Effects of 5-hydroxykynurenamine (5-HK), produced from serotonin by the action of indoleamine 2, 3-dioxygenase, and its anologues on platelet functions were investigated. Platelet aggregation was studied by using a dual sample aggregometer (Sienco, Model DP-247) and serotonin uptake was examined by incubating platelet-rich plasma with 1μM ¹⁴C-serotonin at 37°C for 2min. 5-HK showed regulatory effects on human platelet functions both by antagonizing against the promoting effect of serotonin on arachidonic acid-induced platelet aggregation as well as that on ADP-induced aggregation and by inhibiting ¹⁴Cserotonin uptake by platelets. LASS production by incubation of platelet microsomes with arachidonic acid was estimated by the maximal changes in light transmission of citrated human platelet-rich plasma which was added to the incubation mixture, containing microsomes and arachidonic acid, and was found to be enhanced by 5-HK. Kynurenamine, N, N′-dimethyl-5-HK, N, N′-dimethyl-3-HK, 2-(3′-aminopropyl) aniline and 5-methoxykynurenamine inhibited both ¹⁴C-serotonin uptake by platelets and serotonin-induced aggregation, while N-acetyl-5-methoxykynurenamine, kynurenine and 5-hydroxykynurenine affected neither of these platelet functions. However, the degree of inhibitory effects of these analogues on serotoninuptake was not always in parallel with that on serotonin-induced aggregation. These findings support the concept that the inhibition of the serotoninuptake by these substances is intimately, if not directory, related to their ability to inhibit serotonin-induced platelet aggregation. LASS production was enhanced by N, N′-dimethyl-5-HK, N, N′-dimethyl-3-HK, 5-methoxykynurenamine and 5-hydroxykynurenine, while kynurenamine, 2-(3′-aminopropyl) aniline, N-acetyl-5-methoxykynurenamine and kynurenine showed no effect upon it. These studies with analogues indicated that the substitutions of o-aminobenzyl moiety with hydroxy or methoxy groups were somewhat tolerated for their inhibitory effects on platelet aggregation and serotoninuptake, whereas such groups were essential for their promoting effect on the LASS production by platelet microsomes. When the amine was masked by N-acetylation, both the inhibitory activities on serotonin-induced aggregation as well as on serotonin uptake and the promoting activity on LASS production were completely lost, suggesting that the alkylamine in the side chain is essential for these activities.

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Recent studies have indicated that platelet aggregation might be mediated by a decrease in CAMP from basal levels and prostaglandins (PGs) play a major role in mediating platelet aggregation. On the other hand, platelet function is altered in atherosclerosis and thrombosis. In these diseases, platelet adhesion is incleased, platelet's sensitivity to aggregating agents is increased and platelet secresion is enhanced. The administration of antiaggregating drugs is thought to modify and supress one or more of these functions and consequently prevents or delays the formation of thrombi, but in the treatment of these diseases, anticoagulants were not effective sometimes.
One of these reasons is that probably mechanism of inhibition of platelet function by antiaggregating agents is varied. In consideration of this point, it is important to clarify the modes of action of antiaggregating drugs in the treatment of these diseases. Drugs such as Heparin, Trasylol, Dextran sulfate, Carbocromen, Prenylamine, Dipyridamole, CDP-choline and α-Tocopherol were incubated with human platelet suspension. The effects of those drugs on platelet aggregation were studied and those on the formation of PGs and cAMP.
Material and Methods
Human platelets were obtained from platelet rich plasma by centrifugation and were suspended in 134mM NaCl-15mM Tris-HCl buffer pH 7.4. After treatment with drugs, the platelet suspension was separated into two.
One was used for platelet aggregation test by aggregometer. The other was further separated into supernatant and platelet fraction by centrifugation, which was used for assays of PGs and cAMP by RIA methods.
Results
Heparin, Dextran sulfate, and Carbocromen showed the dosedependent inhibitory effect on platelet aggregation induced by thrombin as well as aspirin or PGE₁ did.
On the other hand, Trasylol, Prenylamine, CDP-choline and Dipyridamole did not show inhibitory effect in vitro at concentrations lower than those of clinical use.
Prenylamine and CDP-choline showed inhibitory effect in vitro at concentrations higher than those of clinical use, but α-Tocopherol did not show inhibitory effect in vitro.
(Fig. 1) PGs formation showed stepwise decrease both in supernatant and platelet fraction by Heparin and Dextran sulfate.
(Fig. 2) PGs formation showed stepwise decrease in platelet fraction by Aspirin and Trasylol, and no change in supernatant fraction.
(Fig. 3.) In platelet fraction, PGs formation showed decrease by the high concentration of CDP-choline and stepwise decrease by Carbocromen. In supernatant fraction, Carbocromen showed slight increase of PGs formation at pg order.
(Fig. 4.) The high concentration of Prenylamine depressed PGE, PGF₂α formation in platelet fraction. On the other hand, it increased cAMP production.
(Fig. 5) Group I did not supress the PG formation, and was further separated into subgroups A and B by the ability of cAMP formation. Group II inhibited the PG formation and was further separated into subgroups 1 and 2 by the ability of PG formation in platelet fraction.
Conclusion
The mechanisms of inhibitory effect in platelets by antiaggregating drugs were varied.
It is important to consider the modes of action of each antiaggregating drug in the clinical treatment of thromboembolism.

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The use of drugs which inhibit platelet function as potential antithrombotic agents has gained considerable support with recognition of the important role of platelet in thrombus formation. The identification of drugs that suppress platelet function and retard thrombosis in experimental models are also recognized. It is very difficult to assess correctly whether or not platelet function inhibiting drug is efficacious as antithrombotic agent. But it would be clinically required to have laboratory method for an evaluation of efficacy and dosage of the drugs.
From this point of view, studies were done to establish the criteria for an assessment of the effects to suppress platelet functions.
Method: Platelet adhesiveness was measured using Salzman-Yasunaga modified method. Platelet retention rate in a glass bead column was expressed as an adhesiveness (%). Platelet aggregation was measured by a densitometric technique and its strength was presented as a maximum aggregation rate at final concentration of ADP; 2×10^-6M, collagen; 10μg/ml and adrenalin; 9×10^-3M respectively.
Results and Discussion: Variation of adhesiveness (%) and that of ADP-, collagen- and adrenalin- induced maximum aggregation rates during the 6 month period were tested in 14 cases treated with platelet function inhibiting drugs. The coefficients of variation were 9.3% to 39.4% (mean 22.0%±SD 10.3%) in adhesiveness, 12.4 % to 55.4% (30.7±13.7) in ADP, 6.3% to 73.5% (25.3±17.6) in collagen and 12.15 to 82.8% (39.9±20.6) in adrenalin induced maximum aggregation.
The relationships between adhesiveness (%) and maximum aggregation rates induced by ADP, collagen and adrenalin were studied in 94 samples tested during 1 month period before and after the drug. The coefficients of correlation were 0.49, 0.48 and 0.48 respectively with statistically significanse.
The platelet adhesiveness (%) in normal subjects (50 males and 50 females) was determined. The mean values and standard deviations were 46.0±11.3 (%) in male, 40.7±13.6 (%) in female and 43.4±12.8 (%) in total subject respectively.
We found that Salzman-Yasunaga modified method is simple and rapid technique. This method may be a reasonable clinical screening test to detect platele tadhesiveness and showed stable data in comparison with aggregation test. Therefore, platelet adhesiveness by this method and the mean ±SD in normal subjects were set as a criteria for an asessment of platelet function inhibiting efficacy.
The efficacy of platelet function inhibiting drugs was determined as follow:
A=times of measurement which showed below 56.2 (%)/total times of measurement×100
80%≤A: good control “G”
80%>A≥50%: fair control “F”
A<50%: poor control “P”
According to this criteria, adhesiveness inhibiting efficacies of gliclazide (antidiabetic agent), dipyridamole and aspirin in vivo were assessed respectively. The gliclazide (80-240mg/day) conducted “G” in 6, “F” in 1 and “P” in 3 of 10 patients, and the dipyridamole (150-500mg/day) did “G” in 5 and “P” in 5 of 10 patients, at 3 month after these drugs. The aspirin inhibited platelet adhesiveness in all of 4 patients at 1 month after (1.0-3.0g/day). Those who showed poor control at 3 months after gliclazide and dipyridamole remained poor even after 6 to 12 months.

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The purpose of this communication is to describe a transient change in platelet volume in relation to a change in its aggregability in an experimental pulmonary thromboembolism in the rabbit.
MATERIALS AND METHODS
Twenty-five rabbits (5 splenectomized in advance) were catheterized in the bilateral femoral arteries and the central vein under heparin anticoagulation (100U/Kg). The electrocardiogram, respiration, arterial and central venous pressures were recorded throughout the experiment. Adenosine diphosphate (ADP; Sigma) 2 to 5mg/Kg was injected into the central vein in 22 rabbits and saline as a sham procedure in 3 rabbits. Citrated blood samples were obtained before, 30 seconds, 3, 10 and 45 minutes after an injection of either ADP or saline and platelet rich plasma was obtained by centrifugation.
Platelet count and volume were measured using a Coulter Counter ZbI coupled with a Channelyser C-1000. An aperture size of 30μ was used and calibration was done using with latex particles. The platelet aggregability was measured by the optical density method as induced by 100μmol of ADP using a Sienco aggregometer. Platelet morphology was studied by a scanning electron microscope (Hitachi S-500).
RESULTS
Within several seconds after completion of ADP infusion the animal developed bradycardia, premature beats, ischemic ST-T wave changes, hypotension, bradypnea, apnea and convulsion.
Histology of the lung revealed the small arteries to be obstructed with platelet aggregates.
Platelet count decreased 30 seconds after and increased 10 minutes after ADP injection (Fig. 1). No significant increase in platelet count was noted in the splenectomized rabbits. A significant increase in platelet volume was noted after ADP injection (Fig. 2, 3). No significant change in platelet shape was noted when its volume showed the maximal change (Fig. 4). Platelet aggregability increased at 3 and 10 minutes after ADP infusion (Fig. 5). When the platelet volume at 10-minute was expressed as the increment from the control value and the aggregability at 10-minute as the increment from the control, the correlation between two deviations was significant (r=0.60, P<0.05(Fig. 6)).
COMMENT
Large platelets appeared in the ADP induced pulmonary thromboembolism showed an increase in platelet aggregability and there was a significant correlation between platelet volume and aggregability. This experiment demonstrated that the platelet changes its volume transiently in a certain circumstance in vivo.

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We investigated the platelet volume in perinatal period using Coulter Counter ZB.
1) When EDTA was used as an anticoagulant, we found the platelet volume was larger than that in cases using citrate, oxalate, heparin, ACD, and CPD.
2) Platelet volumes in cord blood and newborn's blood were smaller than those in adult male's and their mother's blood just after delivery.
3) Adult female's platelet volume was larger than adult male's platelet volume.
4) The platelet volume in the early and late stage of pregnancy were smaller than adult female's platelet volume.
Just before the delivery, the platelet volume became larger, became smaller again just after the delivery, and got to as large as adult female at one month after the delivery.

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In previously papers, we had reported that platelets had a contractile protein prossessing calcium ion sensitivity, and that clot retraction was made by its activation. In this paper it is reported whether the contractile protein participates in platelets adhesion and aggregation.
(Materials and Methods) Human platelets obtained from citrate blood were incubated on a resin (VCD, Polyscience) plate and embedded. Adhered and aggregated platelets were treated with glycerol-HMM and observed electronmicroscopically by the vertical and the horizontal section.
(Results) 1. Platelets adhered on the resin plate showed many thin filamentous bundles in its cytoplasm of the pseudopod on the substratum. The bundles run toward the pseudopod and contained tick filaments and microtubles. These filaments were arranged in pararell and started from cell membrane. The thin one could react with HMM to form a HMM-decorated filament.
2. Aggregated platelets showed filamentous network in some part in their cytoplasm. The filament started from a cell membrane to the center of cytoplasm and had reactivity to a HMM. The characteristics of these filaments were similar to that of skeletal F-action.
3. It may be presumed as follows: (1) the above electronmicroscopic figures of the intracellular filament are similar to those of platelets in the retracted clot, (2) a bundle of thin filaments was originated from a cell membrane accompanied with cell transformation and could form HMM-decorated filaments, and then (3) adhesion and aggregation of platelets resulted in activation of a cell membrane and in polymerization of intracellular contractile protein.

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Urokinase (UK) has been immobilized on nylon monofilaments, tubes and sheets, by the method of Edelman et al. (1971). The quantity of immobilized UK by this method was estimated to be 0.05-0.06U/cm².
The fibrinolytic activities were observed on the standard fibrin plates after incubation at 37°C, for 12-24 hours.
The immobilized UK showed fibrinolytic activity even after preservation at 4°C, for long term and even after heat-treatment at 70°C for 15min. These results have been published previously.
The fibrinolytic activity of immobilized UK was prevented by “antiplasmin” which exists in normal human plasma. So, we have devised a modified technique to immobilize UK on nylon for the purpose of increasing the quantity of binding UK on nylon and of suppressing the activity of antiplasmin. In our technique, Gantrez (G) (copolymer of maleic anhydride and methylvinyl ether) was used to increase quantity of UK (immobilized) on nylon and Nitrophthalic acid (NP) as antiplasmin inhibitor.
The samples we had produced by the devised method revealed remarkable fibrinolytic activities in the existence of human plasma.
The fibrinolytic activities of immobilized UK decreased gradually by repeated incubations. After several repetitions, they settled to a constant value, which was measured at 10-25% of the initial activity. The constant value is considered to represent the quantity of UK which had coupled covalently on the nylon surface.
Antithrombogenicity of immobilized UK was observed employing the method of chandler, by taking measure of thrombus formation time (TFT). Two kinds of anticoagulants were used to test the antithrombogenicity.
The anticoagulants were FP-522 and EG-626, which had been tested as anti-platelet-aggregation medicaments.
The results of these studies were summarized like follows:
1) TFT of non-treated nylon tubes were within ten min.
2) TFT of UK-immobilized nylon tubes with G and NP, were over 45min.
3) TFT of non-treated nylon tubes with clinical dosage of FP-522 solution were over 45min.
Antithrombogenecity of immobilized UK and clinical dosage of FP-522 were found to be effective.
Possibilities of immobilization of another fibrinolytic or thrombolytic agents must be examined. We are carrying out the next studies in vivo, employing the immobilized UK.

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To clarify interrelationship between myocardial infarction and coronary thrombosis, Japanese autopsy hearts with myocardial infarction were morphologically studied. For prevention of myocardial infarction, this kind of basic study would be imperative for the internists and surgeons who are interested in coronary bypass surgery.
The materials consisted of 215 cases with 244 myocardial infarctions which were divided into 129 men and 86 women. Five age groups were set: young (20-39 y-o), middle aged (40-59 y-o), old (60-69 y-o), older (70-79 y-o) and oldest (over 80 y-o). Grade of coronary sclerosis was macroscopically estimated according to WHO standard and coronary thrombosis was both macro- and micro-scopically diagnosed.
The overall incidence of coronary thrombosis in the infarction was 53% and the incidence was higher in the massive necrosis (M) type infarction, to 70% in male and 63% in female. In the M type infarction, the highest incidence was recorded in the old male (84%) and in the older female (69%). The anterior infarction fairly matched thrombosis in the anterior descending branch, but the posterior one which was relatively increasing after 70 y-o, did not show such an intimate relation to thrombosis in the right coronary artery. This tendency was especially marked in the older female group. The thrombosis appeared in only 3/7 cases with the M type infarction within the initial 24 hours after the clinical signs started.
Thrombosis on moderate stenosis in the left coronary stem and proximal anterior descending branch was noted in the middle aged male. The site of thrombosis in the Ad branch shifted distalwards and thrombosis just above and distal to the severe atherom atous stenosis became remarkable with advancing age. Thrombosis was predisposed at the median portion of the right coronary artery and circumflex branch with a shift both proximal- and distal- wards with aging.

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Seven patients with progressive diabetic retinopathy developed extensive retinal areteriolar and capillary obstruction.
The clinical features shared to some extent by all of these patients were summarized as follows;
1) Retinal edema, haemorrhages and soft exudates were observed in all cases and thread-like arterioles in 4 cases.
2) Arterial vascular disease was complicated in 6 of 7 cases.
3) Loss of visual acuity in all cases and usually visual loss may be of sudden onset.
4) Visual field defects also were seem in all cases, central and paracentral scotomas corresponded to the areas of retinal ischemia.
5) Optic disc pallor and neovascularization of the optic disc was seen in 4 of 7 cases.
6) Retinal ischemia was especially severe at the posterior pole. Fluorescein angiogram showed severe arteriolar and capillary obliteration.
7) Granular blood flow was observed in some cases showing severe retinal vascular stasis.
8) Higher platelet aggregation enhancing activity and Malondialdehyde value in the plasma was found in all subjects than in the normals.
9) These findings may suggest the retinal intravascular coagulation syndrom.

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Purpose: It was the purpose of this present study to determine the biocompatibility, especially antithrombogenic and antihemolytic properties, of vinyl chloride and polyurethane, which are used as material for artificial hearts.
Methods: An LV-Ao hypass-pump driven by air was installed in rabbits between the apex of the left ventricle and the abdominal aorta at which time heparin at a dose of 300units/kg was intravenously injected. One hour later, Protamine was intravenously injected at a of mg/kg and one hour thereafter the prothrombin-time of the blood was determined. Eight hours after injection of heparin, the driving of the device was stopped and immediately thereafter prothrombin-time of the blood and the concentration of free hemoglobin in the serum were determined. The grade of thrombosis on the surface of the device and the percentage of infarct regions on the kidney surface were grossly evaluated and the kidneys were examined pathologically. A comparison os the results was made among the pumps made of vinyl chloride, only the sacks made of polyurethane and only the valves made of vinyl chloride and the remaning parts coated with polyurethane.
Results: Prothrombin time ranged from 10 to 20 secnds and the counteraction to heparin was relatively favorable. The grade of the thrombosis on the pump surface made of vinyl chloride was higher than than those made of polyurethane. The perdentage of infarctregion on the kidney surface and concentration of free hemoglobin in the serum were also similary higher.
It was thus demonstrated that polyurethane had higher effectiveness than vinyl chloride in anithrombosis, antihemolysis and renal infarction.

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Platelets have been known to be an important factor in thrombogenesis. In an early stage of thrombosis, platelets adhere to exposed subendothelial collagen eliciting release of ADP and aggregate each other. ADP seems to be a very important substance in platelet thrombosis. In this experiment ADP was injected into the vein in 7 control Wistar rats and 6 spontaneously hypertensive rats (SHR). SHR were administered with 0.1% NaCl as drinking water over a period of 10 weeks and the blood pressure showed to be 200mmHg or above.
ADP 1mg/kg was injected rapidly into the jugular vein of the anesthetized rats. Blood samples were collected from carotid cannula before and 30sec, 3, 10, 20 and 30min after the injection. EKG and respiration were monitered and recorded continuously. ⁵¹Cr-labeled platelets were infused in 2 rats. They were sacrificed 1min after ADP injection and the organs were studied for the radioactivity and the histological observation. In control animals, platelet count decreased immediately after the ADP injection. Variable A-V block, premature beats and apnea developed immediately after the injection and subsided by 1min. Arterial hypotension and rise in the central venous pressure were also observed for a period of 10min and gradually subsided. In the analysis of platelet radioactivity, platelets were revealed to accummulate unproportionally in the lungs. Histological examination revealed platelet thrombi in the pulmonary microvas-culature.
In SHR rats, decrease in platelet count and heart rate, degree of arrhythmia, apnea, systemic hypotension and elevation in central venous pressure after ADP injection were less than in control rats. In the platelet volume study, the mode related volume was at 2.7μ³ in SHRs as compared with the mode of 2.4μ³ in controls. The volume distribution curve in SHR shifted to the left indicating a relative increase in smaller platelets in the population.
In summery, ADP injected into the central vein produced platelet aggregation which were trapped in the pulmonary microvasculature causing pulmonary thromboembolism. Arrhythmia and respiratory distress seems to be due to transient pulmonary thromboembolism. These changes were less severe in SHRs. These results would suggest that hypertension and/or possible association of the vascular change cause different attitude of platelets to ADP.

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Concerning the pathogenesis of microangiopathy, it is possible to assume that the metabolic failure due to an insufficient insulin action appears first and then vascular lesion follows, to this state. The relationship between an insufficient insulin action and morphological and chemical changes in capillaries, paticularly in glomerulus, retina, muscle and skin, have been pointed out and studied as the causes of microangiopathy. Of late, however, attention has been paid to microcirculatory failure as a possible cause; abnormal viscous flow, intravascular red cell aggregation, hypercoagulability and low fibrinolytic activity of blood are being regarded as important factors in the development of microcirculatory failure and investigation from the novel viewpoint of hemorheology has been made. This paper concerns some studies that attempt to correlate blood sludging in eye bulbar conjunctiva with the extent of the hemorheological abnormalities and the severity of retinopathy in patients with diabetes mellitus. The degree of sludging in bulbar conjunctiva and its correlation with blood and plasma viscosity, platelet adhesion and aggregation, blood clotting curve, plasma fibrinogen concentration and erythrocyte sedimentation rate were investigated in 16 normal healthy subjects and 48 diabetics. For the observation of blood sludge in conjunctiva vessels, our newly-developed biomicroscope (magnification ×50) was used, and then classified arbitarily into four categories according to the extent of intravascular red cell aggregation in arterioles, capillaries and venules.
The patients with diabetes were divided into two groups: DM-I, a group without retinopathy and/or with mild retinopathy, consisted with O-II stages in Scott's classification of diabetic retinopathy, and DM-II, with advanced retinopathy (Scott III-V). Diabetes groups showed significantly increasing of the parameters such as blood and plasma viscosity, platelet aggregation, maximum dynamic rigidity modulus of blood clotting and fibrinogen concentration than those of normal subjects. However, no significant differences of these parameters were found between DM-I and DM-II groups, though latter having a tendency for more abnormalities in blood rheology. Significant sludging in conjunctival vessels were observed in most of DM-II group and half DM-I group: It was assumed that there were some correlation between blood sludge and the severity of diabetic retinopathy. And in the relation of clinical factors high incidence of sludging grade 2 or higher was observed in diabetics with long duration, albuminuria and high triglyceride. Other factors such as blood sugar level in fasting, body weight and total cholesterol have no significant correlation with degree of sludging.

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Intratubular coagulation study, as in vitro model experiment of disseminated intravascular coagulation (DIC), and a case study of a patient with DIC, were performed for effects of the blood viscosity and the hemolytic pattern by coil planet centrifuge (CPC) on DIC.
The time changes of blood viscosity and hemolytic pattern by CPC, together with partial thromboplastin time (PTT), prothrombin time (PT), platelet counts, fibrinogen level and some hematological test were observed during coagulation process of flowing blood in small polyethylene tube (diameter 0.5mm).
The PTT gradually shortened after 10, 20, 30min. flowing respectively, but prolonged after 40min., and the blood in tube completely coagulated after 50min. flowing. The PT prolongedd after 20min., and extremely prolonged after 40min. flowing in small tube. Platelet counts and fibrinogen level were gradually reduced according to the progress of coagulation process, and showed nearly 0 after 50min. Namely, intratubular coagulation resembled closely DIC, though was not so effective on the enhancement of fibrinolysis.
On the relation of blood viscosity to hematocrit (Ht) level, there was a sharp increase in viscosity with increase in Ht level. The blood viscosity in intratubular coagulation process gradually increased till 30min. after flowing, when PTT was the shortest, but the viscosity thereafter decreased gradually in accordance with the prolongation of PTT and the decrease of fibrinogen and Ht level.
Hemolytic pattern by CPC showed specific waving curve after 20min. to 30min. flowing, and then extention of hemolysis with poikylocytosis in red blood cells.
On the case study of post hepatectomy with DIC, the blood viscosity increased in stage of hypercoagulability, decreased in period of consumption coagulopathy or with heparin therapy. The hemolytic pattern by CPC showed waving curve in hypercoagulable state, wide low curve in consumption coagulopathy and normal pattern with heparin therapy.
It was considered that measurements of blood viscosity and observation of hemolytic pattern by CPC had clinical significance for diagnosis of hypercoagulable state in early DIC, and prognosis of DIC.

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Hyperfibrinogenemia has been found in the cases with cancer, inflammatory disease and several types of injuries. In previous observations, the hyperfibrinogenemia associated with cancer was striking in view of the rapid turnover rate of ¹³¹I-labeled fibrinogen. It seems of interest to study the regulation of fibrinogen production in hyperfibrinogenemia.
Recently, Pickart et al. reported that fibrin-fibrinogen degradation products (FDP) which potentiated epinephrine effect on beta-adrenergic receptors in fat cells, would enhance the mobilization of free fatty acids (FFA) from depot fat and saturated FFA stimmulated the production of fibrinogen in the liver.
We studied the effect of beta-adrenergic blockade on fibrinogen synthesis in rats with tumor, inflammation and intraperitoneal injection of toxohormone. The serum FDP level and the FFA/Albumin ratio were rised as much as the plasma fibrinogen level in rats following subcutaneous injection of turpentine oil and intraperitoneal injection of toxohormone.
The enhanced fibrinogen synthesis was suppressed significantly in the rats treated with the beta-blocker every 4 hours as measured by incorporation of ¹⁴C-L-leucine at 12 hours after recieving of turpentine or toxohormone. However, the beta-blockade did not alter the incorporation of labeled amino acid into the serum albumin in these experiments.
The effects of beta-blocker on the concentration of plasma fibrinogen in tumor bearining rats were examined. The rats were given the beta-blocker every 6 hours by stomach sonde between 5th and 8th day after the subcutaneous inoculation of Yoshida sarcoma cells. On 8th day, fibrinogen, albumin and FFA in the blood were assayed. The plasma fibrinogen level and the FFA/Albumin ratio in tumor bearing rats were reduced by the administration of beta-blocker.
On the other hand, the beta-blockade did not interrupt the activation of fibrinolysis in rats with inflammation induced by turpentine oil but enhanced the phagocytic activity of reticulo-endotherial system when measured by carbon clearance in normal animals.
In this study, it was postulated that beta-adrenergic receptor might relate to enhancement of fibrinogen synthesis in hyperfibrinogenemia associated with inflammation, tumor and injection of toxohormone.
Furthermore FDP and FFA might involve in the regulating factors of fibrinogen synthesis.

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We studied experimentally factors of regulating fibrinogen in rat.
Result: Liver cirrhosis induced by CCl₄ decreased fibrinogen of plasma.
Subcutaneous transplantation of Bacteria and tumor cells (AH130, AH 166F), I. V. injection of tumor cells and tumor extracts and injury of skin increased fibrinogen. But transplantation of egg albumin and i. v. injection of antiplasmin did not increased fibrinogen.
Increase of fibrinogen was inhibited by corticosteroid, but antihistamin agents had no effect.
Conclusion: as the result.
We suggested some inflammation stimulating fibrinogen production in liver, and the promoting factor is not related with histamin system, and corticosteroid inhibited this mechanism.

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Clottable fibrinogen in human plasma was determined by messuring spectrophotometrically the increase in turbidity with time due to the fibrinogen-fibrin conversion with thrombin. From the maximal absorbance, A_max, at 450nm obtained 2min or less after the addition of thrombin to plasma, the fibrinogen concentration was estimated in plasma of normal subjects and patients. Analysis of the rate of the absorbance increase yielded the K_m value, 1.6×10^-5M, which closely agrees with K_m=1.2×10^-5M obtained by analysis of the fibrinopeptides released from fibrinogen.

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Prevention of postoperative vein thrombosis was accomplished by means of either low amount of heparin administration or physical treatments.
Heparin treatment groups
Group I: 160 cases of hysterectomy received total amount of 30, 000 IU heparin during 50hrs. postoperatively as shown in table 1.
Group II: 38 cases of vascular surgery were injected 5, 000 IU heparin 2hrs. prior to surgery, and postoperatively the same amount of heparin were administered as in group I.
Physical treatment group
Group III: 220 cases of hysterectomy were treated postoperatively by such physical therapies as raising up her legs, active and passive excercise and massage.
Results
Group I: 6 out of 160 cases showed hemorrhagic tendency and another 3 cases showed vein thrombosis.
Group II: This group showed no vein thrombosis, however a hematoma formation was encountered in 2 cases and postoperative bleeding on the suture line was in 1 case.
Group III: Vein thrombosis was appeared in 4 cases.
Conclusion
The administration of a low amount of heparin during both pre and postoperative phase (Group II) is effective enough to prevent the occurrence postoperative vein thrombosis. The effectiveness of the different two methods (Group I, III) in prophylaxis of the vein thrombosis is not so enough as in Group II.
Pain or sudden swelling on the legs during the physical treatment are very useful informations to detect a venous thrombosis in the early postoperative phase.
Immediately after a venous thrombosis is detected, heparin and urokinase therapy should be initiated. This kind of anticoagulant therapy can prevent the pulmonary embolism and postphlebitic syndrome.

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Effects of bencyclane on the human platelet in aggregates induced by adenosine diphosphate (ADP) and latex were studied by transmission electron microscopy in combination with freeze-etching technique.
Platelet-rich plasma (PRP) was obtained from heparinized blood by centrifugation at 1, 000r. p. m. for 10min. at 4°C. Bencyclane was used as either one of the solution containing 500, 250, 125 or 62.5μg/0.1ml. Latex was used in a concentration of 2.0%. ADP was obtained at the concentration of 2×10^-4M.
The Chandler loop was used for the production of aggregated platelets in a 21°C water bath. In the experimental group, each loop contained 0.7ml of PRP, 0.1ml of either one of bencyclane solution and 0.1ml of latex suspension. The loop had started to rotate for 10min. One tenth ml of ADP was added to the contents to continue further rotation for 10min. In the control group, bencyclane was replaced with same amounts of its placebo.
In the control, many latex particles were visible in the open channel system (OCS) and in the α-granule of platelets in solid aggregates. After uptake of latex particles by the platelet had progressed, matrices of the α-granule intervened in the spaces between the particles and the limiting membrane of the OCS and also in the interplatelet spaces. This suggests that both release reaction of the platelet and uptake of the particles by the cell may happen through the same OCS. Microtubular structure probably connecting the α-granule with OCS revealed in a replicated platelet may participate in uptake of particles.
In the experiment, no clump or tiny aggregates were formed. The contents of the loop, therefore, were centrifuged to separate a ribbon of platelets. Bencyclane was found to be an effective inhibitor of the aggregation and adhesion of platelets respondent to latex and ADP. In addtion, bencyclane inhibited release reaction of the platelet and also uptake of the latex particles by the cell. Effects of bencyclane on the platelet were concentration dependent; reliable effects were obvious at the concentration of 250μg/ml. Mild effects wer also seen at 125μg/ml.
In conclusion, bencyclane inhibits the platelet function probably by acting not only to the cytoplasmic membrane but also to the membrane of the OCS including microtubular structure and by reorganizing the membrane system. Whether uptake of the latex particle by the platelet is real phagocytosis or not remains to be dissolved.

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Our findings obtained by use of Coagulation Profiler PAP-3 show a nice reproductivity in the short time experiments as prothrombin time or thrombin, time but there was seen a considerable fluctuation in the long time experiments like as PTT or recalcification test.
The Predominance of manual assay in the reproductivity was demonstrated in the comparison between the coagulation profiler PAP-3 and manual assay technique.
The results obtained from use of Platelet Aggregation Profiler CAP-3 show a tolerablea good reproductivity in the maximum aggregation induced by ADP, Collagen, Epinephrine…etc. It seems to be necessary determination of optimal concentration of aggregation inducers in reaction mixtures.

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In this study the factors affecting on Factor XIII (FXIII) levels of human plasma were investigated in 185 patients (98 male, 87 female) from 16 to 86 years of age, using single radial immunodiffusion method developed in our laboratory. Citrated plasma were sampled from antecubital vein by centrifugation at 3, 000r. p. m. for 10min. and stored at -20°C until use. Preparation for immunoplate was described in our preceding report. Results obtained were represented as percentage of Standard Human Plasma (from Behringwerke) employed for standard.
Subjects, male and female, were divided respectively according to age into three groups; to 40 years old, from 40 to 59, over 60, and mean value (MV) in each group was estimated. MV of FXIII for subunit A (sub A) in younger male and female were 103.73 and 84.92, and for subunit S (sub S) were 97.93 and 92.33 respectively. In the other groups of male and female, MV for both subunits were almost in the same levels respectively.
From observations analysed on the diurnal changes in sixteen patients with mild diabetes mellitus and on the seasonal alternations in ten patients with stable hypertension, no remarkable variabilities of both subunits levels were obtained. This finding indicates that sub A which is bound to sub S is kept stable under physiological conditions, and that it is hardly affected by exercise, meals and climate.
On the other hand, following the changes of levels in seven CVA patients who were within seven days after accidents, it was observed that sub A decreased from early stage of the accidents, while the decrease of sub S appeared about ten days after onset. It seems that the decrease of sub A is caused by consumption which is brought about by coagulative events e. g. CVA, menstruation, and other hemorrhagic diseases, and by subsequent healing process after rapid activation of FXIII. In addition, the view is allowable that sub S is being retained in form of “free” for a time, thereafter disappearing gradually from plasma.
The experiments to determine the relation between FXIII subunits and other biochemical plasma constituents were carried out. There were significant correlations between both subunits and total cholesterol (p<0.05).
However, no significant correlation between both subunits and other lipids; triglyceride, β-lipoprotein, FFA and phospholipid, were obtained.
Plasma fibrinogen had a correlation with sub S but not with sub A. As previously reported, a high correlation between sub A and sub S (r=0.440, p<0.05) was otained. Moreover, it has been generally accepted that about 30% of total sub S in plasma is present in form of free.
From our findings and other reports, authors came to the concept that sub S may be present in forming complex with fibrinogen in plasma, and that it may bind with fibrinogen in preference to fibrin monomer.
We suppose that sub S, especially “active free sub S” which releases sub A would be under labile condition, and that this “sub S” would react with fibrinogen, resulting in forming sub S-fibrinogen complex.

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It was reported that there were two forms of factor VIII procoagulant activity in rabbit plasma. In this work we studied the properties of these two forms, high molecular weight factor VIII (HMW FVIII) and low molecular weight factor VIII (LMW FVIII). The results were as follows.
1) Rabbit plasma was chromatographed on 6% agarose at physiologic ionic strength. In agreement with previous reports, the factor VIII activity eluted in the void volume and in early fractions of the third protein peak.
2) At -5°C, ice-cold 53.3% ethanol was added to rabbit plasma to a final concentration of 3%. The resulting cryoethanol precipitate was dissolved in barbital-saline buffer, then chromatographed on 6% agarose. The factor VIII activity was detected only in the void volume, suggesting that rabbit HMW FVIII was concentrated in the cryoethanol precipitate but LMWF VIII was not.
3) Chromatography of rabbit plasma adsorbed with Al(OH)₃ showed no activity of LMV FVIII, indicating that LMW FVIII was removed from plasma by Al(OH)₃.
4) Chromatography of rabbit HMW FVIII in the presence of 0.25M Ca²⁺ demonstrated that factor VIII activity was separated from the void volume and eluted later. From the elution position the molecular weight of Ca²⁺-dissociated LMW FVIII subcomponent seemed to be somewhat higher than that of native LMW FVIII in rabbit plasma. After the removal of Ca²⁺, the late-eluting fractions having factor VIII activity were mixed with void volume fractions and the mixture was rechromatographed at physiologic ionic strength. The FVIII activity was found in the void volume, which indicated that LMW FVIII subcomponent was recombined with void volume protein. These results were similar to those reported for human factor VIII.
5) When rabbit serum was chromatographed on 6% agarose, the factor VIII activity was not found in the void volume but found in the same elution position in which rabbit plasma LMW FVIII was detected by chromatography using the same column. This indicated following three possibilities:
a) During blood coagulation HIM FVIII was consumed but LMW FVIII wag not.
b) LMW FVIII was produced from HMW FVIII and remained in serum.
c) Procoagulant activity of later fractions was due to procoagulant active substance, which might be factor Xa or thrombin, other than LMW FVIII.

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It is widely known that tissue thromboplastin (T. Tbp) is one of the direct trigger factors in DIC. By electron microscopy, purified T. Tbp consists of two types of structures, concentric membrane and amorphous substances, as reported by M. Hvatum et al. We have studied the ultrastructures of T. Tbp to demonstrate how it changes during coagulation.
[Materials and Methods] T. Tbp from lungs of rabbits (Mochida Ltd.) was used for these studies. T. Tbp has been studied on the purificant mixed in plasma, in plasma added with Ca⁺⁺, in plasmin solution and in plasma added with Ca⁺⁺ and Urokinase.
Then, attempts were made to demonstrate ultrastructural changes of T. Tbp after intravenous injection, as a model experiment on the pulmonary microthrombi formation induced by T. Tbp circulating from venous return.
The specimens were fixed in phosphate buffered formalin, followed by post-fixation in osmium tetroxide solution. They were examined by transmission electron microscope (TEM) or scanning electron microscope (SEM).
[Results] Morphological appearance of concentrically arranged membrane structure in T. Tbp remained nearly intact during blood coagulation process. T. Tbp, which existed in the sediments following dissolution of fibrin clot by the application of UK, showed an appearance of fine granules adhering to the surface of aggregates of particles through SEM. Through TEM, T. Tbp in the sediments was found to have retained its concentrically arranged membrane structures in some places, while, in some other places the appearance of fused membranes, smaller single vesicles and long sheets of membranes, and the formation of “blebs” etc. were observed. Various morphological changes caused by fibrinolytic substances accompanied the loss in coagulation activities.
Our results showed that coagulation activities of T. Tbp must be completely dependent upon the presence of the membrane structures.
Concentrically arranged membrane structures of the injected T. Tbp disappeared in extremely short time after the injection of te T. Tbp in rabbits. The long sheet membrane of the injected T. Tbp was frequently seen as adhered to the vascular endothelium or to the surface of blood corpuscles. Furthermore, fibrin fibers were formed in contact with the long sheet membrane of T. Tbp.

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We reported that thrombokinetic study was useful to detect early stage of platelet plug formation and adhesion in vivo on the cases of thrombocytosis Blood and Vessel 8 (2), 193-198, 1977. Afterward we added more cases with risk factors of thrombogenesis and discussed about their significance by means of throm bokinetic study.
Clinical studies. A total of 28 cases were studied, composed of 24 cases of cardio-valvular replacement, 2 cases of vascular disorders and 2 cases of diabetes mellitus with retinopathy. The result of this study showed that platelet survival in cases without thrombosis was almost normal but was significantly shortened in cases with thrombogenic episodes (2 cases of chronic myelogenous leukemia (CML), 1 case of myelofibrosis with myeloid metaplasia (MMM), 1 case of primary thrombocythemia). The shortened platelet survival time implied increased platelet consumption for thrombus formation.
We measured von Willebrand factor activity, and FVIII related antigen (FVIII AGN) in cases of vascular disorders (34 cases of diabetes mellitus with retinopathy, 10 cases of hypertension). Diabetes mellitus with retinopathy were proved to have high vWF activity and high VIII AGN value. On the other hand, hypertension with retinopathy showed high vWF activity but normal FVIII AGN value.
In 2 cases of diabetes mellitus with retinopathy, platelet survival time was significantly shortened.
These results suggested that increased FVIII AGN might initiate and produce vascular change and resulted in platelet consumption and thrombus formation.
Experimental studies.
SHR (spontaneously hypertensive rat) was used as the experimental model of hypertension. We studied platelet survival time on spontaneously hypertensive ratstroke prone (SHR-SP) and spontaneously hypertensive rat-stroke resistant (SHRSR) by means of in vivo labelling with ⁷⁵Se-Selenomethionine.
The result of this study showed that significantly short survival was observed in SHR-SP of 10 weeks of age compared with SHR-SR.
Platelet seems to have some concern with elevation of blood pressure and producing vascular alteration on SHR-SP.

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We have noticed the elevated dynamic viscoelasticity during process of blood coagulation in patients with various kinds of thrombotic disease, observed by using the viscoelastorecorder. We have confirmed that dynamic viscoelasticity of whole blood clots correlates with the concentration of fibrinogen but opposing with hematocrit level. We studied the effect of platelet count and platelet aggregation inhibitors on clotting curve.
Dynamic viscoelasticity was measured by the viscoelastorecorder and was shown by absolute values, namely dynamic elastic modulus (G′) and loss modulus (G″). Experimental conditions were: temperature, 25.0°C; oscillation of outer cylinder, 3c/s, amplitude; 60μm from peak to peak; and citrate and CaCl₂ solutions were used as an anticoagulant and for recalcification.
According to experiment, the elevated dynamic elastic modulus of plasma clots were obtained in cases of increased platelet count. Also other physical characteristics, such as dynamic loss modulus, maximum clotting gradient (MCG′) and tangent delta (G″/G′) correlated with platelet count.
In order to observe the effects of platelet aggregation inhibitors, we recorded clotting curve of platelet rich plasma with dipyridamole and aspirin respectively. Dipyridamole reduced by 50% of maximum dynamic elastic modulus (G′m) of clots to the control under the final concentration of 3×10_-4Mol for 15min. incubation at room temperature. However clotting time and maximum clotting gradient were not reduced. From these observation, it is supposed that dipyridamole does not affect on the thrombin formation and fibrin polymerization but does on fibrin polymer cross linking mechanism. Under the final concentration of 2×10_-4 Mol for 15min. incubation at room temperature, aspirin has no significant effect on dynamic viscoelasticity of clots.

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