Hiroyuki Sumi

Enhancement of the Fibrinolytic Activity in Plasma by Oral Administration of Nattokinases

The existence of a potent fibrinolytic enzyme (nattokinase, NK) in the traditional fermented food called natto, was reported by us previously. It was confirmed that oral administration of NK (or natto) produced a mild and frequent enhancement of the fibrinolytic activity in the plasma, as indicated by the fibrinolytic parameters, and the production of tissue plasminogen activator. NK capsules were also administered orally to dogs with experimentally induced thrombosis, and lysis of the thrombi was observed by angiography. The results obtained suggest that NK represents a possible drug for use not only in the treatment of embolism but also in the prevention of the disease, since NK has a proven safety and can be massproduced.

Urinary trypsin inhibitor and urokinase activities in renal diseases.

Using an improved method of determination, urinary trypsin inhibitor (UTI) activities were assayed in relation to urokinase (UK) activities in a large group of patients with various renal diseases. In normal subjects (n = 50), the mean 24-hour values of the UTI and UK activities were 4.29 +/- 1.44 U/ml and 9.80 +/- 3.81 IU/ml, respectively. Data for renal diseases such as renal stone, hydronephrosis, renal cancer, and chronic glomerulonephritis (UTI, 5.51 +/- 2.29 U/ml (p less than 0.005) and UK, 6.88 +/- 2.64 IU/ml (p less than 0.001); n = 40), and particularly uremia (UTI, 9.90 +/- 5.68 U/ml (p less than 0.001) and UK, 3.85 +/- 2.36 IU/ml (p less than 0.001); n = 30), showed that the UTI level was increased whereas the UK level was decreased. The UTI/UK ratio more clearly demonstrated the difference between these diseases.

Modulation of the receptor for urokinase-type plasminogen activator in macrophage-like U937 cells by inflammatory mediators.

Macrophages in the tissues have been shown to express receptor for urokinase-type plasminogen activator (uPAR) on their cell surface which plays an important role in cell invasion and attachment. We examined the effects of inflammatory mediators on the expression of uPAR employing U937 cells which have monocyte/ macrophage-like characteristics. U937 cells were incubated with various mediators such as interleukins (IL), tumor necrosis factors (TNF), dexamethasone, thrombin, fibrin fragment D, bradykinin, complement C5a, and components of the extracellular matrix. The uPAR expression on the cell surface was then analyzed by radio-ligand binding assay using125I-scuPA. The strongest enhancement of uPAR was observed in the cells stimulated by TNFα and TNFβ. IL-1β, IL-6, and C5a also increased the uPA binding sites with various patterns of affinity change. Dexamethasone decreased the uPA binding sites without changing the affinity. Fibrin fragment D and IL-3 reduced the affinity without changing the number of receptors. These findings suggest that the expression of uPAR in inflammatory cells could be modulated by various inflammatory mediators.

Purification and partial characterization of two forms of urinary trypsin inhibitor

Crude urinary trypsin inhibitor was obtained by DEAE-cellulose column chromatography from normal fresh urine. By the purification of the crude urinary inhibitor on successive chromatography methods using Sephacryl S-200, DEAE-cellulose, CM-Sepharose CL-6B and Sephadex G-100, we detected two forms of urinary trypsin inhibitor: form I and form II. The specific activity of form I increased approx. 4-fold with a recovery of 60%, as compared to that of crude urinary trypsin inhibitor. N-terminal amino acids of form I and form II were determined to be alanine and valine, respectively. Molecular weights of forms I and II were estimated to be 67000 and 28000 by gel filtration on Sephadex G-100 and to be 43000 and 19000 by SDS-polyacrylamide gel electrophoresis. S-carboxymethylated form I migrated as a single band corresponding to a molecular weight of 59000 in SDS-polyacrylamide gel electrophoresis. From the results of the determination of a single N-terminal amino acid of form I and a single band of S-carboxymethylated form I, it is indicated that it is composed of single polypeptide chain. And the present study suggests that form I is a native form of trypsin inhibitor in normal human urine and form II is a fragmented product from form I in the purification steps.

Distribution of acid stable trypsin inhibitor immunoreactivity in normal and malignant human tissues

The distribution and localization of acid stable trypsin inhibitor (ASTI) in normal and malignant human tissues from various organs were examined using immunohistochemical techniques that used goat antibody raised against highly purified ASTI from human urine. Tissues were assessed as positive only when they were stained by both the biotin‐avidin‐peroxidase complex system and biotin‐streptavidin‐β‐galactosidase complex system, and the staining was abolished by absorption with purified ASTI. Under normal conditions, ASTI immunoreactivity was observed in only a few organs. Positive tissues for ASTI immunoreactivity included the kidney proximal tubules, glial cells of the cerebrum, fibrillar structures of the lamina propria of the stomach and colon, and bronchial epithelial cells. No ASTI immunoreactivity was observed in the cardiovascular system, reproductive system, or other tissues examined. As is not the case for normal tissues, ASTI immunoreactivity was found to be widely distributed in malignant tumors. Staining was observed in the extracellular space, i.e., in the stroma of the tumor and in connective tissues around the tumor invasion, whereas no ASTI immunoreactivity was detected in the malignant cells. Considering the identity of the first 36 NH2‐terminal residues of ASTI purified from plasma or urine with a recently reported endothelial cell growth factor, the present findings suggest that ASTI could play an important role, not limited to its function as a protease inhibitor, in the invasive growth of malignant neoplasms.

Fibrinolysis relating substances in marine creatures

1. Extracts with physiological saline solution were obtained from about 20 species of invertebrates and seaweed. Tosyl-L-Arg-MeOH hydrolysing and fibrin plate lytic activity were detected in the invertebrates Stichopus japonicus, Crassost gigas, Tapes japonica, and Kintai-gai as well as the seaweed Codiales codium. 2. These activities were all labile against heat (at 65 degrees C for 1 hr). Except for the extract from Stichopus japonicus, lytic activities against fibrin plates with and without plasminogen were similar. 3. The extract from S. japonicus showed plasminogen activating potency as well as the existence of urokinase (UK) activity enhancing factor. 4. On the other hand, the extract of the seaweed Hizikia fusiformis showed a strong UK inhibiting activity. 5. A fraction of fibrinolytic enzyme was obtained from the extract of S. japonicus by absorption to the celite affinity chromatography. It was orally administered to rabbits at a dosage of 40 mg/kg/day. 6. Fibrinolytic activity was determined periodically on the eugloblin fraction of plasma samples collected from these animals. 7. As compared with the pretreatment value, the activity increased about 2 times (P less than 0.01) and 3 times (P less than 0.005) after 4 and 8 weeks, respectively, of the treatment. 8. After 8 weeks of treatment, the kidney of treated rabbits was extracted with 2 M KCl. The activity of tissue plasminogen activator (free-type TPA) was revealed to be enhanced significantly (P less than 0.001) in the extracts. 9. The fibrinolytic enzyme increased in the blood was recognized by zymography to be mainly the UK type plasminogen activator with mol. wt of 53,000.

Clearance and distribution of acid-stable trypsin inhibitor (ASTI)

The clearance, organ distribution and metabolic pathway of the acid-stable trypsin inhibitor (ASTI) were studied in mice using 125I-labeled urinary trypsin inhibitor (UTI), the most typical ASTI in the urine. Following intravenous injection of 125I-UTI, the radioactivity disappeared rapidly from the circulation with a half-life of 4 min for the initial part of the curve. Gel filtration of plasma samples revealed that the rapid disappearance of the radioactivity was due to elimination of free inhibitor from the plasma. 125I-UTI was cleared primarily in the kidney. Gel filtration of urine samples showed that part of the radioactivity in the urine appeared at the same elution volume as 125I-UTI in the plasma, indicating that the origin of UTI was ASTI in the plasma.

A very stable and potent fibrinolytic enzyme found in earthworm Lumbricus rubellus autolysate.

Abstract 1. 1. The autolysate of earthworms was found to exhibit powerful fibrin and thrombin substrate hydrolyzing activity. 2. 2. It also showed a clot-forming activity in the fibrinogen- or plasma-added system. 3. 3. Zymography revealed that there were three active components with mol. wts of 40,000, 21,000 and 15,000 in the autolysate. 4. 4. The major form with a mol. wt 35,500 (by SDS-PAGE) was further purified. The N -terminal amino acid sequence of this enzyme (16 residues) was similar to that of the swine pancreatic proelastase.

Development and application of an enzyme-linked immunosorbent assay for acid stable trypsin inhibitor (ASTI) in human plasma

An enzyme-linked immunosorbent assay (ELISA) for acid-stable trypsin inhibitor was developed using goat antibodies raised against urinary trypsin inhibitor purified from normal human urine. The assay was highly sensitive and precise, and gave reproducible results in the range of 1-1,000 ng/ml. The specificity and sensitivity of this assay permitted measurement of a low content of acid stable trypsin inhibitor in different tissue extracts and body fluids. Using the new method, the mean level in plasma samples taken from healthy individuals was found to be 54.9 +/- 17.4 (mean +/- SD) micrograms/ml and that of acid-treated plasma was 11.5 +/- 3.0 micrograms/ml (n = 27). The calculated specific activity of acid stable trypsin inhibitor in acid treated plasma was 300 U/mg, while that of highly purified inhibitor is 2,100 U/mg. These results suggest the presence of both acid unstable protein having the same antigenicity as urinary trypsin inhibitor and the inactive form of acid stable trypsin inhibitor.

Distribution and localization of inter-α-trypsin inhibitor and its active component acid-stable proteinase inhibitor: Comparative immunohistochemical study

Inter-α-trypsin inhibitor (ITI) is a complex that consists of three components. One of these is the acid-stable proteinase inhibitor (ASPI), which is an acutephase reactant and a broad-spectrum inhibitor. The tissue distribution of ITI and ASPI were investigated and compared using immunohistochemical methods. ITI immunoreactivity was revealed only in the liver and plasma, while ASPI immunoreactivity was found to be distributed in the brain, liver, kidney, gastrointestinal tract, plasma, and urine. Both immunoreactivities were demonstrated in Kupffer cells of the liver, which is thought to be an ITI-producing organ. From these results, it seems unlikely that ASPI is distributed as a part of the ITI molecule. The residual component of ITI may act as a carrier protein of ASPI, or ASPI in the tissues may be produced independently of ITI.