A novel fibrinolytic enzyme from Neanthes japonica (Iznka), named NJF, was purified to electrophoretic homogeneity using ammonium sulfate precipitation, hydrophobic interaction, ion exchange and gel-filtration chromatography. NJF consisted of a single polypeptide chain with a molecular weight of 28-32 kDa, which was determined by MALDI-TOF mass spectrum and SDS-PAGE. The isoelectric point of NJF determined by isoelectric focusing electrophoresis (IEF) was 4.4, and the maximum activity of the enzyme was observed at 60 degrees C and pH 9.0. The cleavage speed of fibrinogen by NJF affected the A alpha-chain first, followed by the B beta-chain and finally the gamma-chain. NJF activity was strongly inhibited by PMSF, indicating that it is a serine protease. Partial amino-acid sequences of its fragments were different from those of other known fibrinolytic enzymes. N.japonica may thus represent a potential source of new therapeutic agents to treat thrombosis. (C) 2009 Elsevier Ltd. All rights reserved.