Purification and characterization of novel fibrinolytic proteases as potential antithrombotic agents from earthworm Perionyx excavatus

Tram Thi Bich Phan,Dung Thi Xuan Nguyen,Lambertus Am Van Den Broek,Tien Duy Ta,Giang Thi Huong Duong

doi:10.1186/2191-0855-1-26

Tram Thi Bich Phan, Dung Thi Xuan Nguyen + Show 3 more

Open Access

https://doi.org/10.1186/2191-0855-1-26

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Abstract

Six protease fractions, namely FI, FII, FIII-1, FIII-2, FIII-3 and FIV, were isolated from Perionyx excavatus earthworm biomass by acetone precipitation, followed by serial chromatography using anion exchange, hydrophobic interaction and size exclusion chromatography. All fractions exhibited strong hydrolytic activity towards casein. The activity of six fractions towards fibrin, determined by fibrin plate assay, ranged from 44 to 831 plasmin unit.mg-1 and ranked as FIII-3 > FIII-2 > FI > FIII-1 > FIV > FII. Casein degradation was optimal at pH 7 and 11, and at 45-60°C. All fractions were considerably stable at high temperature and wide pH range. They were completely inhibited by phenylmethylsulfonyl fluoride (PMSF). The molecular weights (MW) and isoelectric points (pI) determined by 2D-electrophoresis were 27.5-34.5 kDa, and 4.3-5.2, respectively. Tandem mass spectrometry (MS) analysis was used to deduce the amino acid sequences of some peptides from FIII-1 and FIII-2. The sequences shared 16.9% and 13.2% similarity, respectively, with the fibrinolytic enzymes from two related earthworm species, Lumbricus rubellus and Eisenia fetida. The P. excavatus proteases were classified as serine proteases. They could perform rapid hydrolysis on both coagulated fibrous fibrin and soluble fibrinogen monomers without the presence of activators such as tPA or urokinase.

Highlights

Cardiovascular diseases have become one of the biggest concerns all over the world (Grundy et al 1999)
Purification of P. excavatus proteases The hydrolytic activity on casein of the crude extract before autolysis was determined as 0.02 U.mg-1
To examine the further activation of these proteases, earthworm biomass was subjected to autolysis at 45°C in 4 hours, following by a 15-day autolysis at room temperature

Summary

Introduction

Cardiovascular diseases have become one of the biggest concerns all over the world (Grundy et al 1999). Thrombosis is the most widespread within the elderly population. The disease results from severe blood-clotting, which leads to obstruction of the blood flow circulation. Fibrin and platelets are utilized for clotting to prevent blood loss from injuries in a process called hemostasis (Furie and Furie 2008). A serine protease called plasmin acts to digest blood clots via fibrinolysis to properly terminate the hemostasis. Plasmin deficiency is one reason that leads to thrombosis due to insufficient clots degradation

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