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Purification of serine protease from polychaeta, Lumbrineris nipponica, and assessment of its fibrinolytic activity

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Abstract

Ischemic stroke and cardiovascular disease can occur from blockage of blood vessels by fibrin clots formed naturally in the body. Therapeutic drugs of anticoagulant or thrombolytic agents have been studied; however, various problems have been reported such as side effects and low efficacy. Thus, development of new candidates that are more effective and safe is necessary. The objective of this study is to evaluate fibrinolytic activity, anti-coagulation, and characterization of serine protease purified from Lumbrineris nipponica, polychaeta, for new thrombolytic agents. In the present study, we isolated and identified a new fibrinolytic serine protease from L. nipponica. The N-terminal sequence of the identified serine protease was EAMMDLADQLEQSLN, which is not homologous with any known serine protease. The size of the purified serine protease was 28 kDa, and the protein purification yield was 12.7%. The optimal enzyme activity was observed at 50°C and pH 2.0. A fibrin plate assay confirmed that indirect fibrinolytic activity of the purified serine protease was higher than that of urokinase-PA, whereas direct fibrinolytic activity, which causes bleeding side effects, was relatively low. The serine protease did not induce any cytotoxicity toward the endothelial cell line. In addition, anticoagulant activity was verified by an in vivo DVT animal model system. These results suggest that serine protease purified from L. nipponica has the potential to be an alternative fibrinolytic agent for the treatment of thrombosis and use in various biomedical applications.

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References

  • Ahn MY, Hahn BS, Ryu KS, Kim JW, Kim I, Kim YS (2003) Purification and characterization of a serine protease with fibrinolytic activity from the dung beetles, Catharsius molossus. Thromb Res 112:339–347

    Article  CAS  PubMed  Google Scholar 

  • Astrup T, Müllertz S (1952) The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 40:346–351

    Article  CAS  PubMed  Google Scholar 

  • Bi Q, Han B, Feng Y, Jiang Z, Yang Y, Liu W (2013) Antithrombotic effects of a newly purified fibrinolytic protease from Urechis unicinctus. Thromb Res 132:e135–e144

    Article  CAS  PubMed  Google Scholar 

  • Brady L, Brzozowski AM, Derewenda ZS, Dodson E, Dodson G, Tolley S, Turkenburg JP, Christiansen L, Huge-jensen B, Norskov L (1990) A serine protesase triad forms the catalytic centre of a triacylglycerol lipase. Nature 34:797–770

    Google Scholar 

  • Carter P, Wells JA (1988) Dissecting the catalytic triad of a serine protease. Nature 332:564–568

    Article  CAS  PubMed  Google Scholar 

  • Choi JH, Sapkota K, Kim S, Kim SJ (2014) Starase: a bi-functional fibrinolytic protease from hepatic caeca of Asterina pectinifera displays antithrombotic potential. Biochimie 105:45–57

    Article  CAS  PubMed  Google Scholar 

  • Chou JH, Sapkota K, Park SE, Kim S, Kim SJ (2013) Thrombolytic, anticoagulant and antiplatelet activities of codiase, a bi-functional fibrinolytic enzyme from Codium fragile. Biochimie 95:1266–1277

    Article  Google Scholar 

  • Collen D (1999) The plasminogen (fibrinolytic) system. Thromb Haemost 82:259–270

    CAS  PubMed  Google Scholar 

  • Deng Z, Wang S, Li Q, Ji X, Zhang L, Hong M (2010) Purification and characterization of a novel fibrinolytic enzyme from the polychaete, Neanthes japonica (Iznka). Bioresour Technol 101:1954–1960

    Article  CAS  PubMed  Google Scholar 

  • Hahn BS, Cho SY, Wu SJ, Chang IM, Baek K, Kim YC, Kim YS (1999) Purification and characterization of a serine protease with fibrinolytic activity from Tenodera sinensis (praying mantis). BBA-Protein Struct M 1430:376–386

    Article  CAS  Google Scholar 

  • Isaev NK, Stelmashook EV, Plotnikov EY, Khryapenkova TG, Lozier ER, Doludin YV, Silachev DN, Zorov DB (2008) Role of acidosis, NMDA receptors, and acid-sensitive ion channel 1a (ASIC1a) in neuronal death induced by ischemia. Biochem Mosc 73:1171–1175

    Article  CAS  Google Scholar 

  • Iversen SL, Jørgensen MH (1995) Azocasein assay for alkaline protease in complex fermentation broth. Biotechnol Tech 9:573–576

    Article  CAS  Google Scholar 

  • Kim DW, Choi JH, Park SE, Kim S, Sapkota K, Kim SJ (2015) Purification and characterization of a fibrinolytic enzyme from Petasites japonicus. Int J Biol Macromol 72:1159–1167

    Article  CAS  PubMed  Google Scholar 

  • Kotb E (2015) Purification and partial characterization of serine fibrinolytic enzyme from Bacillus megaterium KSK-07 isolated from kishk, a traditional Egyptian fermented food. Appl Biochem Microbiol 51:34–43

    Article  CAS  Google Scholar 

  • Lee CK, Shin JS, Kim BS, Cho IH, Kim YS, Lee EB (2007) Antithrombotic effects by oral administration of novel proteinase fraction from earthworm Eisenia andrei on venous thrombosis model in rats. Arch Pharm Res 30:475–480

    Article  CAS  PubMed  Google Scholar 

  • Libby P (2006) Inflammation and cardiovascular disease mechanisms. Am J Clin Nutr 83:4565–4605

    Google Scholar 

  • Moon SM, Kim JS, Kim HJ, Choi MS, Park BR, Kim SG, Ahn H, Chun HS, Shin YK, Dk K, Lee SY, Seo YW, Kim YH, Kim CS (2014) Purification and characterization of a novel fibrinolytic α chymotrypsin like serine metalloprotease from the edible mushroom, Lyophyllum shimeji. J Biosci Bioeng 117:544–550

    Article  CAS  PubMed  Google Scholar 

  • Nordt TK, Bode C (2003) Thrombolysis: newer thrombolytic agents and their role in clinical medicin. Heart 89:1358–1362

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Park JW, Park JE, Chou HK, Jung TW, Yoon SM, Lee JS (2013) Purification and characterization of three thermostable alkaline fibrinolytic serine proteases from the polychaete Cirriformia tentaculata. Process Biochem 48:979–987

    Article  CAS  Google Scholar 

  • Phan TTB, Ta TD, Nguyen TX, Van Den Broek LA, Duong GTH (2011) Purification and characterization of novel fibrinolytic proteases as potential antithrombotic agents from earthworm Perionyx excavatus. AMB Express 1:26

    Article  PubMed  PubMed Central  Google Scholar 

  • Sherry S, Lindemeyer RI, Fletcher AP, Alkjaersig N (1959) Studies on enhanced fibrinolytic activity in man. J Clin Inest 38:810–822

    Article  CAS  Google Scholar 

  • Victor J, Sol S (1988) Thrombolytic therapy: current status. N Engl J Med 318:1512–1520

    Article  Google Scholar 

  • Wang CT, Ji BP, Li B, Nout R, Li PL, Ji H, Chen LF (2006) Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi. J Ind Microbiol Biotechnol 33:750–758

    Article  CAS  PubMed  Google Scholar 

  • Wang S, Deng Z, Li Q, Ge X, Bo Q, Liu J, Cui J, Jiang X, Zhang L, Hong M (2011) A novel alkaline serine protease with fibrinolytic activity from the polychaete, Neanthes japonica. Comp Biochem Physiol B: Biochem Mol Biol 159:18–25

    Article  Google Scholar 

  • Wang YF, Tsirka SE, Strickland S, Stieg PE, Soriano SG, Lipton SA (1998) Tissue plasminogen activator (tPA) increase neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice. Nat Med 4:228–231

    Article  CAS  PubMed  Google Scholar 

  • Wardlaw JM, Murray V, Berge E, Zoppo G, Sandercock P, Lindley RL, Cohen G (2012) Recombinant tissue plasminogen activator for acute ischaemic stroke: an updated systematic review and meta-analysis. Lancet 379:2364–2372

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wiman B, Collen D (1978) Molecular mechanism of physiological fibrinolysis. Nature 272:549–550

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Shi H, Wang W, Ke Z, Xu P, Zhong Z, Li X, Wang S (2011) Antithrombotic effect of grape seed proanthocyanidins extract in a rat model of deep vein thrombosis. J Vasc Surg 53:743–753

    Article  PubMed  Google Scholar 

  • Zivin JA (2009) Acute stroke therapy with tissue plasminogen activator (tPA) since it was approved by the U. S. Food and Drug Administration (FDA). Ann Neurol 66:6–10

    Article  PubMed  Google Scholar 

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Acknowledgements

This work was supported by INHA U Research Grant.

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  1. ** Ha Hwang

    Present address: ST Pharm Co., Ltd., Gyeonggi-do, South Korea

Authors and Affiliations

  1. Department of Biological Engineering, Inha University, Incheon, South Korea

    Seung Ju Yeon, Goo Yong Chung, Jae Sang Hong, ** Ha Hwang & Hwa Sung Shin

Authors
  1. Seung Ju Yeon
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  2. Goo Yong Chung
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  3. Jae Sang Hong
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  4. ** Ha Hwang
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  5. Hwa Sung Shin
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Correspondence to ** Ha Hwang or Hwa Sung Shin.

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Editor: Tetsuji Okamoto

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Yeon, S.J., Chung, G.Y., Hong, J.S. et al. Purification of serine protease from polychaeta, Lumbrineris nipponica, and assessment of its fibrinolytic activity. In Vitro Cell.Dev.Biol.-Animal 53, 494–501 (2017). https://doi.org/10.1007/s11626-017-0137-2

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  • DOI: https://doi.org/10.1007/s11626-017-0137-2

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