Discovery of Anticoagulant Active Peptides from the Venom Glands of the <i>Draconarius digitusiformis</i>

doi:10.13523/j.cb.2306009

China Biotechnology

2024, Vol. 44

Issue (2/3): 76-84 DOI: 10.13523/j.cb.2306009

Discovery of Anticoagulant Active Peptides from the Venom Glands of the Draconarius digitusiformis

HUANG Biao,WEN Qiao,LONG Chenbo,GU Zhixin^*(

)

Chengdu Pepbiomedical Co., Ltd., Chengdu 610219, China

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Abstract

Objective: The venom glands of toxic animals secrete a large number of structurally diverse and functionally rich active peptides, representing a natural treasure trove for peptide drug development. At present, only a small portion of the active peptides from toxic animals have been studied, so there is a need to establish a more efficient method of active peptide discovery. Methods: The anticoagulant peptides were developed by transcriptome sequencing, data analysis and polypeptide selection, peptide preparation by recombinant expression, in vitro activity screening, and in vivo activity evaluation in animal models of the Draconarius digitusiformis. Results: PDBPE-001, an inhibitor of coagulation Factor Xa, was screened. The peptide has a molecular weight of 9 889.82 Da, consisting of 92 amino acid residues and 4 pairs of disulfide bonds. This peptide can be efficiently prepared through recombinant expression, and its inhibitory activity against Factor Xa is concentration dependent, with an IC₅₀ ~ 0.807 μmol/L. In the mouse thrombus model, 30 mg/kg of PDBPE-001 has a good anti-thrombotic effect. Conclusions: A novel Factor Xa inhibitor has been obtained for the first time from the Draconarius digitusiformis, providing a new lead peptide molecule for the development of new anticoagulants.

Key words： Toxic animals Active peptides Factor Xa Anticoagulants

Received: 05 June 2023 Published: 03 April 2024

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	Biao HUANG
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Cite this article:

HUANG Biao, WEN Qiao, LONG Chenbo, GU Zhixin. Discovery of Anticoagulant Active Peptides from the Venom Glands of the Draconarius digitusiformis. China Biotechnology, 2024, 44(2/3): 76-84.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2306009 OR https://manu60.magtech.com.cn/biotech/Y2024/V44/I2/3/76

Fig.1 Sequence analysis process of venom gland transcriptome

Table 1 Structure and function prediction information of five candidate peptides

Fig.2 Construction of recombinant plasmid PDBPE-001-PET-32a (+)

Fig.3 Identification of recombinant plasmid PDBPE-001-PET-32a (+) Lane M: 10 kb ladder;Lane 1: PDBPE-001 plasmid; Lane 2: PDBPE-001 plasmid digestedby Mlul and HindIII

Fig.4 Expression of peptide PDBPE-001 A: HPLC anallysis of recombinant peptide PDBPE-001 B: SDS-PAGE identification of recombinant peptide PDBPE-001. Lane 1: Bacterial liquid supernatant;Lane 2: Fusion protein PDBPE-001; Lane 3: Digestion of fusion protein PDBPE-001 C: MS identification of recombinant peptide PDBPE-001

Table 2 Inhibition of Thrombin、Kallikrein、Factor XIa and Factor Xa by five candidate peptides

Fig.5 Results of PDBPE-001 activity evaluation A:The inhibitory effect of different concentrations of PDBPE-001 on Factor Xa B:IC₅₀ of PDBPE-001 on Factor Xa

Fig.6 Evaluation of antithrombotic efficacy in vivo A:Black tail images of different treatment groups after 24 hours after carrageenan treatment B: Black tail length diagram of different treatment groups after 24 hours of carrageenan treatment C: Black tail ratio of different treatment groups 24 h after carrageenan treatment


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