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.