Effects of nattokinase, a pro-fibrinolytic enzyme, on red blood cell aggregation and whole blood viscosity
The vegetable cheese-like food, natto, is extremely popular in Japan with a history extending back over 1000 years. A fibrinolytic enzyme, termed nattokinase, can be extracted from natto; the enzyme is a subtilisin-like serine protease composed of 275 amino acid residues and has a molecular weight of 27.7 kDa. In vitro and in vivo studies have consistently demonstrated the potent pro-fibrinolytic effect of the enzyme. However, no studies to date have evaluated the effects of nattokinase on various hemorheological parameters and thus we have begun to assess the effects of the enzyme on RBC aggregation and blood viscosity. Blood samples were incubated with nattokinase (final activities of 0, 15.6, 31.3, 62.5 and 125 units/ml) for 30 minutes at 37 degrees C. RBC aggregation was measured using a Myrenne MA-1 aggregometer and blood viscosity assessed over 1-1000 s(-1) with a computer controlled scanning capillary rheometer (Rheolog). Our in vitro results showed a significant, dose-dependent decrease of RBC aggregation and low-shear viscosity, with these beneficial effects evident at concentrations similar to those achieved in previous in vivo animal trials. Our preliminary data thus indicate positive in vitro hemorheological effects of nattokinase, and suggest its potential value as a therapeutic agent and the need for additional studies and clinical trials.
Clinical Hemorheology and Microcirculation 35 (2006) 139–142 139
Effects of nattokinase, a pro-ﬁbrinolytic
enzyme, on red blood cell aggregation and
whole blood viscosity
Eszter Pais a, Tamas Alexy a,∗, Ralph E. Holsworth, Jr. band Herbert J. Meiselman a
aDepartment of Physiology and Biophysics, Keck School of Medicine, University of Southern
California, Los Angeles, CA 90033, USA
bN-ZymeCeuticals Inc, Pagosa Springs, CO 81147, USA
Abstract. The vegetable cheese-like food, natto, is extremely popular in Japan with a history extending back over 1000 years.
A ﬁbrinolytic enzyme, termed nattokinase, can be extracted from natto; the enzyme is a subtilisin-like serine protease composed
of 275 amino acid residues and has a molecular weight of 27.7 kDa. In vitro and in vivo studies have consistently demonstrated
the potent pro-ﬁbrinolytic effect of the enzyme. However, no studies to date have evaluated the effects of nattokinase on
various hemorheological parameters and thus we have begun to assess the effects of the enzyme on RBC aggregation and
blood viscosity. Blood samples were incubated with nattokinase (ﬁnal activities of 0, 15.6, 31.3, 62.5 and 125 units/ml) for
30 minutes at 37◦C. RBC aggregation was measured using a Myrenne MA-1 aggregometer and blood viscosity assessed over
1–1000 s−1with a computer controlled scanning capillary rheometer (Rheolog R
). Our in vitro results showed a signiﬁcant,
dose-dependent decrease of RBC aggregation and low-shear viscosity, with these beneﬁcial effects evident at concentrations
similar to those achieved in previous in vivo animal trials. Our preliminary data thus indicate positive in vitro hemorheological
effects of nattokinase, and suggest its potential value as a therapeutic agent and the need for additional studies and clinical trials.
Keywords: Nattokinase, red blood cell, aggregation, hemorheology
Cardio- and cerebrovascular diseases are the leading cause of morbidity and mortality in the devel-
oped nations and pose a major burden to developing countries as well. It is notable, however, that age-
standardized morbidity rates vary widely between populations. Data from the World Health Organization
indicate that people in western cultures have a signiﬁcantly higher risk of developing a thrombotic event
than those in Asian countries, with cardiovascular mortality rates much lower in Japan than in the west-
ern countries . Several studies have emphasized the importance of disparities in genetic background,
as well as conventional risk factors including dietary habits, as the basis for the observed geographical
Soybean-fermented foods, such as the Korean chunggok-jang, the Chinese dou-shi and the Japanese
natto have served a major dietary role in these countries for at least 1000 years. Of particular interest is
the vegetable cheese natto, which is produced from boiled soybeans by fermentation using Bacillus sub-
tilis natto. Natto has been widely consumed in Japan for its popular, characteristic taste and has also been
*Corresponding author: Dr. Tamas Alexy, Dept. Physiology and Biophysics, Keck School of Medicine, 1333 San Pablo
Street, MMR 230, Los Angeles, CA 90033, USA. Tel.: +1 323 442 1267; Fax: +1 323 442 1617; E-mail: firstname.lastname@example.org.
1386-0291/06/$17.00 2006 – IOS Press and the authors. All rights reserved
140 E. Pais et al. / Nattokinase and RBC aggregation
utilized as a folk remedy to relieve fatigue, treat the symptoms of beri-beri, and to prevent cardiovascular
diseases. Sumi appears to have been the ﬁrst to recognize the ability of natto to dissolve ﬁbrin clots ;
he isolated and puriﬁed the enzyme and named it nattokinase rather than its previous designation as
Bacillus subtilis protease. Natto is a serine endopeptidase composed of 275 amino acid residues with
a molecular weight of 27.7 kDa, and exhibits a high molecular homology with subtilisins . It has
been shown to have an approximately four-times stronger ﬁbrinolytic activity than plasmin in clot lysis
assays . In addition to its direct effect on ﬁbrin, the enzyme promotes the conversion of plasminogen
to plasmin  and inactivates plasminogen activator inhibitor type-1 (PAI-1) by limited proteolysis of
its reactive site . Functionally active enzyme has been reported to be absorbed from the rat intestinal
tract following intra-duodenal administration . These latter observations suggest the possibility that
oral use of nattokinase could serve as a potent natural agent supporting existing thrombolytic remedies.
2. Previous in vitro and in vivo studies of nattokinase treatment
The pro-ﬁbrinolytic effects of nattokinase have been tested in over a dozen in vitro and in vivo studies,
including two small-case human trials. Sumi and colleagues induced blood clots in male dogs under
experimental conditions. Following the procedure, the animals orally received either 1000 mg natto-
kinase as capsules (Group 1) or placebo capsules (Group 2). Angiograms revealed that reperfusion oc-
curred within ﬁve hours of treatment in all animals in Group 1, while no clot lysis could be observed
in Group 2 . Japanese researchers compared the efﬁcacy of nattokinase and plasmin in dissolving
carotid artery thrombi in rats; a signiﬁcantly higher percentage of the original blood ﬂow was regained
in the natto treated group (62.0% vs. 15.8%) . In a human crossover study, 12 healthy Japanese vol-
unteers received 200 g of natto cheese and the ﬁbrinolytic activity of their plasma serially measured.
The orally consumed natto signiﬁcantly enhanced the ability of plasma factors to dissolve blood clots
for up to 2–8 hours. After the ﬁbrinolytic activity of the volunteer’s plasma returned to control, the sub-
jects received boiled soybeans with no enzyme activity; serial measurements in this situation indicated
that the ﬁbrinolytic activity of their plasma was essentially unchanged . Recent reports have also
indicated the beneﬁcial effects of nattokinase in hypertension: human and animal studies conﬁrmed that
both systolic and diastolic blood pressures are reduced following the oral administration of the enzyme,
with average decreases of 10.9% (systolic) and 9.7% (diastolic) . Since deep vein thrombosis and
edema development in the lower extremities are important issues for passengers during long ﬂights, an
international study was conducted in order to evaluate the value of nattokinase among high-risk individ-
uals: orally administered enzyme was found to signiﬁcantly reduce swelling of the extremities and the
incidence of deep vein thrombosis .
As noted above, several trials have been conducted to explore and evaluate the pro-ﬁbrinolytic prop-
erties of orally administered nattokinase. However, there do not appear to be published studies that have
assessed possible effects of the enzyme on various hemorheological parameters. We have thus begun to
examine its in vitro rheologic effects and present herein a brief overview of our work to date.
3. Effects of nattokinase on hemorheological parameters
Increasing evidence from several investigations indicates signiﬁcant changes in the rheologic behav-
ior of pathologic blood [3,5,11]. Most importantly, hemorheological parameters, such as high ﬁbrinogen
levels, enhanced RBC aggregation, and elevated blood and plasma viscosity, have been shown to be
E. Pais et al. / Nattokinase and RBC aggregation 141
associated with the development and progression of cardiovascular diseases [6,12,14,15]. Hence, favor-
able modiﬁcation of these factors might help to reduce the incidence of serious adverse clinical events.
Our preliminary in vitro experiments therefore focused on the effects of nattokinase on red blood cell
aggregation and whole blood viscosity proﬁles.
Blood was drawn from healthy adult individuals by sterile antecubital venipuncture into vacuum tubes
containing EDTA (1.5 mg/ml). Blood samples were centrifuged at 1750 ×gfor 10 minutes, following
which RBC and autologous plasma were recombined to achieve a 41% hematocrit. Lyophilized nattok-
inase powder was dissolved in phosphate buffered saline (PBS) at various concentrations and added to
aliquots of the 41% RBC +plasma suspensions to yield samples having a constant hematocrit of 40%
and the following ﬁnal enzyme activities: 0 (PBS dilution control), 15.6, 31.3, 62.5 and 125 units/ml.
The samples were incubated and gently stirred for 30 minutes at 37◦C. RBC aggregation was assessed
at room temperature using a Myrenne MA-1 erythrocyte aggregometer (Myrenne GmbH, Roetgen, Ger-
many). Blood viscosity proﬁles were measured at 37◦C over the shear rate range of 1–1000 s−1with a
newly developed, computer controlled scanning capillary rheometer (Rheolog R
Our in vitro results indicated signiﬁcant, dose-dependent decreases of erythrocyte aggregation with
enzyme treatment: at the activities employed (i.e., 15.6, 31.3, 62.5 and 125 units/ml), mean decreases
from control were 21.9%, 25.9%, 49.7% and 62.0%. Thus, at the highest level utilized, nattokinase di-
minished RBC aggregation remarkably. Importantly, reduced erythrocyte aggregation could be achieved
at concentrations similar to the serum levels achieved in previous animal trials . These aggregation
data were conﬁrmed via light microscopy, which indicated a dose-dependent decrease of aggregate size.
Blood viscosity was also affected by nattokinase, with the greatest effects observed at the lower shear
rates; minimal or no changes of high shear viscosity were observed.
Our preliminary in vitro results to date indicate that nattokinase has marked, dose-dependent hemorhe-
ological effects, including reduced RBC aggregation and low shear viscosity. Given that ﬁbrinogen is
probably the most important plasma protein promoting RBC aggregation and rouleaux formation, our
experiments seem to conﬁrm the potent ﬁbrinogenolytic activity of nattokinase. That is, in addition to its
enzymatic action on ﬁbrin (i.e., ﬁbrinolytic activity), it also affects soluble plasma ﬁbrinogen. In turn,
reduced ﬁbrinogen levels result in decreased RBC aggregation.
Although our ﬁndings suggest a possible therapeutic role of nattokinase as an orally active hemorhe-
ological agent, several additional studies are required. Among these are an evaluation of the enzyme’s
possible effects on RBC properties (e.g., rigidity, surface charge, glycocalyx structure) and plasma vis-
cosity. Detailed studies of its ﬁbrinogenolytic actions and its potential ability to generate ﬁbrinogen
degradation products should also be evaluated. In vivo studies, including quantitative microcirculatory
measurements and relations between oral dose and enzyme plasma levels will be important for under-
standing its physiological effects. Finally, clinical trials will be required in order to determine possible
beneﬁcial outcomes associated with nattokinase administration.
Supported by NIH Research Grants HL 15722 and HL 70595. Dr. Pais received salary stipend support
from N-ZymeCeuticals Inc.
142 E. Pais et al. / Nattokinase and RBC aggregation
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