- Fibrin – Directly hydrolyzes fibrin and plasmin substrate [1]
- Plasminogen activator inhibitor-1 (PAI-1) – Degrades PAI-1 [1]
- Prourokinase – Converts endogenous prourokinase to urokinase (uPA) [1]
- Tissue plasminogen activator (t-PA) – Increases t-PA levels [1]
Nattokinase targets fibrin, the insoluble protein matrix of blood clots. It is a potent fibrinolytic enzyme that breaks down blood clots by directly hydrolyzing fibrin and plasmin substrates. Nattokinase also degrades plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue plasminogen activator (tPA), thereby promoting fibrinolysis. It inhibits platelet aggregation and has anticoagulant properties.

- Nattokinase exhibits strong fibrinolytic activity; it can dissolve artificial fibrin and degrade plasmin substrate [1]
- Nattokinase from Pseudomonas aeruginosa CMSS UV60 mutant showed 94% blood clot lysis at 10 minutes in an in vitro blood clot lysis assay [2]
- Partially purified nattokinase (70% ammonium sulfate precipitate) from mutant UV60 strain showed maximum fibrin clot liquefaction after 2 hours of incubation at 37°C in fibrin plate assay [2]
- Casein digestion assay: nattokinase activity was determined using 0.1 mL of 2% casein, 0.7 mL of 0.1 M sodium phosphate buffer and 0.1 mL of enzyme, incubated for 5 minutes at room temperature; reaction terminated with 0.1 mL of 1.5 M trichloroacetic acid; absorbance measured at 560 nm; one unit of caseinolytic activity defined as amount of enzyme releasing 1 μM tyrosine equivalent per minute [2]
- Fibrin degradation assay (modified fibrin plate method): Petri dishes containing 9 mL of 0.2% fibrinogen solution with 0.2 mL plasminogen (10 U) and 0.2 mL thrombin (20 U) incubated at 37°C for 15 minutes to form clot; enzyme solution placed as droplets on fibrin clot surface; incubated at 37°C for 2 hours; visually inspected for liquefaction [2]
- SDS-PAGE confirmed molecular mass of nattokinase from P. aeruginosa CMSS UV60 as 21 kDa [2]

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Ninety-four percent of blood clots disintegrate in ten minutes when nattokinase is added, and fibrin clots breakdown after two hours [2].

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In vitro, nattokinase is a potent fibrinolytic enzyme that can break down blood clots by directly hydrolyzing fibrin and plasmin substrates. It dissolves 94% of blood clot at ten minutes and degrades the fibrin clot after two hours of incubation. Nattokinase exhibits potent fibrinolytic activity and is used for research on cardiovascular diseases. Its ability to degrade fibrin and PAI-1 makes it a valuable tool for studying thrombosis and fibrinolysis.

- In a rat model of chemically-induced thrombosis (carrageenan injection into toes), oral administration of nattokinase (150 mg/kg or 250 mg/kg twice daily for 2 days) resulted in thrombolysis; high-dose nattokinase (250 mg/kg) reduced thrombus area to 38.76% of vessel cross-section (compared to 62.68% in negative controls); low-dose nattokinase (150 mg/kg) reduced thrombus area to 47.15% [3]
- High-dose nattokinase (250 mg/kg) significantly increased plasma FDP levels (48.60 ± 7.10 pg/mL in low-dose, higher in high-dose) compared to saline controls (15.10 ± 4.38 pg/mL, p < 0.05); D-dimer levels were also increased (32.51 ± 2.11 ng/mL in low-dose vs 29.14 ± 0.23 ng/mL in controls, p > 0.05 for low-dose; significant for high-dose) [3]
- High-dose nattokinase showed comparable thrombolytic effect to vermis kinase (positive control, 200 mg/kg) with no significant difference in FDP or D-dimer levels (p > 0.05) [3]
- In dogs, oral administration of four nattokinase capsules (2000 FU/capsule) completely dissolved chemically-induced thrombi in the major leg vein within five hours and restored normal blood flow [1]
- In a rat common carotid artery thrombosis model, nattokinase-treated rats recovered 62% of arterial blood flow; this was considerably stronger than plasmin (15% recovery) or elastase (0% recovery) [1]
- Nattokinase inhibited ferric chloride (FeCl₃)-induced oxidative thrombosis and platelet aggregation; effects similar to aspirin but without bleeding or gastric ulcer adverse effects [1]
- In a κ-carrageenan-induced inflammatory thrombus model in rat tails, 12 hours after gavage administration of nattokinase, higher levels of FDP fragments and D-dimers were detected; greater than 50% decrease in thrombosis was observed in blood vessels by biopsy analysis [1]
- In a human trial, healthy volunteers, patients with cardiovascular risk factors, and patients undergoing dialysis orally administered two capsules of nattokinase (2000 FU/capsule) daily for two months showed significant decrease in factor VII, factor VIII, and fibrinogen; no adverse effects detected; heart rate, body weight, and uric acid levels remained stable [1]
- In 12 healthy young males, a single oral capsule of nattokinase (2000 FU) resulted in significantly increased antithrombin concentration at 2 hours; FDP fragments observed at 4 hours; D-dimers observed at 6 hours; factor VIII activity declined at 4 hours [1]
- Nattokinase can be absorbed from the rat intestinal tract in intact form and degrade fibrinogen in plasma [1]
- In healthy humans, intact nattokinase was detected in serum after a single oral dose (2000 FU/100 mg) in a capsule [1]

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In vivo, nattokinase (150–250 mg/kg; orally administered twice daily for two days) demonstrates fibrinolytic activity and breaks up blood clots[3].

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In vivo, nattokinase exhibits oral bioavailability and fibrinolytic/anticoagulant properties. It degrades fibrin and PAI-1 to dissolve thrombi while inhibiting platelet aggregation. Nattokinase may be utilized to study cardiovascular disease, including thrombosis, atherosclerosis, and stroke. It has also been investigated for its potential in Alzheimer's disease research. Nattokinase is classified as an investigational compound with ongoing clinical evaluation, including clinical trial NCT00749801 assessing its efficacy and safety.

- Casein digestion assay: Nattokinase activity was determined using 0.1 mL of 2% casein, 0.7 mL of 0.1 M sodium phosphate buffer and 0.1 mL of partially purified enzyme or crude extract, incubated for 5 minutes at room temperature. The reaction was terminated by adding 0.1 mL of 1.5 M trichloroacetic acid and centrifuged at 8,000 rpm for 10 minutes. Absorbance of the supernatant was measured at 560 nm. One unit of caseinolytic activity (U) was defined as the amount of enzyme releasing 1 μM of tyrosine equivalent per minute [2]
- Fibrin degradation assay (modified fibrin plate method): Petri dishes containing 9 mL of 0.2% fibrinogen solution were placed on a horizontal glass plate. 0.2 mL of plasminogen (10 U) was added and mixed well. Clotting was induced by the addition of 0.2 mL of thrombin solution (20 U). Plates were incubated at 37°C for 15 minutes to speed up clotting. A known quantity of enzyme solution was placed as small droplets on the surface of the fibrin clot. Plates were then incubated at 37°C for two hours and visually inspected for liquefaction [2]
- Blood clot lysis assay: Venous blood (500 μL) was transferred to pre-weighed sterile microcentrifuge tubes and incubated at 37°C for 45 minutes to form clots. Serum was aspirated, and tubes with clots were reweighed to determine clot weight. 100 μL of crude enzyme or ammonium sulfate precipitated enzyme was added to clots. Tubes were incubated at 37°C for 90 minutes and observed for clot lysis. After incubation, fluid was removed and tubes reweighed. Weight difference before and after clot lysis was expressed as percentage of clot lysis [2]

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In vitro enzyme assays for nattokinase typically involve measuring its fibrinolytic activity using fibrin plate assays or chromogenic substrate assays. In a fibrin plate assay, the enzyme is applied to a fibrin-containing agar plate, and the diameter of the zone of lysis is measured. In chromogenic substrate assays, nattokinase is incubated with a synthetic peptide substrate (e.g., S-2251, a plasmin substrate), and the release of p-nitroaniline is measured spectrophotometrically. The enzyme's ability to degrade PAI-1 can be assessed using Western blot or activity assays.

In vitro cell-based assays for nattokinase involve culturing cells (e.g., endothelial cells, smooth muscle cells) and treating them with the enzyme to assess effects on fibrinolysis, cell migration, or inflammatory responses. The enzyme's ability to degrade fibrin clots can be assessed in cell culture models of thrombosis. Cytotoxicity is assessed using MTT or LDH release assays. The effects of nattokinase on platelet aggregation can be assessed using platelet-rich plasma and aggregometry.

Animal/Disease Models: SD (Sprague-Dawley) rats (180-220 g) are injected κ-Carrageenan[3]
Doses: 150, 250 mg/kg
Route of Administration: po (oral gavage) twice (two times) daily for 2 days
Experimental Results: Increased the plasma concentration of fibrin/ fibrinogen degradation products (FDPs) and D-Dimer in a dose-dependent manner. diminished the fraction of the vessel cross section occupied by thrombosis.

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- Rat thrombosis model (carrageenan-induced): Healthy female Sprague-Dawley rats (180-220 g, tail length >13 cm) were fasted for 8 hours. κ-Carrageenan was dissolved in saline (4 mg/L) and injected subcutaneously into toes at a concentration of 2.0 mg/kg. Approximately 12 hours later, tail appearance was observed. Rats were randomly divided into 4 groups (n=6 per group): low-dose nattokinase (150 mg/kg by gavage twice daily for 2 days), high-dose nattokinase (250 mg/kg by gavage twice daily for 2 days), positive control (vermis kinase 200 mg/kg by gavage twice daily for 2 days), and negative control (saline by gavage twice daily for 2 days). Twelve hours after the last administration, rats were anesthetized with 20% urethane (5 mL/kg intraperitoneal), blood was drawn from postcaval vein into anticoagulant tubes containing 0.109 mol/L sodium citrate, centrifuged at 3,000 rpm for 20 minutes, and plasma collected for FDP and D-dimer ELISA. Rats were euthanized by spinal dislocation, and ear tissue was collected for histopathological examination [3]
- Rat common carotid artery thrombosis model: Rats were orally administered nattokinase; chemically-induced thrombi were assessed for blood flow recovery [1]
- Dog leg vein thrombosis model: Dogs were orally administered four nattokinase capsules (2000 FU/capsule); chemically-induced thrombi in the major leg vein were assessed for dissolution within five hours [1]
- Rat tail inflammatory thrombus model (κ-carrageenan): Rats were administered nattokinase by gavage; 12 hours later, blood samples were analyzed for FDP fragments and D-dimers; tail vessels were biopsied for thrombosis assessment [1]
- Human trial (healthy volunteers, cardiovascular risk factor patients, dialysis patients): Subjects orally administered two capsules of nattokinase (2000 FU/capsule) daily for two months; factor VII, factor VIII, fibrinogen, heart rate, body weight, and uric acid levels were measured [1]
- Human single-dose study: 12 healthy young males were randomly administered a single capsule of nattokinase (2000 FU); blood samples were collected at various time points for antithrombin concentration, FDP fragments, D-dimers, and factor VIII activity [1]

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In vivo animal studies for nattokinase have been conducted in various models of thrombosis and cardiovascular disease. Standard protocols involve oral or intravenous administration of nattokinase in rodents, followed by assessment of thrombus formation and dissolution in models such as FeCl3-induced arterial thrombosis, middle cerebral artery occlusion (stroke), or venous thrombosis models. Blood samples are collected for measurement of fibrinolytic parameters, including fibrin degradation products and PAI-1 levels.

- Nattokinase can be absorbed from the rat intestinal tract in intact form [1]
- In healthy humans, intact nattokinase was detected in serum after a single oral dose (2000 FU/100 mg) in a capsule [1]
- Nattokinase can resist high temperature (50°C) and pH up to 10, which contributes to its ability to remain intact in the gastrointestinal tract [1]
- Nattokinase has a long half-life compared to other thrombolytic agents (which have 3-20 min half-lives) [3]
- Nattokinase is resistant to pancreatic enzymes [3]
- Specific PK parameters (Cmax, Tmax, AUC, exact half-life) were not reported in these papers [1][2][3]

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The pharmacokinetic properties of nattokinase include oral bioavailability, which is unusual for a protein enzyme. Following oral administration, nattokinase is absorbed from the gastrointestinal tract and reaches the systemic circulation. It has a relatively short half-life and is cleared by the liver and kidneys. The enzyme's activity in plasma can be measured by its fibrinolytic activity. Detailed PK parameters such as Cmax, Tmax, and half-life are available from clinical and preclinical studies.

- In a human trial (two months, two capsules daily of 2000 FU/capsule), no adverse effects were detected; heart rate, body weight, and uric acid levels remained stable [1]
- Unlike aspirin which often triggers bleeding or gastric ulcers, nattokinase improves blood flow without any adverse effects [1]
- Nattokinase exhibits little to no side effects compared to t-PA and uPA which can produce various side effects such as bleeding [1]
- Acute toxicity study in rats: Single dose of nattokinase (2000 mg/kg) by gavage; no significant adverse signs or mortality observed within 14-day study period [1]
- Repeat-dose toxicity study in rats: Daily single dose of nattokinase (1000 mg/kg) for 90 days; no abnormal clinical observations detected relative to control groups [1]
- No-observed-adverse-effect-level (NOAEL) in humans: Healthy volunteers orally consumed nattokinase (10 mg/kg) daily for 28 days; no significant changes in urine, blood pressure, or pulse [1]
- Bacillus subtilis (natto) inoculation in rats (1.51×10⁹ CFU/mL) produced no signs of toxicity; 14 days after treatment, no viable bacteria observed in lungs, liver, brain, or kidneys by histopathological examination [1]
- No clastogenic or mutagenic activity observed in vitro after nattokinase treatment in GLP-compliant studies [1]

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Nattokinase is generally considered safe and well-tolerated. It has been evaluated in clinical trials for its safety and efficacy. As a fibrinolytic enzyme, the primary safety concern is bleeding risk, particularly at high doses or when co-administered with other anticoagulants or antiplatelet agents. No significant toxicity has been reported in clinical studies. Standard toxicology assessments include acute and sub-chronic toxicity studies in rodents and dogs.

[1]. Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease. Int J Mol Sci. 2017 Feb 28; 18(3):523.

[2]. Exploring the In Vitro Thrombolytic Activity of Nattokinase From a New Strain Pseudomonas aeruginosa CMSS. Jundishapur J Microbiol. 2015 Oct 26; 8(10): e23567.

[3]. Thrombolytic effects in vivo of nattokinase in a carrageenan-induced rat model of thrombosis. Acta Haematol. 2014; 132(2): 247-53.

- Nattokinase is a serine protease produced by Bacillus subtilis during the fermentation of soybeans to produce natto, a traditional Japanese food [1]
- Nattokinase is not related to any known kinases [1]
- Nattokinase was discovered in 1980 by Hiroyuki Sumi at Chicago University Medical School [1]
- Nattokinase can break down blood clots by directly hydrolyzing fibrin and plasmin substrate, converting endogenous prourokinase to urokinase (uPA), degrading PAI-1, and increasing t-PA [1]
- Nattokinase is considered a safe, powerful, low cost, and all-natural supplement for treatment of heart and cardiovascular disease [1]
- Commercial nattokinase products are widely used in Japan, China, Korea, European Union countries, Canada, and the United States as a food supplement to thin blood, prevent blood clots, and improve blood circulation [1]
- Studies indicate nattokinase can ameliorate hypertension, stroke, Alzheimer's disease, and atherosclerosis [1]
- Nattokinase is currently undergoing a Phase II clinical trial in the USA for atherothrombotic prevention (ClinicalTrials.gov Identifier: NCT02080520) [1]
- The recommended usage for nattokinase is two capsules (100 mg/capsule) daily [1]
- Nattokinase has a molecular weight of 27.7 kDa (mature peptide) and is encoded by the aprN gene [1]
- The amino acid sequence of nattokinase is 99.3% homologous to Subtilisin E [1]

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Nattokinase is a serine protease enzyme derived from natto fermentation. It is a potent fibrinolytic agent with oral bioavailability. Nattokinase degrades fibrin, PAI-1, and plasmin substrates to dissolve thrombi and inhibit platelet aggregation. It is used for research on cardiovascular diseases, including thrombosis, atherosclerosis, and stroke, and has also been investigated for Alzheimer's disease. Nattokinase is classified as an investigational compound and is not approved as a therapeutic agent in most countries.

133876-92-3

White to off-white solid powder

1.5±0.1 g/cm3

1.580

ClC1C([H])=C([H])C(=C([H])C=1C(N([H])C([H])([H])C(N([H])[C@]([H])(B1OC(C([H])([H])C(C(=O)O[H])(C([H])([H])C(=O)O[H])O1)=O)C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)=O)Cl

Kinase, Natto fermentation

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: ~100 mg/mL

Solubility in Formulation 1: 50 mg/mL (Infinity mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)