Role of Ca⁺⁺ and calmodulin in PGI₂ generation of cultured human vascular endothelial cells was investigated using ⁴⁵Ca and W-7, including the mechanism of the enhancement of PGI₂ generation by a Ca antagonist, diltiazem.
We have previously reported that PGI₂ generation was dependent on intracellular Ca⁺⁺ mobilized from Ca⁺⁺ storage sites, and that extracellular Ca⁺⁺ was also essential for the enhancement of PGI₂ generation by diltiazem or A23187. In this study, net influx and efflux of ⁴⁵Ca were rapidly increased and reached at plateau phase after 10min of the incubation, following PGI₂ generation. While A23187 increased both of them, diltiazem did not show significant effect on both of them. W-7 inhibited PGI₂ generation in a dose dependent manner, and also decreased PGI₂ generation by arachidonic acid or PGH₂ in the pretreatment with mepacrine or aspirin. Moreover PGI₂ generation induced by diltiazem was also inhibited by W-7.
Through these results, it was suggested that 1) PGI₂ generation and its enhancement by A23187 were triggered by the influx of extracellular Ca⁺⁺ inducing the mobilization of Ca⁺⁺ from storage sites. 2) Calmodulin might be contributed to activation of not only phospholipase, but also cyclooxygenase or PGI₂ synthetase probably via modulation of kinetics of intracellular Ca⁺⁺ 3) The enhanced PGI₂ generation by diltiazem was considered to be brought by the influx of a small amount of extracellular Ca⁺⁺ which was not detected in this experimental system, and calmodulin was contributed to this enhancement.

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The effects of angiotensin I (AI)-converting enzyme (ACE) linked substances upon PGI₂ generation were investigated using cultured human vascular endothelial cells and rat aortic rings.
Endothelial cells were obtained from human umbilical cord vein and cultured by the modified method of Jaffe et al. PGI₂ level was measured by radioimmunoassay of its stable metabolite 6-keto PGF_1α, and ACE activity was assayed by Lieberman's method.
In the endothelial cells, addition of AI or bradykinin (BK) enhanced PGI₂ generation and also increased ACE activity in the culture medium, but angiotensin II (AII) did not show any effect. Captopril inhibited both of PGI₂ generation and ACE activity in a dose dependent manner, but the enhancement of PGI₂ generation by AI or BK was not affected by the pretreatment with captopril. Meanwhile in the rat aortic rings, AI, AII, and BK enhanced PGI₂ generation but captopril decreased PGI₂ generation in its higher concentration (10^-3M), and at the same time mechanical stimulation enhanced PGI₂ generation. But in the aortic rings whose endothelium was rubbed with blade, PGI₂ generation and AII induced PGI₂ enhancement remarkably decreased.
Through these results it was suggested that conversion of AI to AII by ACE was suspected not to cause the enhancement of PGI₂ generation in the endothelial cells, and that the enhanced PGI₂ generation of aortic rings by AII was considered to be due to interaction of smooth muscle cells and endothelial cells. The previously reported hypothesis that captopril enhanced PGI₂ generation by the accumulation of AI or BK via ACE inhibition, was not confirmed in this experimental system, and captopril was proved to inhibit PGI₂ generation directly. It was rather suggested that the enhanced PGI₂ generation in the endothelial cells by AI or BK was probably controlled by their ACE activation, as an autoregulation mechanism.

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Human plasma antithrombin III (AT III) shows heterogenous banding pattern by agarose gel isoelectric focusing (AGIEF) and its heterogeneity is considered to be closely related to the development of activity.
Among 300 citrated plasma, 298 samples show the same AGIEF pattern (AT IIIc, c; common), however 2 of them exhibited the different pattern (AT IIIv, v; variant). AT IIIc consists of 5 major bands (pH 4.82-5.12) and the amount of band with isoelectric poit (pl) 4.87 accounts for 61.7% of total AT III. In contrast, AT IIIv shows 7 major bands (pH 4.76-5.12) and the bands with pl 4.84 and 4.87 account for 36 and 37%, respectively. Moreover, some bands of AT IIIv possess same pls as those of AT IIIc. After the treatment with neuraminidase, AT IIIc and AT IIIv decreased their heterogeneity to 1 major (pl=5.72) and 2 mayor (pl=5.60, 5.72) bands, respectively. From the analysis of AGIEF pattern of these AT III, AT IIIv was suggested to be phenotypically heterozygous and combination of the band-group of AT IIIc and variant bandgroup which have pH 0.12 lower than AT IIIc. AT IIIc and AT IIIv inhibited thrombin in a similar manner in the absence or the presence of excess heparin, However at lower heparin concentration, AT IIIv inhibits thrombin more slowly than AT IIIc.
Through these studies it is concluded that the variant type of AT III detected by AGIEF represents a qualitative abnormality and that it is suspected to be responsible for the pathogenesis of thromboembolism.

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We studied the fibrinolytic activity, amount and localization of plasminogen activator (PA) in bovine vitreous extract (VE) and aqueous humor (AH). Fibrinolytic activity of VE and AH was quenched by adding anti-human t-PA IgG, but not by anti-human HMW u-PA IgG. After SDS electrophoretic analysis, enzymographic distributions of PA in VE and AH were detected at about Mr. 66K and 110K. These findings suggested the fact that the main PA in VE and AH was t-PA. Estimating fibrinolytic activity of VE and AH by amidolytic assay, activity was 0.12±0. 10IU/ml (n=10) in VE and 0.04±0.07IU/ml (n=10) in AH. Measuring the amount of t-PA by RIA, t-PA was 4.1±0.9ng/ml (n=10) in VE, and 3.3±0.8ng/ml in AH. Investigating the PA activity by Todd's fibrinolysis autography, clear lytic areas could be seen on the small vessels of the iris, and the subepithelial stroma and along the endothelium of the cornea. In immunohistochemical studies using anti-human t-PA IgG, vascular endothelial cells and corneal epithelial cells showed the positive reactivity.

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Protein C is a vitamin K-dependent anticoagulant plasma protein, and its deficiency is known to be associated with a risk for thrombosis.
We measured plasma protein C levels of healthy Japanese adults and patients with cerebral infarction, to study whether there is any relationship between the level of protein C and the occurrence of cerebral infarction.
In healthy subjects there was no significant difference of plasma protein C level with age. However, the protein C level was significantly lower in patients with cerebral infarction than healthy adults.
The protein C level was not correlated with clinical courses, location of infarction, or severity of disease: any significant difference was not recognized in the protein C level between the patients with acute and subacute cerebral infarction, with the lesions in the cortical and perforating arteries, or with severe and mild manifestations at onset. But the protein C level was significantly decreased in patients with bad course in comparison with those with good course.
It was suggested that the low level of plasma protein C has some relevance to occurrence of cerebral infarction and furthermore the low protein C level observed at the first attack may indicate poor prognosis with likelihood of relapse.

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The serial pathologic studies of placenta examined in Nepal of high mountain country since 1977 were compared with those of Japanese placentas. As the most characteristic findings of Nepalese placentas, massive subchorionic fibrin deposits were observed. In this paper, in order to find a cause of such a high incidence of intraplacental coagulation, coagulation and fibrinolytic states of 160 Nepalese pregnant women from 20 to 40 weeks of gestation were investigated and compared with those of Japanese pregnant women.
Results: 1) Levels of fibrinolytic inhibitors of α₁-AT, α₂-MG, α₂-PI and C₁-INA in Nepalese group were higher than those of Japanese group, while AT-III levels were lower than those of Japanese women group. 2) No significant differences of PTT as well as concentrations of fibrinogen, plasminogen and FDP were found in both groups, but FVIII value showed significantly high in Nepalese group. 3) Prekallikrein and kallikrein levels in Nepalese group were much higher than those of Japanese group. 4) Protein C levels in Nepalese pregnant women were less than those of Japanese group, while levels of PIVKA-II over 1μg/ml in Nepalese group amounted to 45%.
In conclusion, in general, Nepalese pregnant women at high land have a tendency of hypercoagulable state which might lead to intraplacental hypercoagulation.

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To evaluate the effect of intracoronary thrombolysis (ICT) using urokinase (UK) in acute myocardial infarction, 79 patients were studied. ICT was performed within 6 hours (4.1±1.2 SD) after onset of symptom (Table 1-A and 1-B). Infarction size was estimated by ΣCK (Norris's method) and ECG QRS score (Wagner's method) (Table 2). Cardiac function at the late period (4-7 weeks) was evaluated by left ventricular ejection (LVEF) (Dodge's method). Incidence of total death, re-infarction, post-infarction angina and congestive heart failure (CHF) were observed for the mean period of 18.1±12.8 months.
The obstructed coronary artery had been recanalized spontaneously before ICT in 13 patients (group A), was successfully recanalized with ICT in 34 patients (group B) and was not reperfused with ICT in 32 patients (group C). Accordingly successful reperfusion of the obstructed coronary artery with ICT was obtained in 34 out of 66 patients (51.5%) and when recanalization with residual 99% stenosis and delayed filling was inclued in the group of successful reperfusion the rate increased to 63.6%.
In 16 patients whose initial QRS score was more than 5, ΣCK and the score at 28-day were highest and LVEF was lowest, regardless of the result that the obstructed coronary artery was recanalized or not (Figure 1, 2 and 3).
Among the patients of the first MI with initial QRS score less than 4, ΣCK and 28-day QRS score were lower and LVEF was higher in groups A and B than non-recanalized group C. If the subject was limited to those patients with anterior MI (lesion at #6 or #7) and initial score less than 4, ΣCK and 28-day score were lower and LVEF was higher in groups A and B as compaired to group C, and these difference were statistically significant (p<0.05-0.02) (Figure 1, 2 and 3).
Incidence of total death, re-infarction, angina and CHF were 6%, 10%, 32%, and 24% respectively, and the difference among 3 groups was showen in Table 4.
In conclusion, ICT seems to be effective on reducing infarction size and preserve cardiac function only in patients with initial QRS score less than 4. The hospital mortality was 5%, post-infarction angina noted in 54% of group A and CHF was most frequently seen in group C.

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For an analysis of relationship between platelet sialic acid and platelet associated IgG (PA-IgG), PA-IgG and sialic acid level in both platelet and plasma were measured. PA-IgG was measured by immunfluorescent assay. This immunofluorescent assay is based on the treatment of platelets with neuraminidase. Sialic acid levels of platelets and plasma were measured by the thiobarbituric acid method.
In patients with thrombocytopenia (ITP, acute leukemic patients who received platelet concentrates from random donors), the PA-IgG positive rates (% PA-IgG) in neuraminidase-treated and non-treated platelets were 37.0±4.18% and 7.9±2.33%, respectively. The results indicated that % PA-IgG was significantly elevated by neuraminidase treatment of platelets.
Platelets from normal controls had a mean sialic acid of 0.72+0.30mg/10⁸ platelets. Platelets from the patients had a mean sialic acid of 0.86+0.44mg/10⁸ platelets. Sialic acid levels of platelets were significantly elevated in the patients. Sialic acid levels of plasma were elevated in the patients, but there was no correlation between platelet and plasma sialic acid in the patients.
These findings suggest that the higher quality of sialic acid on the patient platelets might inhibit the detection of the PA-IgG.

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Nine commercially available intravenous immunoglobulin preparations (IVIG) produced by different manufacturers, such as alkylated, sulphonated, pepsin-, plasmin-, polyethyleneglycol- and pH 4 treated ones in 42 different lots were examined their human immunodeficiency virus (HIV) antibody, employing three ELISA test kits (Abbott, Wellcome, Du Pont), an immunofluorescence test procedure and a Western blot technique.
Eight preparations out of nine were revealed to be antibody positive, and they were tested first by ELISA for screening and then confirmed their results by immunofluorescence and Western blot tests. Although HIV antibody positive results do not necessarily mean that. HIV is present in the products, our finding suggests that there might be potential infectivity for HIV by IVIG, and so careful donor selection and devised preparative processing of IVIG for inactivation of HIV are needed. Furthermore screening fo HIV itself in the products would become indispensable in the near future.

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The effect of molecular weight and sulfate amount of sulfated polysaccharide on the thrombin inhibitory activity of heparin cofactor II was investigated by using various dextran sulfate fractjons with different molecular weight and sulfur content. The dextran sulfate fractions of over-10kDa and 18% sulfur showed the strongest effect. The maximum second order rate constant of heparin cofactor II-thrombin reaction in the presence of these fraction was 2.7×10⁸M^-1min^-1 that was almost same as in the presence of heparin (2.5×10⁸M^-1min^-1) or dermatan sulfate (3.8×10⁸M^-1min^-1). On the other hand, dextran sulfate accelerated antithrombin III-thrombin reaction only about 40-fold less than heparin. These results indicate that a large molecular size and significant amount of sulfate groups are only essential in the acceleration of the thrombin inhibitory activity of heparin cofactor II, whereas a specific sequence of heparin is required to that of antithrombin III.

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MK-447 has been reported to scavenge oxygen-derived free radicals, which are generated in conversion of PGG₂ to PGH₂. Furthermore, we have shown that the agent stimulates PGI₂ formation from endogenous and exogenous arachidonic acid by isolated rat aortae, acting as a tryptophan-like cofactor of PG endoperoxide synthetase. The present paper shows that MK-447 increased plasma 6-keto PGF_1α level in rabbits.
Pentobarbital-anesthetized rabbits were injected with angiotensin II (A II, 5μg/kg, i. v.) before and after treatment with MK-447 (1mg/kg, i. p.). Blood samples were drawn from carotid artery, immediately after maximal hypertensive reaction to A II. Plasma levels of 6-keto-PGF_1α and TXB₂ were measured by radioimmuoassay, after cleaning up of the samples by SEP-PAK C₁₈ and HPLC.
Levels of 6-keto-PGF_1α and TXB₂ in control plasma were 131±24 and 121±46pg/ml (n=4), respectively. Injection of A II caused increase in plasma 6-keto-PGF_1α level, which was further significantly enhanced by pretreatment with MK-447 (*p<0.05). MK-447 alone did not affect the level. The plasma TXB₂ level was not significantly affected by these treatments.
These results suggest that MK-447 selectively enhances PGI₂ generation in vivo.

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We attempt to examine to qualitative comparison of plasminogen activator (PA) in transitional cell carcinoma of the urinary bladder and normal bladder mucosa as control. PA was purified by three procedures of 30% ammonium sulfate precipitation, acid treatment and concanavalin A-Sepharose affinity chromatography. Purified PA was analysed by using electrophoretic enzymography, fibrin film method and chromogenic substrates (s-2444 and s-2288) reaction. Purification factor of PA from bladder tumors (tumor PA) and PA from normal bladder mucosa (normal PA) was about 80, 000 and 325, 000, respectively. Enzymography revealed that tumor PA adsorbed to concanavalin A-Sepharose showed a single lytic zone at 55, 000 and normal PA adsorbed to the same column 72, 000. Both PAs lost fibrinolytic activity in the absence of plasminogen. The PA activity was inactivated by DFP. Tumor PA reacted with anti-UK IgG but not with anti-t-PA IgG. On the other hand, normal PA reacted with antit-PA IgG but not with anti-UK IgG. Both PAs cleaved s-2288 to a greater extent than s-2444, although UK cleaved s-2444 to a greater extent than s-2288. So both PAs were thought to show t-PA type amidolytic activity.

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The thrombolytic effect of proform of urinary plasminogen activator (Pro-PA) was examined in the rabbit using a jugular vein thrombosis model. Infusion of a low dose (120, 000IU/kg) of either urokinase (UK) or Pro-PA did not produce any significant thrombolysis. However, UK administration at such a dose caused 20% degeneration of circulating fibrinogen. A high dose (480, 000IU/kg) caused significant thrombolysis. The degree of fibrinogenolysis was about 20% in Pro-PA, but about 80% in UK. The thrombolytic efficiency, which is obtained by the ratio of fibrinolysis (thrombolysis) per fibrinogenolysis, was higher in Pro-PA than that of UK. Analysis of fibrinolytic parameters such as plasminogen, α₂-plasmin inhibitor etc. suggested that UK caused systemic activation of the fibrinolytic system, but Pro-PA, locally limited activation on the fibrin surface (fibrinolysis). These results indicate that Pro-PA represents a highly efficient thrombolytic agent without producing fibrinogenolysis.

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This study examines the effect of anticoagulant and antithrombotic drugs on blood pressure (BP), plasma renin activity (PRA) and renal glomerular and vascular lesions in malignant hypertensive rats. Whole-heparin, low molecular weight (LMW)-heparin, or defibrinotide were administered for 4 weeks to rats following complete aortitic legation between the renal arteries. BP and PRA in whole-heparin treated rats were significantly lower than those in control rats (p<0.001, p<0.001). Renal morphology also revealed a reduced number of epithelial cell droplets (ECD) in glomeruli and less severe vascular lesions (p<0.05, p<0.005). A decreased BP and PRA were seen in rats receiving LMW-heparin, but there was no improvement of vascular damage in this group.
From these results, it was suggested that an increased anti-Xa activity or reduction of PRA is not enough to prevent these lesions in malignant hypertensive rats, and that antiinflammatory action or enhancement of negative electric potential of vascular wall included in whole-heparin also may contribute to prevent vascular damage. The rats receiving defibrinotide showed reduced BP only.

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We investigated the platelet volume and other blood coagulation parameters in early postoperative periods in patients who underwent cardiac surgery. They were 11 (coronary artery bypass grafting, 5; prosthetic valve replacement, 6) in total numbers, and their mean age was 52.3 (41-61).
We tested their blood for platelet numbers, and mean platelet volume (MPV) as well as plasma levels of beta-thromboglobulin (β-TG), fibrinogen (Fbg), antithrombin III (ATIII) and plasminogen (Plg). The tests were done on the preoperative day, immediately after the operation, 1d, 2d, 5d, 1w, 2w, 3w and 4w after the operation.
The platelet number fell from 21.2±5.7×10⁴ (M±SD)/μl to 7.9±2.1×10⁴/μl immediately after the operation and showed its lowest value (5.3±3.1×10⁴/μl) two days after the operation. Then it showed dramatic increase through 1st and 2nd weeks and reached its maximum value of 39.3±7.1×10⁴/μl two weeks postoperatively. Plasma β-TG reached its highest value of 107.6±35.3ng/ml immediately after the operation. MPV changed from preoperative values of 8.96±0.73μm³ to 8.11±0.5μm³ immediately after the operation. MPV changed from preoperative values of 8.96±0.73μm³ to 8.11±0.5μm³ immediately after the operation and then gradually increased. The value reached its maximum value of 9.27±0.63μm³ on the fifth postoperative day. Fibrinogen levels increased mono-exponentially after the surgery and returned to its preoperative value very slowly.
These results suggested the destruction and consumption of the platelet within extracorporeal circulation circuit during cardiac surgery. Rapid rebound increase in platelet numbers and changes in coagulation parameters may explain the hypercoagulable state in the early postoperative period following cardiac surgery.

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In our country, the various hemostatic medicines have been used after the operation. On the contrary, in Europe and USA, the anticoagulent medicines have been used before and after operation, because of the high incidence of the complications of deep vein thrombosis and pulmonary infarction after operation. So we made inquireies about how to use them in 566 main surgical facilities in Japan.
On the results, in the above of half surgical departments these medicines have usually been used yet, but in 17% of surgery, 9.5% of obsterics and gynecology and 29.4% of orthopedics, they have never been used at all. On surgery, in 42% of cardiovascular, 32.1% of respiratory and 10.8% of general surgery, they have not been used absolutely. For the patients with liver cirrhosis, obstructive jaundice and the massive transfusion on the operation, in over the 80% of facilities the hemostatic medicines have been used.
The using medicines are usually adrenochrome, anti-plasmin, vitamin K, the conjugated estrogens and hemocoagulase etc.
We have pointed out that there are no necessity to use the hemostatic medicines on the operation, because of hyper-coagulatic state, hypo-fibrinolytic state of blood, hyper function of platelet and high viscosity of blood after the operation, especially on the cancer operation. Also we investigated how difference of the volume of bleeding and hemorrhagic complications after operation were between in the groups of using the hemostatic medicines and no using them. No significant difference was between using and no using. Therefore, we issue the warning to against the various hemostatic medicines after the operation.

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