A Novel Fibrinolytic Protein From Pheretima vulgaris: Purification, Identification, Antithrombotic Evaluation, and Mechanisms Investigation

Hai Liu; Jianqiong Yang; Yamei Li; Yunnan Ma; Wenjie Wang; Wanling Zhong; Pengyue Li; Shouying Du

doi:10.3389/fmolb.2021.772419

A Novel Fibrinolytic Protein From Pheretima vulgaris: Purification, Identification, Antithrombotic Evaluation, and Mechanisms Investigation

Front Mol Biosci. 2022 Jan 24:8:772419. doi: 10.3389/fmolb.2021.772419. eCollection 2021.

Authors

Hai Liu^{1

2}, Jianqiong Yang³, Yamei Li², Yunnan Ma¹, Wenjie Wang¹, Wanling Zhong¹, Pengyue Li¹, Shouying Du¹

Affiliations

¹ School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China.
² College of Pharmacy, Gannan Medical University, Ganzhou, China.
³ The First Clinical Medical College, Gannan Medical University, Ganzhou, China.

Abstract

Thrombotic diseases have been considered major causes of death around the world. Treatments with thrombolytic drugs, such as recombinant tissue-plasminogen activator, urokinase, and streptokinase, are reported to have a life-threatening bleeding tendency. On the contrary, lumbrokinase, identified from Lumbricus rubellus, is specific to fibrin and does not cause excessive bleeding. It possesses fibrinolytic activity and activation of plasminogen to dissolve fibrin. Hence, the purification of fibrinolytic protein monomer from earthworm and antithrombotic evaluation and investigation of mechanisms are needed. In this study, a novel fibrinolytic protein EPF3, with strong fibrinolytic activity, was purified from Pheretima vulgaris by ion exchange and size exclusion chromatography. SDS PAGE, bottom-up proteomics analysis, de novo sequencing, and circular dichroism (CD) analysis were carried out for identification and characterization of it. EPF3, with a molecular weight of 25136.24 Da, consisted of 241 amino acids and contained various forms of secondary structures, including α-helix (3.9%), β-sheet (42.8%), β-turn (21.2%), and random coil (32.1%). It was a trypsin-like serine protease and stable at pH 7.0 to 11.0 and below 40°C. EPF3 was confirmed to possess an antithrombotic effect by ex vivo clot lysis test and fibrinogen-thrombin time (Fib-TT) assay. The three-dimensional structure of EPF3 was predicted by SWISS-MODEL. Molecular docking analysis predicted that EPF3 could directly interact with antithrombotic target proteins (fibrin, fibrinogen, and plasminogen), which was further confirmed by further studies. The antithrombotic mechanism of EPF3 was clarified to be outstanding direct fibrinolysis, fibrinogenolytic activity, and certain activation of plasminogen. EPF3 possesses the potential to be developed into a promising antithrombotic agent.

Keywords: Pheretima vulgaris; antithrombotic evaluation; de novo sequencing; fibrinolytic protein; molecular docking; purification.