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Undariase, a Direct-Acting Fibrin(ogen)olytic Enzyme from Undaria pinnatifida, Inhibits Thrombosis In Vivo and Exhibits In Vitro Thrombolytic Properties

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Abstract

A direct-acting fibrinolytic serine protease named undariase possessing anticoagulant and antiplatelet properties was purified from Undaria pinnatifida. Undariase showed a molecular weight of 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. It displayed a strong fibrin zymogram lysis band corresponding to the same molecular mass. The N-terminal sequence of undariase, LTATTCEELAAAPTD, does not match with any known fibrinolytic enzyme. The enzyme was stable and active at high temperatures (35–70 °C). The fibrinolytic activity of undariase was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and 4-(amidinophenyl) methanesulfonyl fluoride (APMSF). The K m and V max values for substrate S-2251 were determined as 6.15 mM and 90.91 mM/min/ml, respectively. Undariase resulted in clot lysis by directly cleaving α and β chains of fibrin. Similarly, it preferentially acted on the Aα chain of fibrinogen followed by cleavage of the Bβ chain. It significantly prolonged the PFA-100 closure times of citrated whole human blood. In addition, undariase delayed the coagulation time and increased activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). Undariase exerted a significant protective effect against collagen plus epinephrine-induced pulmonary thromboembolism in mice. It prevented carrageenan-induced thrombus formation in the tail of mice. It also resulted in prolongation of APTT ex vivo. In conclusion, these results suggested a therapeutic potential of undariase for thrombosis.

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Acknowledgments

This research was supported by the Technology Development Program for Fisheries, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

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Authors and Affiliations

  1. Department of Life Science & BK21-Plus Research Team for Bioactive Control Technology, Chosun University, 375 Seosuk-dong, Dong-gu, Gwangju, 501-759, Republic of Korea

    Jun-Hui Choi, Kumar Sapkota & Sung-Jun Kim

  2. Department of Bio-food Technology, Chonbuk National University, Iksan, 570-752, Republic of Korea

    Myung-Kon Kim

  3. Department of Alternative Medicine, Gwangju University, Gwangju, 503-703, Republic of Korea

    Seung Kim

  4. Central Department of Zoology, Tribhuvan University, Kirtipur, Kathmandu, Nepal

    Kumar Sapkota

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  1. Jun-Hui Choi
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Correspondence to Seung Kim or Sung-Jun Kim.

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Choi, JH., Sapkota, K., Kim, MK. et al. Undariase, a Direct-Acting Fibrin(ogen)olytic Enzyme from Undaria pinnatifida, Inhibits Thrombosis In Vivo and Exhibits In Vitro Thrombolytic Properties. Appl Biochem Biotechnol 173, 1985–2004 (2014). https://doi.org/10.1007/s12010-014-0981-4

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