The Profibrinolytic Enzyme Subtilisin NAT Purified fromBacillus subtilis Cleaves and Inactivates Plasminogen Activator Inhibitor Type 1

Tetsumei Urano,Hayato Ihara,Kazuo Umemura,Yasuhiro Suzuki,Masaki Oike,Sumio Akita,Yoshinori Tsukamoto,Isao Suzuki,Akikazu Takada

doi:10.1074/jbc.m101751200

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.

Highlights

We demonstrate an interaction between subtilisin NAT, a profibrinolytic serine proteinase from Bacillus subtilis, and plasminogen activator inhibitor 1 (PAI-1)
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 (␣2AP) [6]
In the present study we examine the possible interaction between subtilisin NAT and PAI-1 or ␣2-AP to clarify the mechanism for subtilisin NAT to enhance fibrinolytic activity

Summary

The mechanism for this enzyme to potentiate fibrinolysis is

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 (␣2AP) [6]. These inhibitors are both members of the serine protease inhibitor superfamily (SERPINs). The fibrinolytic activity determined by the balance between tPA and PAI-1 can be altered by changing the gene expression of either molecule under a variety of physiological or pathological conditions [13] Their balance could be altered as a consequence of the interaction of PAI-1 with serine proteases other than plasminogen activators in plasma [14]. We found that subtilisin NAT cleaved PAI-1 at the P1-P1Ј peptide bond by limited proteolysis, resulting in the effective enhancement of tPA-induced clot lysis of PAI-1-enriched fibrin

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION