The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis cleaves and inactivates plasminogen activator inhibitor type 1

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 similar to0.1 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 +/- 1.48 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.