The Profibrinolytic Enzyme Subtilisin NAT Purified fromBacillus 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 ϳ0.1 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.Subtilisin NAT (1) (formerly designated BSP, or nattokinase), a serine proteinase from Bacillus subtilis, has been reported to have potent fibrinolytic activity (1, 2). The enzyme is composed of 275 amino acids with a molecular mass of 27.7 kDa in its mature form (1). DNA sequence analysis showed that subtilisin NAT was 99.5 and 99.3% homologous to subtilisins E and Amylosacchariticus, respectively (3). It is also homologous to other members of the subtilisin family (BPNЈ 86% and Carlsberg 72%), and sequences are conserved especially around the three amino acids (serine 221, histidine 64, and aspartic acid 32) essential for the catalytic center of serine proteinases.The mechanism for this enzyme to potentiate fibrinolysis is not fully understood. Subtilisin NAT is reported not to possess plasminogen activator activity but appears to directly digest fibrin by limited proteolysis (4). However, this direct cleavage of fibrin does not seem to account for all of the enhancement of the fibrinolytic activity that has been observed without affecting the fibrinolytic cascade. To explore other possible mechanisms, we have looked for interactions between subtilisin NAT and the physiological inhibitors of fibrinolysis, plasminogen activator inhibitor type 1 (PAI-1) 1 (5) and ␣ 2 -antiplasmin (␣ 2 -AP) (6). These inhibitors are both members of the serine protease inhibitor superfamily (SERPINs). The SERPINs are proteolytically cleaved and inactivated by a variety of proteases including members of the subtilisin family (7).PAI-1 is the primary inhibitor of tissue-type plasminogen activator (tPA) and regulates fibrinolytic activity in the vasculature at the initia...

A Novel Antithrombotic Role for High Molecular Weight Kininogen as Inhibitor of Plasminogen Activator Inhibitor-1 Function

“…(54); (v) the enzyme subtilisin NAT purified from Bacillus subtilis (55). However, in these approaches the concept of destabilizing the complex between VN and PAI-1 was hardly considered.…”

Enhanced production of nattokinase from UV mutated Bacillus sp.

“…(Suzuki et al, 2003) found that dietary supplementation of Natto suppressed intimal thickening and modulated the lysis of mural thrombi after endothelial injury in rat femoral artery. Furthermore, NK can cleave active recombinant prokaryotic plasminogen activator inhibitor into low molecular weight fragments (Urano et al, 2001). Those researches imply that NK is useful as a promising thrombolytic agent (Sumi et al,1990).…”

Vitronectin in host pathogen interactions and antimicrobial therapeutic applications

“…Similarly, the role of PAI-1 in bacterial pathogenesis is also supported by the increased level of the plasminogen inhibitor during sepsis and other severe pathological conditions [21][22][23]. Some bacterial pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis cause degradation of PAI-1 during invasion [24][25][26]. Bacterial proteases also degrade Vn during invasion [27,28], but there is no information available on degradation of the PAI-1/Vn complex.…”