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

We aimed to comprehensively determine the quantitative effects of a single dose of Nattokinase (NK) administration on coagulation/fibrinolysis parameters in healthy male subjects. A double-blind, placebo-controlled cross-over NK intervention study was conducted 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 a 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 to enhance fibrinolysis and anti-coagulation via several different pathways simultaneously.

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Introduction

Fibrinolytic enzymes are derived from various microorganisms, including those from the genus Bacillus, which are present in traditional fermented foods. One notable enzyme is nattokinase (NK), found in the sticky component of natto—a cheese-like food made from soybeans fermented with Bacillus subtilis. Natto has a rich cultural history, dating back over 1,000 years in Japan. The fibrinolytic mechanism of NK has been studied more extensively than that of other microbial fibrinolytic enzymes.

Nattokinase is a serine protease comprised of 275 amino acid residues, with a molecular weight of approximately 28 kDa. It exhibits a high degree of similarity to subtilisins E (99.5%) and subtilisins J (Amylosacchariticus, 99.3%). In vitro clot lysis assays show that NK cleaves cross-linked fibrin six times more efficiently than plasmin, as measured by kcat/Km. At an equivalent molar dose (0.12 μmol/kg), NK is four times more effective than plasmin at dissolving thrombi in rats. These findings indicate that NK possesses robust 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), transforming inactive plasminogen into active plasmin. Additionally, NK enhances fibrinolysis by cleaving and inactivating plasminogen activator inhibitor-1 (PAI-1), which is the primary inhibitor of tPA. This regulation affects overall fibrinolytic activity based on the relative ratio of PAI-1 to tPA.

Several animal studies have also demonstrated the efficacy of NK in thrombolysis. The restoration of blood flow in rats was directly proportional to the amount of NK injected into chemically induced thrombi. Another study illustrated that blood circulation was completely restored after 5 hours of oral NK administration in dogs.

Although NK has been recognized as a promising enzyme derived from microorganisms for reducing the risk of thrombosis, the precise mechanisms by which it accelerates fibrinolysis remain not fully understood, and data regarding its effects in humans are still limited. The purpose of this double-blind, placebo-controlled crossover study was to comprehensively assess the effects of a single dose of NK administration on the coagulation and fibrinolysis profiles in healthy young Japanese individuals.

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

blood coagulation cascade by inhibiting thrombin, Factor Xa, and Factor IXa. Notably, after antithrombin treatment, the activity of antithrombin was a predictor of prognosis in patients with septic disseminated intravascular coagulation. Moreover, antithrombin exhibits anti-inflammatory effects through the elevation of cyclic AMP. Therefore, NK administration may improve outcomes in patients facing infectious or inflammatory diseases.

Currently approved drugs and those under clinical investigation for thrombolysis primarily act as plasminogen activators, leading to the proteolytic degradation of fibrin clots through plasmin alone. In contrast, NK activates multiple fibrinolytic and antithrombotic pathways simultaneously, either directly or indirectly. Generally, thrombolytic agents require small doses and relatively short treatment periods. Given NK's unique ability to provide strong fibrinolytic and anticoagulant activity, alongside its stability in the gastrointestinal tract and prolonged bioavailability in vivo, it holds potential advantages over conventional agents for the treatment and prevention of specific disease processes.

Previous studies have also demonstrated the efficacy of oral NK administration in reducing both systolic and diastolic blood pressure in 73 subjects with pre-hypertension or stage 1 hypertension after eight weeks of consuming 2,000 FU daily. Furthermore, the velocity of arterial blood flow after acute exercise improved with NK supplementation in healthy participants. Thus, NK may influence not only fibrinolytic and anticoagulant pathways but also other risk factors for thrombosis, suggesting its potential for the prevention and treatment of various 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).

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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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