Description

The Problem; Thrombosis

Thrombosis accounts for one in four deaths worldwide(1). It is a general term for diseases caused by blood clots obstructing blood vessels, including myocardial infarction, stroke, pulmonary embolism, and cardiogenic stroke.

This is a significant issue in Japan, which is experiencing an aging society. Among the G7 countries, Japan has the highest percentage of elderly people within its total population. According to data from 2022, while the rate of individuals aged 65 and older in the U.S. is 17%, it is estimated to be around 30% in Japan(2).

Statistics show that 66% of stroke deaths occur in individuals aged 70 and older(3). Age is a primary risk factor for Thrombosis, and with the aging population in Japan, the burden of cardiovascular diseases is increasing. The second leading cause of death in Japan is heart disease, including myocardial infarction, while cerebrovascular disorders rank third. It is believed that approximately 30% of deaths in Japan are due to Thrombosis.

There are various types of thrombosis, but here we will focus on two particularly severe conditions.

Strokes

“Stroke” is a general term for diseases caused by blood clots’ obstruction of blood vessels in the brain, leading to insufficient blood supply to brain cells and subsequent cell necrosis. According to WHO data from 2021, stroke ranked as the third leading cause of death globally(4), with approximately 6.6 million deaths predicted in 2022(3). In Japan, cerebrovascular diseases accounted for 6.6% of deaths in 2023, with over 10,000 fatalities(5).

The severity of stroke is not only reflected in the death toll. Survivors may experience significant physical and mental burdens, such as tremors, speech disorders, and the risk of recurrence years later. Cognitive and emotional dysfunction can lead to deteriorating social relationships and increased social isolation(6). Physical disabilities can further limit mobility and lead to job loss, contributing to this isolation(6).

Furthermore, Post-stroke depression (PSD) is one of the sequelae that negatively impact recovery and quality of life (QOL), complicating rehabilitation and placing a heavy burden on caregivers(7). In caregiving settings, cerebrovascular disorders pose a significant challenge. In Japan, 16.1% of patients requiring care cite cerebrovascular disorders as the primary cause of their condition(8).

Acute Myocardial Infarction

Acute myocardial infarction occurs when blood vessels are blocked by plaque or blood clots, preventing blood flow and resulting in insufficient delivery of nutrients and oxygen to the heart muscle, leading to its necrosis. In 2023, over 30,000 deaths due to acute myocardial infarction were confirmed in Japan(5). Additionally, the economic losses associated with myocardial infarction are substantial. The economic burden of ischemic heart diseases, including myocardial infarction, was estimated at 1.619 trillion JPY in Japan in 2017(9).

Moreover, myocardial infarction is the leading cause of sudden death. There are numerous cases where individuals, previously living normally, suddenly experience severe chest pain that leads to death. About half of those who experience an acute myocardial infarction have no prior symptoms or warning signs, making it a particularly frightening disease(10).

Additionally, between one-third and one-half of hospitalized patients with acute myocardial infarction are elderly individuals aged 70 and older(11). 80% of deaths from myocardial infarction occur in those aged 65 and older, similar to strokes, indicating that the majority of patients affected by these conditions are elderly(11).

In this way, thrombosis represents a critically significant issue in Japan’s rapidly aging society, as well as a major global challenge that must be addressed.

Current Issues with Cardiovascular Treatment Methods; What’s Missing?

Currently, the treatment of cardiovascular diseases can be broadly categorized into two types: pharmacotherapy and invasive procedures.

In pharmacotherapy, antiplatelet drugs such as aspirin and clopidogrel, as well as newer anticoagulants like dabigatran and rivaroxaban, are used to prevent thrombus formation and reduce the risk of heart attacks and strokes(12). However, these medications can have serious side effects. For example, aspirin and clopidogrel increase the risk of gastrointestinal bleeding, while the new anticoagulants, dabigatran, and rivaroxaban, carry risks of excessive bleeding and renal or liver function impairment. Additionally, commonly used heparin carries the risk of heparin-induced thrombocytopenia (HIT), which can lead to severe bleeding or thrombotic events(13).

On the other hand, invasive procedures such as stent placement and bypass surgery are also effective treatment options, but they are costly, require significant recovery time, and carry risks of postoperative complications, which can impose a considerable burden on patients.

Our Solution; The New and Revolutionary

Natto has long been recognized in Japan as a health-promoting food and is a traditional product made by fermenting soybeans with natto bacteria. We focused on nattokinase, which is found in natto, as a treatment for thrombosis.

Nattokinase demonstrates strong thrombolytic activity by enhancing the effects of the fibrinolytic enzyme plasmin present in the body. Research has shown that it can rival the effects of aspirin(14), a currently used antiplatelet agent, and it has been demonstrated to be effective in preventing the progression of strokes in patients with acute ischemic stroke in clinical settings(15).

Furthermore, nattokinase has recently garnered attention as a thrombolytic agent with few side effects. Despite being consumed for centuries, very few side effects have been reported. Recent studies have shown that it can inhibit thromboxane formation, thus preventing platelet aggregation without causing bleeding side effects(14). However, delivering this traditional food, natto, to everyone at risk of thrombosis remains challenging.

- A single pack of natto does not provide the necessary amount for thrombolysis(15).
- Natto is a food that divides opinions, similar to cilantro.
- As a traditional Japanese food, most people overseas dislike natto.
- Only a very small number of people consume natto daily.
- Natto is not offered in elderly care facilities.

Given these circumstances, there is a reality where natto is not accessible to everyone who should benefit from it.

Thus, by utilizing synthetic biology, we aim to stably and mass-produce nattokinase, overcoming the drawbacks of natto. We believe this can provide seniors with a more affordable and user-friendly approach that is effective against thrombosis, leading to the launch of VitaliNK.

Why Nattokinase?

We decided to use Bacillus subtilis natto because it has been used since ancient times and is easy for people to eat. There are a very limited number of bacteria that are less resistant to being put into the mouth, and Bacillus subtilis natto is one of them. We chose Bacillus subtilis var. natto to eliminate consumer resistance.

Currently, little progress has been made in the genetic modification of Bacillus subtilis natto. Compared to E. coli, there are fewer studies on substance production with Bacillus subtilis var. natto, but it is suitable for substance production in that it is orally available and safe. However, the challenge is that the potential of Bacillus subtilis natto has not been fully exploited. If the Bacillus subtilis natto could be more readily genetically modified, it would contribute not only to our project but also to the fields of iGEM and synthetic biology.

Furthermore, this was a very challenging task for the past iGEM teams, as few projects have used Bacillus subtilis natto. Since it is difficult to perform genetic modification in Bacillus subtilis as it is, we instead performed genetic modification in Bacillus subtilis and introduced the plasmid into Bacillus subtilis by conjugative transmission.

Modeling Overview

In our iGEM project, we focused on modeling to improve the quantification of nattokinase and to understand its production pathways in Bacillus subtilis subsp. natto cultured in the NB medium.

First, we proposed a new method for estimating Fibrinolytic Units (FU)from the degradation radius in the fibrin degradation assay, a standard method for quantifying NK. Traditionally, FU estimation relied on standard curves based on the degradation area, which lacked sufficient explanatory power and posed a risk of undetected discrepancies between the estimated FU and its true values. To address this issue, we developed a novel mathematical model incorporating the dynamics of the enzyme reaction (Reaction-Porous Medium Equation Model), providing a more accurate and explanatory approach to FU estimation than previous methods.

Second, we analyzed the metabolic pathways involved in NK production in Bacillus subtilis subsp. natto cultured exclusively in NB medium using Flux Balance Analysis (FBA). Since there were no prior studies on obtaining NK by culturing Bacillus subtilis subsp. natto solely in NB medium, it was necessary to elucidate which metabolic pathways are active under these conditions.

Why Green Tea, the famous “Japanese” Tea?

In our search for a product that could provide nattokinase as an alternative to natto, we engaged in discussions with various stakeholders. We collaborated with Tsuruya Kasei Co., Ltd., which specializes in developing sweeteners and thickeners, and conducted surveys with the general public visiting nursing homes, as well as discussions with healthcare professionals.

Through these activities, we deepened our understanding of thrombosis and considered what types of foods would be easy for seniors to consume. Initially, we planned to create a product using thickeners to make it easier for elderly individuals to ingest. However, after speaking with a variety of people, we decided to shift our product focus from thickeners to tea, visiting facilities for seniors, and engaging in human practice activities (for more details, please see the Human Practice page).

Tea is a beverage that anyone can enjoy, regardless of age or location, allowing us to target not only seniors but also working individuals who may be at risk for myocardial infarction or stroke.

Additionally, catechins and epigallocatechin gallate found in green tea are known to have antiplatelet effects, contributing to its anti-thrombotic properties, as referenced in(16).

For these reasons, we believe that green tea is the optimal product for self-managing thrombosis.

Reference

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2. H. Dyvik, Elnar. “G7: Age Distribution by Country 2022 | Statista.” Statista, 2022. Accessed 2 Oct. 2024.
3. World Stroke Organization. “Global Stroke Fact Sheet.” World Stroke Organization, 2022. Accessed 2 Oct. 2024
4. WHO. “The Top 10 Causes of Death.” Who. int, World Health Organization: WHO, 7 Aug. 2024. Accessed 2 Oct. 2024.
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10. Watanabe. “心筋梗塞とは？原因・症状・治療・手術方法をわかりやすく解説｜ニューハート・ワタナベ国際病院.” ニューハート・ワタナベ国際病院｜NewHeart Watanabe Institute, 2024. Accessed 2 Oct. 2024.
11. McMechan, S. R, and A A J. Adgey. “Age-Related Outcome in Acute Myocardial Infarction.” BMJ, vol. 317, no. 7169, 14 Nov. 1998, pp. 1334–1335. Accessed 2 Oct. 2024.
12. Bai, Ying, et al. “Rivaroxaban versus dabigatran or warfarin in real-world studies of stroke prevention in atrial fibrillation: systematic review and meta-analysis.” Stroke 48.4 (2017): 970-976.
13. Warkentin, Theodore E., and Andreas Greinacher, eds. Heparin-induced thrombocytopenia. CRC Press, 2012.
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16. “Japan NattoKinase Association What Is Nattokinase?” J-Nattokinase.org, 2024. Accessed 2 Oct. 2024.
17. Arts, Ilja CW, et al. “Catechin intake might explain the inverse relation between tea consumption and ischemic heart disease: the Zutphen Elderly Study.” The American journal of clinical nutrition 74.2 (2001): 227-232.