Yoko Nagamatsu

Effect of Physical Training on Thrombotic Tendency in Rats: Decrease in Thrombotic Tendency Measured by the He-Ne Laser-Induced Thrombus Formation Method

The effect of physical training on thrombotic tendency was assessed in rats. Exercise was done on a flat treadmill for 30 min at a rate of 1,400 m/h (submaximal speed), 5 times a week for either 1.5 or 3 months. The thrombotic tendency was measured by the He-Ne laser-induced thrombus formation method in microvessels of mesentery, i.e. measurement of the number of laser irradiations necessary to induce stasis of blood flow by occlusive thrombus formation. An increase in the number of irradiations necessary to induce occlusive thrombus formation was observed in arterioles, but not in venules after physical training for 1.5 and 3 months.

Determination of Leukocyte Elastase Concentration in Plasma and Serum by a Simple Method Using a Specific Synthetic Substrate

The concentration of leukocyte elastase (ELP) in plasma and serum was determined by an amidolysis method using a specific synthetic substrate for ELP, Suc-Ala-Tyr-Leu-Val-pNA. Results were compared with those using ELISA. ELP levels in plasma from healthy donors were similar to those determined by ELISA; however, the levels in serum were lower than those determined by ELISA. Correlation coefficients of ELP levels in plasma and serum as measured by the two methods were 0.75 (amidolysis) and 0.90 (ELISA). On the other hand, the correlation coefficient between serum ELP by the two methods was 0.83. Half of the ELP levels in plasma from 150 patients and serum from 400 patients were significantly elevated when compared with those from healthy donors, and the ELP elevation determined by amidolytic assay correlated with some variables in blood, namely fibrin(ogen)-degraded products, fibrinogen, GOT, GPT, gamma-GTP, LDH and leucine aminopeptidase. Despite the fact that the amidolysis method detects the alpha 2-macroglobulin-ELP complex while ELISA detects the alpha 1-antitrypsin-ELP complex, a comparative study showed amidolysis to provide sufficiently sensitive measurement of both plasma and serum ELP.

Synthesis of peptide fragments related to eglin c and examination of their inhibitory effect on human leukocyte elastase, cathepsin G and α-chymotrypsin

Various kinds of peptide fragments related to eglin c were prepared by the conventional solution method and their inhibitory effects on human leukocyte elastase, cathepsin G and alpha-chymotrypsin were examined. Peptide (31-40) inhibited cathepsin G (Ki = 2.3 x 10(-4) M), peptide (41-49) potently inhibited cathepsin G and alpha-chymotrypsin (Ki = 4.2 x 10(-5) M and 2.0 x 10(-5) M, respectively), and peptide (60-63) inhibited leukocyte elastase (Ki = 1.6 x 10(-4) M), whereas, peptide (31-35) weakly inhibited both elastase and cathepsin G (Ki = 2.1 x 10(-3) M and 7.3 x 10(-4) M, respectively).

A new polypeptide substrate, Suc-Tyr-Leu-Val-pNA, specific for spleen fibrinolytic proteinase (SFP)

Summary In a search for an adequate synthetic substrate for human spleen fibrinolytic proteinase (SFP), Suc-Tyr-Leu-Val-pNA was newly synthesized. Its degradation by SFP was compared with that of other synthetic polypeptide substrates. Suc-Tyr-Leu-Val-pNA was degraded by SFP with a high K cat /K m , but it was not degraded by the following serine proteinases, plasmin, trypsin, thrombin, chymotrypsin, urokinase and milk plasminogen-activator, or by pancreatic elastase to any practical extent.

Synthesis of a trihexacontapeptide corresponding to the sequence 8–70 of eglin c and studies on the relationship between the structure and the inhibitory activity against human leukocyte elastase, cathepsin G and α-chymotrypsin

A trihexacontapeptide corresponding to the sequence 8–70 of eglin c and its related peptides were synthesized by the conventional solution method and their inhibitory activity against human leukocyte elastase, cathepsin G and α‐chymotrypsin was examined. Although synthetic eglin c (41–49) inhibited cathepsin G and α‐chymotrypsin (K i = 4.0 × 10−5 M and 2.0 × 10−5 M, respectively) but not leukocyte elastase, the synthetic trihexaconta‐peptide potently inhibited cathepsin G, α‐chymotrypsin and leukocyte elastase (K i = 1.8 × 10−9 M, 1.4 × 10−9 M and 2.2 × 10−9 M, respectively). The relationship between the stucture of eglin c and the inhibitory activity against the above enzymes is also described.

Antithrombotic effect of ticlopidine on He-Ne laser-induced thrombus formation in rat mesenteric microvessels.

The inhibitory effects of ticlopidine and acetylsalicylic acid (ASA) on thrombus formation in rat mesenteric microvessels were studied. The results were compared with the effect of the drugs on platelet aggregation in citrated whole blood. He-Ne laser-induced thrombus formation in arterioles and in venules was significantly inhibited by 100 mg/kg ticlopidine. In contrast, a higher dose of ASA (300 mg/kg) was needed to inhibit thrombus formation and the effects of ASA were observed only in arterioles and not in venules. In addition, although the inhibition by ASA in arterioles was not strong it followed a dose-dependent manner, suggesting that the differential effect of ASA on platelets and endothelium may not be evident in vivo. Ticlopidine and ASA strongly inhibited ADP-induced whole blood platelet aggregation, but not collagen- or thrombin-induced platelet aggregation.

Effect of Long-Term Aerobic Exercise on Helium-Neon-Laser-Induced Thrombogenesis in Rat Mesenteric Arterioles and Platelet Aggregation

We have previously investigated the antithrombotic effect of aerobic exercise in rat arterioles and venules under conditions of variable exercise load. In the present study, rats were subjected to constant exercise of 90% maximal oxygen uptake for 30 min, 5 times a week for 2, 4, 8 or 18 weeks. Thrombotic tendency was assessed by the He-Ne-laser-induced thrombus formation method. Platelet aggregation, platelet adhesion, whole blood clotting time, fibrinogen levels and blood cell count were also measured. The thrombotic tendency in arterioles decreased significantly after 18 weeks training, but not in venules. Thrombotic tendency increased slightly after 2 weeks training though the differences were not significant. A significant decrease in collagen-induced platelet aggregation was observed after 18 weeks training. The results demonstrated that long-term aerobic training decreased the thrombotic tendency in rat arterioles and that this was partly due to decreased platelet aggregability.

Increase of Nonplasmin Fibrinolytic Activity in the Lung and Spleen of Streptozotocin-Induced Diabetic Rats

In streptozotocin-induced diabetes in rats, nonplasmin fibrinolytic activity was found to be significantly increased in the lung and spleen, but not in the liver and kidney. The plasminogen-activator activities, either soluble or insoluble, were almost simultaneously increased in the lung, suggesting the possible release of plasminogen-activator from the lung to the blood. It seems likely that plasminogen-activator of the usual type and nonplasmin protease are also involved in thrombotic disorders in the experimental diabetes.

Fibrinolytic and Amidolytic Activities of Elastase- and Chymotrypsin-Like Proteases in Spleen and Leukocytes of Some Mammals

Nonplasmin-mediated fibrinolytic activities were extracted with 2 M NaCIO4 from the spleen and leukocytes of nine species of mammals. The amidolytic and fibrinolytic activities of extracts were measured using Suc-Ala-Tyr-Leu-Val-pNA for elastase-like protease (ELP) and Suc-Tyr-Leu-Phe-pNA (or Suc-Ile-Pro-Phe-pNA) for chymotrypsin-like protease (CLP). ELP and the fibrinolytic activity in the human spleen and leukocytes exhibited the highest values among the nine species. The relative order of amidolysis by CLP was as follows: hamster greater than rat greater than mouse greater than dog in the spleen and hamster greater than rat = ox greater than dog in leukocytes. Neither fibrinolytic nor amidolytic activity was measurable in spleens and leukocytes of pigs and rabbits. The ratios of ELP to CLP activity in the spleen or leukocytes of each of the species varied significantly from those observed in humans. The amidolytic activity of the spleen in control mice was low; however, that of mice which had been intraperitoneally inoculated with sarcoma 180 cells was enhanced and further accompanied by an increase in the number of granulocytes and in fibrinolytic activity. These results suggest that the enhancement of spleen amidolytic and fibrinolytic activities is induced by an increase of granulocytes in the circulation.

The level of granulocyte elastase (ELP) in children suffering from inflammatory diseases.

In order to study on the elastolysis and fibrinolysis in inflammatory process, the level of granulocyte elastase (ELP) was assayed in inflammatory diseases. The assay was carried out in children suffering from inflammatory diseases such as Kawasaki disease (MCLS), pneumonia, allergic purpura and others.ELP was extracted from granulocytes by 2mol/l KSCN and measured by using the artificial substrate, Suc-Ala-Tyr-Leu-Val-pNA. The biological activity was expressed as amidolytic activity per cell. The ELP activity of normal newborn babies was 9.0±3.1 nU/cell and it increased by age, attaining to the level of 16.1±5.8 nU/cell of normal adults. In MCLS, pneumonia and aseptic meningitis, the level of ELP decreased during the 2nd and 3rd weeks of illness, and it returned to the normal range up to the 4th or 5th week. On the other hand, the level of ELP was not reduced in upper respiratory diseases in which the inflammation was moderate. In allergic purpura, the level of ELP increased and returned to the normal range after the 5th week. Although the causes of the decreased level of ELP in inflammatory diseases are not clear, it can be suspected that ELP is released from granulocytes and consumed during inflammatory process.