A single-dose of oral nattokinase potentiates thrombolysis and anti-coagulation profiles

The nutritional components of natto: During the production process, the raw soybean is fermented to break down soy protein, increasing the digestibility of natto from 50% to 85%, making it easier to absorb. Natto has high nutritional value, with higher levels of protein, fiber, calcium, iron, vitamin B, vitamin PP, and vitamin E compared to steamed soybeans. In particular, the content of vitamin B Chemicalbook is more than 6 times higher than that of steamed soybeans. Natto also contains various substances such as protease, multiple vitamins, gamma glutamyl transpeptidase, gamma polyglutamic acid, etc. It also contains highly active substances such as nattokinase, isoflavones, soybean phospholipids, amylase, superoxide dismutase (SOD), saponin, tocopherol, pyridine dicarboxylic acid, vitamin K2, etc.

Abstract

Our aim was to determine the quantitative effects of a single-dose of Nattokinase (NK) administration on coagulation/fibrinolysis parameters comprehensively in healthy male subjects. A double-blind, placebo-controlled cross-over NK intervention study was carried out in 12 healthy young males. Following the baseline blood draw, each subject was randomized to receive either a single-dose of 2,000 FU NK (NSK-SD, Japan Bio Science Laboratory Co., Ltd) or placebo with subsequent cross-over of the groups. Subjects donated blood samples at 2, 4, 6 and 8 hours following administration for analysis of coagulation/fibrinolysis parameters. As a result, D-dimer concentrations at 6 and 8 hours and blood fibrin/fibrinogen degradation products at 4 hours after NK administration elevated significantly (p < 0.05, respectively). Factor VIII activity declined at 4 and 6 hours (p < 0.05, respectively), blood antithrombin concentration was higher at 2 and 4 hours (p < 0.05, respectively) and the activated partial thromboplastin time prolonged significantly at 2 and 4 hours following NK administration (p < 0.05 and p < 0.01, respectively). All the changes, however, were within the normal range. In conclusion, thus, a single-dose of NK administration appears enhancing fibrinolysis and anti-coagulation via several different pathways simultaneously.

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Introduction

Fibrinolytic enzymes have been derived from different microorganisms, including the genus bacillus which occurs in traditional fermented foods. Nattokinase (NK) is an enzyme contained in the sticky component of natto, a cheese-like food made of soybeans fermented with Bacillus subtilis. Natto has a long cultural history, extending back more than 1,000 years in Japan and the fibrinolytic mechanism of NK has been explored more extensively than of other microbial fibrinolytic enzyme¹. NK is a serine protease that has 275 amino acid residue with a molecular weight of approximately 28 kDa² and exhibits a high homology with subtilisins E (99.5%)³ and subtilisins J (Amylosacchariticus, 99.3%)⁴. Using a clot lysis assay in vitro, the cleavage of cross-linked fibrin by NK was 6 times more efficient than by plasmin as measured from kcat/Km⁵. At an equivalent molar ratio of doses (0.12 μmol/kg), NK is four times more effective than plasmin at dissolving a thrombus in rats in vivo⁶. These facts suggest that NK has strong fibrinolytic activity both in vitro and in vivo. In in vitro studies, NK not only directly cleaves cross-linked fibrin, but also activates the production of tissue-type plasminogen activator (tPA), resulting in the transformation of inactive plasminogen to active plasmin⁵. NK also enhances its fibrinolysis through cleavage and inactivation of plasminogen activator inhibitor-1 (PAI-1) in vitro, which is the primary inhibitor of tPA and regulates total fibrinolytic activity by its relative ratio with tPA⁷. Several animal studies also demonstrated the efficacy of NK on thrombolysis in vivo. Restoration of blood flow was directly proportional to the amount of NK injected into chemically induced thrombi in rats⁶. Another study demonstrated that blood circulation was recanalized completely after 5 hours of oral NK administration in dogs⁸. Although NK has been promoted as a most promising microorganism-derived enzyme for reducing thrombosis risk, the mechanism by which NK accelerates fibrinolysis has not been fully determined and the data in humans is still limited. The purpose of this double-blind, placebo-controlled cross-over study was to comprehensively determine the effects of a single-dose of NK administration on coagulation/fibrinolysis profile in healthy young Japanese.

Materials and Methods

The study was approved by the Institutional Review Board for Human Experiments, Ritsumeikan University, in accordance with the ethical principles contained in the Declaration of Helsinki. Written informed consent was obtained from all twelve participants. All subjects were healthy, young male Japanese (age; 22.3 ± 0.6 years old, Height; 169.3 ± 1.0 cm, body weight; 61.9 ± 1.5 kg, BMI; 21.6 ± 0.5, %body fat; 12.7 ± 0.9%) with no medication. None of the participants had a history of hematologic disease, symptoms of venous or arterial diseases. All subjects stated they had never taken NK supplement before and were not consuming food natto within 2 months before the experiments started. The same researchers performed all the procedures using identical techniques.

Experimental Procedures

A double-blind, placebo-controlled cross-over NK intervention study was carried out in twelve healthy subjects. Baseline blood samples were collected between the hours of 8:30 AM and 9:30 AM to minimize potential diurnal variations. Following the baseline blood draw, each subject was randomized to receive either a single-dose of 2,000 FU NK in a soft gel capsule form (NSK-SD, Japan Bio Science Laboratory Co., Ltd, Osaka, Japan) or soft gel capsule containing the placebo (P). Subjects donated blood samples at 2, 4, 6 and 8 hours following administration for coagulant/fibrinolysis parameters analysis. After a washout period more than 2 weeks, the second trial commenced with the alternate group assignment. During the experiment, the time and quantity of water and caloric intake were identical in both groups (Fig. 1). No side effects were declared.

Figure 1

Results

In this study, we measured each blood parameter until 8 hours after administration, in reference to our preliminary trials which represented all the average peak values in blood appeared within 8 hours after NK intake in 6 young males.

D-dimer, the subunit of the specific degradation products of cross-linked fibrin and FDP elevated following NK administration (+44.5 ± 12.9% for 6 hours, +38.2 ± 19.2% for 8 hours in D-dimer, +21.2 ± 6.3% for 4 hours in FDP compared with baseline values, p < 0.05 for placebo, respectively, Fig. 2A,B). The aPTT prolonged significantly at 2 and 4 hours following NK supplementation (p < 0.05 and p < 0.01, respectively, Fig. 2D). Factor VIII activity declined at 4 and 6 hours following NK intake (−7.4 ± 1.9% and −7.6 ± 1.6% on average compared with baseline data, p < 0.05 for placebo, respectively, Fig. 2E). Antithrombin concentration increased significantly after NK administration (p < 0.05 for 2 and 4 hours, Fig. 2F). All the blood data changed after NK administration, however, were within the normal range. No differences were observed in any other parameters between NK and placebo groups at any time points before and after supplementation (Fig. 2C, Table 1).

Table 1 Blood parameters before and after a 2,000 F.U. of NK administration or placebo in twelve healthy young males

Full size table

Figure 2

Discussion

This study provides the first evidence of NK’s ability to enhance fibrinolysis and antithrombosis contemporaneously after a single-dose of oral NK administration in human.

Bioavailability

NK is effectively absorbed across the rat intestinal tract inducing fibrinolysis after intraduodenal administration⁶. Recently, Ero and colleagues presented the first bioavailability data of NK in human by enzyme-linked immunosorbent assay⁹. Following 2,000 FU NK administration (the same amount as in our current study), they demonstrated NK serum activity between 2 through to 24 hours in healthy subjects. Our data, which confirmed an increase in activity of fibrinolysis and anticoagulant parameters between 2 and 8 hours after NK intake, is consistent with their results.

Fibrinolysis parameters

NK’s fibrinolytic potential was first identified in vitro study using fibrin plate¹⁰. The NK had 6 times stronger activity in vitro⁵ and approximately 4 times greater efficiency for cleavage of cross-linked fibrin as compared with plasmin in rats⁶. Of note, among subtilisins, only NK demonstrates high substrate specificity for fibrin¹, despite high homology^2,5. In this study, we found that a single-dose of NK administration enhances fibrinolysis via cleavage of cross-linked fibrin and its effect lasted for a relatively long period of time (over 8 hours), compared with tissue-type plasminogen activator’s (t-PA) and/or urokinase’s 4–20 minutes half-life in human blood.

Coagulation parameters

The decline of factor VIII activity after NK intake were similar with previous data in which three different subject groups (healthy individuals, patients with cardiovascular risk factors and patients undergoing dialysis) took 4,000 FU of NK daily for 2 months and factor VIII antigen declined significantly after 2 month of NK intake in all three groups¹¹. The precise mechanism of this NK action is not yet clarified. However, to the extent that elevation of Factor VIII level is known to be risk factors for cardiovascular and related diseases¹², we propose here further potential for acute effects of NK to reduce the risk of thrombosis.

In this study, we found NK administration increased blood antithrombin concentration after 2 and 4 hours of administration. To the best of our knowledge, this is a novel pathway of NK’s antithrombotic action and there is no data available to reveal the precise mechanism how NK enhances blood antithrombin levels. Only few studies were published which demonstrated soybean fermented with basidiomycete facilitated antithrombotic activity indirectly via elongation of prothrombin time, however, the mechanism is still unknown¹³. Esmon, et al. emphasize that there is a strong molecular links between inflammation and coagulation and this crosstalk creates a cycle that progresses to vascular injury as occurred in septic shock¹⁴. Thrombin, Factor Xa and tissue factor are the main targets for the treatment of this disease. Antithrombin is one of the most important physiological regulators for blood coagulation cascade, with the role of inhibition of thrombin, factors Xa and IXa¹⁵. In fact, antithrombin activity after treatment of antithrombin administration was the predictor of prognosis in patients with septic disseminated intravascular coagulation¹⁶. Antithrombin’s anti-inflammatory effects via elevation of cyclic AMP^17,18, therefore, NK administration might improve prognosis in patients who suffer from infectious or other inflammatory diseases.

All drugs currently approved and/or under clinical investigation for treatment of thrombolysis function as plasminogen activators, which lead to proteolytic degradation of fibrin clots mediated by plasmin alone. However, NK activates multiple fibrinolytic and anti-thrombotic pathways simultaneously, either directly or indirectly (Fig. 3). In general, a thrombolytic agent requires small amount and relatively short period of administration for treatment. Based on NK’s unique, comparatively strong fibrinolytic/anticoagulant activity, stability in the gastrointestinal tract and long bioavailability in vivo, NK would appear to offer potential advantages over other currently used agents for treatment and/or prevention of selected diseases processes. Previous study also demonstrated the efficacy of oral NK administration on reduction of systolic/diastolic blood pressure in 73 subjects with pre-hypertension/stage 1 hypertension after 8 weeks of NK intake 2,000 FU daily¹⁹. Furthermore, velocity of arterial blood flow after acute exercise was enhanced accompanied by NK supplementation in healthy participants²⁰. Thus, NK might have an impact on not only fibrinolytic/anticoagulant pathways but also other risk factors for thrombosis, which imply as a NK’s possibility for prevention and/or treatment of the diseases.

Figure 3

Additional Information

How to cite this article: Kurosawa, Y. et al. A single-dose of oral nattokinase potentiates thrombolysis and anti-coagulation profiles. Sci. Rep. 5, 11601; doi: 10.1038/srep11601 (2015).

References

Dabbagh, F. et al. Nattokinase: production and application. Appl Microbiol Biotechnol. 98, 9199–9206 (2014).

Article CAS Google Scholar

Fujita, M. et al. Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan. Biochem Biophys Res Commun. 197, 1340–1347 (1993).

Article CAS Google Scholar

Stahl, M. L. & Ferrari, E. Replacement of the Bacillus subtilis subtilisin structural gene with an in vitro-derived deletion mutation. J Bacteriol. 158, 411–418 (1984).

CAS PubMed PubMed Central Google Scholar

Kurihara, M., Markland, F. S. & Smith, E. L. Subtilisin Amylosacchariticus. 3. Isolation and sequence of the chymotryptic peptides and the complete amino acid sequence. J Biol Chem. 247, 5619–5631 (1972).

CAS PubMed Google Scholar

Fujita, M., Ito, Y., Hong, K. & Nishimuro, S. Characterization of nattokinase-degradaded products from human fibrinogen or cross-linked fibrin. Fibrinolysis. 9, 157–164 (1995).

Article CAS Google Scholar

Fujita, M. et al. Thrombolytic effect of nattokinase on a chemically induced thrombosis model in rat. Biol Pharm Bull. 18, 1387–1391 (1995).

Article CAS MathSciNet Google Scholar

Urano, T. et al. The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis Cleaves and inactivates plasminogen activator inhibitor type 1. J Biol Chem. 276, 24690–24696 (2001).

Article CAS Google Scholar

Sumi, H., Hamada, H., Nakanishi, K. & Hiratani, H. Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase. Acta Haematol. 84, 139–143 (1990).

Article CAS Google Scholar

Ero, M. P., Ng, C. M., Mihailovski, T., Harvey, N. R. & Lewis, B. H. A pilot study on the serum pharmacokinetics of nattokinase in humans following a single, oral, daily dose. Altern Ther Health Med. 19, 16–19 (2013).

PubMed Google Scholar

Sumi, H., Hamada, H., Tsushima, H., Mihara, H. & Muraki, H. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet. Experientia. 43, 1110–1111 (1987).

Article CAS Google Scholar

Hsia, C. H. et al. Nattokinase decreases plasma levels of fibrinogen, factor VII and factor VIII in human subjects. Nutr Res. 29, 190–196 (2009).

Article CAS Google Scholar

Grant, P. J. Diabetes mellitus as a prothrombotic condition. J Intern Med. 262, 157–172 (2007).

Article CAS ADS Google Scholar

Fukuda, S., Matsui, T., Tachibana, H., Tomoda, T. & Ohsugi, M. Development of functional soybean foods produced by fermentation with mushroom mycelia. Bull Mukogawa Women’s Univ Nat Sci. 55, 53–59 (2007).

CAS Google Scholar

Esmon, C. T. et al. Inflammation, sepsis and coagulation. Haematologica. 84, 254–259 (1999).

CAS PubMed Google Scholar

Butenas, S. & Mann, K. G. Blood coagulation. Biochemistry (Mosc). 67, 3–12 (2002).

Article CAS Google Scholar

Iba, T., Saitoh, D., Gando, S. & Thachil, J. The usefulness of antithrombin activity monitoring during antithrombin supplementation in patients with sepsis-associated disseminated intravascular coagulation. Thromb Res. (2015) pii:S0049-3848(15)00113-9. 10.1016/j.thromres.2015.03.007.

Taylor, F. B., Emerson, T. E., Jordan, R., Chang, A. K. & Blick, K. E. Antithrombin-III prevents the lethal effects of Escherichia coli infusion in baboons. Circ Shock. 26, 227–235 (1988).

CAS PubMed Google Scholar

Ji, H. et al. Mechanisms of Nattokinase in protection of cerebral ischemia. Eur J Pharmacol. 745, 144–1451 (2014).

Article CAS Google Scholar

Kim, J. Y. et al. Effects of nattokinase on blood pressure: a randomized, controlled trial. Hypertens Res. 31, 1583–1588 (2008).

Article CAS Google Scholar

Henry, L., Stevens, M. & Shore, S. Effects of nattokinase on blood flow and blood composition after cute exercise. Cardiopulmonary Physical Therapy J. 19, 138–139 (2008).

Article Google Scholar

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Acknowledgements

The authors thank Dr. Thomas A. Tomsick (University of Cincinnati Neuroscience Institute, Ohio, USA) for his scientific advice. This work was supported by funding from the Grant-in-Aid for Scientific Research (KAKENHI) Japan.

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Authors and Affiliations

Department of Sport and Health Science, Ritsumeikan University, Kusatsu, Shiga, Japan

Yuko Kurosawa, Shinsuke Nirengi, Toshiyuki Homma & Takafumi Hamaoka

Department of Economics, Tokuyama University, Shunan, Yamaguchi, Japan

Kazuki Esaki

Department of Clinical Chemistry, Kobe Pharmaceutical University, Kobe, Hyogo, Japan

Mitsuhiro Ohta

Department of Neurology, University of Cincinnati, Cincinnati, Ohio, USA

Joseph F. Clark

Contributions

Y.K., M.O. and T.H. designed the research. Y.K., S.N., T.H. and K.E. performed the literature search and analyzed data. Y.K., M.O. and T.H. reviewed the articles for inclusion. Y.K. wrote the manuscript. J.F.C. and T.H. interpreted data and critically reviewed the manuscript. All authors revised the manuscript and approved the final version.

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Kurosawa, Y., Nirengi, S., Homma, T. et al. A single-dose of oral nattokinase potentiates thrombolysis and anti-coagulation profiles. Sci Rep 5, 11601 (2015). https://doi.org/10.1038/srep11601

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Received28 August 2014

Accepted29 May 2015

Published25 June 2015

DOIhttps://doi.org/10.1038/srep11601

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