held together by disulfide bond(s). The N-terminal sequences of the individual subunits of BF-AC1 and BF-AC2 showed that the larger subunit is homologous to phospholipase A2, while the smaller subunit is homologous to Kunitz type serine proteinase inhibitor. Functionally, in addition to their anticoagulant activity, these proteins showed presynaptic neurotoxic effects in both in vivo and ex vivo experiments. Thus, BF-AC1 and BF-AC2 are structurally and functionally similar to β-bungarotoxins, a class of neurotoxins. The enzymatic activity of phospholipase A2 subunit plays a significant role in the anticoagulant activities. This is the first report on the anticoagulant activity of β-bungarotoxins and these results expand on the existing catalogue of haemostatically active snake venom proteins.

Keywords

Anticoagulant - extrinsic tenase complex - β-bungarotoxin - phospholipase A2 - Bungarus fasciatus

# These authors contributed equally to the publication.


 

  • References

  • 1 Furie B, Furie BC. Mechanisms of thrombus formation. N Engl J Med 2008; 359: 938-949.
    CrossrefPubMedGoogle Scholar
  • 2 Bates SM, Weitz JI. New anticoagulants: beyond heparin, low-molecular-weight heparin and warfarin. Br J Pharmacol 2005; 144: 1017-1028.
    CrossrefPubMedGoogle Scholar
  • 3 Hart RG, Pearce LA, Aguilar MI. Meta-analysis: antithrombotic therapy to prevent stroke in patients who have nonvalvular atrial fibrillation. Ann Intern Med 2007; 146: 857-867.
    CrossrefPubMedGoogle Scholar
  • 4 Singer DE, Chang Y, Fang MC. et al. The net clinical benefit of warfarin anticoagulation in atrial fibrillation. Ann Intern Med 2009; 151: 297-305.
    CrossrefPubMedGoogle Scholar
  • 5 Wein L, Wein S, Haas SJ. et al. Pharmacological venous thromboembolism prophylaxis in hospitalized medical patients: a meta-analysis of randomized controlled trials. Arch Intern Med 2007; 167: 1476-1486.
    CrossrefPubMedGoogle Scholar
  • 6 Hirsh J, Anand SS, Halperin JL. et al. Guide to anticoagulant therapy: Heparin : a statement for healthcare professionals from the American Heart Association. Circulation 2001; 103: 2994-3018.
    CrossrefPubMedGoogle Scholar
  • 7 Link KP. The discovery of dicumarol and its sequels. Circulation 1959; 19: 97-107.
    CrossrefPubMedGoogle Scholar
  • 8 Gould MK, Dembitzer AD, Doyle RL. et al. Low-molecular-weight heparins compared with unfractionated heparin for treatment of acute deep venous thrombosis. A meta-analysis of randomized, controlled trials. Ann Intern Med 1999; 130: 800-809.
    CrossrefPubMedGoogle Scholar
  • 9 Quinlan DJ, McQuillan A, Eikelboom JW. Low-molecular-weight heparin compared with intravenous unfractionated heparin for treatment of pulmonary embolism: a meta-analysis of randomized, controlled trials. Ann Intern Med 2004; 140: 175-183.
    CrossrefPubMedGoogle Scholar
  • 10 Eikelboom JW, Anand SS, Malmberg K. et al. Unfractionated heparin and low-molecular-weight heparin in acute coronary syndrome without ST elevation: a meta-analysis. Lancet 2000; 355: 1936-1942.
    CrossrefPubMedGoogle Scholar
  • 11 Hirsh J, O’Donnell M, Eikelboom JW. Beyond unfractionated heparin and warfarin: current and future advances. Circulation 2007; 116: 552-560.
    CrossrefPubMedGoogle Scholar
  • 12 Schulman S. Advantages and limitations of the new anticoagulants. J Int Med 2014; 275: 1-11.
    CrossrefPubMedGoogle Scholar
  • 13 Shameem R, Ansell J. Disadvantages of VKA and requirements for novel anticoagulants. Best Pract Res Clin Haematol 2013; 26: 103-114.
    CrossrefPubMedGoogle Scholar
  • 14 Kazmi RS, Lwaleed BA. New anticoagulants: how to deal with treatment failure and bleeding complications. Br J Clin Pharmacol 2011; 72: 593-603.
    CrossrefPubMedGoogle Scholar
  • 15 Salim I, Al Suwaidi J, Ghadban W. et al. Anticoagulation in atrial fibrillation and co-existent chronic kidney disease: efficacy versus safety. Exp Opin Drug Saf 2013; 12: 53-63.
    CrossrefPubMedGoogle Scholar
  • 16 Goel R, Srivathsan K. Newer oral anticoagulant agents: a new era in medicine. Curr Cardiol Rev 2012; 8: 158-165.
    CrossrefPubMedGoogle Scholar
  • 17 Schulman S, Kearon C, Kakkar AK. et al. Dabigatran versus warfarin in the treatment of acute venous thromboembolism. N Engl J Med 2009; 361: 2342-2352.
    CrossrefPubMedGoogle Scholar
  • 18 Connolly SJ, Ezekowitz MD, Yusuf S. et al. Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med 2009; 361: 1139-1151.
    CrossrefPubMedGoogle Scholar
  • 19 Bauersachs R, Berkowitz SD, Brenner B. et al. Oral rivaroxaban for symptomatic venous thromboembolism. N Engl J Med 2010; 363: 2499-2510.
    CrossrefPubMedGoogle Scholar
  • 20 Patel MR, Mahaffey KW, Garg J. et al. Rivaroxaban versus warfarin in nonvalvular atrial fibrillation. N Engl J Med 2011; 365: 883-891.
    CrossrefPubMedGoogle Scholar
  • 21 Uchino K, Hernandez AV. Dabigatran association with higher risk of acute coronary events: meta-analysis of noninferiority randomized controlled trials. Arch Intern Med 2012; 172: 397-402.
    CrossrefPubMedGoogle Scholar
  • 22 Mega JL, Braunwald E, Wiviott SD. et al. Rivaroxaban in patients with a recent acute coronary syndrome. N Engl J Med 2012; 366: 9-19.
    CrossrefPubMedGoogle Scholar
  • 23 Faiz A, Ghose A, Ahsan F. et al. The greater black krait (Bungarus niger), a newly recognized cause of neuro-myotoxic snake bite envenoming in Bangladesh. Brain 2010; 133: 3181-3193.
    CrossrefPubMedGoogle Scholar
  • 24 Liu CS, Hsiao PW, Chang CS. et al. Unusual amino acid sequence of fasciatoxin, a weak reversibly acting neurotoxin in the venom of the banded krait, Bungarus fasciatus. Biochem J 1989; 259: 153-158.
    CrossrefPubMedGoogle Scholar
  • 25 Heilbronn E, Jiang MS, Zhou H. et al. Neurotoxins from Agkistrodon halys (pallas) and Bungarus fasciatus venom. Prog Clin Biol Res 1987; 253: 265-275.
    PubMedGoogle Scholar
  • 26 Banerjee Y, Mizuguchi J, Iwanaga S. et al. Hemextin AB complex--a snake venom anticoagulant protein complex that inhibits factor VIIa activity. Pathophysiol Haemost Thromb 2005; 34: 184-187.
    CrossrefPubMedGoogle Scholar
  • 27 Banerjee Y, Kumar S, Jobichen C. et al. Crystallization and preliminary X-ray diffraction analysis of hemextin A: a unique anticoagulant protein from Hemachatus haemachatus venom. Acta Crystallograph F 2007; 63: 701-703.
    PubMedGoogle Scholar
  • 28 Ward M. Pyridylethylation of Cysteine Residues. In: The Protein Protocols Handbook Humana Press; 2002. pp. 461-463.
    Google Scholar
  • 29 Pratt CW, Monroe DM. Microplate coagulation assays. BioTechniques 1992; 13: 430-433.
    PubMedGoogle Scholar
  • 30 Greenberg CS, Miraglia CC, Rickles FR. et al. Cleavage of blood coagulation factor XIII and fibrinogen by thrombin during in vitro clotting. J Clin Invest 1985; 75: 1463-1470.
    CrossrefPubMedGoogle Scholar
  • 31 Koyama T, Noguchi K, Aniya Y. et al. Analysis for sites of anticoagulant action of plancinin, a new anticoagulant peptide isolated from the starfish Acanthaster planci, in the blood coagulation cascade. Gen Pharmacol 1998; 31: 277-282.
    CrossrefPubMedGoogle Scholar
  • 32 Diaz-Oreiro C, Gutierrez JM. Chemical modification of histidine and lysine residues of myotoxic phospholipases A2 isolated from Bothrops asper and Bothrops godmani snake venoms: effects on enzymatic and pharmacological properties. Toxicon 1997; 35: 241-252.
    CrossrefPubMedGoogle Scholar
  • 33 Pawlak J, Mackessy SP, Fry BG. et al. Denmotoxin, a three-finger toxin from the colubrid snake Boiga dendrophila (Mangrove Catsnake) with bird-specific activity. J Biol Chem 2006; 281: 29030-29041.
    CrossrefPubMedGoogle Scholar
  • 34 Sajevic T, Leonardi A, Krizaj I. Haemostatically active proteins in snake venoms. Toxicon 2011; 57: 627-645.
    CrossrefPubMedGoogle Scholar
  • 35 Rowan EG. What does beta-bungarotoxin do at the neuromuscular junction?. Toxicon 2001; 39: 107-118.
    CrossrefPubMedGoogle Scholar
  • 36 Kini RM, Banerjee Y. Dissection approach: a simple strategy for the identification of the step of action of anticoagulant agents in the blood coagulation cascade. J Thromb Haemost 2005; 3: 170-171.
    CrossrefPubMedGoogle Scholar
  • 37 Kruck TP, Logan DM. Neurotoxins from Bungarus fasciatus venom: a simple fractionation and separation of alpha- and beta-type neurotoxins and their partial characterization. Biochemistry 1982; 21: 5302-5309.
    CrossrefPubMedGoogle Scholar
  • 38 Hanley MR, Eterovic VA, Hawkes SP. et al. Neurotoxins of Bungarus multicinctus vernom. Purification and partial characterization. Biochemistry 1977; 16: 5840-5849.
    CrossrefPubMedGoogle Scholar
  • 39 Chang CC, Lee CY. Isolation of neurotoxins from the venom of Bungarus multi-cinctus and their modes of neuromuscular blocking action. Arch Int Pharmacodyn Ther 1963; 144: 241-257.
    PubMedGoogle Scholar
  • 40 Kondo K, Toda H, Narita K. Characterization of phospholipase A activity of beta1-bungarotoxin from Bungarus multicinctus venom. I. Its enzymatic properties and modification with p-bromophenacyl bromide. J Biochem 1978; 84: 1291-1300.
    CrossrefPubMedGoogle Scholar
  • 41 Kondo K, Narita K, Lee CY. Chemical properties and amino acid composition of beta1-bungarotoxin from the venom of Bungarus multicinctus (Formosan banded krait). J Biochem 1978; 83: 91-99.
    CrossrefPubMedGoogle Scholar
  • 42 Kondo K, Narita K, Lee CY. Amino acid sequences of the two polypeptide chains in beta1-bungarotoxin from the venom of Bungarus multicinctus. J Biochem 1978; 83: 101-115.
    CrossrefPubMedGoogle Scholar
  • 43 Kini RM, Evans HJ. The role of enzymatic activity in inhibition of the extrinsic tenase complex by phospholipase A2 isoenzymes from Naja nigricollis venom. Toxicon 1995; 33: 1585-1590.
    CrossrefPubMedGoogle Scholar
  • 44 Condrea E, Yang CC, Rosenberg P. Lack of correlation between anticoagulant activity and phospholipid hydrolysis by snake venom phospholipases A2. Thromb Haemost 1981; 45: 82-85.
    PubMedGoogle Scholar
  • 45 Condrea E, Yang CC, Rosenberg P. Additional evidence for a lack of correlation between anticoagulant activity and phospholipid hydrolysis by snake venom phospholipases A2. Thromb Haemost 1982; 47: 298.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 46 Ouyang C, Jy W, Zan YP. et al. Mechanism of the anticoagulant action of phospholipase A purified from Trimeresurus mucrosquamatus (Formosan habu) snake venom. Toxicon 1981; 19: 113-120.
    PubMedGoogle Scholar
  • 47 Kerns RT, Kini RM, Stefansson S. et al. Targeting of venom phospholipases: the strongly anticoagulant phospholipase A(2) from Naja nigricollis venom binds to coagulation factor Xa to inhibit the prothrombinase complex. Arch Biochem Biophys 1999; 369: 107-113.
    CrossrefPubMedGoogle Scholar
  • 48 Kini RM. Structure-function relationships and mechanism of anticoagulant phospholipase A2 enzymes from snake venoms. Toxicon 2005; 45: 1147-1161.
    CrossrefPubMedGoogle Scholar
  • 49 Inada M, Crowl RM, Bekkers AC. et al. Determinants of the inhibitory action of purified 14-kDa phospholipases A2 on cell-free prothrombinase complex. J Biol Chem 1994; 269: 26338-26343.
    PubMedGoogle Scholar
  • 50 Kondo K, Toda H, Narita K. et al. Amino acid sequence of beta 2-bungarotoxin from Bungarus multicinctus venom. The amino acid substitutions in the B chains. J Biochem 1982; 91: 1519-1530.
    CrossrefPubMedGoogle Scholar