Study on molecular mechanisms of nattokinase in pharmacological action based on label‐free liquid chromatography–tandem mass spectrometry

Abstract Cardiovascular diseases (CVDs) are the leading causes of premature death and disability in people around the world. Therefore, the prevention and treatment of CVDs has become an important subject. In this study, we verified the thrombolytic activities of a nattokinase‐like protease named NK‐01 in vivo. Label‐free liquid chromatography–tandem mass spectrometry (LC‐MS/MS) technique was used in our study. NK‐01 could inhibit the activity of coagulation factors though the up‐regulation of proteinase C inhibitors and protein S. NK‐01 also could inhibit the angiotensinogen conversion to AngII and promote the degradation of kininogen to reduce the blood pressure. In addition, NK‐01 could increase the content of paraoxonase 1, which could prevent atherosclerosis. In our study, we found that NK‐01 cloud effect some key proteins which participant in CVDs associated metabolic processes such as coagulation system, blood pressure, and atherosclerosis. Taken together, the underlying molecular mechanisms for the biological beneficial of NK‐01 were investigated. Our proteomic study will provide further theoretical basis for application of NK in prevention or adjuvant treatment in biomedicine areas.

long-term medical treatment. The patients usually have poor adherence to drugs, and the benefits of medications cannot be fully realized (Haynes, McKibbon, & Kanani, 1996;O'Flaherty, Buchan, & Capewell, 2013). In addition, most drugs have some side effects (Beltowski, Wojcicka, & Jamroz-Wisniewska, 2009;Hankey & Eikelboom, 2006;Klegerman, 2017;Toh et al., 2012). For example, thrombolytic agents could lead to the unwanted internal bleeding and their half-lives are short in vivo Nordt & Bode, 2003). Statins could lower the plasma low-density lipoprotein (LDL) cholesterol, and it could also cause sympathy and severe rhabdomyolysis (Beltowski et al., 2009). As an antiplatelet drug, aspirin plays an important role in the prevention of CVDs. But the curative effects of aspirin vary from person to person and some people showed poor antiplatelet effect, which was called "aspirin resistance" (Georgiadis et al., 2013).
Nattokinase is a kind of serine protease with strong thrombolytic activity (Sumi, Hamada, Tsushima, Mihara, & Muraki, 1987). It was extracted from fermented beans such as natto, douche, and tempeh and can be absorbed through the intestine .
Nattokinase was widely studied as a dietary supplement and nutritional food, which has potential to prevent and treat CVDs (Dabbagh et al., 2014;Weng, Yao, Sparks, & Wang, 2017). Hongjie Chen et al. reported that nattokinase was a promising alternative in prevention and treatment of cardiovascular diseases by possessing a variety of favorable cardiovascular effects, such as fibrinolytic activity, antihypertensive, antiatherosclerotic, and lipid-lowering, antiplatelet, and neuroprotective effects (Chen et al., 2018). The consumption of natto has been linked to a reduction in CVD mortality. Compared with other thrombolytic drugs, nattokinase has higher thrombolytic activity, which was about 4× higher compared with urokinase . Nattokinase is more sensitive to cross-linked fibrin than fibrinogen, which could effectively prevent internal bleeding . Ji Young Kim et al. found that nattokinase resulted in a reduction in systolic blood pressure (SBP) and diastolic blood pressure (DBP) of hypertensive patients (Kim et al., 2008). Ren nina et al. had proved that nattokinase also played an important role in preventing atherosclerosis of patients (N. N. Ren, Chen, Li, Mcgowan, & Lin, 2017).
Recently, we found a nattokinase-like protease named NK-01, which shared 99% sequence identity with the nattokinase (Genebank number: AHZ12722.1) and composed of multiple fragments. Through casein plate method, we showed that NK-01 has better fibrinolytic activity than urokinase (Y. Ren, Pan, Lyu, & Liu, 2018). In this study, we confirmed the in vivo thrombolytic activity of NK-01. Based on label-free technology, we studied the effects of NK-01 on the proteomic profiling of plasma proteins in rats and further analyzed the possible mechanisms of its biological functions in the prevention of cardiovascular diseases.

| Chemicals
Nattokinase NK-01 was prepared in our laboratory. Urokinase was obtained from Nanda Co., Ltd, Pelltobarbitalum Natricum was purchased from Solarbio. Trypsin was purchased from Promega, and iodoacetamide was purchased from Sigma. MeOH, formic acid, CAN, and TCEP were purchased from ThermoFisher Scientific.

| Animals
Male SD rats (~300 g, 8 week) were purchased from DaRenFuChen Animal Co., Ltd. Rats were customized in cages in air-conditioned animal room under a photoperiod schedule of 12-hr light/12-hr dark cycles at 25 ± 2°C. The rats were fed with normal food and tap water for 1 week before the experiments. Rats were fasted overnight before experiments with free access to tap water. All experiments with animals were carried out in accordance with the guidelines of Care and Use of Laboratory Animals published by China National Institute of Health.

| Thrombolytic activity of NK-01 in vivo
Fifteen rats were divided into negative control group, NK-01 group, and positive control group. Rats were anesthetized by 3% Pelltobarbitalum Natricum (1 ml/kg body weight). Rats in negative control group and NK-01 group were injected with 0.5 ml 0.9% NaCl and 0.5 ml NK-01 (5,000 FU) into duodenal. Rats in positive control group were treated with urokinase (50,000 U) by tail vein injection. Four hours after injection, the blood of all rats was collected with tubes containing 3.2% sodium citrate solution (9:1, v/v).
Blood was centrifuged at 3,000 g for 15 min at room temperature.
Subsequently, the citrated plasma was used for analysis of thrombotic indexes (PT, APTT, TT, and FIB).

| Preparation of proteomic samples
Twelve rats were divided into two groups: negative control group and NK-01 group. Rats were anesthetized as mentioned above and were injected with 0.5 ml 0.9% NaCl and 0.5 ml NK-01 (5,000 FU) into duodenal. Four hours after injection, the blood of all rats was collected with tubes containing 3.2% sodium citrate solution (9:1, v/v) and was centrifuged at 3,000 g for 15 min at room temperature.
A moderate amount of plasma, PBS, and protease inhibitors were added into 0.22-μm membrane filter centrifuge tube with loaded packing (Thermo 191085305) and incubated for 1 hr at room temperature. Then, the tubes were centrifuged at 500 g for 1 min and the filtrates were collected. The packing was cleaned with PBS twice to collect the filtrates. All of the filtrates were concentrated by 3K ultrafiltration. The concentrated plasma samples were replaced with 8 M urea for three repeated times to appropriate volume. Finally, the protein concentrations of plasma samples were determined by bicinchoninic acid (BCA) method.

| Protein digestion and peptides quantification
The plasma samples were digested with trypsin solution according to the standard procedure. The peptides were re-suspended with 2% ACN, 0.1% TFA, and Sep-Pak desalination. Then, each sample was vacuum dried. The peptides were finally quantified by Thermo Fisher Scientific (Thermo 23275).

| Mass spectrometry analysis
Experiments were performed on a Q Exactive mass spectrometer coupled with Easy-nLC 1,200. Peptide of each sample (0.5 μg/μl) was injected for nano LC-MS/MS analysis. The peptide (2 μg) was loaded onto a C18-reversed phase column (75 μm × 25 cm, Thermo) in buffer A (2% acetonitrile and 0.1% Formic acid) and separated with a linear gradient of buffer B (80% acetonitrile and 0.1% Formic acid) at a flow rate of 300 nl/min. In order to fully separate the peptides in plasma, the gradient elution program was set as follows: The concentration of (B) in the mobile phase was 0% at the initial, and then a linear increase from 3% to 6% (B) from 0 to 2 min, followed by a linear increase from 6% to 23% (B) from 2 to 105 min, and a linear increase from 23% to 29% (B) from 105 to 130 min, further a linear increase from 29% to 100% (B) from 130 to 149 min,

| Statistical analysis
All results were expressed as mean ± standard deviation (X ± SD), and statistical analysis was performed using Student's t test for comparison of two groups. In order to avoid problems such as the omission of identification results, the peptide error of the database search needs to be controlled within ± 10 ppm. To define the differentially expressed proteins, either a fold change (FC)>1.2 or a FC < 1/1.2 was set as the threshold. p-value (<.05) was set as significant level.
It was shown in Figure 1 that the levels of PT, APTT, and TT were significantly increased in the male rats treated with NK-01 compared with the negative control group (p < .05). However, the level of FIB in the rats treated with NK-01 was significantly decreased compared with the negative control group (p < .05). The result indicated that NK-01 had potential in vivo antithrombotic activities.

| Identification of proteins from quantitative proteomic analysis
A total of 254,117 spectra were obtained from the LC-MS/MS proteomic analysis. After data filtering, a total of 64,302 spectra were matched to 4,086 peptides, which were mapped to 665 proteins and 516 protein groups. Among all the identified proteins, approximately 57% of the proteins included 1-3 peptides and 43% of the proteins included at least 4 peptides (Figure 2a). In addition, the proteins were identified with high sequence coverage, 65% of proteins were identified with more than 10% of the sequence coverage, and 54% of proteins were identified with more than 20% of the sequence coverage (Figure 2b). Also, 42% of proteins were F I G U R E 3 Identified proteins associated with fibrinolysis and coagulation system. (a) Showed the seventeen proteins associated with fibrinolysis and coagulation system. The dot lines mean the thresholds of the significantly differentially expressed proteins. (b) Showed the metabolic pathway which the proteinase C1 and protein S participate in and the function of them F I G U R E 4 Identified proteins associated with regulation of blood pressure. (a) Showed the five proteins associated with regulation of blood pressure. The dot lines mean the thresholds of the significantly differentially expressed proteins. (b) Showed metabolic pathway which the Angiotensinogen and T-kininogen 2 participant and the function of them identified with the molecular weight less than 40 kDa and 58% of the proteins were identified with the molecular weight larger than 40 kDa (Figure 2c).  Table 1.

| Influence of NK-01 on key serum proteins involved in the thrombolysis and blood coagulation process
As shown in Figure 3a, a total of 17 proteins that are associated with thrombolysis and coagulation processes, such as prothrombin, plasminogen, and coagulation factors, were detected. The proteinase C1 inhibitors and protein S were up-regulated in normal rats after treatment with NK-01, and others had no significant changes in normal rats after treated with high dose of NK-01.
The roles of proteinase C1 inhibitors and protein S were illustrated in Figure 3b (Castoldi & Hackeng, 2008;Schurmann et al., 2014). Our data indicated that NK-01 could affect the process of blood coagulation by influencing the activities of coagulation factors.
In addition, most of the 17 proteins that were detected had no significant fold change, suggesting that high dose of NK-01 treatment cannot cause disorder in the thrombolytic process. Therefore, NK-01 had good biosafety in terms of thrombolysis.

| Influence of NK-01 on key proteins involved in blood pressure regulation
Among the proteins detected, there were five proteins that were related to the regulation of blood pressure. The angiotensinogen was significantly up-regulated, and the T-kininogen 2 was significantly down-regulated as shown in Figure 4a. Angiotensinogen and T-kininogen 2 all played important roles in regulating blood pressure (Figure 4b).
Renin plays an important role in renin-angiotensin system (RAS) and could convert angiotensinogen to angiotensin I. Then, angiotensin I was converted to angiotensin II, which could raise the blood pressure directly (Lavoie & Sigmund, 2003). In addition, the kallikrein-kinin system (KKS) could lower blood pressure and negatively regulate RAS. In the KKS, kininogen is degraded into kinin, which is helpful for controlling the blood pressure (Regoli & Gobeil, 2017). It was reported that nattokinase could lower the blood pressure and had impact on the renin activity (Kim et al., 2008).
Nattokinase could inhibit angiotensin I converting enzyme by a mixed type (the decrease in Vmax and the increase in Km value) to suppress blood pressure. (Murakami, Yamanaka, Ohnishi, Fukayama, & Yoshino, 2012). In our study, angiotensinogen was up-regulated and the kininogen was down-regulated in rats treated with NK-01.
This implied that nattokinase could inhibit the conversation of angiotensinogen and promote the degradation of kininogen. In this way, nattokinase could regulate the blood pressure.
F I G U R E 5 Identified proteins associated with regulation of blood lipids. This figure showed the thirteen proteins associated with regulation of lipids. The dot lines mean the thresholds of the significantly differentially expressed proteins

| Influence of NK-01 on key proteins involved in blood lipid regulation
It was shown in Figure 5 that a total of 13 proteins that were associated with regulation of blood lipids were detected. Paraoxonase/ arylesterase 1, apolipoprotein N, and apolipoprotein E were all upregulated, while apolipoprotein A-IV was down-regulated ( Figure 5) Paraoxonase/arylesterase 1, a high-density lipoprotein (HDL)-associated enzyme, could inhibit the oxidation of LDL (Mackness, Quarck, Verreth, Mackness, & Holvoet, 2006). After treatment with NK-01, the paraoxonase/arylesterase 1 was significantly up-regulated in rats. Therefore, NK-01 had the potential to prevent atherosclerosis. According to our results, the Apo E was up-regulated and the Apo A-IV was down-regulated in normal rats after treatment with NK-01. The possible reason might be because that the rats were fasted overnight and need more LDL to maintain normal metabolic.

ACK N OWLED G M ENTS
This work was supported by the National Natural Science

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest. All the authors approve the submission of this manuscript and will take responsibility for any conflict of interest.

E TH I C A L A PPROVA L
This study was approved by the Institutional Review Board of Ocean University of China, and the study's protocols and procedures were ethically reviewed and approved by China National Institute of Health.