The aim of this report is to clarify the significance of platelet aggregation in arteriosclerosis compared to healthy subjects as well as the effect of smoking on the platelet aggregation. Fifty five arteriosclerotic patients including ischemic heart disease and cerebral infarction in chronic state and 44 controls were examined. Serum cholesterol, triglyceride, free fatty acid, plasma fibrinogen and euglobulin lysis time were also measured at the same time. The response of platelet rich plasma to 2×10^-6M of ADP, collagen solution using bovine tendon, and 1×10^-6M of adrenaline were observed by the optical density method of Evans (EEL-169). All of these three types of aggregation were increased in arteriosclerosis compared to control (p<0.001). The maximum optical density, which showed most significant difference, in ADP was 67.8±2.4% in arteriosclerosis and 48.8±3.8% in controls. Among controls, heavy smokers (smoking over 20 cigarettes daily) showed a significant increase in adrenaline-induced aggregation compared to non-smokers, but there was no difference between heavy smokers and non-smokers in arteriosclerosis.
The effects of smoking on the platelet aggregation as well as serum lipid, plasma fibrinogen and euglobulin lysis time were examined in the heavy smokers after 12 hours of breaking off with smoking. The control showed an increase of platelet aggregation to ADP, collagen and adrenaline, but there was no definite change in arteriosclerosis. Serum free fatty acid increased 25 minutes after smoking, but did not show any correlation with the change of platelet aggregation. Plasma fibrinogen and euglobulin lysis time caused no significant change, but fibrinolytic activity (fibrinogen/euglobulin lysis time) showed a tendency to decrease in arteriosclerotic patients.
These results suggest that smoking-induced potentiation of platelet aggregation might be considered to be a factor causing the high incidence of thrombosis and arteriosclerotic change in heavy cigarette smokers.

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This report concerns some studies that attempt to correlate sludging in bulbar conjunctiva with the extent of the hemorheological abnormalities in patients with varying diseases.
Sludging of the conjuncivum was observed by a biomicroscopy and photography and then classified into four categries according to the extent of intravascular red cell aggregation. Hemorheological examination included blood and plasma viscosity measurement, recording of viscoelastic curve in blood clotting, erythrocyte sedimentation rate and platelet function test. There is a correlation between the degree of the sludging and the values of plasma vicosity, maximum viscoelastisity of blood clotting, plasma fibrinogen concentration and erythrocyte sedimentation rate, although no correlation was observed between sludging and platelet adhesiveness or aggregation. In general, patients with raised ESR showed a tendency of remarkable increase of red cell aggregation. However, it was interesting that moderate sludging in diabetic patients who had not any raised ESR was frequently observed. We postulate that increased intravascular red cell aggregation as seen in the conjunctivum leads to the development of microcirculatory disturbance, together with high plasma viscosity and hypercoagulability and thereby it plays a significant role in the genesis of the obstructive microangiopathy.

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Disseminated intravascular coagulation has been suggested as a cause or an important secondary mechanism in toxemia of pregnancy. In order to clarify this evidence platlet count, aggregation, FDP, and serum lipids were studied on 30 normal pregnant women and 18 women with severe toxemia of pregnancy.
The following results were obtained:
1. The platelet count decreased slightly in severe toxemia of pregnancy as compared with the third trimester of normal pregnancy.
2. In severe toxemia of pregnancy, the rate of platelet aggregation induced by ADP was lower than of normal pregnancy.
3. In one case with abruptio placentae, platelet aggregation rate had fallen considerably at the time of hemorrhagic shock.
4. FDP and serum lipids levels showed a marked increase in severe toxemia of pregnancy than in third trimester of normal pregnancy.

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A case of bilateral cortical necrosis (BCN) was reported because of her abnormal platelet count and aggregability. She was 26 years old primipara.
In her past history, at the age of 11, she was operated because of papillary adenocarcinoma of the thyroid gland, when no abnormal bleeding was reported.
After cesarean section, atonic hemorrhage occurred and anuria developed. Because BCN was suspected heparin and urokinase therapy were started (later, patchy BCN was verified by renal biopsy). Uremia was controlled by hemodialysis (3/week). Platelet count (Plt.) increased gradually to normal level within a week. After heparin cessation, Plt. continued to increase and exceeded 60×10⁴/mm³. In spite of thrombocytosis, bleeding time (BT) was prolonged to more than 15 min. On 31st hospital day when Plt. was 40×10⁴/mm³and BT more than 15 min., platelet aggregability was investigated by screen filtration pressure (SFP) method (normal range: 264.9±14.3mmHg when induced by 3μM ADP). Her aggregability was no more than 20mmHg. On 34th hospital day, she was attacked by generalized convulsion and tracheotomy was done. Despite 17 units of blood transfusion bleeding was not discontinued. Therfore, 6 units of packed platelet were transfused and bleeding was dramatically stopped within 2 hours. After this episode, BT was normalized while thrombocytosis persisted. On 59th hospital day when Plt. showed 48.1×10⁴/mm³ and BT was 3.5min., platelet aggregability was investigated by SEP method and optical density (O. D.) method. Using SFP method 3μM ADP induced aggregability was 400mmHg. By O. D. method, 10μM ADP induced maximum aggregability (ADP max) was 32.8% (normal 70.2±2.6), 1μg/ml adrenaline induced maximum aggregability (adrenaline max) 90.8% (normal 40.6±4.0), and 1mg/ml collagen induded maximum aggregability (collagen max) 69.6% (normal 74.8±5.6), respectively.
She discharged on 14th hospital day, when her creatinine clearance was 17L/day, BUN 31mg/dl, and creatinine 3.3mg/dl, and conservative management was indicated. On 178 th day after BCN, when Plt. revealed 27.6×10⁴/mm³ and was 3min., platelet aggregability was investigated again. Using SFP method, 3μM ADP induced aggregability was 390mmHg, while by O. D. method, ADP max was 63.2%, adrenalin max 98.5%, and collagen max 82.4%, respectively.
Throughout her clinical course, fibrin degradation products were 40μg/ml or less, fibrinogen 370-900mg/dl, and euglobulin lysis time 24 hours or more. CB-PC and cephalothin, which were administered when platelet aggregability decreased, were investigated in vitro by O. D. method, with no effect to platelet aggregability being found.

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Batroxobin is a thrombin like enzyme originate from the snake venom of Bothrops atrox. Effects of defibrinogenation therapy using Batroxobin (Bothrops atrox marajoensis) on 2 cases of peripheral vascular occlusive diseases were reported.
Case 1.
The patient was a 74 year old Japanease female. She noticed a dullness or right lower extremity for 5 years. Since six months, she developed ulcers on her right toes. On January 1975, she was admitted to our hospital with intermittent claudication and ulcers of her right toes. Pulses of popliteal and dorsalis pedis artery were not palpable on the right leg. Angiography revealed occlusion of the right femoro-popliteal artery. Examination of peripheral blood and coagulation systems revealed hypercoagulable state (RBC 652×10⁴, Ht 54%, platelet count 89.6×10⁴, fibrinogen 600mg/dl). She was diagnosed as arteriosclerosis obliterans and thrombectomy of right femoro-popliteal artery was done. Popliteal arterial pulse could be palpable after the operation, but it was gradually decreased. The artery seemed to be rethrombosed caused by the hypercoagulability. Batroxobin (Defibrase) was given in doses of 30 to 50μl/kg/day diluted in 200ml of physiological NaCl-solution as infusion for 1 hour. RBC, Ht, and platelet count were not changed after each infusion. Fibrinogen level were decreased to 160mg/dl after 4th infusion. The maximum concentration of FDP was found 4 hours after the initial infusion of Batroxobin. Prothrombin time was not changed. Partial thromboplastin time was shortened 24 hours after initial infusion, but it returned to normal level after that. Coagulation factor II, VIII and IX were increased 24 hours after the initial infusion. These changes seemed to be related to the shortened PTT. Factor V and VII-X were not changed. Plasminogen and antiplasmin were decreased parallel. α₁-antitrypsin, α₂-macroglobulin and antithrombin III were not changed. Pain of her right leg was restored and she was discharged well.
Case 2.
The patient was a 68 year old Japanese female. She was complaining of discomfortable of her right extremity from August 1975. On October 27, she admitted to our hospital complaining of edema at her right leg. There were not any abnormal findings on the laboratory examination except fibrinogen level. Phlebography revealed thrombi in the right iliac vein. Fibrinogen level was 560mg/dl at the time of her admission.
Batroxobin were given in doses of 50 to 85μl/kg 3 days before the vascular reconstruction for prevention of rethrombosis after the operation due to the hyperfibrinogenemia. The fibrinogen level was 80mg/dl at the time operation. FDP was increased markedly 4 hours after initial infusion and it gradually returned to normal concentration. Thrombectomy was done during defibrinogenation therapy. There was no bleeding during and after the operation. Defibrinogenation was continued for 4 days after the operation and fibrinogen level varied between 50 to 100mg/dl. RBC, Ht, platelet count and partial thromboplastin time were not changed. Prothrombin time was prolonged at fibrinogen level of 80mg/dl. Edema of her right leg disappeared in 19 days after the operation.

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Ultrastructual changes and localization of tissue plasminogen activator during the preparation of human saphenous vein graft were studied employing transmission and scanning electron microscope and histochemical fibrin slide technique of modified Todd's methood.
Samples of great saphenous vein were excised from five patients who underwent stripping for varicose veins of mild degree. Preparation media for storage of vein graft included (1) heparinized normal saline at room temperature, (2) heparinized normal saline at 4°C, and (3) heparinized whole blood at room temperature. Veins were subjected to 30-, 60-, 90-, 120-, and 180-minutes storage in each medium at which times segments were removed for study.
Apparent endothelial cell swelling and focal cellular disruptions were common after 60-minutes storage in normal saline at room temperature. These changes were minimal in whole blood and cold saline storage. After 120-minutes storage even in the cold saline, the most favorable preservative, cell swelling of endothelium, degenerative changes of endothelial mitochondria, disruption of basal lamina and accumulation of intercellular debris were observed frequently. Smooth muscle was minimally damaged in all the preservatives comparing with the endothelial changes.
Scanning electron microscopic study revealed striking endothelial cell swelling, intimal ulceration and exposure of subendothelial connective tissue, especially in normal saline at room temperature after 60-minutes storage. Vessels preserved in heparinized whole blood showed platelet-fibrin net formation frequently after 120-minutes storage.
Presence of fibrinolytic activity was demonstrated in all three layers of cold normal saline and whole blood stored specimens, however diminished activity was present in the intima of normal saline storage at room temperature after 60-minutes.
From the results mentioned above, it was concluded that cold normal saline could be preferable for vein preservation under conditions studied in this investigation.

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Synthetic polymers suitable for use in the cardiovascular system are needed as biomaterials for heart valve prostheses, artificial heart, veins, arteries and extracorporeal circulatory devices as heart-lung and kidney machines. Despite the wide variety of synthetic polymers that are available in the form of plastics or elastomers, none has proven eligible to clinical application for this purpose as a perfectly nonthrombogenic material. Currently, most investigations seem to be directed toward clarification of the blood biomaterial surface interaction, especially the mechanism whereby thrombi are formed in contact with such prosthetic. In many of these studies the surface properties of synthetic materials are being investigated from the angle where the foreign surface: blood interaction is taken as interface reaction, with the attention focused upon hydrophobicity, hydrophilicity and electronegativity of the materials. Findings obtained in our studies aimed at development of antithrombogenic materials have indicated importance of a definite distribution density of hydrophobicity and hydrophilicity for such polymers; namely, it appears unlikely that no surface factor is involved in the biomaterial: blood interface.
Supposing, for example, that a synthetic venous prosthesis with a surface of about 15cm² be introduced into the vascular system, this newly added surface of about 15cm² undoubtedly would be so slight as to fall within the limits of errors as compared with the entire inner surface of the cardiovascular system and hence as viewed in terms of the concentration of heparin injected for inhibition of the clotting system. Thus it would be rational to introduce into the study the findings on the surface structure of cells exposed to the cardiovacular lumen. Our studies are based on this ground, pursued through physico-chemical analysis of the interface comprised of the prosthetic surface and the circulating plasma surface. By comparative evaluation of various synthetic polymer prostheses in terms of antithrombogencity, the interfacial condition of the vascular inner surface of the organism was determind to consist of γ_C(Zis.)=20 to 30erg/cm², γ_C=68.5erg/cm² and γ_SL(H₂O)=0erg/cm², or the surface endowed both with hydrophilicity and it was demonstrated that a synthetic polymer fulfilling the prerequisites of γ_C(Zis.)=29 to 30erg/cm², γ_C=29 to 30erg/cm² and γ_SL(H₂O)=0erg/cm² is remarkably antithrombogenic. The results, stress prime importance of the function of interface to protein at a synthetic polymer surface and indicate the presence of biomate rials with minimal bearing upon the clotting system of blood. Further studies aimed at clarification of the interaction with protein will be pursued, using such materials.

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1) One tenth volume of 2000U/ml UK was added to the normal human plasma kept at 37°C. The decrease of fibrinogen in this plasma was approximately 50mg/ml even after 60min. incubation, but the fibrin clot lysis time was completed within 15min., when aliquots were taken from the mixture successively for the assay of plasma fibrin clot lysis. On the contrary, the grade of digestion of fibrinogen and fibrin by plasmin proceeded at the same rate when purified fibrinogen and plasminogen were used.
It was suggested that the digestion between fibrinogen and fibrin in norml human plasma was differentiated by the presence of plasmin inhibitors. The same differentiating digestion was observed with the use of purified fibrinogen, plasminogen and α₂-macroglobulin or Trasylol as the plasmin-inhibitor, whereas it was not with t-AMCHA as the activator-inhibitor.
2) Immediate as well as progressive inhibitor activity in normal human plasma was assayed by fibrin clot lysis time method and fibrinogenolysis method. It was found that the immediate activity was 15CTAU/ml and the progressve one was 36CTAU/ml by fibrinogenolysis method, while the former was 8CTAU/ml and the latter one was only 5CTAU/ml by fibrin clot lysis time mothod. The result indicated that the plamin inhibitor, immediate as well as progressive, acts more intensively on the process of fibrinogenolysis than fibrinolysis.
3) Normal human plasma was chromatographed on Sephadex G200 column and the inhibitor activities in the eluted fractions were assayed by both fibrin clot lysis and fibrinogenolysis methods. It was revealed that the immediate inhibitor activity on fibrinolysis was eluted in three-peaked profile and the most of the activity was localized in the eluttion region of α₂-M, while the activity of progressive inhibitors formed three-peaked profile with the same activity between them. With the use of fibrinogenolytic method, it was revealed that the immediate form were eluted in two-peaked profile, while the progressive form was eluted in probably in four-pearked profile in which the highest activity was localized in the region of α₁-antitrypsin.
It was concluded that the differentiating digestion between fibrinogen and fibrin in plasma was performed at the presence of plasmin inhibitors. The main role of fibrinolysis inhibition was played by α₂-M, while that of fibrinogenolysis by α₁-antitrypsin.

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The role of thrombosis on the genesis and progress of human aortic atherosclerosis were discussed.
Fourty-four human aortas were dissected res pectively into about 30-50 step sections and were studied morphologically on the existence of thrombi and the relation to intimal lesion.
Thrombosis on human aorta were frequent and more frequent in advancing age. In the aortic sections without atherosclerotic lesion “non-pathologic” the frequency of thrombosis was 7.3% and all of them were microthrombi.
The areas with “cloudy thickening” (early atherosclerotic lession) revealed thrombi in 16.8% cases and 23% of thrombi were membranous-fibrin thrombi or organized thrombi. The frequency of thrombus formation on atherosclerotic plaque was 61% and 36% of them were membranous-fibrin or organized thrombi.
From these results, it is concluded that some of the microthrombi developed on the “non-pathologic” area (no lesion) could play a role in atherogenesis by incorporation into aortic intima and some of them might disappear. The moderate sized membranous-fibrin or organized thrombi would play an important roles for the development of aortic atherosclerosis.

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ADP, collagen and epinephrine induced platelet aggregation was measured by the optical density method in patients with cerebral arteriosclersis (45 cases) and controls (25 cases) in order to clarify that patients with cerebral arteriosclerosis have a thrombotic tendency. In addition, platelet count, platelet adhesiveness, fibrinogen, cholesterol and triglyceride were measured. The results obtained are as follows.
1) The time and rate of collagan induced platelet aggregation were higher in the patients than the controls (P<0.05).
2) Neither ADP nor epinephrine induced platelet aggregation was significantly different in both groups.
3) Platelet adhesiveness was higher in the patients than the controls (P<0.05).
4) Epinephrine induced platelet aggregation correlated with serum cholesterol (r=0.469, P<0.05).
These data have probably reflected a thrombotic tendency in patients with prolonged cerebral arteriosclerosis.

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The blood coagulation factor XII which brings about the activation of the intrinsic coagulation system has more intricate significance in hemostatic balance than other clot promoting factors, because the factor is closely associated with kinin release and activation of the fibrinolytic system which inversely relates to the coagulation system in thrombus formation.
In this study the significance of contact factors, which include factor XII and XI, were observed clinically by assessment of the activity in plasma of cerebrovascular patients.
Contact activity was determined in plasma of 89 cerebrovascular patients without acute infection and hepatic disorders, and of 20 healthy controls following Waalar's method using exhausted plasma prepared by adding 30mg of celite 503 to 1.0ml of plasma at the same time.
The mean values and standard deviations of activity in 89 patients and 20 controls were 140±39.0 and 106±22.0%, significantly higher activity was indicated in patients than in controls. The activity in patients with normal ECG findings was 153±38.1%, and 131±40.2% in those with abnormal EGG findings, this difference was significant. In patients with normal ECG findings the activity of the hypertensives was higher than that of the normotensives, in patients elapsed over 4 months after their admission it was higher than that in one month, in patients with good marks in the test of activities of daily livings (ADL-T) it was higher than that with poor marks.
The activity was not associated with other clinical findings. In normotensive patients with abnormal ECG findings the activity was inversely proportional to serum cholesterol and β-lipoprotein fraction levels respectively.
The high activity in patients with normal ECG findings, and few marked atherosclerotic changes in contrast to patient with abnormal ECG findings, and the inverse correlations between the activity and two lipids are unresonable, considering only the participation of the coagulation system triggered by factor XII.
Few articles on the effect of long-term physical exercise on the fibrinolytic system have been published, except our previous study which demonstrated the lower serum α₁-antitrypsin level in cerebrovascular patients with good ADL-T marks than in those with poor marks. However, activation of the fibrinolytic system by exercise of short duration has been reported frequently. Therefore, the high activity must be regared as the result of a participation of the fibrinolytic system activated by factor XII. Although the high activity in patients with normal ECG findings and hypertension may be regarded as the protective effect of the fibrinolytic system to development of atherosclerosis induced by hypertension, and may be apperciated as the direct effect of kinin release by factor XII in hypertension.
These results indicated the complex association of the contact factors with three different systems, which are closely related to the pathophysiolosic status of cerebrovascular disorders, and the necessity of further intensive investigation of contact factors, especially of factor XII.

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(1) In order to dissolve left ileo-femoral vein thrombus, 50, 000 unit of urokinase in 500ml of Dextran was infused continuously from saphena magna vein of left thigh for 8 hours a day.
(2) While collateral veins were not sufficiently developed yet, femoral vein thrombus was dissolved and FDP appeared. However, no more dissolution of femoral vein thrombus and appearance of FDP occured, after collateral veins were developed enough and blood could come easily back through those collateral veins.
(3) It was suspected that an appearance of FDP was due to the dissolution of collateral veins thrombi as well as femoral vein thrombus.
We concluded as follows: This therapy could not make complete dissolution of femoral vein thrombus, but worked effectively for reopening of collateral veins and keeping good blood circulation.

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血小板外形を半定量的に検討できる簡単な光顕法 (Zucker と Borrelli の変法) を述べ, 採血からグルタルアルデヒド固定までの各種の条件によって外形が影響されることを明らかにした. 正常人40名 (男女各20) について, 可及的に in vivo に近い条件で血小板を固定すると, 外形は円板形88.6%, 半球形6.6%, 球形1.3%, その他3.7% (双極形0.6%を含む) であった. 偽足をもつ血小板は9.2%あった. 男女間に有意の差はみられなかった. 外形の意義や成立機序についてふれ, 今後の問題を述べた.

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The effects of testosterone and fluoxymesterone (fluoxy) on erythroid colony (CFU-E) formation in human bone marrow cultures were studied in vitro using the plasma clot technique of McLeod et al. Both testosterone and fluoxy directly increased the number of erythroid colonies with increasing concentrations ranging from 10^-12 to 10^-6 M in vitro. The optimal steroid concentrations were found to be 10^-8 and 10^-6 M for the enhancement of CFU-E growth. However, testosterone was more effective than fluoxy in growing colonies in vitro. In addition, a synergistic effect of a combination of these steroids and erythropoietin (0.5U/ml) on erythroid colony formation was seen at a concentration of 10^-8 M in vitro.
In this system, the similarity of the enhancement of CFU-E growth by both agents suggests that testosterone and fluoxy may act on erythropoiesis in vitro with a similar fashion.

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Effects of androgens on hematopoiesis in vivo were studied using the growth of endogenous spleen colony forming units (CFU-s) in subleathal (600rad) irradiated mice.
Androgens, testosterone and fluoxymesterone, were injected on 1, 3, 5 days after the irradiation and colonies counted on 10 days.
Both androgens were found to produce a significant increase in endogenous CFU-s in subleathal irradiated mice, although more stimulation was seen in the mice treated with testosterone. In addition, the type of colonies formed in spleens stained with H. E. were analysed to see effects of androgens on granulopoiesis in vivo. Testosterone stimulated the growth of granuloid colonies as well as erythroid ones in the spleen's, but there was no stimulation seen in the mice treated with fluoxymesterone at the dose used in this study.
These results suggest that testosterone, given in vivo, may act on pluripotent stem cells directly to enhance their differentiation into erythroid and granuloid commited stem cell compartments causing an increase in the numbers of erythroid and granuloid cells in spleen.

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Since an incidental observation of retarded clot retraction noticed in the blood of a patient with liver cirrhosis by an addition of tranexamic acid, effects of this drug on blood coagulation and platelet function have been studied by means of thrombelastography (TEG) and screen filtration pressure method (SFP) respectively.
No significant changes of coagulation of native or citrated blood were obtained by an addition of tranexamic acid followed by immediate starting of coagulation on TEG. However, preincubation of citrated blood from a healthy individual before or after the addition of tranexamic acid induced elongation of r+k and s on TEG. The citrated blood samples each from 5 healthy subjects and 5 patients with liver cirrhosis were incubated with tranexamic acid at 25mg/ml and tested for TEG. Marked prolongation of s was observed in blood from both healthy subjects and the patients, and a diminution of ma was observed in bloods only from the patients. This results suggested the possibility that susceptibility of the platelets to tranexamic acid is different between patients with liver cirrhosis and healthy subjects.
Tranexamic acid did not decrease the number of platelets and the amount of fibrinogen of specimen from a healthy subject.
In the tests of SFP, the inhibition of ADP-induced aggregation of human platelets was definitely observed by an administration of tranexamic acid in vitro at concentrations between 0.4mg/ml and 3.2mg/ml. Fifty % inhibition was observed at an appropriate concentration of 1.6mg/ml respectively.
The mechanisms of inhibition of platelet aggregation is still unknown. However, the inhibition observed immediately after the addition of tranexamic acid on SFP without significant changes on TEG could suggest that this drug influenced directly on the surface membrane but not the intracellular metabolism of the platelets.

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Fibrinogen is one of the important factors of the coagulation and haemostasis. We have investigated in several cases the method of measuring the plasma fibrinogen and ¹³¹I-labelled fibrinogen disappearance curve.
The plasma fibrinogen was assayed by the following method; thrombin time, tyrosine, and immuno partigen.
1) The normal range of the plasma fibrinogen was from 250 to 350mg/dl.
2) Definite correlation was observed among these methods, thrombin time, tyrosine and immuno partigen.
3) DM and malignant tumor showed the hyperfibrinogenemia, and liver disease showed the hypofibrinogenemia. However ¹³¹I-labelled fibrinogen half life, in all of them, was shortened.
4) In some cases with liver cirrhosis that showed hypofibrinogenemia, ¹³¹I-labelled fibrinogen half life was prolonged remarkably and the plasma fibrinogen increased after the injection of heparin. We consider it very important to measure the plasma fibrinogen and ¹³¹I-labelled fibrinogen half life in order to pick up knowledge on the role of haemostatic blance on haemostasis.

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The relationship between activity as transglutaminase and molecular composition or antigenicity of Factor XIII has still some discrepancty among the data of several researchers. In order to unveil this puzzle some quantitative estimation method is needed for measuring the immunological entities of subunits A and S. For this purpose a new device of double layer rocket electroimmunodiffusion technique based on Laurell's method (details were shown in Table 1) was introduced and the results with various plasmas and sera through this technique compared with that of ordinary two dimension rocket electroimmunodiffusion to prove that some advantages in estimation accuracy and consuming time was realized to the former which was adequate to analysis of protein of same antigenecity, particularly detection of abnormal fractions. By means of this technique it was also found that sera had no antigenecity against subunit A in spite of that they still remained F. XIII activity to some extent, but showed a certain antigenecity against subunit S. The same pattern of antigenecity behavior was observed with plasmas of defibrination syndrome (DIC), malignant tumors including leukemia and some of hepatopathia. However, the quite contrary circumstance in connection with antigenecity particularly of subunit A and activity was also realized in plasmas of these disorders.
When thrombin was added to plasma (with or without calcium) experimentally, the antigenecity against subunit A was lost nevertheless its F. XIII activity was more depressed at higher concentration of thrombin and recovered later at its lower concentration, but not at its higher, and the antigenecity against subunit S was remained. All these data on activity and antigenecity of F. XIII suggested complexity in composition of its subunits and their arranging order as well as their relationship with activity, which needed further investigation.

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The congenital and inherited abnormalities of fibrinogen is differentiated into two different types. One is the quantitative abnormality called afibrinogenemias or hypofibrinogenemias, and the other is the qualitative abnormality belonged to dysfibrinogenemias, and both types of abnormalily may be present together.
In 1958 Imperato and Dettori reported the first case of a congenital functional defect of fibrinogen as fibrinogen Parma and then about 30 cases of dysfibrinogenemia have been described in the literature.
The process in fibrin convertion can be devided into three phases, consisting the proteolytic phase with the release of fibrinopeptide A and B from the A α-chain and B β-chain of the fibrinogen molecules, the polymerization phase to aggregate to the visible fibrin clot, and finally the stabilization phase to be stabilized with cross-linking by the transglutaminase action of factor XIII, resulting in a stable clot in dispersing agent.
In all cases of the reported dysfibrinogenemias except fibrinogen Oklahoma, their functional defects lie in the first and second phases.
However, the abnormality in this case of fibrinogen Tokyo described here lies in the third phase.
It is discovered a new type of congenital dysfibrinogenemia in Tokyo characterized by abnormal stabilization of fibrin with normal concentration in plasma factor XIII.
The formation of cross-linkage in the fibrin clot seemed to be formed normally, but the fibrin clot could be dissolved in 1% monochloroacetic acid or 5M urea solution.
Plasma fibrinogen level is in normal level by the use of both thrombin dependent method and immunological method. Also, thrombin time and reptilase time are in normal range.

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Human citrated plasma was treated with ammonium sulfate at 30-50% saturation, and applied to the column of sephadex G-200. Macroglobulin XaI, which was obtained by gel filtration on Sephadex G-200, was separated to two fractions by CM-cellulose chromatography. One fraction (Fr. A) was eluted with pH 6.0, 0.02M acetate buffer, and another fraction (Fr. B) with 0.06M NaCl in the same buffer by 0→0.2M NaCl gradient elution on CM-cellulose. Rechromatography of Fr. A and Fr. B resulted in the same elution pattern as the first chromatography, namely it was obtained Fr. A and Fr. B from Fr. A, and Fr. B (a) and Fr. B (b) from Fr. B.
These observations have given rise to the speculation that anti-Xa substance in Fr. A may be identical with Fr. B.
Fr. A showed one main band and many other bands on polyacrylamide gel disc electrophoresis, and three precipitin lines besides α₂-macroglobulin on immunoelectrophoresis. While, Fr. B (Fr. B (a), Fr. B (b)) with anti-Xa activity showed one main band and two other thin bands on polyacrylamide gel disc electrophoresis, but only one distinct precipitin line (α₂-macroglobulin) on immunoelectrophoresis. These observations indicate that anti-Xa substance in macroglobulin fraction is α₂-macroglobulin.

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Heparin itself has little or no antithrombin activity and it requires some of plasma protein fractions to neutralize the thrombin activities in its inhibitory mechanism (Antithrombin II, Heparin Cofactor). Thrombin activity was progressively inhibited by Antithrombin III without existence of heparin. Antithrombin II or Heparin Cof actor has an immediate type of antithrombin activity with the existence of Heparin.
Identity of Heparin Cofactor and Antithrombin III, however, is still questionable and there is some discussions on this point, and the assay methods of both Heparin Cofactor (Antithrombin II) and Antithrombin III are not established yet.
Authors established these assay methods according to their definition of both Antithrombins. Heat defibrinated plasma was fractionated by Sephadex G-200 column, and both activities were checked with these assay methods. The highest activities of Heparin Cofactor and Antithrombin III were observed on the third protein O. D. peak. Another peaks were also seen in the first protein peak of the plasma fraction, although both activities in this fraction were lower than those observed in the third protein peak, and each peaks of Heparin Cofactor and Progressive Antithrombin activities were observed in the different fractions.
³H-Heparin was utilized on Gel-filtration with Sephadex G-200. The ³H-Heparin treated plasma or heat defibrinated plasma showed two peaks of radioactivities. The first one is the heparin-protein complex and the other is noncomplexed free-heparin radioactivity. The first radioactivity peak is approximately corresponded with the peak of the first Heparin Cofactor activity which is apart from the first Progressive Antithrombin Activity peak, while this first radioactivity peak was not detected in the elution of ³H-Heparin alone. The Heparin-protein complex in the third protein fraction where the highest Heparin Cofactor and Antithrombin III activity was detectable, was not definite.
According to our experimental results, Heparin Cofactor and Progressive Antithrombin activities appeared in the fractions of late first protein peak. Recently proposed affinity chromatographic technique for the purification of Heparin Cofactor seems to yield the principally different plasma protein fraction from the fraction by gel-filtration and regular chromatographic techniques. However the binding affinity of heparin to plasma protein varies depending on the different heparins, and it is speculated that the Heparin Cofactor or Antithrombin II separation from plasma by affinity chromatography is related to their affinity of the used heparins as a ligand.

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Leukopenia is common in collagen disease, especially systemic lupus erythematosus (SLE). However, the pathogenesis of leukopenia in SLE has not as yet been resolved.
In order to make clear granulopoiesis in SLE, the ability to grow in vitro granulocyte colonies (CFU-c) in 9 patients with SLE including a patient with progressive systemic sclerosis was studied using a semi-solid double agar culture method of Robinson et al. and compared to that of 12 normal controls.
The number of colonies formed in vitro was variable in non-treated SLE. However, the number of colonies formed per plate seemed to increase in the proportion to the increase in marrow M/E ratio. In addition, a case with myeloid hyperplasia was able to generate normal levels of CFU-c indicating that leukopenia in SLE may be due to the abnormality of colony stimulating factor production or release.
On the other hand, the number of CFU-c was markedly reduced in patients with SLE who were during treatment, suggesting that immunosuppressants may affect the growth of CFU-c even in culture.

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