We have now investigated the haemontasis in hepatitis and liver cirrhosis, especially about the the platelet.
1) Coagulability decreased, while fibrinolytic activity increased.
2) ¹³¹I-labelled fibrinogen turnover rate shortened especially in liver cirrhosis. And in several cases, we recognized the prolongation of fibrinogen half life after i. v. the injection of heparin.
3) The enlargement of platelet volume measured by Coulter Counter ZB was found in liver diseases, especially in liver cirrhosis.
4) And we could find the significant negative correlation between the platelet volume and platelet number.
5) We often found the appearance of endotoxin especially in chronic hepatitis and liver cirrhosis, while decreased in inactive stage.
From the above results, we came the conclusion that in liver deseases the thrombotic tendency and the intravascular coagulation are closely related to the prognosis of hepatitis.

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Recently von Willebrand Factor (VIII VWF) has been proved to be localized in vessel wall and in platelets, and synthesized in endothelical cells. We studied the effects of temperature and the time course of VIII VWF movement from platelets in vitro and discussed its significance in thrombogenesis.
We observed ristocetin induced aggregation of 4 times washed platelets suspended in Tris buffer and stored at 4°C or 20°C for 48hrs. Platelet suspension stored at 20°C was induced slight aggregation by 1.5mg/ml of ristocetin in two hours and aggregability continued to increase gradually for 20hrs. After 24hrs. this aggregation decreased. Suspention stored at 4°C exhibited, however, a low grade of aggregation induced by 1.5mg/ml of Ristocetin after 20hrs. Aggregation increased after 24hrs. and 48hrs., but it was not intense as that of suspension stored at 20°C.
The supernatant of these suspension, collected on 3, 5 and 24hrs. of both temperature was assayed on VIII VWF, VIII-antigen (VIII AGN) and antihemophilic factor (VIII AHF). The platelets from the same source was washed 4 times and disrupted by freezing and thawing. The supernatant of these disrupted platelets were also assayed on the VIII VWF, VIII AGN and VIII AHF (Table 1). Approximately 10% of VIII VWF activity and VIII-antigen were proved to bederivedfrom disrupted platelets. VIII VWF activity in the supernatant of stored platelets was demonstrated time dependent as showing in Table 1 and in Fig. 2.
No antigenic activity was demonstrated in the supernatant by the assay method applied, however, we observed the antigenic activity in the supernatant of platelets stored at 20°C for 24hrs. it the concentration of platelets was high enough (10⁶/μl).
We also observed the inhibitory effects on its excretion by ASA, and glyclazide; the new antidiabetic agent. ASA (10^-3, 10^-4M) which inhibit potently the release reaction, did inhibit slightly and glyclazide (10^-3, 10^-4M) inhibited well this excretion. This result prompted us to imagine of some relation to the mechanism of the effect of this drug on the diabetic retinopathy, whose VIII VWF and VIII AGN increased significantly. We concluded that 1) there is fairly high VIII VWF in platelets, 2) this factor was excreted out of platelet by some mechanism other than the release reaction, 3) this excretion was inhibited by high concentration of ASA, and by glyclazide.

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Studies from many laboratories have indicated that dextran sulphate (DS) enhanced the fibrinolytic system, as shown in shortening of euglobulin lysis time. For the activation mechanisms of DS on fibrinolytic activity, it has been proposed that DS enhanced the fibrinolytic activity induced by contact activation, or accelerated the action of plasminogen activators. In this paper, we investigated the activation mechanisms of DS on fibrinolytic activity in vitro and in vivo system.
We discovered that DS, orally administrated into rabbits (ranged 50-300mg/kg), suppressed the plasmin inhibitory activity, as measured by the diameter of lysis area on plasminogen-free fibrin plates. The suppressive action of DS on plasmin inhibitory activity was related to the dosage of DS administrated, and occasionally the fibrinolytic activity of euglobulin was enhanced.
Furthermore, 2 groups of 19 subjects, 14 patients with hyperlipemia and 5 healthy volunteers were administrated orally 1, 800mg of DS daily from 4 to 47 weeks. Abnormal high levels of plasmin inhibitory activity in patient peripheral blood were reduced to normal levels in a few weeks after the DS medication, but normal levels of that in healthy subjects were not significantly changed. In vitro system, we could not estimate that DS (ranged 10-500μg/ml) might enhance the action of urokinase, nor that DS might accelerate the fibrinolytic activity induced by contact activation.
This result suggested that the activation mechanisms of DS on fibrinolytic activity could depend upon the reduction of plasmin inhibitors.

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Total antiplasmin was measured in vitro and in vivo. In the former case, rabbit plasma was mixed with various concentrations of UK and the least concentration for appearance of fibrinolytic activity was estimated. This concentration was multiplied by the plasma volume of the rabbit to give the in vitro total antiplasmin. The mean value for 14 rabbits was 4, 068.6 units.
In order to estimate the total antiplasmin in vivo, UK solution was infused into rabbits. The infusion speed was multiplied by the time of frst appearance of fibrinolytic activity to give the total antiplasmin, although when the infusion speed was low, fibrinolytic activity did not appear during infusion. The mean in vivo total antinlasmin calculated from values of 6 cases in which the infusion speed was high and fibrinolytic activity was observed, was 28, 699.8 units, i. e. about 7 (ranged, 3-11) times as much as the in vitro value.

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Since it was shown that anti-coagulant therapy suppressed glomerular lesions and intraglomerular fibrin deposits refeying to the activity of glomerular abnormality, intraglomerular coagulation has been considered significant as one of the glomerular injurying factors.
This experiment was to study the effects of fibrinolytic therapy with urokinase on Masugi nephritis in rabbits, in which a proliferative glomerulonephritis with fibrin deposits was induced by intravenous injection of anti-rabbit-kidney duck serum.
The following results were obtained from the treatment with urokinase.
(1) plasma fibrinogen levels were not increased,
(2) serum FDP levels decreased,
(3) fibrin deposits and proliferative changes in the glomeruli suppressed histologically.
This study suggested that urokinase suppressed the developmental process of Masugi nephritis.

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The present study was undertaken to clarify the clinical significance of urinary fibrin/fibrinogen degradation products (U-FDP) measured by tanned red cell hemagglutination inhibition immunoassay in renal disease.
The relationship of U-FDP to urinary protein (U-P) and renal functions was studied. In addition, the effect of anticoagulant therapy to renal diseases was evaluated. The results are summarized as follows:
1) The relationship between U-FDP and U-P showed characteristic patterns of individual renal diseases: A) In lipoid nephrosis and membranous nephropathy, small amounts of U-FDP was found in spite of large amounts of U-P. B) In acute glomerulonephritis large amounts of U-FDP but small amounts of U-P was recognized. C) In chronic glomerulonephritis and lupus nephritis, the relationship between U-FDP and U-P was variable.
2) In cases with large amounts of U-FDP in spite of small amounts of U-P, their renal functions showed a decreasing tendency, whereas no such a tendency was seen in cases with small amounts of U-FDP in spite of large amounts of U-P.
3) U-FDP was markebly reduced and the decreased GFR recovered by heparin administration in cases with large amounts of U-FDP and small amounts of U-P, although no such an effect was observed in cases with reversed excretion of U-FDP and U-P, U-FDP and GFR were not modified at all by warfarin administration in both types
The following results were concluded:
A) In lipoid nephrosis and membranous nephropathy, U-FDP appeared to be derived chiefly from plasma fibrinogen and serum FDP through the increased permeability of the glomerular basement membrane. On the other hand, in acute glomerulonephritis, U-FDP appeared to be derived chiefly from fibrin deposition in kidney tissue.
B) Heparin administration may be an effective therapy for renal disease in cases with large amounts of U-FDP in spite of small amounts of U-P.

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The FDP in blood was assayed in 24 patients with nephritis by both methods of hemagglutination inhibition test (HIT) and staphylococcal clumping test (SCT). In cases with nephritis, a remarkable discrepancy between SCT and HIT values was observed, having the ratio with SCT to HIT of 7.8, whereas this ratio was 1.7 in other diseases, such as liver cirrhosis, purpura, cancer or hypertrophy of prostata, leukemia, myocardial infarction, pregnancy, Abruptio placentae and others (Fig. 1). The discrepancy was generally more pronounced in the active phase of nephritis than in the phase of remission. As these results seemed to be indicative for the significance in clinic as well as phathophysiology of nephritis, more studies in vitro were carried out in this report.
Three kinds of samples such as patient's plasma with high discrepancy (ratio 8.0), normal plasma and plasmin digested normal plasma (TP-plasma) by the addition of thrombin and plasmin were flowed through DEAE Sephadex A-50, respectively. The TP-plasma was obtained by the procedure in which normal plasma was incubated with 0.02 units of thrombin for 5min at 37°C, followed by the incubation with 0.5 units of plasmin for 15min (Fig. 4). In the elution of patient's plasma through DEAE Sephadex A-50, the fraction giving storongly positive SCT but negative HIT was obtained at the gradient of 200mM NaCl (Fig. 2) from TP-plasma (Fig. 5). However, this fraction could not be obtained by the column procedure for untreated normal plasma (Fig. 3). These results indicated that the plasma of cases in active phase of nephritis might involve lot amount of soluble fibrin.
Further studies had been carried out by using soluble fibrin by Felten's method or fibrin monomer by Largo's method.
The soluble fibrin obtained by the former method in which thrombin treated EDTA plasma was precipitated by 50% ethanol in saline, gave positive SCT (640μg/ml) and negative HIT (below 20μg/ml). The fibrin monomer obtained by the latter method which was started from purified fibrinogen, followed by defibrination with thrombin, disolution in 5M Urea and purification by precipitation, gave no discrepancy between the result on SCT and HIT.
According to our present results, it could be assumed as follows. The soluble fibrin which was made in vitro experiments showed the similar pattern to the plasma of nephritis, having discrepancy between SCT and HIT. However the purified fibrin monomer failed to show the remarkable discrepancy between them. The present results would indicate that the soluble fibrin or soluble fibrin-fibrinogen complex might contribute to the significance in FDP assay for cases with nephritis.

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The coagulation and fibrinolysis in renal failure were studied.
The k value of thromboelastogram were shorten in patients with chronic nephritis, diabetic nephropathy accompanied by uremic symptom The ma value also was increased. Similary tendency was observed in nephrotic syndrome. While these were within normal limits in acute nephritis. Fibrin and/or fibrinogen degradation products (FDP) in serum correlated positively with blood urea nitrogen concentration in patients with chronic nephritis. On the other hand plasminogen activity was markedly decreased in most of cases with chronic nephritis, diabetic nephropathy accompanied by uremic sign.
Levels of FDP in urine correlated positively with protein concentration of urine in patients with acute and/or chronic renal failure.
From those results, the presence of hypercoagulability and low fibrinolytic activity in patients with chronic renal failure and nephrotic syndrome were realized.
After haemodialysis, in seven out of 11 cases with uremic patients showed an increase of fibrinolytic activity and the decrease of FDP in serum. Then, in chronic nephritis, tissue activator activity of necropsy kidnies was elevated.

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An increased coagulability of blood could be induced not only by nephrotic syndrome itself but by the therapeutic administration of corticotropine (ACTH) and cortisone. The use of modern diuretic agents and of immunosuppressive ones seemed to make the coagulation mechanism more complexed. As a result it was not so infrequent that nephrotic patients did die suddenly from the thromboembolic complications.
This paper reports two cases of pulmonary artery thrombosis and cerebral artery thrombosis in nephrotic patients who died suddenly.
Case 1. 25yrs. old male; There was edema in legs at the age of 23yrs. Two years had passed without any symptoms, but marked edema in legs reappeared and he developed edema on the face and discomfortness in anterior chest on exertion. He was admitted to our hospital with a diagnosis of nephrotic syndrome. Laboratory examination revealed marked proteinuria, hypoalbuminemia, hyperlipemia and the decrease in renal functions. During the hospitalization he was given diuretics, decholesterol agents, predonisolone and rinderon. However, serum total protein and cholesterol level were not changed. On the 115'th hospital day he suddenly complained discomfortness of chest and expired after three hours. Autopsy revealed extensive fresh and organized thrombosis of pulmonary arteries and focal glomerulonephritis.
Case 2. 53yrs. old, female; There was edema in legs. Five months previously she had noticed abdominal extension. She was admitted to our hospital with a diagnosis of nephrotic syndrome. Predonine was administrated for one month, improving her symptoms gradually. She was discharged on the fourtieth hospital day. A few weeks after discharge abdominal extension, edema on the face developed progressively and she was readmitted to our hospital again. Laboratory examination revealed moderate proteinuria, hyperlipemia and slight decrease in renal function tests. During the hospitalization she was given predonine, imuran, decholesterol agent, diuretics and anabolic steroid, with a slight recovery of edema and proteinuria. On the 196'th hospital day she suddenly became shock state after passing the water. She gradually became stuporous. After 26 hours she expired. Autopsy revealed multiple, petechial hemorrhage with encephalomalacia in bilateral thalamus and slight membranous glomerulonephritis.

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(Object) There are many reports on the fibrinolytic dynamics in blood as to the development of peptic ulcers. However, these studies have not been completed and we have many problems to be solved on this subject. In the present study we reexamined the correlation between the fibrinolysis and activity of the coagulation system.
(Method) Ulcers were induced by contact freezing of the left gastric artery on the gastric serosa in Mongorel dogs for materials. The basis of ulcers was confined within the limit of the submucosal layer in all cases and fibrin deposit blood vessels were maintained comparatively intact. The plasminogen activator was observed at necrotic portions, but activity was determined at the margin, peripheral elevation and distal mucosa of ulcers and the plasminogen and plasmin inhibitors in the circulating blood were analyzed by affinity chromatography at the same time. The tissue was observed by antifibrinous staining.
(Result) At the margin of ulcer: Free plasminogen activator was proved at the local peripheral circulatory insufficiency but it was diministred after this maniplation. In the peripheral elevation of ulcer: Free activator was detected when circulatory insufficiency was invited and a secondary liberation of the activator due to the elevation of membrane permeability of peripheral cells containing quinines was observed. In the distal mucosa: The same correlation as in the peripheral elevation was noticed, but the secondary liberation of activator showed considerably wide variation. The activity of plasminogen or plasmin inhibitors was correlated with that of plasminogen activator in blood. The coagulation system had an inhibitory action on factors V and VIII, but it was correlated with neither PT nor PTT.
(Conclusion) The activity of plasminogen activator in the initial lesions of acute peptic ulcers showed clear biphasic peaks and was well correlated with that of plasmin inhibitor or of the coagulation system at the same time.

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Liver diseases are frequently one of the causes of gastrointestinal hemorrhage that might be associated with clinically infavorable prognosis. But the pathogenesis of gastro intestinal hemorrhage in liver diseases has tno been clearly understood.
The aim of the present study is to assess the significance of fibrinolytic activity of the esophago-gastric mucosa in the development of the upper alimentary hemorrhage in liver dis eases.
Tissue blocks of liver, esophagus and stomach were obtained at routine autopsies, mostly within 6 hours after death. The group with liver deseases was consisted of 4 cases of liver cirrhosis and 2 cases of liver cirrosis with liver cell carcinoma, and the group without liver disease was consisted of 3 cases of ma lignant lymphoma, 2 cases of gastric cancer and one case of chronic myelocytic leukemia. Each tissue block was immered, cut at 4 to 6μ on a cryostat microtome and covered with fibrin film made by 2% human fibrinogen rich in or free from plasminogen. Autographs were in-cubated for 30 minutes at 37°C, fixed and stained. In a comparison of local fibrinolytic activity of the group with liver diseases and the group without liver disease, the diameter of lytic area was measured in 50 small vessels in each autographs of esophagus and stomach (cardia and pylorus).
Results obtained were as follows. 1) Cirrhotic liver had a high fibrinolytic activity in the small vessels in the bands of proliferated connective tissue. This might be one of the causes of the hyperfibrinolysis in liver diseases.
2) Local fibrinolysis of the gastric mucosa was higher in cases with liver diseases than those without liver disease. High fibrinolytic activity was found in the wall of esophageal varices. High fibrinolytic activity of the esophago-gastric mucosa might play an important role in the upper alimentary hemorrhage in liver diseases.

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The authors have reported that platelet sensitivity to ADP-aggregation, measured by our new method which was devised to be performed without centrifugation of blood (Sano et al. 1974), correlated significantly with age in healthy males, while intensity of aggregation measured by the optical density (O. D.) method or the screen filtration pressure (SFP) method did not correlate with age. The further study disclosed that intensity of platelet aggregation measured by the O. D. or the SFP method did not change significantly when human citrated blood was filtrated through glass filter. But, platelet sesitivity measured by our own method was enhanced significantly after the filtration. These findings suggested that injury or activation of platelets induced by their contact with rough surface such as arteriosclerotic vessel wall or glass surface was reflected more on platelet sensitivity than on intensity of aggregation.
The present study was carried out to investigate how the enhancement of platelet sensitivity seen after the filtration of citrated blood through glass filter was affected by the changes in flow rate, contact time and contact area of rough surface. Human citrated blood was flltrated through glass beads column two times with the column renewed each time with flow rate of 1ml/60sec or 1ml/30sec. After the platelet counts of the pre-and post-filtration samples were equalized, platelet sesitivity to ADP-aggregation was assessed using our method as follows. Platelet-rich plasma (PRP) was separated without centrifugation. Then the PRP was mixed with serially two-fold diluted ADP (2^-5 to 2^-15mg/ml) prepared in a Microtiter tray. After agitating 15 seconds, minimum effective ADP concentration to give platelet aggregation was detected microscopically. Platelet sensitivity to ADP-aggregation was expressed by the absolute value of the exponent of the ADP concentration endpoint (Fig. 1).
The degree of the enhancement of platelet sensitivity became significantly greater when the contact area increased by means of increasing flow rate or contact time. When flow increased to double while contact time decreased to the half so that the contact area did not change, the degree of enhancement of platelet sensitivity did not change significantly (Table 1, Fig. 2 & 3).
The enhancement of platelet sensitivity by aging or exercise in arteriosclerotic patients, obserbed by our previous studies, might be considered to be analogous to the situations of the present experimental model (Table 2).
These findings may suggest that measurement of platelet sensitivity by our method could be one of the important means to investigate platelet-vessel wall interaction.

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Platelet aggregation test has been widely used to detect congenital and acquired abnomalities of platelet functions. However, it is difficult to measure platelet aggregation in patients with thrombocytopenia, such as idiopathic thrombocytopenic purpura, acute leukemia, aplastic anemia, SLE, and liver cirrhosis.
We demonstrated the effect of low platelet number in platelet rich plasma (PRP) on platelet aggregation using new model of aggregometer, Dual Sample Aggregometer Model DP-247-E (SIENCO) and recorder, Unicorder U-225-MS39 (NIHON DENSHI KOHGAKU).
Blood samples were taken from eight healthy volunteers without ingestion of any drugs known to affect platelet aggregation at least ten days prior to study.
PRP was diluted with autologous platelet poor plasma (PPP) to make various consentrations of PRP as follow; 30×10⁴/mm³, 15×10⁴/mm³, 10×10⁴/mm³, 7.5×10⁴/mm³, 5.0×10⁴/mm³, 3.0×10⁴/mm³. As aggregating reagents, was used ADP (final concentration: 1×10^-5M, Siguma), Epinephrine (1×10^-4M, DAIICHIKAGAKU), and Collagen (25μg/ml, Sigma). We measured the maximum percentage of aggregation and the pattern of aggregation curve.
With ADP, no significant change was seen at or above 5.0×10⁴/mm³ of platelet consentration in PRP. However, aggregation was significantly diminished at 3.0×10⁴/mm³. When epinephrine and collagen were used, aggregation remained normal at or above 7.5×10⁴/mm³ of platelet concentration.
It was concluded that platelet aggregation was measurable with this aggregometer when the platelet concentration was more than 5.0×10⁴/mm³. 7.5×10⁴/mm³, 7.5×10⁴/mm³ with ADP, epinephrine and Collagen respectively.

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The aggregation of “gel filtrated platelet” could be induced with ADP, and the relationship between ADP concentration and the initial rate of platelet aggregation was that:
V₀=Vmax([ADP]-a)/Km+([ADP]-a), ([ADP]≥a) where “v₀” was the initial rate of this reaction, “[ADP]”, the concentration of ADP; “Vmax”, the maximum rate of this reaction; “Km”, the ADP concentration which gave 1/2 Vmax, and “a”, the ADP concentration with which the reaction began to be induced for the first time.
When we made the clinical aggregometer indexes to show the degree of platelet aggregation with ADP, not on the aim to search the easy and simple indexes but to search those which expressed the actual phenomenon faithfully, it was evaluated most adequate for us to use above three constants as the clinical aggregometer indexes, that is to say, the first index “a” which was the ADP cencentration to induce the reaction for the first time, the second index “Km” which was the degree of rate variability according to the change of ADP concentration, and the third index “Vmax” which was the maximum rate of this reaction under the condition of excess ADP.

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It is generally accepted that platelets play an important and primary role in arterial thrombosis. Present study was carried out to clarify the platelet aggregability in patients with Buerger's disease (TAO) and Arteriosclerosis obliterans (ASO).
Sixty male cases with TAO aged 25-65 (average and SD: 43.2±9.7), 60 male cases with ASO aged 46-75 (62.5±8.1), and 50 healthy males aged 28-70 (48.5±12.0) were studied with optical density method.
According to the clinical course of the disease, the patients of each group were further classified into 4 stages: stationary, improving, chronically aggravating, and acute occlusive stages in ASO or acute stage of thrombophlebitis migrans (TPm) in TAO.
Four concentrations of ADP and three concentrations of collagen suspension were used to induce aggregation. The maximal aggregation rate (MAR, described as percentage of maximal transmittance change of PRP in 5 minutes after adding the aggregating agents to that of PPP) and the appearance rate of the second aggregation at each ADP concentration were employed as the parameters of aggregability.
In patients with TAO in the aggravating and stationary stages, reduced aggregability was found at the lower concentrations of aggregating agents. In the improving stage the aggregability was almost normal and significantly enhanced in the TPm stage.
In patients with ASO in the stationary and improving stages, enhanced aggregability was found, and it was the most marked in the acute occlusive stage. In the aggravating stage, the aggregability was normal.
In the chronically aggravating stage, chronic narrowing of arterial lumen is suspeited to derelope probably due to repeated thrombus formation and its partial dissolution and partial organization. In the improving stage such a process does not take place and the development of the collateral circulations or recanalization of the thrombosed arteries lead to the improv ement of the peripheral circulation. This difference between the two stages may account for the difference of the platelet aggregability, reduced in the aggravating stage and maintained normal in the improving stage. This relative difference of the platelet aggregability between the two stages were found not only in patients with TAO but also with ASO.
It was of interest that in patients with TAO the platelet aggregability in the stationary stage was the same as in the aggravating stage. This result lead to the speculation that in patients with TAO there exists even in the stationary stage a certain change similar to that in the aggravating stage.
In patients with ASO enhanced platelet aggregability was noticed in the stationary stage. This result cannot be explained only by the effect of aging and the contribution of some plasma factors is probable.
In the acute occlusive stage in ASO and the TPm stage in TAO, markedly enhanced platelet aggregability was observed. This may be explained partially by the relatively increased number of young platelets in the circulating blood secondary to the rapid platelet consumption by the acute thrombus formation. Considering that the patients in such an acute stage are in a stressed condition, many other unknown factors might contribute to the enhanced platelet aggregability.

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Batroxobin is a thrombin-like enzyme from Bothrops atrox venom which causes hypofibrinogenemia by splitting off only fibrinopeptide A from fibrinogen. Defibrinogenation with this enzyme is expected to be a new type of anticoagulation therapy without serious bleeding complications.
In this paper, the changes in blood coagulation systems, specifically platelet function, during defibrinogenation therapy with Batroxobin (B. moojeni) were investigated.
Two out of 3 patients suffered from iliofemoral vein thrombosis were treated with 30 to 40μl/kg/day of Batroxobin and 30, 000u/day of urokinase for 8 to 16 days. In the other one, thrombectomy was performed after the initial administration of Batroxobin, and the defibrinogenation therapy was continued for 15 days after the operation. In these 3 patients, Warfarin sodium was administered 2 days prior to the time when defibrinogenation therapy was discontinued, and it was continued for 1 to 3 months after the therapy.
Red blood cell, platelet count and coagulation factors (II, V, VII-X, VIII, IX) were unchanged during the treatment. Fibrinogen concentration varied between 0 and 80mg% during the defibrinogenation therapy. The maximum level of FDP was found to occure 4 to 6 hours after the initial administration of Batroxobin, and it was gradually restored after that. Prothrombin time was prolonged at a fibrinogen level of below 100mg%. Partial thromboplastin time shortend slightly. Platelet adhesion to glass and ADP induced platelet aggregation were not changed after Batroxobin administration. There was no corelation between platelet function and fibrinogen concentration. No bleeding complications were observed in all patients.
These results suggest as follows:
1) Batroxobin from B. moojeni induces longer lasting defibrinogenation than Batroxobin from B. marajoensis.
2) Platelet adhesion to glass and ADP induced aggregation are not influenced by the decreased fibrinogen concentration nor the increased level of FDP during defibrinogenation therapy with Batroxobin.
3) It is an imporatant factor in certain hemostasis that normal platelet function is maintained during defibrinogenation therapy.
4) It is possible to use defibrinogenation therapy with Batroxobin as a postoperative anticoagulat therapy in vascular surgery.

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Many papers have been reported regarding to the alterations of blood coagulation during and after the extracorporal circulation. Most of them, however, have concerned themselves with the thrombocytopenia during and after the open heart surgery and detailed platelet function studies remained unclear. Platelet functions and their relating coagulation factors including fibrinogen concentration, circulating fibrin degradation products and prothrombin time were studied pre-, during and after extracorporal circulation.
(1) Platelet count: The average preoperative platelet count was 21×10⁴/ml with the range from 13×10⁴ to 37×10⁴/ml. At the end of 1/2 hour of cardiopulmoneary bypass, the average platelet count dropped to 11×10⁴/ml. It was 13×10⁴ml at 1/2 hour after the administration of protamine sulfate and the same level continued until the 3rd postoperative day.
(2) ADP-induced platelet aggregation: Preoperative average maximal aggregation was 73 percent and it dropped to 41 percent at 1/2 hour after the administration of protamine. Gradual recevery was found and the average maximal aggregation was 66 percent after 6 hours postoperatively.
(3) Fibrinogen concentration: The fibrinogen fell from 262 to 159mg/dl at 30min. after neutralization of heparin but recovered daily. In the first postoperative day, the average value was higher (293mg/dl) than the preoperative value and there was a considerable hyperfibrinogenemia during 1 to 2 weeks after the surgery.
(4) FDP: All but 1 patient showed a lower level of FDP less than 5μg/ml preoperatively but a significant increase of FDP with a range from 10 to 20μg/ml was usually observed.
(5) Prothrombin time: The preoperative mean value was 13.6 second and it prolonged up to 17.3 second at 1/2 hour after cardiopulmonary bypass. This change became normal within 6 hours after neutralization of heparin.

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Mechanisms of hemostasis in primary thrombocythemia have still remained unknown. A case with primary thrombocythemia was reported, in whom abnormalities of platelet function were found and thrombokinetics as well as ultrastructural study were performed. From these results, some suggestive data concerning abnormalities of hemostasis were obtained.
Case: Male 62 Yrs. A company employee.
Chief complaints: Bleeding tendency after shaving, easy fatiguability and epigastralgia.
Onset and course of present illness: Leukocytosis was pointed out by medical examination on April 8, 1977.
Admission of present state: There were hepatomegaly (1 finger breath) but no purpura, no lymphadenopathy and no splenomegaly.
Hematological examinations: RBC 548×10⁴/μl, Hb 15.8g/dl, Ht 50%, Ret 7‰, Plt 80.4-162×10⁴/μl, WBC 13, 700/μl.
Neutrophil alkaline phosphatase activity: Rate 100%, score 380. Platelets showed morphologically anisocytosis, aggregated formation and large size over 3.5μ in diameter, and moreover they had a few specific granules, great and small sized mitochondriae and swollen cristae and so on in electron-microscopic level.
Examinations on coagulofibrinolysis: Caolin activated PTT was subnormal. PTT and recalcification clotting time were prolonged slightly. Rumpel-Reede's phenomenon was strongly positive and bleeding time prolonged incidentally to 8.5 minutes. There is no abnormality in fibrinolysis.
Platelet function: Retention rate; 12-24%. Aggregation; No pathological finding but abnormal aggregation curve with ADP (2μM) was observed. In PRP (160×10⁴/μl near native platelet level), secondary aggregation curve and accelerated aggregation were realized. Platelet factor 3 content and platelet availability test were within normal limit. In thrombokinetic study by using ⁵¹Cr labeled platelet, the survival time t 1/2 and t 10% were 3.5 days and 8 days, respectively.
Comments: From above-mentioned characteristic results, his diagnosis was comformed as primary thrombocythemia. It might be suggested that a possibility of intravascular thromboembolic formation would be indicated as one of more important factors, though bleeding tendency in primary thrombocythemia were complicatedly associated with some detects in coagulation, disturbed platelet functions and abnormal morphological features in light-and electron-microscopic levels.

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Eleven cases with marked thrombocytosis over 100×10⁴/cmm were encoutered in the past 10 years. All of them belonged to myeloproliferative disorders, including 6 cases with thrombocythemia hemorrhagica, 3 cases with chronic myelocytic leukemia, each one case of panmyelosis in leukemic stage and of primary myelofibrosis in leukemic stage.
In 4 out of 6 cases with thrombocythemia hemorrhagica, definite clinical hemorrhagic tendency were observed such as gingival, postoperative, gastrointestinal bleeding. In 2 cases, intramuscular hematoma just as a simillar from to classic hemophiliacs were occured. These symptomes were seemed to relate clearly with some of the abnormal laboratory data. That is, prolongation of bleeding time (PTT), plasma clot time (PCT) and platelet function defects were revealed in all 4 cases. Low platelet retention rate and low aggregation rate by ADP and collagen were seen. Treatment with Busulfan (3 cases), Busulfan+P³² (1 case) were done without any side effects nor leukemic transformation.
Platelet disfunctions seemed to be sustained continually even when platelet count decreased after treatment, although clinical hemorrhagic episodes were disappeared with normalization of bleeding time, PTT and PCT. Afterall Busulfan therapy was thought to be effective to control the hemorrhagic diathesis. In other two cases with thrombocythemia hemorrhagica there was no hemorrhagic diathesis.
In 3 cases with CML, no obvious clinical bleeding evidences were seen. On laboratory data, there were no abnormal prolongation of PTT nor bleeding time. Decreased platelet functions were seen in one case which had had bleeding tendency one 13 years ago when she was diagnosed to be CML for the first time.
Etiological consideration of thrombocytosis and/or thrombocythemia in myeloproliferative disorders was discussed.

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A sixty-year-old woman, who complained of headache and monoplegia of left upper extremity, was admitted to the Department of Internal Medicine of Tokai University Hospital in May, 1976.
The spleen was palpable. The lymph nodes were not enlarged. Examination of the blood showed that platelet counts was 219×10⁴ per mm³, white cell counts was 20, 800 per mm³. Bone marrow puncture revealed numerous megakaryocytes. On the hemostatic studies bleeding time, prothrombin time and partial thromboplastin time were prolonged. Ability of platelet adhesion decreased. Platelet aggregation studies showed the response to epinephrine and norepinephrine to be absent and that to ADP decreased. The ultrastructural features of platelets were reduction of α-granules, presence of bull'seye shaped dense granulations and vacuolization of ergastoplasmic reticulum and cytoplasm. The surface of platelet membrane was smooth and few projections from the cell membrane were observed.
A mechanism of bleeding tendency in primary thrombocythemia was disccused, and it was suggested, on the basis of our findings that the cause of abnormality of the platelet function might have the relation with these abnormal ultrastructural findings.
Diagnosis as idiopathic thrombocythemia, 2mg per day of busulfan was administered. Three months after busulfan therapy, she was in good condition with platelet count of 91×10⁴ per mm³.

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It is widely accepted that the platelet which is originally discoid is deformed into spiny spheres by ADP, thrombin or collagen (viscous metamorphosis) and forms thrombus by its function-adhesiveness and aggregation. But we have hardly achieved the quantative analysis of the morphological changes and disclosed the relationship between the deformed platelets and their functions.
In order to clarify this relationship, we examined the influences of the following procedures on human platelets.
1) preservation (non-anticoagulated whole blood, citrated whole blood), 2) stirring (the same blood samples), 3) preservation after stirring (citrated whole blood) and 4) surgical operation with laparotomy on 10 patients with various diseases.
After fixing the platelets by 1% glutaraldehyde, the platelets were classified into disc, hemisphere, sphere and the other form, and checked for pseudopod formation under a light microscope. As a platelet function, platelet retention rate was measured by a modified Hellem-II method.
Platelet shape was easily changed and pseudopods issued by preservation and stirring. These morphological changes were brought about more strikingly by stirring than by preservation. The retension rate decreased when spherification and pseudopod formation had prominently advanced. However, it increased when the incidences of non-discoids and the pseudopods increased to some degree, e. g. when citrated blood was preserved up to 60min. at room temperature and when it was stirred for 1min. These phenomena were similarly observed in vivo condition (during operation).
These results suggested that some relationship existed between the platelet shape and its function and that halfly deformed platelets were in an activated phase and prominently deformed platelets were rather in a refractory phase. This relationship was probably derived fromnot only extra-platelet factors such as activation of plasma coagulation factors (contact factor and prothrombin) but also from internal factors (or character) of platelets. The latter factors were considered to be more important because both shape change and the increase of retention rate were demonstrated in some experiments to occur before the silicon PTT became shortened.

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Adherent platelets on the plate of vinylcyclohexene dioxide resine (VCD) were studied with scanning electron microscopy (SEM) and translucent electron microscopy (TEM) in crosssection.
Each one drop of PRP (from citrated blood) was kept on two VCD resine plates at 37 degrees centigurade for 30 minutes, and platelets were immersed in 2.5% glutaraldehyde solution. Postfixation was proceeded with 1% OsO₄ solution. After dehydration with ethanol series, one plate was dry up by Anderson's method for SEM, another plate was embedded in epon resine and thin cross-sections were made by ultramicrotome with glass knife for TEM.
In SEM, a quater number of platelets were spreaded out membranously and the center part of these platelets were protruded. Other platelets aggregated each other and stick to membranous changed platelets. A few platelets touched to VCD resine surface with pseudopodes.
In TEM, the protruded center part of membranously changed platelets contained almost all of various granules and maked hill top.
Many microtubules ran paralleled to the surface of VCD resine, and large amount of microfilament-like structures were seen in the near part of VCD resine surface. The ultrastructure of membranous part of platelet on VCD resine plate was same as that of pseudopode of aggregated platelet.
The distance between adherant platelet membrane and VCD resine surface was about 400Å and in platelet aggregation (e. g. induced by collagen) the distance between the membrane of platelet and that of neighboring platelets was 700Å in average.

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The spreading of blood platelets after adhesion was first observed by Eberth et al. (1885) and has been studied since by many investigators. The underlying mechanisms, however, are not known in many respects and there has been no report so far of its electron microscopic observation.
PURPOSE: For making observation of the mode of normal platelet adhesion and spreading by scanning electron microscopy as well as to investigate morphological changes of spreading platelets in elected hematologic disorders.
SUBJECTS: The mechanism of platelet spreading was investigated with specimens from healthy male adults, followed by comparison of the spreading of plateles from patients with C. M. L. or I. T. P.
PROCEDURE: Platelets were allowed to spread on plastic plates by the method of Breddin and fixed after intervals of 5, 15 or 30 minutes for microscopic observation. Those from patients were fixed after 30 minutes of spreading. For electron microscopy, specimens were fixed with 1% glutaraldehyde and, after postfixation in 1% OsO₄, they were dehydrated, dried and covered by the vacuum evaporation of carbon and metal.
RESULTS: Mode of spreading—At 5 minutes of spread each platelet was found to have sent out one or two pseudopodia. When 15 minutes had elapsed the pseudopodia were found increasing in number and elongated to shape up a dendritic form. At 30 minutes, the platelet was spread to assume a discoid shape with the peripheral transparent hyalomere and the central portion consisting of a gel-like material. A series of scanning electron micrographs shows that, generally speaking, the platelet sends out pseudopodia which thereafter increase in number and extend to form dendroid structures with a subsequent formation of the hyalomere just like a membrane spreading around neighboring pseudopodia; the platelet thereby gradually spreads into an ellipsoid shape. An occasional platelet shows the formation of a clear zone by a sol-like outflow around the pseudopodia, followed by its confluence with the substance from the central portion to eventually make the platelet discoid. It was also noted that a platelet had a peripheral clear zone formed around it at as early as the initial stage of adhesion and thereafter appeared to continue spreading in all directions; it remains yet to be determined whether or not this represents the consequences of a rapid progress in platelet spreading in either of the foregoing two fashions. Platelets from patients—Most platelets from CML patients were found asteroid with incompletely spread peripheral dendroid structures. In ITP, platelets were ellipsoid and displayed remarkably active spreading; the central portion consisted of a gel-like substance with an irregularly uneven surface and with trabeculations around it.
CONCLUDION: Blood platelets were observed to spread in three different modes, and this finding would be of significance as an aid to elucidation of the process of platelet adhesion. The observation also disclosed a remarkably striking difference in the morphology of platelet spreading between CML and ITP, suggesting likely abnormalities of platelet function in these conditions.

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Cationic polymers such as polylysine, aggregate platelets by reducing the negative surface charge and forming bridges between the adjacent platelet membranes. A controversy however, has arisen about the question whether it can elicit the release reaction.
In the present study, three types of polylysines were tested in platelet rich plasma (PRP) and in washed platelet suspension (WPS) for the ability to cause aggregation and release reaction. We also studied the effects of various inhibitors including negative charged acid mucopolysaccharide, heparin.
All three types of polylysine induced platelet aggregation which showed biphasic aggregation curve in PRP but showed no secondary curve of aggregation in WPS. At the same concentration of polylysine, maximum aggregation was slightly reduced in both system when the lower molecular one was used. Various inhibitors such as NEM, adenosine, aspirin, PGE₁, completely inhibited the aggregation in PRP but had no effect on the aggregation in WPS. Heparin inhibited the both aggregation in PRP and WPS and higher concentration of heparin was needed to inhibit aggregation when the larger molecular one of polylysine was used.
Polylysine caused release reaction from the ¹⁴C-5HT labeled platelets in PRP but could not elicit release reaction in WPS. Although the small amount of radioactivity was detected in surrounding medium in WPS, these were probably due to the increased permeability of membrane because the same amount of radioactivity was also detected when the various inhibitors were added.
Electron microscopic observation of aggregated platelets in PRP and WPS showed the evidence supporting the result of the presence or absence of release reaction in two systems assessed by measuring the radioactivity.
Polylysine aggregates platelets by reducing the negative surface charge of platelets and can elicit release reaction in the presence of Ca⁺⁺ or plasma. The aggregation in PRP and WPS was inhibited by heparin probably due to neutralizing the positive charge of polylysine. In contrast to in WPS, inhibitors other than heparin inhibited the first wave of aggregation in PRP but this mechanism remains unexplained.
The average distance between plasma membrane of the aggregated platelets in WPS did not vary with the degree of polymerization of these polylysine. In this respect, platelet aggregation differs from aggregation of red blood cells by polylysine in which widening of the intercellular space were observed by others with an increase in molecular size.

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We had examined the abnormality of platelets in cirrhosis of the liver from the standpoints of the count of megakaryocytes and its classification, platelet spreading test, platelet volume and its distribution and platelet aggregation, and had found the disturbance of platelet formation in the bone marrow and fragility of the platelets in cinhosis of the liver.
In this paper, to clarity further the qualitative abnormality of the platelets in cirrhosis of the liver, we examined ⁵¹Cr-labeled platelet life-span and its organ distribution, platelet uptake and release of ¹⁴C-labelled hydroxytryptamin (5-HT), ADP content in the platelet (ADP release reaction aceording to Weiss) and platelet antibody.
Conclusively, in cirrhosis of the liver,
1) platelet life. span was shortened to 2.9 days against 4.3 days of normal subjects,
2) radioaccumulation in the spleen was not always to same degree in the patients with same platelet counts, life-span and similar splenomegaly,
3) uptake and release of ¹⁴C-5HT by platelet were decreased to 73.8% and 52.20 against 89.5% and 73.8% of normal subjects,
4) ADP content in the platelets was also decreased to 32.5% against 54.8% of normal subjects,
5) platelet antibody was negative in all the cases, these findings suggest that in cirrhosis of the liver, reduction of platelet counts may owe not only to hypersplenism but also the disturbance of platelet formation in the bone marrow and its selated qualitative abnormary of the platelet tself

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This is a case report of a 24-year-old woman with storage-pool desease associated with albinism. From her childhood, she has often experienced the prolongation of nasal bleeding or bleeding from various sites due to slight injuries, but no blood transfusions were required. At the age of 22 years, she visited a hospital because of subcutaneous bleeding in the upper and lower extremities. At that time it was found shehad prolonged bleeding time, but other examinations on coagulation factors showed no abnormalities.
Since September 1975 she has developed repeatedly painful subcutaneous bleeding with swelling and heat, but recovered usually only by keeping rest. In Feburuary, 1976, she was admitted to the 3rd Department of Internal Medicine, Tohoku University Hospital in order to have precise examination on this hemorrhagic diathesis.
Laboratory examinations revealed no abnormalities except for a mild anemia. The hemostatic screening tests for blood coagulability and fibrinolytic activity were almost normal. However, bleeding time was markedly prolonged to 15 minutes, and platelet function tests showed some characteristic abnormalities, that is, platelet retention rate showed 23-29%, indicating normal limit, whereas platelet aggregation induced by collagen was markedly impaired. The aggregation of normal PRP induced by supernatant which was produced by freezing and thawing of patient's PRP was more markedly decreased than that of normal control PRP. Other platelet function tests were normal. Because of the above mentioned hemostatic findings the diagnosis of ‘storage-pool disease associated with albinism’, probably the first case in Japan, was made.
It is plannd to examine the patient's platelet morphology by electronmicroscopy and the contents of the patient's platelet adenine nucleotides biochemically.

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The platelet volume distribution and the appearance rate of megathrombocytes were measured with Coulter counter Model-B in 20 patients with ITP during platelet kinetic studies.
Normal range of platelet volumes in EDTA-blood is as follows: mode; 5.2±0.3μm³ (mean±1 S. D.), median; 7.1±0.7μm³ and mean volume; 9.0±0.8μm³.
In 13 patients with ITP platelets were increased in mean volume, 6 patients showed normal size and one patient decreased in mean volume. Increased percentage of megathrombocytes (volume more than 14μm³) was noted in 13 cases. Remaining 7 cases showed normal rate appearance (13±3.8%).
In 5 patients, highest peak was deviated to the smaller elements although the mean volume was remained in normal range or increased. This finding suggests that at least two population of platelets are present in the blood in these cases.
No close correlation was noted between platelet counts, platelet mean survival time or platelet turnover and platelet volume. The appearance rate of megathrombocytes had also no correlation with the results of platelet kinetics.
From above results, it could be concluded that young platelets could be of variable size in ITP. And, in ITP it might be impossible to suspect the amounts of effective platelet production in the bone marrow from the data of platelet volume or the appearance rate of megathrombocytes.

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The thromboembolism following the artificial valve replacement in patients with acquired heart valve diseases is one of the most important complications. The studies have been aimed to clarify the specific abnormalities of the platelet function and the coagulofibrinolytic system after the valve replacement.
Methods and Materials
Twenty-six patients with heart valve diseases were divided into 2 groups as follows, in 11 of them the valve repair has been performed (group I), and 15 the valve replacement has been done (group II). Laboratory data have been chronologically checked 2 months after the operation. All patients of group II and some of group I had anticoagulant therapy postoperatively. Platelet aggregation was studied with an aggregometer of Bryston's Co., with the modified method of O'Brien's technique. ADP and collagen were used as aggregating agents. Fibrinogen and plasmin activity in plasma were measured by ordinary methods. FDP in plasma was estimated by the HIT method. Factor VIII procoagulant activity (VIII-AHF) was determined by a one-stage method, and the factor VIII related antigen (VIII-AGN) and antithrombin III in plasma were quantitatively determined by electrophoresis in agarose gel containing antibodies (Laurell's method).
Results
The value of T1/2 of the platelet aggregation induced by both of ADP and collagen were shorter in group II than group I, and the value of R was smaller in group II than group I. The small R means that the aggregability of platelet was enhanced by the activated factor XII. High values of FDP were noticed more frequentry in group II than in group I but plasmin activity of plasma was not detected by standard fibrin plate method. VIII-AHF ranged from 50 to 3000, and significant difference was not found between the two groups. VIII-AGN also ranged from 50 to 400%, but the mean of group II and group I were 197±28%, and 151±17% respectively. The amount of VIII-AGN in group II was slightly increased and it is interesting to find that the high value of VIII-AGN was shown in patients with Starr-Edwards ball valves, but not in patients with Hancock Xenograft valve.
The mean ratio of VIII-AHF to VIII-AGN for both groups were almost same value but about half of the examined samples in group II showed significantly less value than the normal range but only 1/3 in group II.
In these studies in patient with heart valve disease, there was no consistent correlation between ratio of VIII-AHF to VIII-AGN and FDP or LDH.
Antithrombin III was all normal in group I patients but in group II patients some samples showed reduced value out of the normal range and the mean value of group II was smaller than that of group I.
The reduced value of ratio of VIII-AHF to VIII-AGN and AT-III were found in patients with artificial heart valve and this indicates that VIII-AHF and AT-III may be consumed by the formation of microthrombus and VIII-AGN may be produced reactively. In addition we reported previously that hyperaggregability of platelet and FDP production were found more frequentry in patients with artificial heart valve. It would be also important to be pointed out that such a hypercoagulable state was induced even in these patients in good controlled state given by clofibrate and warfarin.

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Hemostatic tests such as; thrombotest, hepaplastin test, prothrombin time, activated-partial thromboplastin time, factor VIII and IX, fibrinogen and platelet counts were studied in 91 full-term appropriate-for-date (AFD) newborn infants, who were considered to be mature healthy newborn, and 39 preterm AFD newborn infants, who were born with short gestational period and immature functions, and 37 fullterm small-for-date (SFD) newborn infants, who were considered with disturbed fetal growth. At the same time, some liver function tests such as; total bilirubin, TTT and ZTT, alkaline phosphatase, LAP, LDH, GOT and GPT, choline esterase and total protein were examined.
Hemostatic tests revealed moderate disturbances in full-term AFD and severe hypofunction in preterm AFD during 1 to 3 days of age. Then, they increased their levels gradually. In full-term SFD, hemostatic abilities were a little different from other two groups; they did not show a gradual increase with days of age.
Functions of liver were not reflected in routine liver tests; they had high bilirubin level due to physiological hemolysis and elevated LDH and GOT originated in RBC. But, decreased choline esterase activity indicates poor protein synthesis in the liver.
The correlation between hemostatic and liver function tests were not so definite in newborn infants.
To evaluate liver functions in the neonatal period, correctly, it would be proper to examine these hemostatic tests, such as thrombotest, hepaplastintest and prothrombin time, before and after the administration of vitamin K.

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