The body of evidence concerning endothelial cell injury and the development of atherosclerosis and its associated clinical complications is compatible with vessel injury being important not only in the thromboembolic events associated with the late stages of atherosclerosis but also with vessel injury being involved in the early stages of atherosclerosis. In addition, vessel injury may cause some episodes of coronary artery spasm. Spasm may cause some clinical manifestations of coronary artery disease and contribute to the formation of occlusive thrombi in diseased coronary arteries.
At present, although there is some evidence that dietary saturated fat and cholesterol and smoking may be able to contribute to endothelial cell injury, the data are not strong enough to draw firm conclusions. It is clear, however, that reducing the risk of endothelial injury should have a significant impact on the development of atherosclerosis and the clinical complications caused by spasm and thrombosis.

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The protein C anticoagulant pathway represents a newly described system for investigating the regulation of blood coagulation. A role for protein C in clinial thrombosis has been suggested by recent findings that patients with 50% normal levels of protein C are at rick of thrombosis and that protein C levels decrease markedly during intravascular coagulation. The data presented here suggest that deficiencics in protein S, thrombomodulin, or the platelet receptor for activated protein C might also result in a thrombotic tendency. The interplay among the cellular, vascular and humoral components in this system suggest that the protein C anticoagulant pathway may be perturbed by a variety of disease processes. Certainly, the system provides a new approach to investigate the molecular basis of thrombotic disease.

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Three growth factors (acidic, neutral and basic) were partially purified from platelet lysate by DE 52 cellulose chromatography. The basic factor was proved to be identical to PDGF hitherto reported. The growth promoting activity of these three factors for variouse cell lines was studied in vitro.
Basic factor (PDGF) promoted growth of fibroblast (Swiss 3T3, SV 3T3) as has been described previouly, but had relatively less activity of growth promotion to malignant tumor cell lines (K562, HeLa and RPMI 4788).
On contrast, acidic factor strongly stimulated the proliferation of tumor cells and, to less extent, of fibroblasts.
Malignant cells (K562) were synchronized to pre S phase by addition of excess of thymidine and hydroxyurea, and thus synchronized cells were also proved to be more sensitive to acidic factor than to PDGF, indicating that the sensitivity is not related to cell cycle.

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The influence of forskolin, an adenylate cyclase activator, and of dibutyryl cyclic AMP (Bt₂cAMP) on the thrombin-induced [³H] glycerol incorporation into glycerolipids was investigated in human platelets. It was found that preincubation with 2.5mM of Bt₂cAMP produced 2-4-fold increase in thrombin-induced incorporation into phospholipids compared to platelets activated by thrombin alone. Pretreatment with forskolin, which increased cellular cAMP content, also resulted in an increase in thrombin-stimulated [³H] glycerol incorporation into phospholipids. These findings demonstrate that a rise in platelet cAMP can accentuate thrombin-induced de novo synthesis of phospholipids from [³H] glycerol. Since the content of cellular cAMP was correlated with their ability to inhibit platelet activation monitored by serotonin release, it seems likely that glycerolipids, in particular phospholipids, biosynthesis is somehow involved in controlling platelet activation by thrombin.

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Aspirin (ASA) has been used as an antiplatelet drug in clinical trials to treat and prevent thrombosis. However, high doses of ASA have disadvantage not only inducing gastric lesion but also inhibiting prostacyclin (PGI₂) generation in vessel wall. The possibility of useful combined therapy of dilazep (DZP), a coronary vasodilator, and low doses of ASA was described.
Platelet aggregation in platelet-rich plasma (PRP) was measured according to the method of Born et al. using the platelet aggregometer (NKK, Hematracer-1). 6-keto-PGF_1α in reaction medium where isolated aortic rings had been incubated at 37°C for 10min. was assayed for estimation of aortic PGI₂ production using the 6-keto-PGF_1α RIA kit (New England Nuclear).
A synergetic inhibitory action of the two drugs was demonstrated on platelet aggregation using rabbit PRP in vitro. In ex vivo experiments using rats 3hr after the oral administration, ASA alone inhibited platelet aggregation at doses more than 6.25mg/kg. However, it prevented aortic PGI₂ generation at 25mg/kg or above. On the other hand, the combined doses of 6.25mg/kg ASA+100mg/kg DZP and 6.25mg/kg ASA+300mg/kg DZP exhibited markedly inhibitory effects on platelet aggregation corresponding to 25mg/kg ASA alone, but had no significant effect on aortic PGI₂ generation.
In time-course experiments using rabbits, the combination of 300mg/kg DZP and 6.25mg/kg ASA showed an enhancement of antiplatelet effect from 3 to 6hr after the oral administration. The combination effect of ASA (30mg) and DZP (100mg) on platelet aggregation was also observed in healthy volunteers from 3 to 6hr after the dose.
Therefore, a desirable antiplatelet therapy may be performed with a combined dose of 100mg DZP and a very low dose of ASA.

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K-MAP (p-Aminobenzoic acid-N-mannoside sodium salt) shows various pharmacological effects such as antihypertensive, antidiabetic and antiinflammatory actions in pathological states. The mechanism of the action of K-MAP is not elucidated yet, but it is thought that this compound acts to alter prostaglandin metabolism. In the present paper, firstly, the antiplatelet action of K-MAP was studied by using human platelets and rat arterial ring, in relation to the prostaglandin metabolism. Secondly, antithrombotic action of K-MAP was studied by using thrombosis models in vivo.
Platelet aggregation was inhibited in a dose dependent manner by pretreatment with K-MAP. Cyclic AMP level and cAMP-dependent protein kinase activity of intact human platelets were augmented by K-MAP in vitro. Phosphorylation of endogenous protein in ³²Pi preloaded human platelets was also enhanced by this compound. But, the activation of cAMP metabolism by K-MAP was not significantly related to the direct action to adenylate cyclase or phosphodiesterase activity of platelet lysate, because these activities were not affected by K-MAP treatment. In washed platelets, K-MAP modulated the metabolism of arachidonic acid to stimulate the production of PGD₂, which is a potent activator of adenylate cyclase activity. Thus platelet cAMP level was increased by K-MAP. Meanwhile, it was found that K-MAP stimulated the release of PGI₂ in rat arterial ring. These results were compatible with the idea that K-MAP regulates cyclic nucleotides metabolism by modifying prostaglandin metabolism. Those metabolic changes might be concerned with multiple pharmacological effects of K-MAP and especially with antiplatelet action.
Furthermore, antithrombotic action of K-MAP was investigated by using three thrombosis models in which platelet aggregation was mainly involved. The models included β-aminopropionitrile-induced hyper-aggregation of platelets, ADP or collagen-induced acute pulmonary thromboembolism and bacterial endotoxin shock. K-MAP was effective in preventing platelet hyper-aggregation in β-aminopropionitrile-treated rats, ADP-or collagen-induced acute pulmonary thromboembolic death in mice and bacteria endotoxin shock in mice. All these results suggest the possibility of clinical application of K-MAP as an antithrombotic drug.

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When platelets were aggregated with an injection of ADP into cerebral artery, cerebrovascular injuries were observed. For analysis of the process of this vascular injury, electron microscopic observation was performed using horseradish peroxidase (HRP) as a marker of vascular permeability change. Two different types of changes; increased vesicular transport and vacuole formation were recognized. Vesicules (0.05-0.2μm) contained HRP reaction product but vacuoles (1.2-2.0μm) did not. In middle cerebral artery, vacuole formation was mainly revealed. Extravasation of HRP reaction products was not seen. In capillaries, arterioles or venules, increased vesicular formation was rare. Extravasation and perivascular edematous changes were remarkable. Namely endothelial cell damage was more prominent in large artery but extravasation and perivascular edematous changes were mainly seen in smaller vessels. And extravasation was more frequently seen in cerebral cortex and hippocampus comparing with basal ganglia. Following the ADP injection, blood levels of TXB₂ and 6-keto PGF_1α revealed marked temporarily increase at 3min after the injection. The results suggest that these vasoactive substances from platelets may play an important role in producing these vascular injuries.
These phenomenon seems to be important to understand the mechanism of vascular damage in various cerebrovascular lesions.

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In order to investigate the influence of hepatic resection on hemostasis, coagulation studies were carried out in patients with hepatocellular carcinoma. Twenty-nine patients were divided into 2 groups designed as group A which underwent 2 segmentectomy or more (13 patients) and group B which underwent 1 or less than a segmentectomy (16 patients).
Preoperative studies of both groups revealed almost normal coagulation studies except for minor degree of thrombocytopenia, but liver function tests were slightly impaired. Postoperative studies revealed thrombocytopenia, prolonged PT, decreased levels of factor II, V, VII, IX, X, fibrinogen, plasminogen, AT-III and α₂-M and increase of FDP. Seven out of the 29 patients showed positive ethanol gelation test in their postoperative course. These findings were more prominent in group A compared with group B and recovery of the levels of factor II, V, VII, IX, X, plasminogen and AT-III to the preoperative levels were significantly delayed in group A.
Increased FDP and positive ethanol gelation test which generally indicate presence of hypercoagulability improved proportionally to the recovery of coagulation abnormalities. DIC was confirmed only in one out of 29 patients by the clinical and laboratory findings.
Consequently coagulation disorders seen in the postoperative period of liver resection are mostly due to impaired liver functions which include impaired synthesis and RES clearance.

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We have studied the effects of urokinase (UK) on concentration changes of α2-antiplasmin (α2-AP) and on fibrinogenolysis. Medium dose (480.000u) or large dose (960, 000u) of UK was given to each of seven normal volunteers by intravenous drip infusion within six hours, and then blood and urine analyses were carried out. Total α2-AP, which includes free α2-AP and α2-AP-plasmin complex, decreased about 50 per cent of the original value with large dose of UK. α2-AP-plasmin complex appeared in the plasma one hour after UK infusion and increased up to 50 per cent of total α2-AP at the end of UK infusion. Bβ1-42, which liberated from fibrinogen at the very early stage of fibrinogenolysis, increased significantly with UK infusion, and was 65 times as much as the normal range at the end of UK infusion. Urinary Bβ1-42 increased as well as plasma Bβ1-41. On the other hand, fibrinogen degradation products (FDP) measured with enzyme immunoassay (ETA) increased only slightly, and moreover, urinary FDP was not detectable at any time. Plasma fibrinogen level did not decrease and it changed within the normal range in both groups. We then gave 960, 000u of UK to three patients with deep vein thrombosis and blood analysis was carried out as with normal volunteers. The most significant observation different from that of normal volunteers was shown in FDP levels. Serum FDP levels of three patients increased significantly in comparison with normal volunteers. Urinary FDP increased as significantly as plasma FDP.
In conclusion, we consider that infusion of 960, 000u of UK is enough to neutralyze α2-AP and enough to express thrombolytic activity. It causes only very early stage of fibrinogenolysis without advanced fibrinogenolysis which may result in bleeding tendency in normal volunteers. On the other hand, in thrombotic patients, advanced fibrinolysis was observed with UK infusion.

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To investigate the role of complement in disseminated intravascular coagulation (DIC), the inhibitory effect of decomplementation with purified cobra venom factor (CVF) was studied in experimental thrombin-induced DIC in comparison to experimental endotoxin-induced DIC.
Complement levels were significantly decreased at 6hrs after an intraperitoneal injection of 200U/kg of CVF (Cordis Lab., Miami). The experimental model of DIC was established by a 1hr sustained intravenous infusion of thrombin (Mochida Co. Ltd., Tokyo) at a dose of 2, 500U/kg in Wistar female rats. During this 1hr infusion, FDP and the number of renal glomeruli with fibrin thrombi (% glomerular fibrin deposits; % GFD) showed a rapid increase after 15min followed by a further increase at 1hr. Platelet count decreased after 15min, being further depressed at 1hr. CH50 and C3 levels showed rapid decrease at 15min, but no further decreases were seen. The infusion of thrombin or endotoxin was started 6hrs after the injection of CVF. In thrombin-induced DIC, %GFD was significantly decreased, while only slight changes were observed in FDP, platelet count and fibrinogen. On the other hand, DIC was significantly prevented by decomplementation in endotoxin-induced DIC.
These results indicate that the complement plays an important role in the pathogenesis of endotoxin-induced DIC, but only moderately influences thrombininduced DIC.

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Fibrinopeptide A (FPA) and fibrinopeptide Bβ15-42 (Bβ15-42) levels were examined in various diseases, according to the methods of Nossel and Kudryk, respectively.
The values elevated in chronic liver disease, chronic hematopoietic malignancy, cerebral thrombosis and chronic renal failure (CRF). Among those diseases remarkable elevation was observed in CRF. During dialysis treatment for CRF relulted in decrease in FPA levels, not in Bβ15-42 levels. The Bβ15-42 levels were well correlated with serum creatinine levels. Significant elevation of Bβ15-42 could be observed in CRP-positive group.
From these findings, it could be concluded that Bβ15-42 was a marker detecting hyperfibrino (geno) lytic condition, but that in renal failure the values should be discussed in relation with serum creatinine values.

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We found urokinase-cofactor (UK-cofactor) which directly increased UK or plasminogen activator activity and purified it from plasma using lysine-Sepharose, salting out, DEAE Sephadex A-50, Sephadex G-100, hydroxyapatite and Amberlite CG-50. However, it remained unclear whether the UK-cofactor acted enzymatically on UK. In the present paper, further characterization of UK-cofactor was performed by the experiments of the reaction between UK and UK-cofactor, and the influence of BAEE (benzoyl arginine ethyl ester) on UK-cofactor were examined.
HMW-UK and UK-cofactor, and LMW-UK and UK-cofactor were incubated at 37°C for 1, 5 and 10min, respectively, then electrophoresed. The UK antigen was not disappeared by incubating with UK-cofactor. The UK-cofactor did not form a precipitaing arc against anti UK-serum. The reaction between UK and UK-cofactor was examined using SDS-PAGE. HMW-UK and UK-cofactor, LMW-UK and UK-cofactor were incubated at 37°C for 1, 5 and 10min, respectively, and the changes of their molecular weights were monitored by SDS-PAGE. HMW-UK was gradually degraded to LMW-UK by UK-cofactor. LMW-UK also degraded to the lower molecular weight protein. It was revealed that the UK-cofactor was not actually a cofactor but a protease which degraded HMW-UK to LMW-UK and to small fragments. This protease was termed UK-converting protease in stead of UK-cofactor. BAEE (2mM) almost inhibited the degradation of UK by UK-cofactor. It is concluded that UK-cofactor is a new protease (UK-converting protease) which degrades HMW-UK to LMW-UK.

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