Concentrations of leukocyte elastase (ELP) and cathepsin G (CLP) in leukocytes, plasma and serum from healthy human blood were measured using synthetic substrates. The best condition of the extraction of both enzymes from leukocytes was first studied, and the normal values of ELP and CLP in plasma and serum were calculated from the decreased ratio of their activities after the addition of plasma and serum to the pure enzymes. The results obtained were as follows; 1) The amidolytic activity of ELP which obtained by repeated freezing and thawing twenty times in Tris-HCl buffer (containing 0.15-2.0M NaCl) showed similar value to the activity extracted with 2M NaClO₄. The amidolytic activity of CLP weakened rapidly in the medium, while the extraction of maximum activity was obtained by repeated freezing and thawing in the above mentioned buffer. 2) The amidolytic activities of ELP and CLP were decreased to 4% and 45%, respectively, by the addition of normal plasma or serum. 3) Normal values obtained were as follows. ELP and CLP were 7.5±1.9μg and 6.0±1.7μg per 10⁷ leukocytes, respectively. ELP and CLP in plasma were 150±66ng/ml and 13±4.4ng/ml, and those in serum were 315±165ng/ml and 26±9.8ng/ml, respectively.

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The response of coagulation and fibrinolysis to venous occlusion test (VOT) was studied in 23 patients with thrombotic disorders before and after 4.7 (3-9) months' administration of elastase (1800 EL. U./day).
The patients were classified into “normal” and “low” responders according to the grade of response to VOT before treatment. After administration of elastase, four of 8 low responders showed improved response, whereas 2 of 15 normal responders changed into low responders. Although plasminogen activator activity before and after VOT did not change after the administration, plasminogen activator inhibitor (PAI) activity at rest decreased after the administration. Tissue plasminogen activator and PAI-1 antigen, crosslinked fibrin derivatives, von Willebrand factor antigen and antithrombin III did not change after the administration.
These results suggest that elastase may improve the reduced fibrinolysis in case of thrombotic disorders due to high PAI activity. Further analysis would be necessory to clarify the mechanisms of the effect of elastase on fibrinolytic system in larger series.

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P-nitrosodimethylaniline (P-NSDA), one of the OH radical (OH·) scavengers, inhibited platelet aggregation by ADP or arachidonic acid (AA), inducing a certain alteration in platelet phospholipid metabolism.
In the platelet aggregation, it has been reported that reactive oxygen species, OH·, is generated in the process of the concomitant AA metabolism, and modified the rate and extent of various reactions in it. Then, we studied the effects of OH· scavengers on platelet aggregation.
Washed rabbit platelets were incubated at 37°C for 4min with OH· scavengers (P-NSDA, thiourea, D-mannitol, urea and methional), lipoxygenase inhibitor (nordihydroguaiaretic acid; NDGA) or cyclooxygenase inhibitor (benzoic acid; BA) before aggregation was induced. P-NSDA inhibited ADP or AA-induced platelet aggregation about 70-80% in comparison with control, but other OH· scavengers had no effects on ADP, AA, thrombin or A23187-induced aggregation. On the other hand, BA and NDGA inhibited AA or ADP-induced aggregation and their inhibition patterns were similar to that of P-NSDA. So, we considered that the inhibition of P-NSDA on ADP or AA-induced aggregation was not induced by its effect of trapping OH· but by altering the phospholipid metabolism associated with the platelet aggregation.
To confirm these considerations, we tried to analyze phospholipid metabolites during AA-induced platelet aggregation treated with P-NSDA by TLC.
In the results, it was found that the pattern of TLC in the platelet treated with P-NSDA was greatly different from those treated with inhibitors of AA metabolism, such as aspirin and NDGA. These findings indicate that the inhibition of the platelet aggregation by P-NSDA relates with other phospholipid metabolism than AA metabolism in platelet associated with platelet aggregation and that P-NSDA may be useful for investigating phospholipid metabolism in platelet aggregation.

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Dynamic evaluation of thrombus formation on vascular catheters has been impossible because thrombi on a catheter are detached when it is taken out. We observed thrombus formation in vivo by using a vascular endoscopic system, and evaluated the inhibitory effects of heparin and antiplatelet agents. During cardiac catheterization a Barman balloon catheter was inserted in the pulmonary artery (PA). When the balloon was inflated and wedged in the main PA, 10-20ml of saline was flushed through to provide a visual field. Observations were done in 13 patients under systemic heparinization. Four of these patients were on antiplatelet agents. On the other hand, observations were done without systemic heparinization in seven patients three of whom were using antiplatelet agents. Fibrin thrombi could be seen in all 13 patients without antiplatelet agents within 6 minutes (5.22±0.14min), while in patients on antiplatelet agents, no thrombi were seen within 10 minutes. We conclude that fibrin thrombi may be formed on vascular catheters within 6min. Antiplatelet agents inhibited this early thrombus formation, while heparin did not.

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Changes in protein C activity, protein C antigen, Vitamin K-dependent coagulation factors (II, VII, IX, X) and protein C induced vitamin K abscense of factor II (PIVKA-II) were investigated in 30 patients treated with Warfarin for long periods. Those included 23 patients with prosthetic heart valves and 7 patients reconstructed peripheral vessels. Protein C activity was measured by rapid assays. amidolytic and clotting time assays using a protein C activator (PROTAC^®) from the venom of Agkistrodon contortrix. Protein C levels measured by amidolytic assay correlated significantly with the immunological assay (ELISA) (r=0.718, p<0.001), but the clotting time assay gave lower protein C values than the amidolytic and immunological assays. Furthermore, the ratio of protein C activity (clotting time assay) to protein C antigen tended to decrease against the increase in PIVKA-II. The comparison of the three PC assays to thrombotest indicated that highly significant correlation was found between clotting time assay and thrombotest (r=0.483, p<0.01), furthermore, protein C values measured by the clotting time assay correlated with prothrombin activity (r=0.598, p<0.001). However a decrease in the ratio of protein C activity (clotting time assay) to prothrombin activity was observed in three cases with prosthetic heart valves (ratios of 0.25, 0.28, 0.34 and the prothrombin activities of these cases were beyond 30%). These results indicate that the protein C activity measured by clotting time assay evaluates the in vivo function of protein C, and that protein C function as well as prothrombin activity is inhibited in patients on stable oral anticoagulant therapy.

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Since the biological role of vitamin K (VK) in the synthesis of blood clotting factors was established, hypothrombinemia in humans and animals has frequently been investigated in relation to VK metabolism. The minimum dose of VK to restore normal plasma clotting factor levels, and its effective duration, were examined in VK-deficient male rats. The prothrombin time (PT) in rats kept on an ordinary diet containing about 500ng/g of VK₁ was 10.6±0.4sec. The PT in rats fed a VK-deficient diet, containing 32ng/g of VK₁, for 7 days was 13.5±0.2sec. When rats were maintained on the VK-deficient diet. a slight but constant VK deficiency was seen from day 7 to day 15. These animals were given single subcutaneous injections of various doses of VK₁ on day 7. The PT, APTT, factor VII, prothrombin and fibrinogen levels, as well as plasma and liver PIVKA-II levels were determined 1, 2, 4, and 8 days after the VK₁ injection. For rats on a VK-deficient diet, the minimum effective dose of VK₁ was 3μg/kg by subcutaneous injection and its effect lasted for 2 days. When 100μg/kg of VK₁, was injected, the effect lasted for almost 8 days. VK synthesized in the large intestine did not seem to be utilized in these rats to prevent the development of hypothrombinemia.

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Plasma levels of crosslinked fibrin degradation products (XDP) in 500 healthy subjects (250 male and 250 female), 18 patients with disseminated intravascular coagulation (DIC) and patients with thrombotic disease were measured by the enzyme linked immunosorbent assay (ELISA) using specific anti D-dimer monoclonal antibody.
Plasma levels of XDP in 500 healthy subjects were 174 (38-309)ng/ml and sex related change was observed (p<0.01). High XDP values were observed in all of 18 patients with DIC. The results were also well correlated with those obtained by the latex agglutination assay.
These results indicate that the measurement of XDP using anti D-dimer monoclonal antibody could be one of the sensitive parameters in thrombotic and fibrinolytic condition especially DIC.

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Circadian fluctuation of plasmatic tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) in healthy 15 adults was examined for 24 hours. In comparison to t-PA level at 16:00, the level significantly increased at 8:00 and 9:00 (p<0.01), then gradually reduced to 1/2 by the late afternoon. PAI-1 level also markedly increased at 8:00 and 9:00 (p<0.01), then rapidly reduced to less than 1/3 of that level by 12:00 and the low level was maintained through afternoon to late at night. There were individuals showing steep drop of PAI-1 level from 8:00 to 12:00. It was concluded that plasma fibrinolysis was regulated by PAI-1 rather than t-PA in physiological fibrinolysis and that interpretation should be careful of the analysis of fibrinolysis particularly of PAI-1 because of its marked circadian fluctuation.

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To study the susceptability of fibrin plate method for determination of t-PA activity, the activity of melanoma t-PA (mt-PA) was compared with that of recombinant t-PA (rt-PA) using the International Reference Preparation for urokinase as a standard.
The dose-response lines of t-PA and urokinase were not parallel. Unexpectedly, in some cases, those of mt-PA and rt-PA were not parallel. The specific activities of them changed with the location on the urokinase standard line at which the t-PA was read and with experimental conditions.
This data suggested that the fibrin plate method was not admissible to compare the activities among t-PA preparations, derived from differnt sources.

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Previous studies revealed that plasma tissue plasminogen activator (t-PA) level was elevated in patients with liver disease. In this study, the correlations between levels of plasma t-PA and serum total bilirubin (T. Bil) and hepatic synthetic functions were investigated in patients with chronic liver diseases (chronic hepatitis CH; N=8, liver cirrhosis LC; N=63) and with hepatocellular carcinoma (HCC; N=35).
The t-PA levels showed significant increase in patients with LC and HCC, especially in the decompensated stage of LC, compared with normal controls (N=20). Moreover, the t-PA levels significantly correlated positively with T. Bil levels and negatively with liver synthetic functions such as serum albumin, protein C in patients with LC.
Serial determination of t-PA levels in 8 HCC cases treated tsanscatheter arterial embolization (TAE) revealed that the values showed rapid and transient rises after TAE and returned to almost the pre-treatment levels on the 7th day after TAE. Therefor, we assume that the elevated t-PA levels in these HCC cases were due not to increased production of t-PA by hepatoma itself, but mainly to impaired liver function.

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Evidence obtained by immunological analysis of plasma from patients with ARDS indicates that polymorphonuclear leukocyte elastase play important role in ARDS and is suggested one of cause of ARDS.

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The third degree burn covering 35% of the total body surface area was made on the back of rabbits, and the animal were divided into 6 groups. The effectiveness of infusion therapy, and some drugs which affect on arachidonate cascade (aspirin, OKY-046, CV-4151 and ONO-3708) after burn injury were studied by examining the changes of arachidonate cascade of the burned tissues. Determinations of tissue thromboxane B₂ (TX) and tissue 6-keto prostaglandin F_1α (PG) showed the increase of tissue TX in infusion therapy groups. On the contrary, tissue TX levels were decreased in ASA, OKY-046 and CV-4151 groups compared to infusion therapy group. Tissue PG levels decreased only in ASA group. On the other hand, tissue TX/PG ratio was decreased in OKY-046 and CV-4151 groups. Examination of wet-dry weight of the burned tissue revealed that only ASA decreased the amount of edema formation after burn injury while other drugs did not affect the increase of wet weight. It is suspected that the changes on arachidonate cascade at burned wound may play a role in edema formation, eventually affect the permeability of vessels on burned tissue.

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The effects of cardiac rehabilitation on blood coagulability were investigated in 30 patients (mean age 55.6 years) with acute myocardial infarction. One month after the onset of acute myocardial infarction, the cardiac rehabilitation was started for about a month, and blood coagulability including fibrinolysis was compared between pre- and post-rehabilitation. The measurements were performed on hematocrit value, prothrombin time, activated partial thromboplastin time, activities of factor VIII, antithrombin III, plasminogen and α2-plasmin inhibitor, and plasma levels of fibrinogen, and α2-macroglobulin. After cardiac rehabilitation, activities of factor VIII lowered from 182% to 123% and activated partial thromboplastin time prolonged, although prothrombin time remained unchanged. Plasma fibrinogen levels decreased from 311mg/dl to 254mg/dl and antithrombin III activities also decreased from 115% to 102% after rehabilitation. Activities of plasminogen as well as α2-plasmin inhibitor decreased after rehabilitation from 96% to 86% and 111% to 86%, respectively., Hematocrit also decreased from 38.5% to 37.1%, These findings appear to suggest that cardiac rehabilitation induces suppression of the coagulation system and activation of the fibrinolysis system without loss of hemostatic balance. Furthermore, there seemed to be no differences in acute response of blood coagulating factors to treadmill exercise between pre- and post-rehabilitation.

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The authors reported previously that an administration of hemostatic drugs including VK has no significant roles on the prevention of hemorrhage during immediate postoperative period. In this article, we studied the effect, of VK on the prevention of hemorrhage in postoperative patients treated with antibiotics.
For this purpose, we measured prothrombin time (PT), Factor II (F-II), Factor, VII (F-VII), protein C, PIVKA II (P-II) and plasma level of VK in two groups of patients with or without VK administration. In the group of patients with VK administration 120mg/day of VK₂ were given intravenously everyday. The patients of both groups were fasted and treated with antibiotics for two weeks after the operation.
In the group without VK administration, PT was prolonged and F-II levels were decreased from the seventh postoperative day. Although F-VII and PC levels were decreased on the first postoperative day and returned subsequently on the seventh day, no significant difference was observed between the levels of two groups. P-II levels were significantly increased in the group without VK administration, whereas no significant changes was observed in the group with VK administration. The plasma level of VK₁ decreased in both groups, but the level of, VK₂, especiall MK-4, was high in the group with VK administration.
These results indicate that patients treated with antibiotics during postoperative fasting period may have a VK deficiency from the seventh day. Although no bleeding disorders were observed, an administration of VK is recommended for these patients.

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Platelet binding IgG (PBIgG) in plasma from a patient with HIT measured with an elevation by the addition of unfractionated heperin rather than LMWH, and the elevation of PBIgG could be suppressed with MD805. It was suggested that both drugs might exert suppressible effect on IgG binding to platelet in patient with HIT.

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To evaluate the antithrombogenic activity of endothelium, we intravenously injected heparin (60U/kg) in patients with thrombotic tendency and healthy controls, and measured both plasma concentration of platelet factor 4(PF4) and β-thromboglobulin(βTG).
The plasma PF4 increased transiently 5 minutes after heparin injection, while βTG did not. PF4 bound to cultured human umbilical venous endothelial cell monolayer was also released by the incubation with heparin (1U/ml) in vitro.
As the heparin exerts its anticoagulant activities by potentiating the antithrombin III (AT III) against the coagulation factor proteinases, more increasing amounts of PF4 released from endothelium neutralize larger heparin-like molecules on the endothelium and decrease the antithrombogenic activities of endothelium.
We designated the heparin-releasable PF4 as ΔPF4 and βTG before heparin injection as pre-βTG respectively. It is assumed that ΔβF4 is one of a marker of the endothelial antithrombogenic activity and pre-βTG is a marker of the platelet activation in vivo.
In healthy controls, there was a direct corelation between pre-βTG and ΔPF4. The patients with thrombotic tendency (cerebral thrombosis, DM, rheumatoid disease, DIC, and malignancy) showed abnormal profile, that is high βTG with increased heparin releasable PF4.
In conclusion, heparin infusion test is appeared to be a useful method for the evaluation of the endothelial antithrombogenic activity.

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