X-ray structure determination and deuteration of nattokinase.

Nattokinase (NK) is a strong fibrinolytic enzyme, which is produced in abundance by Bacillus subtilis natto. Although NK is a member of the subtilisin family, it displays different substrate specificity when compared with other subtilisins. The results of molecular simulations predict that hydrogen arrangements around Ser221 at the active site probably account for the substrate specificity of NK. Therefore, neutron crystallographic analysis should provide valuable information that reveals the enzymatic mechanism of NK. In this report, the X-ray structure of the non-hydrogen form of undeuterated NK was determined, and the preparation of deuterated NK was successfully achieved. The non-hydrogen NK structure was determined at 1.74 Å resolution. The three-dimensional structures of NK and subtilisin E from Bacillus subtilis DB104 are near identical. Deuteration of NK was carried out by cultivating Bacillus subtilis natto in deuterated medium. The D2O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D2O in the medium was gradually increased. NK was purified from the culture medium and its activity was confirmed by the fibrin plate method. The results lay the framework for neutron protein crystallography analysis.

Effect of coffee extracts on plasma fibrinolysis and platelet aggregation.

We have previously reported on study results showing that certain types of coffee have the activity to enhance fibrinolysis. This report covers the activity of 10 types of hot water extracts of coffee on human tissue-type plasminogen activator producing cells. Particularly strong activity (29-35 times the control amount) was observed for Blue Mountain, Yunnan and Kilimanjaro beans. It was found that the hot water extracts have anti-thrombin activity, and that coffee components have anti-platelet aggregation activity, although weak. It was revealed that there is no activity affecting tissue-type plasminogen activator producing cells in the coffee components chlorogenic acid, caffeine, quinic acid, trigonelline hydrochloride, 5-(hydroxymethyl)-2-furfuryl and caffeic acid. It was also revealed that there is activity in fractions with a molecular weight of 10,000 or less. This could also be inferred from the fact that oral administration of such fractions of coffee to human subjects resulted in a shortening of their plasma ELT (p<0.05).

Nattokinase-promoted tissue plasminogen activator release from human cells.

When heated to a temperature of 70 degrees C or higher, the strong fibrinolytic activity of nattokinase in a solution was deactivated. Similar results were observed in the case of using Suc-Ala-Ala-Pro-Phe-pNA and H-D-Val-Leu-Lys-pNA, which are synthetic substrates of nattokinase. In the current study, tests were conducted on the indirect fibrinolytic effects of the substances containing nattokinase that had been deactivated through heating at 121 degrees C for 15 min. Bacillus subtilis natto culture solutions made from three types of bacteria strain were heat-treated and deactivated, and it was found that these culture solutions had the ability to generate tissue plasminogen activators (tPA) from vascular endothelial cells and HeLa cells at certain concentration levels. For example, it was found that the addition of heat-treated culture solution of the Naruse strain (undiluted solution) raises the tPA activity of HeLa cells to about 20 times that of the control. Under the same conditions, tPA activity was raised to a level about 5 times higher for human vascular endothelial cells (HUVEC), and to a level about 24 times higher for nattokinase sold on the market. No change in cell count was observed for HeLa cells and HUVEC in the culture solution at these concentrations, and the level of activity was found to vary with concentration.

Genistein and its analogue enhanced tissue plasminogen activator activity in HeLa S3.

Soybean isoflavones of genistein and biochanin A, its analogue, promote the activity for generating tissue-plasminogen activator (tPA) from human cervical cancer cells (HeLa S3) and human umbilical vein endothelial cells (HUVEC). At a concentration of 50 microM, each of 14 types of isoflavones were added to HeLa culture solution and incubated. After 24 h, the culture solution was replaced, and then incubated for another 24 h. When fibrinolytic activity was checked in the resulting culture solution using the fibrin plate method, substantial fibrinolytic activity was confirmed for two types of isoflavones. Genistein showed the highest level of fibrinolytic activity at 12.4 times the control, and for biochanin A, an analogue of genistein, the level was 3.5 times the control. Checking fibrinolytic activity and molecular weight of the protein bands separated by zymography, a rise in the protein band concentration in proportion to the concentration of the reagents added was confirmed for the protein band with activity in the same position as the standard reference tPA, which has a molecular weight of about 68 kDa. ELISA also demonstrated that the concentration of tPA in the culture solution was higher than that of plasminogen activator-1. Fibrinolytic activity of HUVEC incubated with 25 microM of biochanin A was much higher than that of the control, which suggests that these soybean isoflavones could have beneficial effects on blood circulation in vivo.