Biochemically, DIC is an abnormality consisting of elevated proteolysis in the systemic circulation. The enzymes participating in DIC are serine proteases which involve the systems of coagulation, fibrinolysis and kinin formation. This paper describes a brief review of proteolytic events observed in coagulation·fibrinolysis and kinin-formation. In DIC, there occur stpewise chain reactions of limited proteolysis of each factor of the systems. For activation of the zymogens shown in parentheses, the following clevages of peptide bonds are necessary: Arg-Val (XII), Arg-Ile (XI), Arg-Ala and Arg-Val (IX), Arg-Ile (VII and X), Arg-Ile, A rg-Thr and Arg-Ser (Prothrombin), Arg-Gly (XIII) and Arg-Val, Lys-Lys (Plasminogen). The terminal substrates, fibrinogen in coagulation, fibrino (gen) in fibrin (ogen) olysis and high molecular weight kininogen in kinin formation, recieve their limited proteolysis in such peptide bonds as Arg-Gly of A_α and B_β chains of fibrinogen by thrombin, Arg- or Lys- of fibrin (ogen) by plasmin, Lys-Arg and Arg-Ser of HMW kininogen by kallikrein, generating fibrin, fibrin (ogen) degradation products and bradykinin, respectively. Some of activated enzymes, especially thrombin exert positive or negative control activities, and some of peptide fragments released by limited proteolysis show usually negative ones.

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One of the major differences between fibrinogen degradation products (FgDP) and fibrin degradation products (FDP) exists in the fact that D-dimer can be proouced in the process of late FDP as a results of crosslinkage of γ-γ chains of fibrin and only D-monomer is in late FgDP. Therefore, a new method for detection of D-monomer representing FgDP and D-dimer indicating FDP was described in this paper, and the two dimensional immunoelectrophoresis using SDS-agarose gel in the first dimension step and agarose gel containing anti-D or E serum in the second dimension step was applied for this new method.
D-monomer of late FgDP and D-dimer of late FDP were prepared and studied on our method with anti-D serum, showing characteristic electrophoretic-movilities of those two human materials. Also, from the plasma containing D-monomer or D-dimer the derivatives of FgDP and FDP could be differentiated by our method with anti-D serum.
On the other hand, the fibrinolytic lysates of a clot from a patient with factor XIII deficiency did not demonstrate D-dimer by the use of our method, but presented D-monomer. After the factor XIII concentrates were added into the patient plasma in the 10% final concentration of factor XIII, D-dimer was detectable in the fibrinolytic lysates. Furthermore, when this method was applied for a patient suffering from APL with DIC, no D-dimer was detected in the patient serum. Therefore, it might be suggested that the impairment on the formation of stabilized fibrin in a case of APL with DIC might play an important role as a cause of bleeding tendencies.

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Heterogeneity of fibrinogen molecule in plasma is one of important phenomena in the research of thrombotic disorders. The purpose of this study is to clarify the neecssity of the clinical observation on the fibrinogen heterogeneity in plasma. Venous blood was sampled from 136 healthy persons and 31 cerebrovascular pltients into 3.8% sodium citrate (1:9). Total fibrinogen (TF) and high molecular weight fibrinogen (FI) were determined by the recently developed method using 2.3M glycine with various ionic strength. Low molecular weight fibrinogen (FII) was shown as the difference between TF and FI, and FII/FI was calculated. In healthy subjects, TF and FI increased with advancing age. The level of FII and FII/FI in patients were higher than those in healthy subtects. FII/FI in patients elapsed 13 months or above was higher than those elapsed 3 months or below. In comparison between healthy subjects and patients with 400mg/dl or below, the level of FII and FII/FT were also higher in patients. Patient's FII/FT was inversely proportional to F. XIII-A level determined by Laurell's immunoelectrophoresis. These results comfirm the importance of the fractional observation of plasma fibrinogen separated by molecular weight and clinical usefulness of the glycine precipitation method in the observation of plasma fibrinogen.

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In a patient with congenital afibrinogenemia, prolonged bleeding time is thought to be related to the defective platelet aggregation induced by ADP or poor platelet adhesiveness to glass beads.
Our recent study revealed that the patient's platelets can adhere to subendothelium normally but cannot form thrombi on it.
Accordingly, in a patient with congenital afibrinogenemia we attempted to observe the role of fibrinogen that influenced on platelet adhesion to subendothelium (SE) and thrombus formation on the SE by using Baumgartner's method at various levels of fibrinogen. Fibrinogen was transfused to the patient and the platelet adhesion to SE, thrombus formation, ADP induced platelet aggregation and bleeding time were observed. We also carried out in vitro addition of fibrinogen to the patient's blood samples and observed the platelet adhesion to SE and thrombus formation. The range of fibrinogen in the experiments varied from 0.07mg/dl to 110mg/dl.
Our results revealed that the platelet adhesion to SE was normal. On the other hand, thrombus formation was poor when the level of fibrinogen was between 0.07mg/dl and 1mg/dl. However, it was normalized when the level of fibrinogen was crossed over the level of 2.5mg/dl. This level of fibrinogen was almost equal to the level of fibrinogen that corrected the prolonged bleeding time or defective ADP (2μM) induced platelet aggregation.
This study suggested that the lowest level of fibrinogen consistent with normal platelet function in a patient with congenital afibrinogenemia was about 2.5mg/dl.

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Abnormal fibrinogen in patients with liver diseases, especially severe liver cirrhosis was studied. In these patients, the prolonged thrombin time, the prolonged reptilase time, and the delayed polymerization of fibrin monomer were observed in plasma. These findings suggested the dysfunction of fibrinogen which was related closely to the disability of polymerization of fibrin monomer.
Similar results were identified by using purifired abnormal fibrinogen. From this results, the involvement of the inhibitors againt coagulation in plasma appeared to be unlikely. The total content of sialic acid in purified abnormal fibrinogen was markedly increased as compared to that in purified nomal fibrinogen. Since the prolonged thrombin time was statistically significantly (p<0.01) correlated with the total content of sialic acid in fibrinogen, it may be suggested that sialic acid plays a great role in the dysfunction of abnormal fibrinogen.
To determine whether sialic acid can block the polymerization of fibrinogen monomer, or not, asialofibrinogen was obtained from the purified fibrinogen treated with neuraminidase. When coagulation time was examined by using asialofibrinogen obtained by neuraminidase treatment, the prolonged coagulation time was partially normalized in 3 patients with liver cirrhosis tested. Thus, it was suggested that sialic acid might block the polymerization of fibrin monomer, resulting in the dysfunction of fibrinogen.

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We studied 152 apparently healthy newborns below 30 days of age on crossed immunoelectrophoresis of prothrombin using capillary blood samples, in order to find out the frequency of mild vitamin K deficiency in healthy infants. About 70% of the newborns at 0 to 3 days of age, 47% of the newborns at 4 to 5 days of age and 28.6% of the newborns at 6 to 9 days of age had PIVKA-II on crossed immunoelectrophoresis.
Sixty six infants at one month of age included 28 breast fed infants and 38 either mixed or bottle fed infants. About 14% of these infants had a small amount of PIVKA-II. Although the frequency of mild vitamin K deficiency was very high at birth and quickly decreased to 28.6% by 6 to 9 days after birth, about 14% of apparently healthy infants at one month of age remained mildly deficient from vitamin K, no matter what the type of feeding may be. Through a large scale screening test using Normotest, 16 infants at one month of age were picked up for their value of less than 50% of normal. Among these 16 infants, 87.6% of them were demonstrated to have PIVKA-II and sigificantly larger amount of PIVKA-II than in one month old infants in group D. There was negative correlation between the amount of PIVKA-II and the value of Normotest in these infants.
In conclusion, 70% of newborns at birth are deficient from vitamin K and this deficient state is alleviated gradually and spontaneously with age. However, about 14% of one month old infants are still mildly deficient from vitamin K, no matter what the type of feeding may be.

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We measured prothrombin time (PT) and hepaplastintest (Hpt) of 48 patients (94 materials) with liver diseases(7 of chronic hepatitis, 21 of liver cirrhosis and 20 of hepatocellular carcinoma with liver cirrhosis). Significant difference in numerical value was observed between PT and Hpt as reported other authors, namely PT (75.3±10.7%) was about 30% greater than Hpt (46.4±13.0%). However, there was a little difference between them in the paticnts medicated warfarin (PT: 41.6±13.9%, Hpt: 33.7±14.5%).
Therefore, we measured factor II, V, VII and X, and investigated the correlation of those factors with PT and Hpt. The difference between PT and Hpt-(PT-Hpt)- was correlated with factor II (r=-0.31) and factor VII (r=-0.42). Judging from this correlation coefficient, (PT-Hpt) was in statistically signficant proportion to the decrement of factor II and factor VII (p<0.01, n=94). Besides, Hpt value decreased in proportion to the decrease of factor II and factor VII, but PT existed in higher level than Hpt under same amounts of factor II and VII (Fig. 2). This difference seemed to produce the difference between PT and Hpt. However, the value of PT and Hpt became closely under extremely small amounts of factors (Fig. 2), such as the patients with warfarin medication.
Conclusively, Hpt was much more influenced by factors synthesized in liver than PT (Fig. 2), and had a good correlation with factor VII which reflected a liver function (r=0.806).
These results indicate that Hpt is more reliable and superior test of liver functon as comparison with PT, though Hpt takes low numerical value than PT.

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When vitamin K deficiency occurs, precursor forms of vitamin K dependent coagulation factors which are termed protein induced by vitamin K absence (PIVKAs) appears in the circulating blood.
Crossed immunoelectrophoresis has usually been employed to demonstrate the presence of PIVKAs, but the procedure is time consuming for clinical use.
In the present test method developed by the authors, the abnormal form of prothrombin (PIVKA-II) in the plasma can readily be determined within a period of 30min. This test method is a simple and rapid assay for the detection PIVKA-II.
The principle of this assay is based on the agglutination of antihuman prothrombin rabbit IgG coated latex (AP-Latex) with absorbed plasma which is treated with barium carbonate.
After mixing with 25μl of the plasma and 200μl of the 0.2% bovine albumin saline, in which 100mg/ml of bairum carbonate was suspended, incubation at 37°C or room temperature for 10min was carried out with shaking every 2min. After the incubation, 275μl of the bovine albumin saline solution was added and the mixture was centrifuged at 3000rpm for 5min. Then 100μl of the supernatant placed on a glass slide (2×3cm), one drop of Ap-Latex was added. After the mixture was shaked for 2min, the latex agglutination was checked in daylight against a flat dark background.
The level of PIVKA-II was expressed by the maximum dilution level being agglutinated in the specimen.
This assay is expected to be useful as a screening test to differentiate whether or not vitamin K deficiency is present in various clinical cases.

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Factor VIII-related antigen (VIIIR: AG) in the plasma and cryoprecipitate from 30 newborn cord blood samples was examined by SDS-1.5% polyacrylamide gel electrophoresis followed by crossed immunoelectrophoresis (SDS-PAGE-CIE), which was designed for analysis of high molecular weight proteins.
The pattern of SDS-PAGE-CIE in newborn plasma samples was similar to that in normal plasma, showing a slightly rugged precipitation line which corresponds to molecular weights ranging from 0.8×10⁶ to 8×10⁶ daltons.
With a ten-fold concentrated cryoprecipitate sample from normal plasma, the ruggedness was emphasized, disclosing several distinct arcs. The peaks of these arcs were estimated to correspond to molecular weights of 5×10⁶, 2×10⁶ and 0.8×10⁶ daltons. The patterns of SDS-PAGE-CIE of VIIIR: AG in newborn cord cryoprecipitates could be divided into the following 3 groups: group 1, normal pattern; group 2, three arcs with peaks corresponding to molecular weights of 4×10⁶, 2×10⁶ and 0.8×10⁶; group 3, three peaks with molecular weights of 3×10⁶, 2×10⁶ and 0.8×10⁶ daltons. of 30 newborn infants, 19 belonged to group 1, 7 to group 2, and 4 to group 3.

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Since factor XII deficiency was first described by Ratnoff (1955), more than 100 cases have been reported. Patients with factor XII deficiency are usually not subject abnormal bleeding and have been able to undergo major surgery or delivery without bleeding tendency. A 49-year-old woman was found to have prolonged PTT during routine studies before hysterectomy for myoma uteri. The patient nas never experienced bleeding episodes in deliveries and appendectomy. This report presents our results of coagulative management during myomectomy for such a patient.
Results:
1) In this patient, abnormal prolonged PTT (245sec) and deficient factor XII activity (levels of under 1%) were found, but other coagulation factors and kallikrein were with in normal range.
2) After the trial transfusion of four units of fresh frozen plasma (FFP) to the patient, PTT shortened 97.3 seconds and factor XII activity raised 16.5%. 96 hours after trial injection of FFP, PTT prolonged again 124 seconds and factor XII activity decreased to 2.2%.
3) From these data, the patient was given twelve units of FFP before surgery and four units of FFP twelve hours after operation. As a results, PTT, APTT and SPTT were remarkably reduced. Although factor XII activity raised to 29-30% after twelve units of FFP transfusion, it was reduced again to 6.6% 120 hours after the operation.
In conclusions, blood loss was 233g during myomectomy and no abnormal eqisodes were demonstrated during and after the operation in this patient.

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With the better treatment of burn shock and the improvements in the early care of the burned patients, inhalation injury has become one of the leading determinants of early mortality following burm injury.
Herein, we discuss the hematological and circulatory alterations after inhalation injury by animal experiments. For our experiments, typical inhalation injury was produced by wood smoke inhalation for ten minutes on anesthetized dogs and divided them into three groups; 1) control dogs without any therapy, 2) dogs with infusion therapy and 3) dogs with heparin therapy combined with infusion therapy. Examinations of the control dogs revealed that the platelet counts and fibrinogen decreased after inhalation injury. Examination of the dogs administered only the infusion therapy revealed that almost the same alterations took place as in the dogs without therapy. On the other hand, in the dogs administered heparin in addition to the infusion therapy, there were little decrease in the platelet counts and a slight decrease of fibrinogen. In addition to these results, in the heparin administered dogs decrease of cardiac output which was determimined by thermodilution method and PaO₂ were minimum and their general condition was well controlled compared with the other two groups. These results suggest that hypercoagulation after inhalation injury and consequent microthrombi in the pulmonary vasculatures may play a important role in pathophysiology of inhalation injury, so the heparin therapy improved their condition.

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To reveal the pathogenic role of thromboxane A₂, we examined the effects of thromboxane A₂ synthetase inhibitors, OKY-046, OKY-1581 and imidazol, on the pathophysiological sequelae of endotoxemia. Though intravenous administration of E. Coli endotoxin (3mg/kg) made all rats die within 10 hours following endotoxin injection, pretreatment with thromboxane A₂ synthetase inhibitors markedly improved survival. Thromboxane B₂ level in the venous blood 15 minutes after endotoxin injection in endotoxin shock group increased 354pg/ml (vehicle-treated) to 2, 450pg/ml, but pretreated group with thromboxane A₂ synthetase inhibitors showed no significant increase. 6-keto prostaglandin F_1α level in the venous blood 15 minutes after endotoxin injection showed no significant differences among any experiment group. Microthromboses 2 hours after endotoxin injection in the glomeruli of the kidney were observed in 64% in endotoxin shock group. In pretreated group with thromboxane A₂ synthetase inhibitors, however, occurence of microthromboses showed lower incidence than that in endotoxin shock group.
This study allows us to hypothesize that endotoxin causes a release of arachidonic acid from platelets or other tissue resulting in synthesis of thromboxane A₂, that thromboxane A₂ plays some roles in endotoxin shock through the developement of micrthromboses, and that thromboxane A₂ synthetase inhibitors have protective effects to endotoxin shock.

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1. Increase in procoagulant activity (P-activity) of spleen cells from mouse given 50μg of S. typhimurium endotoxin (post-LPS-cells) was demonstrated as early as 4hrs after endotoxin. Time course of the procoagulant generation in the cells was the same as that of bone marrow cells.
2. P-activity in the post-LPS-cells was higher than that in normal-cells of control, however, no cytotoxic change was observed in the post-LPS-cells.
3. P-activity was destroyed by heating at 56°C for 10min.
4. P-activity of splenic mononuclear cells was higher than that of unfractionated whole nucleated cells.
5. After sonic disruption, splenic cellular fractions were separated by differential centrifugation. Most of the P-activity was found in the 14, 500×g sediment of the sonic lysate.
6. The procoagulant of the spleen cells (14, 500×g sediment or whole nucleated cells) could activate either intrinsic or extrinsic pathway, however, endotoxin could only enhance the activation of extrinsic pathway.
7. Parallelism between the dose response regression lines for post-LPS-cells or normalcells and mouse brain thromboplastin was recognized.
These results suggest that the P-activity is present in the cell membranes and the activity is enhanced as a result of the interaction of the cells with endotoxin in vivo. The procoagulant of post-LPS-cells is considered to be similar or identical to tissue thromboplastin. The participation of P-activity of splenic mononuclear cells would also be suggested in endotoxin-induced DIC.

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1. Procoagulant activity (P-activity) in the intact cells of bone marrow or spleen cells was higher than that in sonic lysate of these cells.
2. Sonic lysate of bone marrow or spleen cells had the anticoagulant activity (A-activity), which was stable against heating at 70°C for 30-60min.
3. A-activity was mainly found in the 40, 000xg supernatant of sonic lysate of the spleen cells.
4. With heat-treated 40, 000xg supernatant prepared from post-LPS-cells or normal-cells, A-activity was also detected in the unactivated partial thromboplastin time (PTT), activated partial thromboplastin time (APTT), prothrombin time (PT) and the thrombin-fibrinogen clotting time (TT).
5. A-activity (prolongation of PTT or TT) of bone marrow or spleen cells was also enhanced by endotoxin injection compared to saline injection.
6. With acid soluble protein (ASP, cationic protein) from bone marrow or spleen cells, A-activity was also detected in the PTT, APTT, and PT, however, was not in the TT.
7. ASP was also stable against heating at 80°C for 30min.
8. In ASP, endotoxin-detoxifying activity was observed.
ASP appears to exist in cytoplasma and to inhibit clotting by blocking the activation of factor X or the formation of plasma thromboplastin. These findings also suggest that the enhancements of P-activity and A-activity of bone marrow or spleen cells in endotoxemia would also regulate the manifestation of DIC.

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The participation of complement in the pathogenesis of DIC was studied using experimental model of DIC in rats.
Experimental DIC was induced by sustained infusion of bacterial endotoxin (ET) (100mg/kg; lipopolysaccharide B, E. coli 055, B5, Difco) into the femoral vein for 4h in female rats of the Wistar strain with weights ranging from 200 to 230g. The severity of DIC was determined with reference to several coagulation parameters, such as fibrinogen (Fbg), fibrin-fibrinogen degradation products (FDP), prothrombin time (PT), partial thromboplastin time (PTT), platelet count (PLT), and number of the renal glomeruli having fibrin thrombi (% glomerular fibrin deposits; % GFD).
At 4h after the sustained infusion of ET, C3 and CH50 levels were markedly decreased compared with those of the control rats treated with saline. C3 and CH50 levels showed rapid decrease by 1h followed by gradual loss by 4h. The rats which showed marked decrease in CH50 levels revealed a tendency of marked increase in % GFD as well as FDP.
These data suggested that there was a close relationship between complement system and ET-induced DIC in rats. The further examination for the role of complement in the pathogenesis of DIC is now under investigation.

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The regulation mechanism of fibrinogen synthesis is unclear. Thrombin, free fatty acids, ACTH, FgDP were reported as stimulators of fibrinogen synthesis. This experiment was done to examine the existence of humoral stimulating factors in DIC serum. In dogs, after continuous infusion of tissue thromboplastin (Lyoplastin) 12.5mg/kg for 3hrs, platelet counts, antiplasmin, fibrinogen decreased to nearly 30%, 35%, 0% and FPA, FDP increased to 10ng/ml, 660μg/ml respectively. After cessation of administration of Lyoplastin, fibrinogen increased rapidly and returned to the value of preinjection at 3 days after. So, these dogs were used as acute DIC model. Serum of these dogs was sampled at this recovery phase of fibrinogen (6, 24, 48, 72hrs) and treated with bentonite to absorb FDP. 3ml of these sera were administered to rabbits and ⁷⁵Se-methionine (⁷⁵Se-M) 20μCi was injected 5hrs after. The effects of DIC dog serum on fibrinogen synthesis in rabbits were evaluated by calculating the incorporated ⁷⁵Se-M into newly synthesized fibrinogen and by circulating fibrinogen concentration. Although the statistical significance was not obtained, the existence of humoral stimulating factors on fibrinogen synthesis in rabbits was suggested by injectien of DIC induced dog serum at 72hrs after Lyoplastin infusion.

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Hypercoagulability or pre-DIC are characterized by the presence of SFMC in plasma. The ethanol gelation test or agarose gel filtration of plasma sample are used for a detection of SFMC. But they were timeconsuming procedure in estimation of SFMC by the gel filtration, in spite of the urgent clinical need. Therefore, we have devised a more rapid methed with a quantitative estimation of SFMC by the high performance liquid chromatography. The normal percentile amount of SFMC of total fibrinogen content showed a level of 2.1±0.8% in plasma. The amount of SFMC in DIC patients showed a level from 6.8% to 16.7%, with a mean value and standard deviation of 10.7±2.7%. A decresed amount of α-chain and the presence of a band of dimeric γ-chains was observed by SDS-PAGE of the eluted fraction of plasma in DIC patients. The level of SFMC after the gastrointestinal surgery without major post-operative complications showed between 3 and 6%. The level returned to normal in the 9th post-operative day. The moderately elevated SFMC levels up to 6% must be shown in the state of hypercoagulability. The measurement system of SFMC gives a very useful method to analyse hypercoagulability and pre-DIC state. It was impossible in the past.

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When protamine was detached heparin from heparinized plasma which exserted a rapid neutralizing activity to thrombin or Xa in vitro, the activity to Xa measured as heparin cofactor activity was completely abolished and the activity to thrombin reduced to one third of original activity. The progressive antithrombin activity measured with incubation of mixture of antithrombin and thrombin or Xa neutralizied slowly to thrombin or Xa and didn't affected with addition of protamine. It was thought that protamine had no binding effect to antithrombin molecule, but it converted the conformation of heparin cofactor to progressive antithrombin form by detachment of heparin from heparin-bound antithrombin.

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For further improvement of the technique to form thrombus in hamster cheek pouch, an arteriole with a diameter of 50-80μm was observed under the microscope and was compressed from lateral side up to 1/5 of the internal diameter by glass capillary with tip of 5μm for 10min. Simultaneously electrical stimulation (square-wave direct-current, 20msec in pulse duration, 50μA in pulse strength and 5Hz) was provided for 80sec at the beginning of the compression. When the compression with electrical stimulation was repeated 5 to 7 times every 30-50μm along the arteriolar wall, the thrombus was formed 5-10μm in height and 150-250μm in length. This thrombus was disaggregated neither spontaneously nor by topical application of PGI₂. An electronmicrograph of the thrombus showed that endothelial cells were desquamated and degranulated platelets were adherent to the denuded sites. Topical application of ADP induced the formation of large thrombus so that the arteriolar lumen was nearly obstructed. The superimposed parts of thrombus induced by ADP were disaggregated by topical application of PGI₂ dose-dependently.

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An experimental arterial thrombus was formed by both of arterial stenosis and de-endothelialization in rabbit carotid artery.
External carotid systolic pressure was decreased about 10-20% by stenosis at the common carotid artery. Five-thirty minutes after, the systolic, diastolic and pulse-pressure were fluctuated, and then the pulse-pressure was disappeared.
Small numbers of platelet aggregates in external carotid arterial blood were counted in both blood samples collected by a polyethylene (2.9±0.48/0.1mm³) or silicone catheter (2.9±1.32/0.1mm³) in the sham control.
Platelet aggregates in blood samples through the injured artery, however, markedly recognized at 1 minutes (20.7±3.26/0.1mm) to 20 minutes (37.5±3.56/0.1mm³) after.
The platelet aggregates grew up from brittle small size to tighly larger size in the course.
These results suggested that platelet aggregates were originated by endothelial injury at the common carotid artery, and the arterial thrombus which repeats to grow and be washed out into the circulation, might grow to enough size occluding lumen when platelet aggregates attached to the arterial wall can withstand to the blood stream.

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In order to elucidate what the circulating platelet aggregate (CPA) ratio by Wu and Hoak means and justify the idea of CPA, we studied experimentally-induced arterial thrombosis in chaired baboons by placing vascular teflon grafts (4mm i. d., 100 or 200mm in length) between femoral A-V shunts examining various parameters of platelet activation. Real aggregated platelets (Agg. Pl.) were counted in fixed whole blood by light microscopy. In this system thrombi occluded about a half of the grafts between 45 and 60min after starting the experiment.
CPA ratio (0.97±0.11 (SD) in control) increased or decreased insignificantly during the experiment. Agg. Pl. increased in some grafts but also insignificantly. The ratio was correlated with neither discoidness index, plasma platelet factor 4 (except case of the long grafts) nor Agg. Pl. It was not affected by preexperimental injection of ancrod, whereas Agg. Pl. seemed to be reduced. The latter were well correlated with platelet count, plasma platelet factor 4, and shape which showed a significant change during the experiment, and decreased at 12min only in drug combinations which suppressed the graft occlusion whereas the former did not. In conclusion CPA ratio neither reflected the platelet activation nor expressed the real Agg. Pl. in this system. Agg. Pl. seem to reflect the process in vivo and its suppression by drugs, suggesting the idea of CPA is essentially acceptable.

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The mechanism of thrombus formation in hyperlipidemia and atherosclerosis was studied using rabbits.
Attempts at artificial creation of arterial thrombus in control rabbits using Maekawa's technique of stenosing the femoral artery by ligature were not successful unless ellagic acid was administered by injection. Howevere, this thrombus formation could be prevented by oral administration of aspirin.
In rabbits with hyperlipidemia, mere creation of stenosis in the femoral artery resulted in a high percentage of thrombus formation with the prophylactic effect of aipirin being considerably decreased.
In rabbits with hyperlipidemia, both thromboxane (Tx) A₂ biosynthesis in platelets and prostacyclin (PGI₂) biosynthesis in the aorta were increased and these changes were noted at the level of cyclo-oxygenase in the arachidonic acid metabolic pathway. A diminished inhibitory effect of aspirin on TxA₂ and PGI₂ production was observed. In hyperlipidemia, although PGI₂ biosynthesis was increased, mere creation of stenosis in the artery resulted in a high percentage of thrombus formation. Therefore, these results indicate that platelet hyperfunction is largely accountable for thrombus formation.
In rabbits with experimental atherosclerosis, TxA₂ bsosynthesis of platelet was increased but PGI₂ biosynthesis of aorta was decreaed.
These results indicate that thrombi are likely to be formed in hyperlipidemia, which may lead to atherosclerosis, and that such thrombus formation is due largely to platelet hyperfunction.

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Platelet adhesion to subendothelium is impaired in Bernard-Soulier syndrome and von Willebrand's disease. Therefore glycoprotein I of platelet membrane and von Willebrand factor in plasma has been reported to have some significant role on platelet adhesion. In this report, platelet adhesion to cellulose ester membrane (millipore membrane filter: MF) as a artificial surface was observed in patients with Bernard-Soulier syndrome, von Willebrand's diseae, afibrinogenemia and normal adults. In normal adults, there was no signigicant difference of adhesion rates between MF and subendothelium, which were 64.7±9.3% (n=5, mean±SD) to MF and 69.4±8.6% to subendotheluim. In Bernard-Soulier syndrome the rate were 7.0±1.4% (n=2, mean±SD) to MF and 11.3±1.8% to subendothelium. However, in von Willebrand's disease, platelet adhesion to MF was almost normal. The adhesion rate was 58±27% (n=4, mean±SD) in contrast to 11.3±1.8% to subendothelium.
The role of von Willebrand factor was not thought to be significant in platelet adhesion to MF, and the effects of other factors, for example electronical charge or microstructures of the surface were thought to relate.

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Platelet adhesion to collagen fibril is important as the first step of the defense against bleeding. However, there are no suitable methods for the measurements of platelet adhesiveness. Various conditions effected to platelet adhesion were studied and a simple method using an aggregometer was determined. The measurement was carried out in the presence of collagen and EGTA with or without Mg²⁺ under stiring at 37°C. After addition of collagen into PRP with EGTA, optical transmittance on an aggregometer decreased temporary and then increased lineary within a few minute. The aggregation of platelets did not occured at this time, which was conformed by SEM observation. Therefore, the adherence was determined by the transmittance change at the initial stage. The error in this method was within 5%, when the method was used at pH 7-8 and platelet concentration was 3×10⁵cells/μl or more. The excellent reproducibility was obtained. In this reaction, the well dispersed, high active and fibrous collagen will be required. The adherence to collagen was increased by Mg²⁺ and inhibited by aspirin. The adhesiveness of PC stored at 22°C decreased 20% for 24 hours and 50% for 48 hours, respectively.

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An effect of plasma components on the maintenance of platelet aggregability was studied. Washed human platelets (WP) were suspended in isotonic buffer containing dialyzed or non-dialyzed plasma, and stored at R. T. The time courses of the aggregability of WP induced by ADP were carried out. The aggregability of all samples at 0 hour, were 80% approximately. However, the aggregability of platelets stored in dialyzed plasma-buffer, decreased more rapidly than that stored in non-dialyzed plasma-buffer. The similar result was obtained when MgCl₂ and glucose were added to the dialysis buffer. Then, dialyzed plasma was reconstituted by adding the filterate from Centriflow 25 which contained the low molecular weight (<2500) components in plasma. The reconstituted plasma indicated the similar efficiency to the non-dialyzed plasma on the maintenance of aggregability.
These results show that there are some dialyzable components in plasma which are not necessary for the aggregating reaction, but are necessary for the maintenance of ADP-induced platelet aggregability. It is suggested that they were not Ca²⁺, Mg²⁺, glucose and proteins, and their molecular weights are less than 2, 500.

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Effects of removal of Na⁺ and Cl^- from the medium were studied on thrombin- and A23187-induced aggregation and 5-HT release from rabbit washed platelets. Platelet aggregation and 5-HT release from platelets were induced by both thrombin and A23187 similarly in normal NaCl and choline chloride medium. Replacement of Cl^- by SO₄^--abolished completely platelet response to thrombin, whereas the response to A23187 remained. Substituting Cl^- with glucuronic anion depressed significantly both aggregation and 5-HT release induced by thrombin and A23187. In mannitol medium, from which Na⁺ and Cl^- were removed, A23187 could not cause aggregation, whereas thrombin-induced aggregation remained irrespective of less responsiveness. Preliminary addition of NaCl to the medium restored the aggregation. These facts may support that thrombin- and A23187-induced platelet activation were fairly independent of the external sodium ion. But chloride ion seems to be more essential for A23187-induced platelet responses than thrombin-induced ones in rabbit washed platelets.

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It has been suggested by several investigators that eicosapentaentaenoic acid (C20: 5 ω3, EPA) might have anti-thrombotic action. Effect of oral administration of EPA rich fish oil concentrate on platelet aggregation and tha release and metabolism of [1-¹⁴C] arachidonic acid and [(U)-¹⁴C] eicosapentaenoic acid by human platelet was studied. Eight healthy male subjects ingested 18 capsules of fish oil concentrate (EPA 1.4g) per day for 4 weeks. Plasma and platelet concentration of EPA were markedly increased, while those of arachidonic acid (C20:4ω6, AA) and docosahexaenoic acid (C22:6ω3, DHA) did not change. Platelet aggregation induced by collagen and threshold dose of ADP was reduced. Collagen induced thromboxane B₂ (TXB₂) formation from [¹⁴C] AA labeled platelets was decreased. There was no detectable formation of TXB₃ from [¹⁴C] EPA labeled platelets, and the conversion of exogenous [¹⁴C] EPA to [¹⁴C] TXB₃ was quite lower than that of [¹⁴C] AA to [¹⁴C] TXB₂. No significant inhibition of platelet cyclooxygenase was observed. The release of [¹⁴C] AA from [¹⁴C] AA labeled platelets by collagen was significantly decreased. Present observations suggest that an inhibitory effect of EPA on platelet aggregation might be partially explained by the impairment of AA release from platelet membrane phospholipids.

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The role of superoxide dismutase (SOD) on human platelet was investigated by utilizing diethyldithiocarbamate (DDC); chelator of copper ion and inhibitor of SOD. DDC inhibited the activity of platelet SOD in a does dependent manner after two hrs incubation at 37°C. Since cytochrome oxydase was not inhibited at low concentrations of the drug, SOD might be inhibited specifically among copper containing enzymes. The platelet aggregations induced by ADP, collagen or arachidonic acid were enhanced at low concentrations of DDC. The N-ethylmaleimide or thrombin-induced MDA production was also enhanced at low concentrations of the drug. These results suggest that SOD plays a stimulatory role on platelet function by affecting the prostaglandin synthesis.

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It is well known that a high dietary intake of sodium chloride is one of the risk factors of hypertension and thrombotic cardiovascular diseases.
There are but few reports on the relationship between the dietary ingestion of sodium chrolide and platelet aggregability wich plays a very important role in the development of thrombotic disorders. Wistar rats and SHR, 5 weeks old, were divided into three groups and were treated for 10 weeks. Group A recieved a high sodium diet, Group B recieved a normo sodium diet and Group C recived a low sodium diet. At the end of 2, 5 and 10 weeks, blood was collected with 1/10 vol. of 3.8% Na citrate from abdominal aorta. PRP and PPP were obtained by centrifugation. Platelet aggregation was carried out with 5μM ADP. In Wistar rat, after 2 and 5 weeks on the study, platelet aggregability of the high sodium group was greater than that of the normo sodium group, which in turn was greater than that of the low sodium group. After 10 weeks on the study, platelet aggregability was same for all diet groups. In SHR the same tendency was observed.
In the present study, a close relation between the ingestion of high sodium diet and accerelated platelet aggregability was observed. Therefore, sodium rich diet may be one of the risk factor for the development of thrombotic disorders.

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The inhibition of trapidil was proportionally to the concentration on human platelet aggregation, induced by ADP, collagen, arachidonic acid, thrombin and thromboxane A₂. Furthermore, trapidil reversed aggregation once the process initiated (Fig. 1 (a)), and inhibited even primary aggregCtion induced by ADP. Subsequent experiments demonstrated trapidil inhibited ATP release from the aggregating platelets on Lumi-aggregometer examination.
So, we investigated the mode of inhibition of trapidil on platelet aggergation and release reaction induced by several inducers. The inhibition pattern was demonstrated that it was almost same on the release reaction, but somewhat different on ADP from the other inducers (Fig. 2).
Based on these findings, it was suggested that trapidil inhibited the lipid metabolism, which brought platelet release reaction. So, we measured the release of arachidonic acid and Tx-B₂ biosynthesis in the aggregating platelets. Trapidil blocked completely Tx-B₂ bio-synthesis with the concentration of 1mM, but did not show so much inhibition for the release of arachidonic acid as Tx-B₂ biosynthesis, namely 39% with 1mM and 81% with 2mM.
Conclusively, trapidil inhibits the biosynthesis of Tx-B₂ from arachidonic acid by inhibiting thromboxane synthetase (unpublished data). However, the inhibition of release reaction does not necessarily follow the inhibition of platelet aggregation.

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A method for platelet factor X activator activity (Pl. FXAA) and its clinical significance were studied. Our method to determine pl. FXAA was based on a measurement of factor Xa formation with a chromogenic substrate (S-2222) in a system containing purified human factor X, gel filtered platered (GFP) and CaCl₂. The factor Xa generation at pH 7.4 was dependent on Ca⁺⁺ concentration and GFP count, While it was not influenced by platelin, ADP, collagen and aspirin. Activation of factor X by GFP from patients with hemophilia A or B occured almost normally. While in a system using factor IX concentrates instead of purified factor X, aspirin inhibited the generation of fabtor Xa. These findings confirm that GFP possess FXAA, and suggest that pl. FXAA may be different from the ability of platelets to catalyze the activation of factor X in the presence of factor VIII, IX and X.
The concentration of Stypven was used as a reference control for pl. FXAA. The mean level of pl. FXAA from 19 healthy subjects was 7.1±2.3 X 10^-7Sty. mg/ml. The platelets from patients with hematopoietic malignancies had various levels of FXAA. We experienced two following patients with thrombocytopenia after chemotherapy. Severe hemorrhage was found in a T-cell lymphoma patient with a low pl. FXAA, while no hemorrhage in an AMMoL patient with a high pl. FXAA.
This study suggests that pl. FXAA may play a role in hemostasis.

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A growth of malignant cell line (K562) by platelet derived growth promoting factor (PDGF) was studied in comparison with that of fibroblast. PDGF was partially purified from platelet lysate by ultracentrifugation and gelfiltration. This factor had a molecular weight of 20, 000-30, 000 dalton and was extremely stable to treatments with heat (100°C 2min), alkli or acid.
3 T 3 fibroblasts at confluent state remained quiescent when they were cultured with PDGF in serum free medium although addition of plasma to this medium promoted these cells to enter into DNA synthesis.
On the other hand, malignamt cells went proliferation by stimulation of PDGF alone without any plasma components.
This independency from plasma requirement, however, was not unique character to tumor cells but was also observed with 3 T 3 fibroblast at logarhythmic phase.
These results indicated that PDGF itself is mitogenic to bring cell cycle into S-phase in tumor cells as well as sparse fibroblasts, and plasma is required for quiescent cells to become competent to synthesis DNA.
In addition to this PDGF activity, platelet lysate was found to contain dialysable factor which also promoted proliferation of tumor cells. Physicochemical properties of this factor are now under investigation.

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Plasmin inhibitors were obtained from Spinacia oleracea root, in purified by perchloric acid fraction, 70% saturated ammonium sulfate fraction, gel filtration on Sephadex G-75, followed by ion exchange chromatography on DEAE-Sephacel and affinity chromatography on Trypsin-porous glass. The peak fractions “P-Inhibitor I” eluted from Trypsin-porous glass in 0.01M HCl were used in the following experiments. Plasmin inhibitor was prepared from “P-Inhibitor-I” by gel filtration using Sephadex G-50 followed by affinity chromatography on Plasmin-porous glass. Plasmin inhibitor was isolated from spinach root and purified 3, 000-fold by affinity chromatography on plasmin-porouse glass. The enzyme prepared from root appeared to be homogenous and exibited a molecular weight of 17, 000±1, 000 by SDS-polyacrylamide gel electrophoresis. Serine proteinase, plasmin and trypsin were inhibited by proteinase inhibitor from spinach roots. Pepsin, papain and bromelain were not blocked by inhibitor from root. The inhibitor may be serine proteinase inhibitor. The inhibitor was stable upon exposure to 60°C for 30min. The hemolytic activity of human complement was also inhibited by serine proteinase inhibitor from root.

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