title: The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis Cleaves and inactivates plasminogen activator inhibitor type 1.
abstract: In this report, we demonstrate an interaction between subtilisin NAT (formerly designated BSP, or nattokinase), a profibrinolytic serine proteinase from Bacillus subtilis, and plasminogen activator inhibitor 1 (PAI-1). Subtilisin NAT was purified to homogeneity (molecular mass, 27.7 kDa) from a saline extract of B. subtilis (natto). Subtilisin NAT appeared to cleave active recombinant prokaryotic PAI-1 (rpPAI-1) into low molecular weight fragments. Matrix-assisted laser desorption/ionization in combination with time-of-flight mass spectroscopy and peptide sequence analysis revealed that rpPAI-1 was cleaved at its reactive site (P1-P1': Arg(346)-Met(347)). rpPAI-1 lost its specific activity after subtilisin NAT treatment in a dose-dependent manner (0.02-1.0 nm; half-maximal effect at approximately 0.1 nm). Subtilisin NAT dose dependently (0.06-1 nm) enhanced tissue-type plasminogen activator-induced fibrin clot lysis both in the absence of rpPAI-1 (48 +/- 1.4% at 1 nm) and especially in the presence of rpPAI-1 (78 +/- 2.0% at 1 nm). The enhancement observed in the absence of PAI-1 seems to be induced through direct fibrin dissolution by subtilisin NAT. The stronger enhancement by subtilisin NAT of rpPAI-1-enriched fibrin clot lysis seems to involve the cleavage and inactivation of active rpPAI-1. This mechanism is suggested to be important for subtilisin NAT to potentiate fibrinolysis.
authors.affiliation: Department of Physiology, Hamamatsu University School of Medicine, 3600, Handa-cho, Hamamatsu, 431-3192, Japan. uranot@hama-med.ac.jp
authors.forename: T
authors.initials: T
authors.lastname: Urano
authors.forename: H
authors.initials: H
authors.lastname: Ihara
authors.forename: K
authors.initials: K
authors.lastname: Umemura
authors.forename: Y
authors.initials: Y
authors.lastname: Suzuki
authors.forename: M
authors.initials: M
authors.lastname: Oike
authors.forename: S
authors.initials: S
authors.lastname: Akita
authors.forename: Y
authors.initials: Y
authors.lastname: Tsukamoto
authors.forename: I
authors.initials: I
authors.lastname: Suzuki
authors.forename: A
authors.initials: A
authors.lastname: Takada
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects; D006801:Humans; D008970:Molecular Weight; D010449:Peptide Mapping; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013381:Subtilisins / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D005337:Fibrin; D005340:Fibrinogen; D013381:Subtilisins; C053771:nattokinase