Indication of OHP treatment on cerebral thrombosis has been widely accepted. The addition of OHP treatment upon other conservative therapy resulted in better neuropsychiatrical improvement. In this paper, the difference between the mechanisms of the therapeutic effect of OHP and oxygentoxicity are mainly discussed from the biorheological point. Fluidity of the RBC membrane was determined by (1) the order parameters of electron spin resonance absorption spectra which were produced by fatty acid spin labels inserted in the membrane double layer, and (2): fluorescent polarization of anisotropic fluorescent dyes (ANS etc) inserted in membrane. The viscosity of the whole blood was measured by viscometry. The plasma MDA and VE lebels which indicate the degree of lipid peroxidation and risk factor for thrombosis were determined by spectrophotometry.
When the improvement of aphasia, agnosia and aplaxia were achieved after 10 times OHP treatment (2-2.5 ATA×1hr OHP treatment was applied with consecutive days), the whole blood viscosity, Ht, MCV, order parameter of ESR spectra and fluorescent polarization of RBC membrane were significantly decreased. These rheological improvement suspected to be effective in the supression of edema. The plasma MDA lebels were high in the beginning of OHP, but low after 10 times OHP. This tendency was more manifest in the patients who were prescribed by VE and GSH during OHP treatment. These indicate that the physiological oxygen consumption was restored and pathological process of oxygentoxicity were supressed.
Overdosis of oxygen with OHP produced inverse rheological effect on experimental animals. Though VE and GSH exerted some protective effects, all animals died from thrombosis by oxygentoxicity.
Oxygen exerts two different effects-in one hand the vicious circle are supressed by OHP therapy but in the other accelerated by oxygentoxicity.

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It is noticed that the decreased peripheral leukocyte and platelet and activation of intravascular coagulation are induced by direct effect of endotoxin to the vascular system. But its mechanism is still uncertain.
We used E-coli 0-26 endotoxin injected into the Donryu rats by intraperitoneal injection, 20mg per kg. and observed the vascular changes of lung, liver, kidney and adrenal, chronologically 30min. 1, 3 and 5hrs. by light and electron microscope. And we also studied the vascular permeability using fesin and horseradish peroxidase as tracers. By electron microscopic examination, we noted the increased pinocytotic vesicle, swelling of mitochondria, widening of intercellular spaces and vacuolization of endothelium and medial cells in the arterial system of lung, liver, adrenal and kidney. These findings were prominent in kidney in the early stage.
And the destruction of polymorphonuclear leukocyte and platelet, deposition of fibrin were observed in sinusoids of the liver, in capillaries of the adrenal and kidney. And, especially, the kidney showed early prominent changes compared with other organs. The degenerative leukocytes were encountered strikingly in pulmonary vessels.
According to the vascular clearance methods, the electron dense tracers and reaction products of peroxidase are also seen in the pinocytotic vesicles and cytosomes of the endothelium through the endothelial intercellular spaces. The extent of increased permeability depends upon the qualitative difference of properties of vesicles and the vesicular system in the endothelium, medial cell and other mesenchymal cells.
The effects of the accumulation and disintegration of polymorphonuclear leukocytes and the break down of their lysosomes in blood vessels may a result of the direct cytotoxic effect of the endotoxin. The authors believe that the endotoxin installed shock may have resulted from the above mentioned vascular changes and the increased vascular permeability which is distinctly substantiated from the investigation of tracer methodology.
We conclude that the activated coagulability, as the direct effect of endotoxin administration is a very important factor in accelerating the pathologic changes of the vessels.

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Disorders of coagulation and fibrinolysis are well known phenomena after exvivo xenogeneic liver perfusion. However, their mechanisms have not been clear so far.
This study is on the coagulation and fibrinolysis in clinical and experimental ex-vivo xenogeneic liver perfusion.
Materials and Methods: Donor liver was taken out from a porcine weighing 10-14kg under intravenous anesthesia, which had been prepared preoperatively by antibioticus for one week, then it was washed out with chilled lactated Ringer's solution via portal vein and hepatic artery. The isolated porcine liver was perfused by an adult mongrel dog experimentally. The same procedures were clinically applied for a patient suffering from hepatic coma. The coagulation and fibrinolysis were avaluated using following various parameters.
Results: A. Experimental studies; RBC was not changed during perfusion. WBC decreased 50% during 2 hour-perfusion. Platelet count markedly decreased (60-80%) within 30 minutes after perfusion and this low level continued for 2 hours. Fibrinogen level showed 30 to 50% decrease within one hour, 50 to 80% decreasing after 2 hour-perfusion. PT after 2 hour-perfusion was 60-15%, PTT was 60-50%, after 2 hours. No active plasmin was found through out the perfusion. SK+plasmin and whole plasmin level did not show any changes. However, FDP after 30 minute-perfusion was 30-60ng/ml.
B. Clinical cases; Prior to the perfusion the platelet count was 46, 000, platelet aggregation was 48.1%, PT was 22.4%, PTT was 10%, TT was 10.5%, and HPT was 5%. After 2 hour-perfusin the platelet count was 28, 000 and platelet aggregation began to increase 30 minutes after perfusion up to 75.3% in 2 hours. Fibrinogen which had been 155mg/dl prior to the perfusion decreased to 92mg/dl after perfusion. PT, PTT, HPT, and ELT did not change during perfusion. The active plasmin level showed negative up to one hour and 30 minutes, however, it changed to positive within next 30 minutes. Prior to the perfusion the FDP was 200ng/dl and did not change during the perfusion.
Conclusion: In the porcine liver which was perfused by recipient dog, a clotting formation was found in each case during first 30 minutes perfusion. The same change was found in the clinical case. This hypercoagulability and clotting formation are assumed to be the result of hyper acute rejection induced by antibody-leuco plugging and antibody-thromboplugging.

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It should be recognized that, the release of proteolytic enzymes about the haemostatic studies are important for solving pathogenesis of pancreatitis.
The effect of coagulation, fibrinolytic system and platelet function were investigated in this paper.
Results:
1) In acute stage of pancreatitis, the increasement of α₁-Antitrypsin and slight decreasement of a₂-Macroglobulin were recognized.
2) The platelet aggregation showed the increasement in acute stage of pancreatitis.
3) The platelet volume showes generally increased in pancreatitis comparing with the platelet volume of normal adults.
4) The shortening of ¹³¹I-Fibrinogen turnover rate were recognized.
5) About the platelet aggregation after injection of Glucagon, pancreatic endocrine hormone, the inhibitory effect of platelet aggregation was smaller in pancreatitis than that in normal adults.
Conclusion:
From the above results, its suggested that the changes of haemostatic balance and platelet functions induced by the release of proteolytic enzymes, esp. trypsin have important relation to the development or progress of pancreatitis.

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Concerning the histogenesis of myocardial infarction, the “mechanical obstruction theory Virchow” was established and is inavoidably believed even now. And moreover, coronary thrombosis is regarded to be one of the most important primary causes of this disease. Based on this classical theory, some following peculiar findings most of which were never pointed out and emphasized can not be explained; such as (1) the existence of a prefered localization of a main coronary thrombus (about 2.5 to 5.0cm distal from the coronary ostium) and of “mechanical destruction accompanied by plasma inbibition” in the portion where a main thrombus attaches, (2) similar lesions in that portion in non-occulusive small thrombus cases and non-occulusive non-thrombotic (pre-thrombotic lesion YAJIMA & KOMOTO), (3) “Dysorie” lesions of systemic intramyocardial coronary arteries and arterioles in a necrotic area and its adjacent portions, being suspectedly regarded as to be due to a sudden elevation of inner pressure caused by arterial hemodynamic disorder, (4) surviving of fibers or of even a single fiber of the muscle cell in a myocardial necrotic part. A new “coronary dysfunction theory YAJIMA” may have to be introduced to explain consistently these peculiar findings in acute death cases due to myocardial infarction.
Detailed demonstrations of each of these findings being apparently contradictory to the classical theory were done and discussed in detail.
According to this theory, the dis-hemodynamic factor may be again seriously emphasized in addition to the coagulability of the blood, concerning the histogenesis of thrombosis.
It was additionally stated that similar arterial and arteriolar lesions could be observed in cases of cerebral and renal infarction diseases. Peculiarity of extramyocardial arterio- and/or athero-sclerosis of the coronary artery was stated, as the most important indirect supporting evidence of the “coronary disfunction theory”.

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It has been well documented that vessel injury elicits a formation of platelet thrombi. When the subendothelium is exposed to blood stream, platelets adhere to subendothelial components, mainly to collagen. On the contrary recent reports indicate that an opposite sequence may take place that platelet aggregation in the circuration may damage a normal vascular wall. However evidence is still scant and the mechanism is not clear. Adenosine diphosphate (ADP) is a principal aggregating agent of the platelet and the ADP-induced platelet aggregation in the circulation is usually reversible. This type of platelet aggregation has been thought to be a frequent clinical occurcnce in various atherosclerotic and thrombotic situations. In this paper effects of ADP-induced intravascular aggregation of the platelet on the pulmonary artery were studied.
Materials and Methods: Twenty-eight male rabbits were used. In 4 of them thrombocytopenia was induced (less than 5×10⁴/μl) by anti-rabbit-platelet guinea pig serum (APS). Seven of 28 were pretreated with acetylsalicylic acid (ASA). All animals were injected with ADP (2-5mg/kg) into the central vein in several seconds under pentobarbital anesthesia. Heart rate, respiration, ECG, systemic arterial and central venous pressures were recorded continuously throughout the experiment. After an observation for one hour animals were sacrificed by overdose of pentobarbital and sections of the lungs were examined by light microscope.
Results: Immediately after ADP injection the rabbits developed marked bradycardia, premature beats, ischemic ST-T changes and convulsion followed by tachypnea for several minutes. Hypotension and rise in central venous pressure were also noted. Platelet count decreased immediately after ADP injection and increased at 10 minutes. In both APS and ASA pretreated animals above changes were absent or very slight and platelet count did not change significantly. Histology of the lungs revealed that platelet aggregates were very frequently present in small and medium sized pulmonary arteries associated with luminal narrowing, thickening of muscular media, swelling of endothelial cells and characteristic micro-cysts in the intima (Figures 1 and 2). Occasional hemorrhagic infarcts were also seen. These changes were totally absent in APS induced thrombocytopenic rabbits and significantly slight in ASA pretreated ones (Table 1).
Comments: The result shown in Figures 1 and 2 would suggest a contraction of the pulmonary artery and a damage of the endothelium associated with platelet aggregation. Recent development in pharmacology of the platelet elucidated that platelet aggregates release potent platelet-aggregating and vasoconstrictive substances, prostaglandin endoperoxides and thromboxane A2. The pulmonary arterial contraction and injury are apparently elicited by the platelet aggregation and the pretreatment with ASA which inhibits cyclooxygenase, the enzyme transforms arachidonic acid to PG endoperoxides, prevented these changes. As a consequence the changes in the pulmonary artery might be induced by PG endoperoxides and thromboxane A2 as well as serotonin.

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It is generally recognized that Antithrombin III plays an important role as an inhibitor on the coagulation mechanism of the blood and is known to be decreased in its amount on the cases of myocardial infarction or arteriosclerosis, in the meanwhile the arteriosclerosis has been considered to be one of thrombogenic factors. This research was designed to investigate the effects of Antithrombin III on the relationship between thrombosis and atherosclerosis. For the purification of Antithrombin III, heat defibrinated plasma was applied to the column of heparin covalentry linked to Sepharose 4B. Non-specifically bound protein was eluted with 0.01M Tris HCl buffer pH 7.5 containing 0.3M NaCl and Antithrombin III subsequently eluted with 0.01M Tris HCl buffer containing 0.08M NaCl. Aliquots of each fractions were assayed for antithrombin activity. The highly purified Antithrombin III was obtained by this affinity chromatography with heparin as the legand. This inhibitor possessed both heparin cofactor and progressive antithrombin activity, and SDS disc electrophoresis showed only single band of molecular weight 62, 000 daltons and rabbit antiserum immunized with this fraction showed also single precipitation arc on IEP. The degree of lipolytic activity was measured by estimation of amount of the released free fatty acids according to the modified method of Boberg and Carlson. The incubation mixture contained various triglycerides as substrates and Antithrombin III as enzyme. The triglyceride substrate, Intralipid fat emulsions were hydrolyzed by the purified Antithrombin III to a certain extent and free fatty acids released were increased pararell with the incubation period and with Antithrombin III amount. The effects of Antithrombin III on the various triglyceride substrates were compared and the triglyceride moiety of these substrates were preferentially hydrolyzed in order of Intralipid, Triolein, Serum Chylomicron.
Atherosclerosis is one of the thrombogenic factors and has been considered to be due to the disorders of lipid metabolism on the vessel wall and also hyperlipemia is emphasized to be one of causative factors. On the other hand decreased antithrombin activity developes hypercoagulable state, which results in fibrin formation on the endothelial cells. Present data indicates that Antithrombin III with lipolytic activity could be postulated as an important inhibitor which intervenes between thrombosis and arteriosclerosis.

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Changes in canine AT III metabolism after i. v. administration of bovine thrombin were studied using I-125-AT III. I-125-AT III was prepared by the method of McFarlane.
(1) On the second day after intravenous injection of I-125-AT III, bovine thrombin (30u/kg/hr) was infused continuously for 3hrs. Plasma fibrinogen level was decreased to 60% of initial level, while plasma AT III concentration and AT III metabolism did not change markedly. AT III. metabolism in the dogs infused larger amount of thrombin was studied. Total amount of 5, 000-15, 000u of thrombin was administered intravenously with simultaneous injection of the same units of heparin. Plasma concentration of AT III and the radioactivity of I-125-AT III decreased to 70-80% of the initial levels, although plasma concentration of fibrinogen did not change significantly.
(2) When, 50μg of I-125-AT III, 1, 000u of bovine thrombin and 1, 000u of heparin were injected simultaneously, the half life of 1st phase of the plasma disappearance curve of I-125-AT III was shortend to 1.6 hours compared to the normal value of 5.5 hours.
From these data, it is suggested that AT III forms a complex with thrombin and thereafter the AT III-thrombin complex is cleared out rapidly from blood stream.

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The formation of heparin precipitable fraction (HPF) is often observed in the chilled plasma of patients with certain infections and inflammations that heparin had been treated. HPF is believed to consist of mainly fibrinogen and cold insoluble globulin (CIG). In this paper, to study the mechanism of formation of HPF in plasma we examined the relationship among CIG, fibrinogen, fibrin monomer and heparin in the formation of HPF.
Heparin formed the precipitate from normal plasma in the cold at the optimal heparin concentration from 10 to 50Uperml of plasma. The further raising of the heparin level reduced the precipitate formation. The washed HPF contained CIG and fibrinogen by the analysis from the SDS-gel pattern of electrophoresis and the immunological test. Furthermore, the NH₂-terminal analysis of HPF by direct Edman's method indicated the presence of alanine and tyrosine. These amino acids were corresponded to NH₂-terminal residues of αand γ-chain of human fibrinogen but not to fibrin monomer, since NH₂-terminus of either CIG and β-chain of fibrinogen were blocked with pyroglutamyl residue. From the experiments by using purified CIG and fibrinogen in the cold, heparin formed HPF with CIG alone or with mixture of CIG and fibrinogen but not with fibrinogen alone. From these results CIG is essential to form HPF. In the mixture composed of purified CIG, fibrinogen and heparin, Ca⁺⁺ increased the formation of HPF at the optimal concentration from 10 to 50mM. Fibrinogen increased the amount of HPF and seemed to reduce the threshold level of CIG that need to form the precipitate.
To clarify the interaction between CIG and fibrinogen or fibrin monomer, CIG was submitted to the chromatographic experiments with fibrinogen-agarose or fibrin monomer-agarose column. CIG had extremely higher affinity to bind the fibrin monomer than fibrinogen, and its affinity of CIG for fibrin monomer was completely disappeared by the addition of heparin (10U/ml) in the eluant. Furthermore, on the affinity chromatography of heparin-agarose column, CIG had a considerably higher affinity for heparin than fibrinogen did.
From these results, it was indicated that CIG is essential factor in the formation of HPF in plasma, although fibrinogen participates mainly in HPF. And the participation of fibrinogen in HPF is seemed to occur by reason of its affinity for CIG through the small amount of fibrin monomer, because fibrin monomer has strong affinity for both fibrinogen and CIG.

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Heparin is one of the acidic glycosaminoglycans (AGAG). And heparin is being used in the capacity of anticoagulant agent, generally. But heparin dose not have anticoagulant activity by itself at medical dosage. But heparin has only cooperative action with AT III in the sense that heparin increases the anticoagulant activity of AT III at the level of general dosage. Although there is no heparin in peripheral blood and layers of human vessels, but this fact has not been widely known.
This paper investigates the cooperative anticoagulant activity of AT III with natural and synthetic AGAG and makes clear the mechanism of the cooperative effect between AT III and AGAG. On the other hand, this paper discusses the meaning of this cooperative anticoagulant mechanism.
The following results were obtained.
1) The cooperative anticoagulant activities of AT III with different AGAG were compared by heparin cofactor method. It was found that 0.0025mg/ml of heparin had 0.6 antithrombin unit (ATU)/ml of anticoagulant activity by itself. Dermatan sulfate, heparan sulfate and keratan sulfate respectively had 0.2ATU/ml of anticoagulant activity. Also 0.0025mg/ml of synthetic amylopectin sulfate had 8.0ATU/ml and 0.0025mg/ml of synthetic chondroitin polysulfate had 2.6ATU/ml.
On the other hand cooperative activity of heparin with AT III produced an increase of 7.9ATU/ml in anticoagulant activity of AT III. This cooperative increase with dermatan sulfate was 5.6ATU/ml. But cooperative actions of synthetic amylopectin sulfate with AT III did not show any increase in anticoagulant activities of AT III (Table 1).
2) Continual observations over a time period of 60 minuets on cooperative activities of heparin or dermatan sulfate with AT III showed that reactions between AT III and thrombin were accelerated by adding heparin or dermatan sulfate to AT III. Besides, final anticoagulant activities of AT III with heparin dermatan sulfate were much greater than that of AT III alone (Fig. 1).
3) By immunoelectrophoretic investigations, it was observed that each AGAG combined with AT III and the AT III-AGAG complex was made (Fig. 2).
4) It was made clear that AT III had allosteric effect, and heparin and dermatan sulfate were allosteric effectors.
5) It is suggested that specific molecular structure of AGAG in needed for causing comformation change of AT III.
6) This allosteric effect of AT III may imply simultaneous regulation mechanism of extravaso-coagulation and intravaso-anticoagulation.
7) In human vessels, dermatan sulfate may be the true allosteric effector on AT III.

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242 patients suffering from peripheral occlusive vascular diseases have been treated with oral anticoagulant, warfarin potassium. Side effects were encountered in 5 cases. They were as follows, epistaxis, left sciatica and bleeding into the groin muscle, left brachial neuralgia and generalized subcutaneous and intramuscular bleeding, bleeding into the calf muscle, and gastrointestinal disorder. Two cases presented in this paper had neuralgia prior to the manifestation of hemorrhagic diathesis which could not be diagnosed at the first time of consultation. One case was vasculo-Behçet disease, 43 year old male who had been treated with 3mg per day of warfarin for the recurrent thrombophlebitis in the right lower limb. Left sciatica and painful swelling in the left thigh occurred approximately two months later after discharge. He was diagnosed as thrombophlebitis and 5mg per day of warfarin were administered, consequently hemorrhagic diathesis was manifested in the left lumbar region and the left ankle joint. Immediately warfarin was discontinued and vitamin K₂ and K₁ was administered. Hemorrhagic diatheses subsided within one week. He became ambulant and discharged. The other case was Buerger's disease, 42 year old male. He underwent thromboendarterectomy for the partial obstruction of right radial artery. After discharge, he had been followed with warfarin potassium 3mg per day. Nine months later, he complained of left brachial neuralgia. At this time, no manifestation of hemorrhagic diathsis was found, in spite of thrombotest less than 5%. He was treated with aminopyrin 1.0g per day. The following day, body temperature rose to 38°C and he complained of severe sore throat. Three days after the first attack of brachial neuralgia, he was admitted. At the admission, subcutaneous bleeding around the neck, myalgia all over the body and submucosal bleeding in the throat and high fever were recognized. Discontinuation of warfarin and administration of vitamin K₁ and tranexamic acid were instituted. Neuralgia, myalgia and hemorrhagic diathesis were all disappered within 2 weeks, and thrombotest returned to 100%. We should keep in mind that neuralgia might occur as the first symptom of side effect prior to manifestation of hemorrhagic diathesis during oral anticoagulant treatment.
During oral anticoagulant therapy, if thrombotest decreased less than 5%, discontinuation of anticoagulant should be recommended and daily laboratory examination must be carried out. The range of control of thrombotest should be 5-15% during addmission and after discharge, 10-25%.

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In our previous studies, UK injected into the general circulation was found to lose its activity very rapidly. The rate constant of such UK inactivation is defined as the inactivation rate constant of UK. Although the value of this inactivation rate constant can be calculated by compartment analysis, its nature and significance remain unclear. The present study was therefore undertaken to clarify the factors which affect the inactivation rate constant of UK.
When the UK solution was injected into the peripheral vein of normal rabbits, the fibrinolytic activity of the plasma showed a rapid decrease, as reported previously. There was marked fibrinolytic activity at 30sec to 1min, slight fibrinolytic activity at 3min, and almost no fibrinolytic activity at 5-6min after the UK injection. When UK solution was injected into the portal vein of the normal rabbits, no detectable fibrinolytic activity developed in the plasma.
When the UK solution was injected into the peripheral vein first, and injected again into the portal vein about one hour later, the fibrinolytic activity of the plasma was markedly enhanced at the second injection, and was remained even at 10min after the injection of UK solution.
When the UK solution was injected into the peripheral vein of rabbits whose liver, spleen and kidneys had been isolated from the general circulation, the fibrinolytic activity of the plasma was marked and sustained for a long period. The half time of decay of fibrinolytic activity obtained from the calibration curve was 4.24±0.98min (mean±standard deviation, n=5). This value is significantly greater than that for UK injection into the peripheral vein of normal rabbits (0.95±0.37, n=5) (p<0.005).
These results suggest that the reticuloendothelial system (RES) plays an important role in UK metabolism, so that the inactivation rate constant of UK may derive largely from the activity of the RES.

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With the use of chromogenic substrate (S-2251), rate assay (kinetics) and end point assay of enzyme activity of human plasmin and human plasminogen, euglobulin and plasma by SK or UK were studied using by auto-analyzer (Hitachi-Co., type-703).
The chromogenic substrate was enzymatically good substrate for plasmin and K_m value of plasmin was 7.6×10^-4mM.. When plasma was activated by SK, the highest value of kinetics was seen at initial time but UK had lag time. From these results, it is suggested that even with the use of small dose of substrate (0.1uM), auto-analyzer could be applied to measure fibrinolytic activity of human plasma (0.05ml) activated by SK (500U) on kinetics.

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Although it has been pointed out by many researchers that the plasmin plays the main role in to lytic ability of the fibrin thrombi in tissues. Thrombi also result from other proteases such as trypsin, chymotrypsin, and cathepsin, etc. When observation was made for changes of Fibrin Degradation Products (FDP) in time variation in patients with Disseminated Intravascular Coagulation Syndrome (DIC) accompanied by hematologic disorders or neoplastic diseases.
We experienced not a few cases where elevated plasmin activity which has been pointed alone can never be responsible. It was found in many of such cases that there was marked elevation in cathepsin activity. In the present study, the fibrinolytic phenomenon was studied in both aspects of general and local fibrinolysis, and resluts of investigation into the role of cathepsin in the latter are reported.
Cathepsin activity is found in 280 patients with various disease, highst in DIC, and high in neoplastic disease, aplastic anemia, chronic nephritis, leukemia, and hemolytic anemia, etc.
A study in autopsy cases of DIC resulted in that fibrin deposits in renal glomeruli and liver sinusoid was noted, non or very little, in many cases where cathepsin activity is high.
This might reflects to some extent local fibrinolysis in the kidney and liver.
In most of patients with acute type of DIC noted an increase in cathepsin activity and FDP levels where significantly higher than those with chronic type.
Moreover, similary tendency was observed in patients, where its prognosis had been poor.
From those results, in patients with DIC, these two parameters were directly proportional to the prognosis.
In the cases of severe tissue injury develops hypoxia locally to make a highly acid enviroment. Since plasmin in an alkaline protease, it has been loose protease activity at pH 6.0 or less, and thus there would be very little possibitity that it lyzes fibrin clot formed in injured tissues. Under such unfavorable conditions, cathepsin of an acid protease would exert fibrinolytic effect on fibrin thrombi.

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On the assay method by the use of chromogenic peptide substrate (Chromozym PK) consisting of Bz-Pro-Phe-Arg-pNA the quantitative observations on plasma kallikrein are performed methodologically and clinically, and the following results are obtained.
1) The minimum incubation time to activate plasma prekallikrein with kaolin requires about 20 minutes at 0°C. However, the incubation time is shorten to 3 minutes at 25°C or 37°C following a rapid decrease of kallikrein activity in the reaction mixture.
2) The standard curve of the PK assay using chromogenic substrate with 0.94mmol/l at the optimal pH (7.9) demonstrates a linear manner within 8 minutes incubation at 37°C.
3) For the purpose of anticoagulant sodium citrate is better than other remedies including sodium oxalate, EDTA and heparin, and it is observed that heparin can not be used for this chromogenic assay because of producing turbid precipitate.
4) The plasma stored at 4°C or -20°C are stable on the amidase assay for one week.
5) When the pooled normal plasma is taken as 100 per cent, the lenear relationship between diluted plasma and ΔOD is obtained in the range to 6.25 per cent.
6) Coefficients of variation is low, normal and high activities are calculated as 13.7%, 2.6%, and 0.95%, respectively.
7) Normal values of plasma prekallikrein and kllikrein in Japanese adults are in the range of 38.5-73.3mU/ml and 0-3.55mU/ml, respectively.
8) Although the levels of plasma prekallikrein and kallikrein in the majority of tested patients are in the normal range, one case with DIC shows extremely low concentration of plasma prekallikrein, and six cases with liver cirrhosis are distributed in the subnormal range.
9) The plasma samples of 11 cases from 14 pregnant women show high levels (42-63mU/ml) of plasma kallikrein accompanying with lower levels of plasma prekallikrein.
10) Furthermore, all plasma samples from 6 cases taking estrogen derivatives as oral contraceptive indicate high levels of plasma prekallikrein (84-110mU/ml), and this fact seems to be worthy to explain the tendency to thrombosis in such conditions created by the pills.

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We observed the hypercoagulation in the blood of the patients after the acupuncture. The causes of the hypercoagulation were clarified by the works of kallikrein.
(1) Urine kallikrein increased the activity of Factor XII and fibrinolysis of the citrated plasma in vitro studies.
(2) Urine kallikrein (20 E. U.) increased coagulation, fibrinolysis and platelet function of the 2.5kg weighted rabbit. Antiplasmin and antithrombin III increased temporarily. Microthrombi were found in the lungs by autopsy.
(3) Factor XII was increased by acu-puncture which activate prekallikrein to kallikrein in the cases with thrombotic disease.

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In order to clarify the mechanism of DIC, the kinin-forming enzyme system which relates to bothcapillary permeability and blood coagulation was studied. Prekallikrein was determined by TAMe-or BAMe-lytic method.
The results obtained were as follows:
1. To obtain sufficient activation of prekallikrein to kallikrein, the concentration of factor XII was necessary to be more than 40%.
2. Since hemolysis caused the activation of prekallikrein, artifact hemolysis should be avoided.
3. BAMe was approximately 10 times more sensitive than TAMe.
4. Prekallikrein content was significantly lowered in the patients with obsterical acute DIC. This might be due to consumption. The activation of prekallikrein to kallikrein causes enhanced capillary permeability and lowered function of blood coagulation, and aggravates DIC with vitious cycle.

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It is essentially important and decisive to find fibrin thrombi in order to diagnose finally as DIC. However, fibrin thrombi is not necessary found so frequently in the cases of DIC clinically diagnosed. Therefore, the authors intended to clarify, 1) the mechanism of lysis or disappearance of thrombi in DIC, 2) to distinguish thrombus in general from that of DIC from morphological and staining points of view.
Fibrin thrombi were in general classified into 2 types of pattern…an uniform pattern (I) and a fibrous pattern (II). Futhermore, (I) was subclassified into coating, bordering, spotty, and massed types. Generally (I) was found in capillary in cases of the acute DIC. Fibrin thrombi easily disappeared in the plasmin-treated paraffin preparations. In the electron microscopical findings, fibrin in (I) was significantly differed from the previously reported ones, and it was composed of the filamentous fibre with irregular arrangements, low densities, and showed homogeneous apperances. (I) was considered to be a soluble fibrin monomer complex or its resembled substances, but not a polymerized fibrin.
(II) was classified into tangled, reticular, and waste thread types, and found frequently in capillary and small sized vessels, mostly in cases of the chronic DIC. Fibrin found in (II) was only slightly affected by the plasmin treatment. Therefore, it seemed to be affected by the action of stabilizing factor. The frequencies of fibrin thrombi found in DIC was generally quite differed from other authors' ones which are now available.
In conclusion, the authors assumed that these results was occurred from remarkable differences in their fibrinolytic activities and to immediate fibrinolysis after postmortem.

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Introduction: The condition observed in coagulation activity during pregnancy was described as a “hypercoagulable state” (Alexander, 1955). Lately, it's reported that acute intracascular coagulation often occurred during pregnancy and delivery, particularly in cases of toxemia of pregnancy, intrauterine death, abruptio-placentae and post-partum haemorrhage. The purpose of the present investigation is to research the extensive DIC in vaginal bleeding.
Patients and Methods: According to bleeding volume in vaginal deliverys 163 cases were divided into two groups; the first group has lesser bleeding volume than 500ml (132 cases) and the second greater than 500ml (31 cases). Their specimen were drawn seriously in the full term or false labor, the first and second stage, 20 minutes after delivery and 3, 6, 12, 24 and 48 hours post partum, and the following determinations were made Fbg, platelet counts, FDP, eug LA, eug+SK LA (Sta), eug+SK LA (Htd), SFMC, PTT, PT, TT and PRT.
Results: eug+SK LA (Std) and eug+SK LA (Htd) decreased from 20 minutes after delivery. SFMC showed arising from the first or second stage to 48 hours after delively. FDP increased from the first stage to 6 hour after delivery and decreased from 12 hours after delivery. Fbg gradually increased from the first stage to 20 minutes after delivery. Eug LA gradually decreased in the first and second stage, but 20 minutes after delivery increased in group I and remained unchanged in group II and gradually increased from 3 hours after delivery. Platelet counts decreased during 3, 6 hours after delivery.
Discussion: Serial coagulation and fibrinolysis studies in vaginal delivery showed a pattern DIC such as decreased platelet count and fibrinogen level with increased ELA and FDP at certain times. These pattern was observed at 6 hours after delivery in group I and at 3, 6 hours after delivery in group II. Thus, at these hours attention about DIC should be paid.

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Case 1: A 28-year-old male was admitted to the hospital complaining of abdominal pain. Since 13 years old, he had been suffering from superficial and deep venous thrombophlebitis of the lower extrimities. Examinations on admission suggested mesenteric venous thrombosis and laparotomy was performed. As a segment of terminal ileum was necrotic, the involved bowel of 105cm in length and its mesentery were resected and a side-to-side anastomosis restored intestinal continuity.
Postoperative recovery was uneventful except fever of 38°C during 23-25 postoperative day. On the 31st day, laboratory examinations revealed fibrinogen was 60mg/dl, prothrombin time more than 60 seconds and platelets count 140, 000 suggesting intravascular coagulation. Heparin was administrated for 3 days until intestinal bleeding began. Despite of transfusion of blood of 4, 300ml for 5 days, hemoglobin decreased to 6.6mg/dl. At that time, clotting time was 6 minutes and whole blood resolution time was 30 minutes, which suggested hypercoagulation and hyperfibrinolysis. After re-administration of heparin, melena subsided and clinical & laboratory findings recovered well. Heparin was changed to warfarin further to aspirin later. During his admission, in spite of positive RA test and high level of gamma globulin, there were no evidence of collagen diseases. Gynecomastia was noted and estrogen value in urine showed 3 times higher than normal.
Case 2: A 41-year-old male. He underwent total gastrectomy for scirrhus gastric cancer. The postoperative course was uneventful until melena occurred on the 13th postoperative day. Purpura was noticed and laboratory examinations revealed fibrinogen was 50mg/dl, prothrombin time 16 seconds and platelets count 50, 000. FDP was, however, negative. Administration of heparin improved clinical and laboratory findings. Bone marrow puncture showed metastatic cancer cells. 73 days after the operation, he expired due to cerebral bleeding.
In these 2 cases, clinical findings, hemorrhagic diathesis, low fibrinogen value and prolonged prothrombin time suggested DIC syndrome improved by administration of heparin. Although tne operative injury is one of the important causative factors of DIC, owing its influence upon coagulation-fibrinolysis system through endcrine reactions, disturbance of local blood flow and so on, the postoperative course of these two cases was both uneventful throughout the convalescent period. Accordingly, it could be assumed that operative injury played little role to the occurrence of DIC in these cases. Otherwise, the basic diseases, recurrent thrombophlebitis in one and metastatic cancer cells in bone marrow in another, might be regarded as a cause of DIC.

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The accident of incompatible blood transfusion is not infrequent in practical medicine. The acute hemolytic transfusion reaction sometimes results severe complications. In authors' experience in the treatment and clinical analysis of this disorder during last one decade, there were some evidences that disseminated intravascular coagulation attributed in considerable extent to the development of such complications as shock, hemorrhagic tendencies and acute renal failure. In this paper, six patients received incompatible blood transfusion are reviewed and some therapeutic approaches are presented.
On the hypothesis that DIC is a primary pathogenetic mechanism involved in these hemolytic reaction, authors have learned that the heparin therapy would be the best at interfering with these pathophysiologic pathways, and have been getting the benifits in prevention of severe complications or of exaggeration of signs and symptoms developed. Of 6 cases presented, five of the incompatibilities were in ABO system and one in Rh system. A 26-year-old female, blood group 0, Rh positive, was given a transfusion with 0 positive blood but she was positive in anti-E antibody. The amount of blood transfused were 50-350ml in ABO incompatibilities and 1, 000ml of E-positive blood in a Rh-incompatible case.
Hemostatic studies were done in 3 patients. One patient received ABO incompatible blood revealed clotting abnormalities even 2 days after the heparin administration. The case with positive E-antibody developed acute renal failure. Severe bleeding tendency was observed at the Cesarian section when she was given more than 1, 500ml of blood. Thrombocytopenia, positive tests in FDP and protamine sulfate test and prolongation of thrombin time and serial thrombin time were seen in these 2 cases. Remaining one patient who was given heparin just after the accident showed no coagulation disturbance when tested during heparin therapy. Five patients received mismatched blood in ABO system were all given heparin as soon as possible after the accident to prevent severe reactions due to DIC. All were symptomatically uneventful, however, the elevation of FDP and thrombocytopenia were noted in one as mentioned above and moderate increase of blood urea nitrogen in three clinical courses. These findings seem to represent the subclinical participation of clotting episodes in mismatched transfusion reactions even after the heparin administration. Therefore, the occurrence of DIC may be more frequent that has been appreciated and prompt treatment with heparin prior to the recognition of signs and symptoms may decrease the morbidity and mortality from incompatible transfusion reactions.

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High blood viscosity is a factor of cause of various thrombotic disease. The blood viscosity changed by Ht level and flexibility of RBC. And their factors has influence on the blood circulation.
In this study the in vitro effects of tocopheryl acetate and tocopheryl nicotinate on the blood viscosity, RBC 2·3 diphosphoglycerate (DPG), RBC fragility and flexibility of RBC in fresh blood and stored blood were investigated.
The visconic EL type viscometer for blood viscosity, a coil planet centrifuge (CPC) for osmotic fragility of RBC an enzymatic method for DPG content of RBC, low shears rate viscosity of packed cells and nucleopore filter with pore size 5μ for flexibility of RBC were employed and measured respectively.
Both tocopherols were added over 50/ml in fresh blood had inhibitory effect on increasing blood viscosity and, on decreasing fragility and flexibility of RBC during preservation. But both tocopherol could not only inhibited on decreasing DPG level of erythrocytes during storage.
On the otherhand, tocopherol added in blood over 14 days after preservation. was not recovered blood viscosity, fragility and flexibility of RBC injured during preservation.
Namely, tocopheryl acetate and tocopheryl nicotinate were inhibition of lipid peroxidation in erythrocytes membrane, maintenance of RBC flexibility in young RBC and blood viscosity in fresh blood, but no effect in aged RBC and in stored blood over 2 weeks.
These result strongly suggest that tocopheryl nicotinate and acetate bring to improvement of blood circulation due to act not only upon platelets and vessel but also upon erythrocytes.

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Close relation of blood viscosity to cerebral thrombosis in the aged has been widely recognized. Recently the medical attension has been paid to the usefulness of pentoxifyllin, which is a xanthine preparation, and elicits the increase of blood flow in sclerotic small arteries by lowering blood viscosity in the treatment of arteriosclerotic disorders. The characteristics of pentoxifylline is suggestive of the effectiveness in the treatment of the symptoms which might be derived from arteriosclerosis of the brain in the aged. The purpose of this study is to observe the effect of pentoxifylline on the blood constituents and to elucidate the relation of the changes in the blood constituents to the improvement of their complaints in the aged.
Twenty three aged subjects with mean age of 77.1 received orally 300mg of pentoxifylline a day for 8 weeks. Analysis of blood constituents was carried out before and after the administration. Levels of fibrinogen, α₁-antitrypsin, antithrombin III, IgG, IgA, IgM, transferrin and haptoglobin were determined by single radial immunodiffusion method.
Changes of blood constituents are shown in Fig. 2. There was significant decrease in the level of hemoglobin, hematocrit, total protein, globulin, fibrinogen and α₁-antitrypsin, and elevation in A/G after the administration for 8 weeks. No change was observed in the levels of FDP, α₂-macroglobulin and antithrombin III. Remarkable effect was observed in heaviness in the head, headache, coldness in the extremities as shown in Fig. 1. Fig. 3 shows the relation of the changes of blood studies to the improvement of the complaints. There was a significant decrease of hemoglobin in cases with improvement of whole symptoms, heaviness in the head and headache, and no statistically decrease in those without improvement of these symptoms. The significant decrease of hematocrit was found only in the cases with improved whole symptoms. The fibrinogen level decreased significantly even in cases without improvement, however, the reduction in the improved cases was higher compared with that in the unimproved cases.
The decreased hematocrit, as shown in a report on pentoxifylline administration, was also demonstrated in this trial, however, the most noticeable finding was the decrease in fibrinogen and globulin levels. Contribution of hematocrit to blood viscosity has been widely recognized. The decrease of hematocrit after administration in our study confirmed further the favorable effect of this preparation on blood viscosity. There are some disputes about the contribution of fibrinogen and globulin to blood viscosity. The decrease in fibrinogen and globulin levels in our study may indicate their important responsibility to the reduction of the visicosity of blood. Change of blood constituents accompanying the improvement of clinical arteriosclerotic symptoms was also noticeable finding in this trial.
It is concluded that these results indicate the important significance of blood constituents in blood viscosity in arteriosclerosis of the brain and the usefulness of pentoxifylline.

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Today, the development of antithrombogenic medical materials is the important points in advance of medical therapy. For this purpose, we prepared the dacron fiber immobilized urokinase which was the most useful fibrinolytic medicament.
This report investigated the influence of disinfection against the fibrinolytic activity of this fiber and presented the two clinical cases who were implanted this fiber in Handley's operation for lymphedema. Figure 1 shows the procedure of immobilization of urokinase to dacron. In this procedure, polyethyleneimine and gantrez (maleic anhydride/methylvinyl ether copolymer) play the role of grafted copolymer in ordor to couple the urokinase in peptide linking. Fibrinolytic activity of this fiber was measured by standard human fibrin plate.
The prepared dacron fiber kept the remarkable fibrinolytic activity in the repeated fibrinolysis test after the 6 months storage. In basic experiment, we checked how the immobilized urokinase was stable to the handling of disinfection. The fiber was sorked in 0.5% chlorhexidine alcohol, boiled saline solution or ethyleneoxide gas. Figure 2 shows these results. The stability of immobilized urokinase was more superior in 0.5% chlorhexidine alcohol.
These experimental data suggest the possibility of clinical use of immobilized urokinase technique.
Then, we applied this immobilized urokinase technique to the lymphangioplasty in lymphedema which was called Handley's operation. We had already two clinical cases implanted dacron fiber immobilized urokinase. The first case had primary lymphedema of left lower extremity and the second case had secondary lymphedema of left lower extremity. These patients were implanted three pieces of dacron fibers of approximately a half meter into subcutaneous tissue from below the knee to above the groin. After the operations, they were injected intravenously the high concentration of urokinase solution once a month. In one patient, however, one of three pieces of dacron fiber was removed for the infection after 45 postoperative days. Figure 4 shows the histological finding of removed dacron fiber. The fiber was surrounded by the granulation tissue with giant cells. In peripheral portion, the spaces among the bundle were filled with granulation tissue, while in central portion, the some spaces were kept open. But the fibrinolytic activity of this removed material had been almost lost.
From these clinical cases, we fully realized the necessity to develop the new material immobilized urokinase which was able to maintain the strong fibrinolytic activity for a long period even in vivo.

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Urokinase (UK), a plasminogen activator was immobilized on nylon and silicone surface by application of a newly devised technique.
The antithrombogenicity of these materials was studied in vitro and UK-immobilized nylon tubes were applied clinically, which proved to be available as antithrombogenic material.
Methods: In our newly devised technique of immobilization of UK, nitrophthalic anhydride (NP) was used as an anti-urokinase inhibitor, polyethylene imine (PEI) and Gantrez (G), a copolymer of maleic anhydride and methyl vinyl ether were used to increase the quantity of UK immobilized on carrier. The nylon tubes immobilized UK in this technique were shown as “nyl-G·NP-UK” and the silicone tubes, as “sil-G·NP-UK”. Details of the immobilization procedures are shown Fig. 1.
The antithrombogenicity of these materials was investigated in vitro, in two different ways: one was lysis areas by standard fibrin plate method and the other was thrombus formation time (TFT) according to the method of Chandler. In the latter studies, a comparison was made with non-treated nylon and silicone tubes, NP-immobilized nylon tubes, G-immobilized nylon tubes and casein-immobilized nylon tubes.
UK-immobilized nylon tubes, 2mm in outer diameter, 1.7mm in internal diameter were applied to surgical drains in 28 patients who had undergone thyroid, breast and thoracic surgery. Three of these were served as IVH tubes. All these tubes had been disinfected using ethylene oxide gas (EOG) before the clinical use.
Silicone tubes, 3mm in outer diameter, 2mm in internal diameter were used as control materials. Fibrinolytic activity was also tested before and after the clinical application of the materials by the standard fibrin plate method.
Results: Thrombus formation time of nyl-G·NP-UK was over 45 minutes.
That of sil-G·NP-UK was various, however, longer than that of other materials. These results are illustrated in Fig. 2.
When the UK-immobilized tubes were clinically applied as drains, blood coagula did not form in the lumina of the tubes for the duration of the clinical application, while in the case of silicone tubes, blood coagula obstructed the lumina within 12-48 hours. The patency of these tubes are summarized in Table 1.
Even after termination of clinical application, the UK-immobilized tubes maintained the plasminogen activating activities.
Discussion: The UK-immobilized nylon tubes showed excellent thromboresistance, both in vitro and in vivo, and such is attributed to the specificity of the surface of the material covered with UK and NP. Silicone is one of the most widely applied clinical materials, however, the degree of antithrombogenicity revealed to be unsatisfactory (Fig. 2).
Antithrombogenicity regarding the UK-immobilized silicone tubes are to be investigated.
In our clinical studies, the drainage with our newly devised tubes was excellent and the patency of the tubes was also satisfactory.
The UK-immobilized nylon tubes show a great promise for application as antithrombogenic materials.

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We have investigated the platelet volume of normal adults and a few cases of several diseases. Coulter Counter ZBI was used in measuring the platelet volume. The results are as follows:
1) About the investigation of normal adults, the platelet volume in females and old persons showed a slight increase.
2) In the advanced stage of ITP, both the mode and the mean platelet volume showed the decrease, while in the recovered stage of ITP, normalization was shown.
3) In chronic active hepatitis and liver cirrhosis, the platelet volume was generally large, and showed the decrease after bleeding.
4) In DM and IHD, the platelet volume showed a slight increase. From the above results we are of the opinion that the measurement of the platelet volume gives a great help in understanding the platelet function. On the other hand the change of the platelet volume is closely related to haemorrhagic diathesis.

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Recent interest has focused on the implication of platelet function in the development and inhibition of thrombogenesis. Ticlopidine is oral durg with a platelet inhibitory effect, being developed by PARCOR CO. in France.
Six patients with diabetic retinopathy were given 200mg/day of Ticlopidine and were clinically studied on the effect of Ticlopidine.
Significant inhibition of platelet function (platelet aggregation and adhesion) was observed.
No significant effect was found in plasma viscosity, whole blood viscosity and maximum dynamic elastic modules G'm in blood clotting curve. One patient with retinopathy showed a clear sign of improvement.

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Examination of fundus provides useful diagnostic information in patients with abnormal vascular walls. Therefore, on-line image processing of fundus is required for the more quantitative and time saving evaluation of vascular abnormalities and hemodynamics in microvascular bed in fundus.
In this study electronic data processing techniques were introduced in order to develope a practical image processing system in ophthalmoscopic photography. Two experiments of different approaches were attempted, that is, firstly, computer image processing of a static photograph and, secondly the development of television picture scanning device to measure blood flow velocities.
1. A 35mm picture film of fluorescence fundus photography was processed by means of a flying spot scanner into a digital picture with 256×256 pixels on a punched tape. This tape was then subjected to a computer image processing with a program to obtain outputs of contour maps and histograms of optical densities. Results showed that capillaries of diameter of as less as 20 micra could be distinguished. It seems that this kind of image processing technique can be used to evaluate quantitatively the leakage of fluorescent tracer through vascular walls.
2. An elecronic data processing unit was deviced to measure blood flow velocities in microvessels. This was designed to sample out instantaneous optical density changes at two different points chosen arbitrarily on the screen of monitor television which was connected directly to the microscope through a television camera. Peaks of the curve of optical density changes showing the passage of a tracer such as fluorescent substance or plasma pocket were recorded on a penrecorder or a CRT. The blood velocity was thus calculated by the measurements of time delay between two peaks and the distance between two sampling points.
Performance of the unit above stated was tested in the animal experiments of intra-vital microscopic observation of capillary blood flow in the rat mesentery. It was found that the capillary blood flow velocities up to 50mm/sec could be measured with enough accuracy.
Both these techniques seemed to provide a basis of on-line image processing of ophthalmoscopic photography with a high sensitive television camera connected directly to the microscope.

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Prostacyclin (PGI₂) production by rat aortae under various conditions was investigated and activities of the aorta to produce this substance in stroke-prone spontaneously hypertensive rats (SHRSP), stroke-resistant SHR (SHRSR) and normotensive control rats from the Wistar-Kyoto (WK) colony were compared.
Except for some preliminary experiments to establish the test system, only age-matched male animals (6 months) before the development of stroke were utilized (5 animals in each group). The descending aorta was removed from each animal, washed and used immediately or stored at -20°C until use. Fine rings of the aorta were routinely incubated in 0.25-1.0ml of pH 9.0 borate-buffered ered saline for 1hr at 20°C. Volumes (1-10μl) of the buffered saline in which the aortic rings had been incubated were added to 0.2ml human plateletrich plasma (PRP) 1 min before the addition of collagen. Platelet aggregation was monitored in a Sienco dual sample aggregometer. The amount of prostacyclin produced by the aorta was estimated by comparison of its anti-aggregatory activity with thay produced by known amounts of authentic prostacyclin-Na.
Rings of the aorta incubated as described above produced increasing prostacyclin-like activity, as shown by the strong inhibition of ADP-, collagen- and arachidonate-induced platelet aggregation for up to 1hr (Fig. 1) and the activity was dependent on the volume of buffered-saline added as well as on the length of aortae incubated. The activity produced by incubation of the aorta in pH 7.4 Tris-buffered saline for longer than 20 min at 20°C decreased progressively during the incubation and disappeared within 1hr (Fig. 1), confirming the lability of prostacyclin at pH 7.4. Incubation of the aorta boiled for 3 min produced no such activity. The spontaneous generation by the aorta of prostacyclip was inhibited by preincubation of the aorta with indomethacin (cyclo-oxygenase inhibitor), while the generation induced by PGH₂ was not (Fig. 2). These results indicate that the aorta has both cyclo-oxygenase and prostacyclin synthetase.
Amounts of prostacyclin generated by the aorta of SHRSP and SHRSR were significantly higher than those of WK rats (Fig. 3). Although the meaning of the enhanced production of prostacyclin by the aorta of these hypertensive rats is not clear at the moment, it could be a physiological defence mechanism against the development of vascular injuries possibly due to hypertension in these animals and stroke might be triggered by the development of any inhibitory mechanisms against prostacyclin synthesis by the vessel of SHRSP.

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Prostaglandins (PGs) play a major role in mediating platelet aggregation. The metabolism of PG endoperoxide is of primary importance to investigate the cell functions, but usually PG endoperoxide is too labile to assay. Then it might be necessary to assay other substrates such as precursor of endoperoxide or classical PG, instead of PG endoperoxide to study the PG metabolism.
Then we studied the ptatelet PG metabotism by the assay of 4 substances: arachidonic acid (AA), endoperoxide intermediate “classical” PGE₂ and F_2α in platelet suspension after adding thrombin, to clarify the regulation of these substances. Method:
Human platelets were obtained from platelet rich plasma treated with EDTA and were suspended in 134mM NaCI- 15mM Tris-HCl buffer.
Aggregation of platelets was studied photometrically continuous recording of light transmission. The aggregating agent investigated was thrombin.
AA, PGE₂, PGF_2α and endoperoxide intermediate were measured at various period after adding thrombin.
1) The contents of aggregometer tube were snap-frozen and were freeze-dried. After extraction of lipids, they were methyl-esterified and fatty acids were assayed by gaschromatography.
2) The contents of the aggregometer tube were decanted into ethanol, both with and without stannous chloride treatment. The procedures had been reported by J. B. Smith.
3) The contents of the aggregometer tube were snap-frozen and dissolved at 4°C. These were separated into supernatant and platelet fraction by centrifugation, and were extracted for PGs as described by Jaffe.
4) The effects of the anticoagulant (Carbocromen 200g/ml) on the formation of PGs were studied. After treatment of the carbocromen, the contents of the aggregometer tube were assayed by the procedure as described (3).
After extraction for PGs by both methods, Smith (2) and ours (3) the PGs were separated into fractions containing E or F types by column chromatography on silicic acid.
PGE₂ was assayed indirectly by ³H PGF_2α radioimmunoassay (RIA) after reduction of PGE to PGF with sodium borohydride, using PGE fraction. PGF_2α was assayed directly by ³H PGF_2α RIA, using PGF fraction.
PG endoperoxide was assayed indirectly by ³H PGF_2α RIA after reduction to PGF_2α with stannous chloride treatment as described by J. B. smith.
Results and Conclusion:
A The reduction in a ratio of fatty acid (C_20:4/C_18:2=AL index) was observed by 60sec. after adding thrombin (Fig. 2.).
PG endoperoxide formation was seen by 60sec. and had a peak at 30 sec. PGE₂ and PGF_2α was increased the amount with time (Fig. 3).
C PGE₂ and PGF_2α formation, measuring by snap-frozen method, showed biphasic pattern. The first peak was seen at 30 sec. and then the titration curve ascended again after 180sec. The amount of PGs in supernatant was greater than that in platelet fraction. The titration curve of PGs in supernatant showed the same pattern as that obtained in platelet fraction (Fig. 4).
D Platelet aggregation was suppressed by the anticoagulant. PGs formation was deppressed completely at any time tested in supernant. In platelet fraction, PGs formation was decreased the amount comparing to control, but the amount was much greater than that in supernatant.
The results were summerized in two conclusions.
1) The consumption of AA, the formation of endoperoxide and first formation of PGs were occured simultaneously.
2) Carbocromen blocked the excretion of PGs from platelet.

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