Characterization of nattokinase-degraded products from human fibrinogen or cross-linked fibrin

Fujita, Mitsugu; Ito, Yasuhiro; Hong, Kee-Jong; Nishimuro, Satoshi

doi:10.1016/s0268-9499(95)80005-0

Cited by 57 publications

(48 citation statements)

References 22 publications

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“…The effect was likely caused by direct digestion of fibrin by subtilisin NAT as previously demonstrated both in vitro (29) and in vivo (4). This is consistent with the fact that subtilisin NAT shows similar activity both in plasminogen-rich and plasminogen-poor fibrin plates.…”

Section: Discussionsupporting

confidence: 89%

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

Urano¹,

Ihara²,

Umemura

et al. 2001

Journal of Biological Chemistry

147

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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...

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Section: Discussionsupporting

confidence: 89%

“…It does not activate plasminogen but is reported to directly digest fibrin especially in its cross-linked form (29). In the present study we found a second mechanism by which subtilisin NAT enhances fibrinolysis through cleavage and inactivation of PAI-1.…”

Section: Discussionsupporting

confidence: 54%

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

Urano¹,

Ihara²,

Umemura

et al. 2001

Journal of Biological Chemistry

147

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“…However, the cleavage of cross-linked fibrin by nattokinase was six times more efficient than by plasmin. These results suggest that nattokinase is less sensitive to the cleavage by fibrinogen, but is more sensitive to the cleavage by cross-linked fibrin as compared to plasmin (Fujita et al 1995b). …”

Section: Soybean Fibrinolytic Activitymentioning

confidence: 88%

“…Natto extracts are known to include nattokinase, a potent fibrinolytic enzyme having approximately fourtimes stronger activity than plasmin in the clot lysis assay (Fujita et al 1993). It is composed of 275 amino acid residues (molecular weight, 28 KDa) and exhibits a high homology with subtilisins (Fujita et al 1995b). Large-molecule proteins usually are inactive because of nonspecific lysis in the digestive organs.…”

Section: Soybean Fibrinolytic Activitymentioning

confidence: 99%

Soybean Products Consumption in the Prevention of Cardiovascular Diseases

Palomo¹,

Guzmán²,

Leiva³

et al. 2011

Soybean and Health

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“…NK는 일종의 microbial serine protease로서 경구 투여 시 체내에 흡수되어 plasminogen activator로 작용하여 plasminogen을 plasmin으로 전 환시키고, 이 plamin이 응고혈액의 fibrin을 용해시켜 fibrinolytic activity을 갖는다 [12,28,37,38]. NK는 in vitro에서 직접적으로 closs-linked fibrin을 분해 할 뿐 아니라 tPA 생 성을 촉진하며 tPA의 상대적 비에 의해 총 혈전용해능을 조 절하고 혈전용해(fibrin clot lysis)의 1차 저해제인 plasminogen activator inhibitor-1 (PAI-1)의 불활성과 분해를 통하 여 혈전용해를 강화시킨다 [8,35]. 일본에서는 2)…”

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Effects of Nattokinase fibrinol supplementation on Fibrinolysis and Atherogenesis

Noh¹,

Park²,

Jang³

et al. 2009

Journal of Life Science

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-Effects of Nattokinase fibrinol (NKF), defined as a fibrinolytic product, on fibrinolytic and atherogenetic markers were studied for healthy adults (20-31 years old), who is smoking more than 20 cigarettes per day. Subjects were divided into 29 for NKF group and 10 for placebo group in a short term study. They were given 2 tablets of NKF (4,000 unit) or placebo tablet and thereafter blood samples were collected at 0, 2, 4 hr prerid. For a 4-week long term study, 15 subjects for NFK group and 10 subjects for placebo group were supplemented one tablet of each NKF (2,000 unit) and placebo per day, respectively. Blood samples were collected at 0, 1, 2, 4 weeks later. The short-term experimental trial showed that NKF remarkably increased fibrinolytic activity at 2hr after consumption, which was maintained up to 4 hr, relative to that of placebo, while NKF reduced the euglobulin clot lysis time (ECLT) and retarded the activated partial thromboplastin time (aPTT), as compared to placebo group. NKF supplementation for 4 weeks elevated fibrinolytic activity, shortened ECLT and retarded aPTT. Furthermore, NKF supplementation increased anti-atherogenic index by decreasing triglyceride (TG) and elevating high-density lipiprotein (HDL)-cholesterol. These results indicate that NKF supplementation for short term or long term might have beneficial effects on preventing and treating cardiovascular disease by increasing fibrinolytic activity and improving atherogenic markers such as hyperlipidemia.

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Characterization of nattokinase-degraded products from human fibrinogen or cross-linked fibrin

Cited by 57 publications

References 22 publications

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

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

Soybean Products Consumption in the Prevention of Cardiovascular Diseases

Effects of Nattokinase fibrinol supplementation on Fibrinolysis and Atherogenesis

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