The participation of coagulation/fibrinolysis system in the pathogenesis of renal disease, particularly glomerulonephritis has drawn much attention among specialists of nephrology as well as of blood coagulation and fibrinolysis. And some evidences have been obtained sofar such as
1) Morphologic and immunopathologic studies demonstrating fibrin and F. VIII deposition
2) Histochemical changes in renal cortical plasminogen activator activity
3) Extraction and isolation of plasminogen activator from renal medullary layer
4) Changes in coagulation parameters (fibrinogen, F. VIII, prothrombin, plasminogen and inhibitors)
5) Injuries by DIC on kidney (microangiopathic hemolytic anemia, thrombotic thrombocytopenic purpura
6) Changes in platelet functions and kinin-kallikrein system
7) Elevation in fibrinogen-fibrin related antigens and FDP in serum and urine
8) Deposition of platelet and FDP in kidney
9) Radioisotopic kinetic studies on fibrinogen, plasminogen and platelet (survival)
10) Analogy to experimental models
However, there are remained still many questions and discrepancies for establishing apparent and concrete causal relationship between these reaction systems and development of glomerulonephritis; for instance, on
1) Existence of so many patients in whom pathoclinical signs and changes of coagulation and fibrinolysis do not accord.
2) Possibility for coagulation and fibrinolysis system to be secondary subsequent results to immunologic reactions in glomerulonephritis.
3) Evaluation of the effects obtained by the administration of anticoagulants and platelet suppressive agents to patients, and their reaction mechanisms inside of body.
4) Explanation for changes in coagulation and fibrinolysis system in patients of renal diseases which are induced by infection, intoxication, malignant tumor and pure hemorheological disorders other than immunological reaction.

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This study was carried out to estimate how platelet aggregation might take a part in pathogenesis of diabetic vascular disease. 296 subjects were chosen in our diabetic clinic and examined platelet aggregation induced by two concentration's ADP solution (high concentration: 2μmol/L, low: 0.4μmol/L) with two channel aggregometer. Aggregation curves were divided into four type according to presence or absence of second wave of aggregation.
1) Patients with retinopathy in degree of Scott I-II were observed to have no significant difference compared with age-sex mached diabetic controls. But an eases with retinopathy in degree of Scott III, IV and V significant hyper-platelet aggregation was recognized. Thus, platelet hyperfunction was thought to be a risk factor to proliferative retinopathy.
2) Relationship between platelet aggregation and duration of diabetes was studied. In proliferative retinopathy, diabetes with duration of more than 10 years had higher frequency of hyper-platelet aggregation patterns than below 5 years duration.
3) Platelet aggregation was changeable in three kinds of antidiabetic therapy. Hyper-aggregation patterns appeared to remain in 52.4% of diet therapy group and in 43% of insulin therapy group. But oral antidiabetic therapy group had 66.6% with hyper-aggregation patterns than the others.
4) Aspirin was given as correcting agent to hyper-platelet aggregation to diabetics with ordinarily diabetic therapy. The effect of aspirin on platelet aggregation was difference according to the kinds of antidiabetic therapy. The improvement of hyperfunction was significant in diet and insulin therapy groups. But significant improvement was not obtained in diabetes with oral antidiabetic therapy.
It is suggested that platelet aggregation of diabetes was much influenced by duration of diabetes, degree and severity of retinopathy and kinds of diabetic therapy. And aspirin had not shown the same effect on the improvement of platelet aggregation of all diabetes.

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Cross-linking of fibrin, which is elicited by Factor XIII, is regarded as an important process connecting blood clotting with thrombus formation. Recently attention has been paid to the significance of Factor XIII in the studies on atherogenesis associated with thrombus formation. The purpose of this study is to observe the participation of Factor XIII in cerebrovascular disorders by the determination of plasma levels.
Ninety-nine patients with sequelae of cerebrovascular accidents and 218 non-cerebrovascular controls were studied. The determination of plasma Factor XIII was performed on subunit A and S by means of rocket immunoelectrophoresis.
Among the some plates prepared with different kinds of agars only the one containing ID-1 agarose gave the shape of equilateral triangle indicating the proportional relationship between the area and the height as shown Fig. 1. The concentration was expressed by the height of the triangular precipitate in millimeters.
The levels of patients and controls are shown in Fig. 2. The level of subunit A was significantly lower in patients compared with controls. As shown Fig. 3, the level of subunit A was significantly lower in patients with poor marks in the test of activities of daily livings (ADL-T) compared with those with good marks. Subunit A was directly proportional to subunit S in patients and controls. Fig. 4 showed inversely proportional relationship between subunit A and fibrinogen in patients.
The low level of subunit A in patients, especially in the patients with poor ADL-T values showed the importance of Factor XIII related to the production of connective tissues and repair of injured tissues. The direct proportion between subunit A and S, and no relation of subunit S to clinical findings indicated the functional difference in two subunits as previously suggested by some authors. Relation of Factor XIII to atherogenesis could not be clarified in this series, and there were many disagreements concerning Factor XIII on sclerotic arterial tissues. Therefore, further versatile observations of Factor XIII relating to arteriosclerotic disorders should be performed intensively.

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Fibrinopeptide A (FPA) is a peptide released from N-terminal ends of Aα chain of fibrinogen molecule in initial stage of fibrinogen-fibrin conversion. It is, therefore, considered that the determination of FPA in blood or urine is able to be a direct proof of fibrin formation occuring in vivo.
The authors studied the radioimmunoassay of FPA of blood and urine in normal adults as well as in patients with renal diseases. In determination of FPA, FPA-antiserum, standard FPA and tyrosyl-FPA obtained from IMCO. Co. Ltd. (Sweden) were used. Procedure of FPA assay was accorded with the method developed by Nossel, et al.. The authors ascertained by preliminary experiment that the Nossel's method could be applied to urinary FPA determination. Our results indicated that, 1) FPA values in clotting reflected strongly to thrombin action. A certain level of FPA-immunoreactivity of fibrinogen split products by plasmin (FgDP) was recognized. In purified in-vitro system, FPA-immunoreactivity of FgDP was 20% when compared to the fibrin clotting system of 100%. While, almost negative (0.3%) immunoreactivity was observed in fibrin split products by plasmin (FDP). 2) Mean and range of blood FPA values of 30 normal adults was 0.3±0.4ng/ml, and this was well closed to the values reported by Nossel, et al.. 3) Urinary FPA values of 33 normal adults were 0.8±0.7ng/ml. It was noted that the urinary FPA of patients with renal deseases showed higher values in many cases in contrast to the normal urine.

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The blood coagulability of 28 chronic non-hemodialysed renal failure patients was investigated. Systemic diseases such as systemic lupus erythematosus or diabetes mellitus were excluded because of their own coagulation abnormalities. All patients were in uremic state. Fasting blood samples were drawn from antecubital vein early in the morning with 3.8% sodium citrate as the anticoagulant. When not measured immediately, samples were stored at -80°C. Following assessments were performed: PT, APTT, TEG (whole blood method), fibrinogen, factor II, V, VII, VIII, IX, X, XI, XII procoagulant activities, factor VIII related antigen (Laurell's method) (VIII_ANG), antigen levels of antithrombin III ATIII), and α₂ macrogloburin (α₂MG) with Mancini's method, serial dilution protamin sulfate test (SDPS), ethanol gelation test (EG), high molecular weight fibrinogen complex (HMWFC) (gel filtration method), fibrin/fibrinogen degradation products (latex aggregation method) (FDP). BUN, creatinine, blood pH, total CO₂ and hematocrit were also determined as the indicators of uremic state. Results were as follows. Shortened results were obtained in APTT (p<0.001), TEG-r (p<0.001) and TEG-K (p<0.001). Increased results were obtained in TEG-ma (p<0.001), fibrinogen (p<0.001), factor V (p<0.05), factor VII (p<0.05), factor VIII (p<0.001), factor IX (p<0.01), VIII_ANG (p<0.001), α₂MG (p<0.02), FDP (p<0.01), HMWFC (p<0. 001). Positive EG is in 14 of 28. SDPS ×5-11, ×10-14, ×20-3. Mean BUN, creatinine, blood pH, total CO₂ and hematocrit were 105±27mg/dl, 12.2±3.9mg/dl, 7.28±0.06, 14.1±3.6mM/L and 20.5±4.8%, respectively. These results indicated that hyper coagulable state and low grade tendency of intravascular coagulation existed in uremia. The precise mechanism of intravascular coagulation was unknown. But increased level of VIII_ANG might be indicative of endothelial damage, because VIII_ANG was synthesized in vascular endothelial cells. More details remained to be elucidated.

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It has been previously reported in our paper that plasma or serum soluble fibrin in the patient with acute stage of nephritis was increased and its changes were related to some extent to disease activity observed by proteinuria and hematuria. In the present paper, the components of soluble fibrin in acute stage of nephritis were studied and it was possible that the soluble fibrin consisted mainly of fibrin monomer and fibrinogen. Furthermore, the clumping mechanism between SCT, which showed high titers in acute stage of nephritis, and fibrin monomer-fibrinogen complex (FF-complex) was investigated.
In order to study the components of soluble fibrin in the serum of nephritis, test samples such as soluble fibrin rich serum (SF-serum) and FF-complex containing solution (FF-solution) were prepared in vitro by the following method and their characteristics were compared with serum of the nephritis patient. The mixture of one tenth volume of fibrin monomer and normal plasma was incubated with thrombin solution (F. C. 5u/ml) containing t-AMCHA (F. C. 4.5%) and the supernatant obtained by centrifugation was used as SF-serum (SCT 320μg/ml, HIT 20μg/ml). FF-solution was made by adding 1/10 volume of fibrin monomer to fibrinogen which was previously fractionated by Sephadex G-200. After treating FF-solution with thrombin and t-AMCHA, fibrin monomer and FF-complex remaining in the supernatant (FF-supernatant) showed 256μg/ml by SCT and 16μg/ml by HIT.
Three samples such as nephritis serum, SF-serum and FF-supernatant were chromatographed by DEAE Sephadex A-50 using a stepwise salt gradient elution technique with 100mM, 200mM and 400mM NaCl, respectively. In each fractions eluted with 200mM NaCl of three samples showed positive (2+) by SCT while negative by HIT (Fig. 1). SDS-polyacrylamide gel electrophoresis of the same fractions displayed protein bands which migrated slower than purified fibrin monomer as well as purified fibrinogen which was obtained by DEAE Sephadex A-50 chromatography (Fig. 2). This high molecular weight bands disappeared after the procedure of clumping by staphylococci. Normal serum did not show protein bands which corresponded to this FF-complex.
Next, using SDS-disc electrophoresis it was studied whether staphylococci could bind fibrin monomer and FF-complex or not. After the interaction between FF-supernatant and staphylococci, the protein bands of both fibrin monomer and FF-complex disappeared as shown by scanning densitometer (Fig. 3).
It is suggested that soluble fibrin in the serum of the patient with nephritis is composed of fibrin monomer and fibrinogen and that this FF-complex could be clumped by staphylococci.

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This study was carried out to know whether abnormal factors of platelet and coagulo-fibrinolysis had influence on the prognosis of acute myocardial infarction. Seventy four subjects admitted to our CCU within 24 hours after attack were selected, and the blood samples were periodically withdrawn more than four times during their hospital stay. Platelet aggregation induced by two different concentration's of ADP, and fibrinogen, plasminogen and proteinase inhibitors as main factors of coagulo-fibrinolysis by single radial immunodiffusion were measured in same blood sample.
ELT and UK activated ELT which was obtained to add each of 0.1, 0.2 and 0.4 units of UK to euglobulin fraction from original plasma, and the percentage of shortening rate of ELT with UK represented as UK-ELT. The results of blood analysis were compared between the survived and the dead cases. Although platelet aggregation showed the hyperaggregation type in almost cases immediately after attack, but the survival cases were improving toward normoaggregation type following clinical course.
However, the platelet aggregation of death cases still remained in aggrevated type and a few returned to normal type. The shortness rate of 0.4 unit's UK activated ELT to exist a significant difference between both groups.
By measurement of 0.4 unit's UK activated ELT, the lysis time of death cases showed less response to UK compared with that of the survival clearly activated with UK.
Although fibrinogen and alpha 1-antitrypsin showed the same progress pattern after attack, two parameters of the death were remaining in higher level than that of the survival. On the contrary, alpha 2-macroglobulin of the survival was decreasing to low level following clinical course and the increase of alpha 2 macroglobulin was suggested to show rather the worse prognosis. Plasminogen and antithrombin III were not valeable to estimate the prognosis because there was no difference between both groups.
The significant findings in the death after attack were found to be the increase of fibrinogen, high level of alpha 2-macroglobulin and no response of UK activated ELT.

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In 6 patients with Fallot (4 cases), endocardial cushion defects (1) and annuloaortic ectasia (1), laboratory evidence of the disseminated intravascular coagulation (DIC) syndrome was found at early time after open heart surgery. Though there have been many of the etiologic factors contributing to the production of DIC following cardiopulmonary bypass, in this study polycythemia, low cardiac output or shock and sepsis in addition to a long term bypass and hemolysis due to it complicated the factors.
In all patients a bleeding tendency occured. The clinical manifestations of this hemorrhagic diathesis included bleeding from venipuncture sites, petechiae, ecchymoses, hematuria, tracheobroncheal bleeding and gastrointestinal bleeding. 2 patients had peripheral vasoconstriction with peripheral cyanosis. In 2 patients renal failure was found and in one necessitated hemodialysis.
All patients were done administration of heparin as well as appropriate therapy of any underlying etiologic factors. 4 of the 6 patients eventually recovered. In laboratory evidence, platelet count, prothrombin time, fibrin degradation products and thrombelastogram returned to normal levels.

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The authors are now engaged in a chronic experiment of left heart bypass on calves weighting 80-100kg. In the conduct of such a mechanical assistance procedure it is important that post-operative thrombogenesis should be prevented and therefore an appropriate anti-thrombogenic therapy should be administered by the use of some method. The authors first attempted general heparinization by one-shot intravenous injection of heparin, but in this method it was difficult to maintain a constant heparin level and thus extensive bleeding tendency was frequency observed. In order to prevent the risk of bleeding due to general heparinization and to remove the possibility of thrombosis within the bypass circuit it would be an ideal method to maintain a non-clottable condition only within the bypass circuit. To satisfy this purpose it was considered that local heparinization by which heparinization would be confined as much as possible to the bypass circuit and blood neutralized by protamine would be circulated throughout the body would be effective. The authors therefore attempted a method in which heparin was continuously injected into the inlet side of the bypass circuit and protamine was continuously injected into the outlet side of the circuit so that heparinized blood would be neutralized. At present, as postoperative anti-thrombogenic therapy this local heparinization method is employed during the first post-operative week. Heparin at a dose of 25U/kg/h and protamine at a dose of 10-25U/kg/h are continuously injected and the prothrombin time is controlled at a level 1.5-2.0 times that of normal value, providing comparatively satisfactory anti-thrombogenic effects. However, this method is somewhat complicated and there is a danger that accident might develop through mixture of air. Therefore, as a safer and simpler method the authors have attempted an anti-coagulation procedure of orally administered Warfarin from the first post-operative week.
This method has been conducted on 7 cases. As it was somewhat difficult to control the prothrombin time within the desired range by Warfarin, the prothrombin time was slightly extended beyond that observed during local heparinization, but bleeding tendency was not observed in the animal. Adequate and safe anti-thrombogenic effect was obtained without any special side effect due to Warfarin.

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Plasma von Willebrand factor (vWF) was determined in various vascvlar diseases.
MATERIALS and METHODS: A new method without utilizing a platelet aggregometer was employed to assess plasma vWF 1) in 20 normal healthy males (21-60 years, 35.7±2.24 (S. E.) years) to know the change in plasma vWF with age, 2) in 15 patients with diabetes mellitus who do not have macroangiopathy (6 males and 9 females, 38-75, 56.9±2.80 years, Scott 0-II: 10, III-IV: 5 cases), 3) in patients with myocardial infarction (6 cases in acute, 5 in convalescent stage), cerebral thrombosis (3 in acute, 2 in convalescence), arteriosclerosis obliterans (ASO) (6 in stage IV (Fontaine), 1 after operation) and with deep vein thrombosis (2 cases) in whom the plasma vWF was compared with the age-and sex-matched controls, and 4) in 16 patients with ischemic heart disease (9 males and 7 females, 39-73, 56.9±2.72 years) before and after isometric handgrip exercise (50% voluntary maximal contraction, 2 minutes). Platelet sensitivity to ADP-aggregation (Sano et al. Thrombosis and Haemostasis 37: 329, 1977) was examined concomitantly in patients with ischemic heart disease.
The new method for measuaring plasma vWF level is as follows. Serially two-fold diluted plasma (2 to 1024 times, in Tris-saline pH 7.2 containing 12mg/ml bovine serum albumin), fixed and washed platelet suspension (6×10⁵/μl, Macfarlane et al. 1975) and 3mg/ml ristocetin were mixed (25μl each) in Microtiter tray and agitated for 15 seconds. The maximal dilution to induce platelet aggregation was obtained microscopically and defined as the titer of plasma vWF (Fig. 1).
RESULTS: 1) Plasma vWF titer correlated with age in healthy males. The regression line was n=2.18+0.06 Age when vWF titer was 2ⁿ (r=0.56, p<0.05).
2) Plasma vWF titer was significantly higher in diabetics with advanced microangiopathy (Scott III-IV) (n=6.0±0.32 when vWF titer was 2ⁿ) comparing to the healthy controls (n=4.3±0.20) (p<0.005). In Scott 0-II group (n=3.7±0.26) no significant difference was observed comparing to the control.
3) In acute stage of myocardial infarction and stage IV of ASO, vWF titer was significantly higher (p<0.05) comparing to the controls (Fig. 2).
4) In ischemic heart disease patients, plasma vWF titer and platelet sensitivity to ADP-ag-gregation became significantly enhanced (p<0.005) after isometric exercise. In healthy subjects no enhancement was observed (Fig. 3).
COMMENTS: The results suggest the close relationship between plasma vWF and vascular diseases.

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Platelets in circulation are not homogenous in size, density, metabolism and responsiveness to ADP or collagen. The platelet carries a net negative surface charge and its change may be an important factor in platelet adhesion and aggregation. Platelet volume has also been related to the functions. In this paper role of the platelet volume and the electrophoretic mobility in thrombus formation was studied.
Materials and Methods: Twelve male rabbits of Japanese white strain were used. Eight were controls and 4 were injected with ADP 2mg/kg into the central vein in several seconds and pulmonary thrombosis was induced under an intravenous pentobarbital anesthesia. Blood, 50ml, was withdrawn from both groups into EDTA containing syringes through a femoral arterial cannula. Platelet rich plasma was washed twice in Tris-NaCl buffer and suspended in 3mM triethanolamine acetate buffer. Platelet electrophoresis was performed with a Continuous Free-Flow Electrophoresis VAP-5 (Bender & Hobein, Munich) at 18°C, 1, 250V and 80mA. Approximately 5×10⁸ platelets were electrophoresed per hour. The fraction #1 is an anode side and #90 a cathode side. Platelets in each fraction suspended in the buffer were studied for platelet count and volume determinations by a Coulter counter ZbI and a channelyzer C-1000.
Results: Control platelets were separated well making a single peak at the 20.9±2.6 fraction (Fig. 1). In acute thrombosis the peak was at the 15.5±1.4 fraction (Fig. 2) and the curve is shifted to the anode side as compared with the control (p<0.10). The mode platelet volume increased as the fraction number increased in the control (Fig. 3), but this trend disappeared in the thrombosis and the mode volume was almost same throughout the fractions at the level of anode side fractions of the control (Fig. 4).
Discussion: Hampton and Mitchell reported that platelets exposed to ADP of low concentration increased the electrophoretic mobility and this might be the explanation of the increased mobility of the platelets which were saved from thrombus formation. The reduction in electrophoretic mobility of the platelets of larger volume could be caused simply by an increase in size and density or a redistribution or a decreased amount of anionogenic groups on platelet surface. Reported experiments (Brinton and Lauffer, 1959) make it less likely that the slow mobility is due to the large size. Disappearance of the platelet with large volume and low electrophoretic mobility would be caused by a selective consumption into the thrombi.
Summary: 1. Increase in electrophoretic mobility was observed in platelets of the ADP-induced thrombosis. 2. Larger platelets carry low electro-phoretic mobility, 3. Platelets with large volume and low mobility were most likely consumed selectively into the thrombi.

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Spontaneous platelet aggregation (SPA) has been recently reported to occur in patients with occulsive diseases. The clinical implication of SPA is however not clarified yet. To investigate the clinical importance and mechanism of this phenomenon, 287 patients who were referred to us for platelet aggregation study were, studied. SPA was positive in 19 patients, 5 in cerebral thrombosis, 3 in Behçet disease, 2 in angina pectoris and 5 in miscellaneous thrombotic disorders.
Platelets from these patients can aggregate with extremely small amount of aggregating agents such as ADP, epinephrine and collagen. Maximum aggregation of SPA depends on platelet counts in platelet rich plasm. On several occasions, SPA became significant only after platelet rich plasma was kept at room temperature for 1-2 hours. SPA was markedly inhibited by apyrase at a concentration of 0.1mg/ml. Aspirin intake at a dose of 500mg daily for one week abolished SPA. Electrophoretic mobilities of platelets from 2 patients with Behçet disease who had SPA were significantly reduced. SPA platelets showed no changes in sialic acid levels or membrane glycoprotein profiles on SDS polyacrylamide gel disc electrophoresis. Malondialdehyde levels of SPA platelets were higher than normal platelets.
Our study clearly indicates that SPA was frequently found in thrombotic disorders. SPA platelets were more sensitive to aggregating agents, and release of ADP plays a role in development of SPA. The mechanism of SPA remained unsolved, but decreased electrophoretic mobility and elevated malondialdehyde level of SPA platelets may give some clue for further study.

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The changes of fibrinolytic factors (especially plasminogen, α₂ macroglobulin, α1 antitrypsin and antithrombin III) were studied in thrombosis, collagen disease and collagen disease like disorders. Five cases of Buerger disease with open ulcer in legs, five cases of SLE (inactive phase) and six cases of collagen disease like disorders (two of MRA, one of arotitis, PSS, Wegener granulomatosis and ulcerative colitis) were investigated. Plasminogen was estimated by means of both enzymatic activities and immunological procedure. Enzymatic activities were estimated by calculating lysis area on bovine plasminogen free fibrin plate, in which euglobulin fraction with added SK or UK was dropped. Immunological procedure was done by single radial immuno-diffusion method. α₂ macroglobulin, α₁ antitrypsin and antithrombin III were also measured by immunological method.
The results were as follows:
(1) In collagen disease and Buerger disease, plasminogen, which being estimated by immunological method, showed a high level. But, in those diseases, it showed a tendency to decrease of plasminogen, which was measured by enzymatic method. In above findings, it could be suggested that, an increase of inhibitors of fibrinolytic system in euglobulin fraction was seen in collagen disease and Buerger disease. The value of plasminogen was within normal limits in collagen disease like disorders.
(2) In Buerger disease, collagen disease and collagen disease like disorders, the value of α₁ antitrypsin and fibrinogen showed a tendency to increase.

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Defibrinogenation therapy is expected to be a new type of anticoagulant therapy without serious bleeding complications. However, it appears to be dangerous to perform surgical procedure in the defibrinogenated state.
In order to investigate the causes of the bleeding tendency which occurs during defibrinogenation therapy, the following clinical study was undertaken.
Twelve patients were treated with Batroxobin (Bothrops atrox moojeni and marajoensis). Eight out of 12 patients were suffering from venous thrombosis of ilio-femoral vein. Another 2 patients were suffering from arteriosclerosis obliterans (ASO). And finally, 2 patients were suffering from cerebral artery thrombosis.
Batroxobin was given in doses of 30 to 115μl/kg/day diluted 200 to 500ml of phisiological NaCl-solution as intravenous infusion for 2 to 8 hours. Eight out of 12 patients were treated concervatively with Batroxobin and Urokinase. On the other 4 patients, surgical procedure was performed during defibrinogenation therapy.
On 8 patients who were treated concervatively with Batroxobin and Urokinase, Red blood cell and platelet counts were not changed. Plasma fibrinogen concentration decreased between 40 to 80mg%. FDP level tended to be moderate after the 3rd or 4th infusion. Platelet adhesiveness and ADP-induced platelet aggregation was not decreased markedly. No bleeding complications were found. The other 4 patients were operated during defibrinogenation therapy. Red blood cell, platelet counts, platelet adhesiveness, and ADP-induced platelet aggregation were not changed. Fibrinogen concentration ranged between 0 and 80mg% at the time of the operation. There was no bleeding tendency on these patients with the exception of patient No. 12.
Tracheostomy was performed as an emergency measure on the patient No. 12, 3 hours after the end of the Batroxobin infusion (It is not considered the Batroxobin to have played any causative role). Fibrinogen concentration was decreased beyond measure ability during and after the operation. There was severe bleeding from the wound.
It is concluded from these results as follows:
(1) Platelet adhesion to the grass and ADP-induced platelet aggregation are not influenced extremely by the decreased fibrinogen concentration during the defibrinogenation therapy.
(2) Batroxobin could be used as a conservative therapeutic drug without haemorrhagic risk even if fibrinogen concentration is markedly decreased.
(3) To insure haemostasis, it is necessary to control the fibrinogen concentration above 60mg% during and after the operation.

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The membrane-associated particle and openings of canalicular system were observed under electron microscopy utilizing a freeze-etching method on the platelets obtained from healthy subjects, diabetic patients and the patients with systemic lupus erythematosus.
The buffy-coats were obtained from venous blood added sodium citrate by centrifugation with 650×g for 3min at room temperature. The materials were fixed with 2.0% glutaraldehyde for 120min and were cryopreserved with 20% glycerol buffer.
A freeze-etched method was performed in the JEOL-FE apparatus under the conditions of 3×10^-6Torr at -155°C for fracture followed by etching for 3min and shadowing with the platinum-palladium-carbon. The replicas were cleaned thoroughly with sodium hypochlorite solution and redistilled water, and then were observed under the JEOL JEM-100C electron microscopy.
The membrane-associated particles distributed diffusely on the surface of platelet membrane of healthy subject as well as of diabetic patient although the diameters of the particles were elongated to 135±18Å (M±SD) in the diabetic cases from 75±27Å in the healthy subjects. The openings of the open canaliculus on the membrane surface of diabetic patients can be recognized as a double-ringed craters having a diameter of 408±117Å (ranged from 170 to 590Å). These openings assembled geometrical patterns which were composed of an angle of 115±32 degree. The openings were recognized by chance to be connected to the canalicular structures on the fractured cytoplasm. The mitochondria and granules located also in the network of these tubular systems. Thin-sectioned electron microscopy revealed an evidence that the opening at the cell membrane was continuous to the open canaliculus. These findings suggests the canaliculus combined three-dimentionally membrane surface with both mitochondria and granules of platelets.
The platelet membranes of the patients with systemic lupus erythematosus had a diameter of 130±30Å showing a larger deviation of the sizes of membrane-associated particles than those of both healthy subjects and diabetic patients. Moreover patched elevations, may be the immune complex, were observed on the external surface as well as on the internal surface associating a ruffled wavy changes of surface itself.
These ultrastructural alterations of platelet membrane might be related to the functional state of platelet in vivo. This should be clarified in near future.

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Fibrin associated with thrombus formation might be derived from both plasma and platelet fibrinogen. In the experiment I, we intended to induce fibrin formation from platelet-fibrinogen using washed human platelets by adding thrombin. The behavior of platelets under the effect of the agent was examined by means of electron microscopy in combination with freeze-etching technique in special referrence to the canalicular system. In the experiment II, inhibitory effect of antimycin A and iodoacetic acid on the aggregation of platelets was investigated by Chandler's loop and aggregometer (Sienco). The detail of experimental materials and methods was shown in table 1-A.
Experiment I: The group numbers used below were corresponding to those described in table 1-B.
In the group (1): The platelet was round or spindle in shape with a few short pseudopods. The cytoplasmic organelles were relatively well preserved except for a slight dilation of the canalicular system.
In the group (2): The platelet had marked pseudopods and discharged the contents of α-granule. Electron dense strands of fibrils and fibrillar structures appeared in the conspicuously dilated canalicular system. Some of the structure had periodic bands about 230Å wide correspoded to those of fibrin. When the structure absent from the periodicity was examined with a goniometer, the band was not observed. The periodic structure about 180Å wide also appeared in a few α-granules. Replica showed a marked dilation of the canalicular system being connected with each other by many branches and a few spheroid resembling α-granule.
In the group (3) and (4): The swollen platelet had almost intact organelles without an appearance of the periodic structure.
Experiment II (table 1-C): When antimycin A (250, 25ng) alone was used, the thrombin-induced aggregation of platelets was moderately inhibited. Both mixture of antimycin A and iodoacetic acid and iodoacetic acid (2, 0.2mM) alone absolutely inhibited the aggregation.
In the process of release reaction induced by thrombin, periodic structures were visible in the canalicular system and α-granule of the platelet. On the other hand, the structure did not appear anywhere when the platelet was exposed to thrombin after the mixture of antimycin A and iodoacetic acid as an inhibitor.
It seems to be concluded that the fibrillar structure with periodic bands was certain fibrin derived from platelet-fibrinogen. It also seems that the canalicular system plays a role as a conduit of release reaction for the structure regardless of the presence or absence of the band.

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Scanning electron microscopic observation of morphological change of platelets when calcium (Ca) was added to platelet-rich plasma (PRP) was carried out in healthy, patients with hemophilia and thrombocythemia.
1) In PRP, many platelets showed pseudopods or aggregation more or less. This may be attributed to stimulation on platelet at blood collection and centrifugation. But platelets with severe abnormality in thrombocythemia showed on pseudopods at all. 2) When fluid was moved after addition of Ca to PRP, platelets was transformed physiologicaly and showed “cherry blossom-like” in generaly. 3) In the case of hemophilia, fibrin-nets were produced independently from the platelets or products of transformed platelets. 4) Morphological change of platelets when Ca was added to PRP of thrombocythemia were multifariousness. When Ca was added to PRP of thrombocythemia, moved and fixed at a proper good time, a few fibrin-like markedly long pseudopods were seen in platelets.
Even morphological changes that could not be recognized in normal could be inferred by using PRP of hemophilia and thrombocythemia.

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The effects of both calcium ionophore (A23189) and prostagrandin E₁ (PGE₁) on the clot retraction were observed in relation with intracellular contractile protein, and the following results were obtained: 1) Calcium ionophore (A23187) intensified the clot retraction under the enough presence of Ca⁺⁺ outside of platelets. 2) Prostaglandin E₁ suppressed the clot retraction and this effect depended upon PGE₁ concentration and the reaction-temperature. 3) When prostaglandin E₁ and calcium ionophore (A23187) were added to platelets simultaneously, the suppressing effect of the former was much stronger than the intesifying effect of the latter. 4) Electron microscopically, platelets in a retracted clot incubated with calcium ionophore (A23187) showed the aggregation in some part and the transformation in other part in accompanied with filamentous network in their cytoplasm which could react with exogenous hevy meromyosin. On the electron micrograph of platelets in a retracted clot incubated with prostaglandin E₁, no significant figures could be observed except the degranulation at some part.
From above results, it is considered that the clot retraction phenomenon is induced by the activation of intracellular contractile proteins depending on Ca⁺⁺ and C-AMP.

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Aceton-dried powder of equine platelets was extracted with 10mM carbonate buffer at pH 10.5 The extracted protein was precipitated at its isoelectric point (pH 4.45) and followed by ammonium sulfate precipitation (15-35%) to make pure.
The purified protein showed the characteristics as follows: 1) The relative viscosity of the pH 10.5 extract was increased after dialysis against the buffer at pH 8.0, but the addition of KCl and MgCl₂ caused an immediate decrease in viscosity. 2) Under an electronmicroscope, the short tubular structure was observed in both positive and negative stained samples at pH 8.0. And the addition of salt caused a side-by-side aggregation. 3) The purified protein incubated with platelet myosin-A showed a low superprecipitation activity at low ionic strength, in the presence of Mg⁺⁺ and ATP, but the degree was much weaker than that of synthetic platelet actomyosin. 4) On the basis of its electrophoretic mobility, the molecular weight was calculated to be about 50, 000. From those results, it seemed that tubulin-like protein was extracted from aceton-dried powder, though an immunochemical demonstration was not carried out.
By our method described in this paper, it became possible to reserve the sample much longer, so that it is expected that the biochemical study about microtubulus in blood cells will be made more progress.

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The number of residual free single platelets was counted in the course of platelet aggregation reaction for the purpose of studying chemical kinetics analysis of platelet aggregation by ADP.
The change in number of single platelets agreed with the function:
single platelets k→aggregates
[P]=[P₀]e^-kt, ([P]: the number of single platelets, [P₀]: the initial number of single platelets, k: rate constant, t: time)
and rate constant “k” was measured to know the degree of platelet aggregation. [P] was measured by electric particle counter (Coulter Counter Model ZBI) under the condition of aperture tube: 70μ, aperture current: 1/4, amplification: 1/4, matching switch: 20K and monometer position: 0.5ml. Upper and lower thresholds were selected to include the main peak on platelet volume distribution curve.
By this method some results were obtained as follows. “k” was constant on the various concentrations of platelets from same blood. “k” changed in proportion to the degree of stir of reaction mixture. “k” was constant when ADP concentration was more than 10^-6M. But “k” was logarithmically decreased between 10^-6 and 10^-7M of ADP, and got toward zero when ADP concentration was less than 10^-7M.

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Preparation procedures of collagen suspension in testing platelet aggregation by Born's method are presented a great deal, but there are few reports on the concentrations of collagen in detail.
Recently, we are able to obtain easily a commercial reagent of collagen suspension (Collagenreagent Horm^R, Hormon-Chemie München, ETC), and then would like to try to be standardized in its method. ETC contains 1mg per ml of native collagen fibrils from equine tendons suspended in an organic isotonic buffer of pH 2.7. Collagen suspension from bovine achilles tendons (Sigma, BTC) treated with 0.03N HCl was prepared as control, which contains the same concentration of collagen as ETC. ETC was used straight from the vial, not diluted with buffer as diluting medium.
1) Each concentration of BTC and ETC requires 20μg, 10μg per ml in platelet rich plasma (PRP) at final concentration respectively, which show about fifty percent of aggregation values after five minutes adding each reagent.
2) Normal ranges (mean±S. D.) of BTC —and ETC—induced aggregation were 48.6±15.6%, 52.0±12.5% each other.
3) We obtained the good reproducibility of these reagents, but there was no difference between BTC and ETC in reliability. The lag times (mean±S. D.) in aggregating process were 47.8±10.2 seconds in BTC and 16.3±1.3 in ETC.
4) ETC was fairly stable against heating or freezing than BTC, and no change in aggregating activity of ETC in storage at 4°C was recognized for at least a half year. In this period, we did not see any coarse fibrils of collagen.
5) Examing the sensitivity of reagents to collagenase, aggregating activity of ETC was more decreased than that of BTC, and then it was suggested that ETC might be microfibrillar collagen.
6) The effects of sucrose on polymerization of monomer collagen were tested and activities in these reagents were reduced with corresponding the dose of sucrose, but no difference was detectable between them.
7) We investigated the sensitivities of these reagents to platelet aggregation in 13 samples from patients with aspirin or other antithrombotic drugs ingested and one patient from Glanzmann's thrombasthenia, but there was no difference in aggregation values between these reagents.
These data suggest that ETC is available to standardize the test af collagen—induced platelet aggregation.

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Since heterogeneity of circulating platelets is present, we have analyzed what kind of platelets and what factors in platelets do play an important role in thrombogenesis. Using 51 healthy young men and women (22.2±4.6 years in mean±SD), 17 patients in acute stage of thrombosis such as myocardial infarction or cerebral thrombosis (The two-week period following the onset of thrombosis was called the acute stage), 19 patients in the recovery stage of thrombosis and 8 DIC patients, their platelet volume, aggregability and adenine nucleotides content were measured. In healthy volunteers, platelet count was 30.1±5.9×10⁴/μl, platelet mode volume was 4.21±0.56, mean volume was 5.32±0.42 and median volume was 4.66±0.47μ³. There was a significant correlation between platelet mode volume and 5 minutes aggregation induced by 3μM ADP (r=0.375, p<0.02). On the one hand, there was not significant correlation between platelet volume and slope of aggregation curve, intensity of primary and secondary aggregation. In acute stage of thrombosis, platelet volume (mode, mean and median) was significantly smaller than those of the healthy (p<0.05). In recovery stage of thrombosis, platelet volume, especially mode was larger than those of the healthy. In DIC, platelet mode volume was significantly smaller than that of the healthy (p<0.05), while the difference was not found in mean and median volume. ADP content in platelets was significantly decreased in acute thrombosis (p<0.05), while ADP and ATP contents were significantly decreased in DIC as compared to those in the healthy. There was a significant correlation between platelet volume and their adenine nucleotides contents (r=0.347, p<0.025 in ATP and r=0.537, p<0.05 in ADP). Platelet aggregation decreased in DIC and increased in acute thrombosis.
These results suggest that large platelets have a large amount of adenine nucleotides and easily are consumed in thrombus formation. In thrombus formation, there may be an enhancement of release phenomenon in platelets.

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Glass bead column methods are widely employed for the estimation of the adhesiveness of the blood platelets. However, light and scanning electron microscopic studies as well as biochemical studies have shown that the retention rate by these methods represents not the pure adhesion but a combination of adhesion and aggregation. In order to devise a method to estimate more pure adhesiveness, a series of experiments were performed on inhibiting the aggregation in the glass bead column and on obtaining an adequate normal range. The platelets on the beads were examined by scanning electron microscopy by our method and the retention rate was calculated as usually.
EDTA-2K (0.1% at final concentration) was proved to almost completely inhibit the aggregation or to dissociate the aggregates on the beads in the columns by Hellem II method as in an aggregometer, whereas adenosine did not so completely. The adhered (spread) platelets, however, were changed to spheres with pseudopods by EDTA, but spread again after washing out EDTA by infusion of saline. This suggests that the platelets retained in the column in the presence of EDTA are adhered ones. The adhesiveness (retention rate) with EDTA-blood was elevated with the increase of length of the column (glass bead amount) and also with the decrease of the infusion speed.
To minimize the blood amount neccessary a short and thick tube, 2.5ml in inner capacity, was connected to a 80cm long column, 3mm in inner diameter, containing 7.8gr of glass beads with diameter of 0.5mm. At measurement about 2ml of EDTA-blood was injected to fill the short tube and a disporsable syringe containing 0.85% saline was connected to the tube and set on a Harvard infusion pump 975. The EDTA-blood was thus indirectly infused by the saline at speed of 27sec/ml into the long column which was held perpendicularly to avoid mixture of blood with saline. The retention rate was calculated from the platelet counts in the control sample and in the first 1ml of the effluent from the column. The retention rate was 50-80% in normal subjects. This method is hoped to give us a new information on the behaviour of normal and pathological platelets.

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Basic problems relating to the clinical application of acetyl salicylic acid (aspirin) as an antithrombotic agent were studied. In patients with cerebral thrombosis, myocardial infarction, diabetes mellitus, etc., collagen induced platelet aggregation and malondialdehyde (MDA) production by their platelets were determined after single ingestion of various amounts of aspirin. Ingestion of 300mg of aspirin resulted in the maximum inhibition of both MDA production and collagen-induced aggregation. (Fig. 1)
In all but one patients who ingested 300mg of aspirin every three days, stable inhibition of collagen-induced platelet aggregation was obtained during whole period of observation. (Fig. 2) Ingestion of more dose of aspirin or more frequent ingestions did not enhance the inhibitory activity for collagen-induced aggregation.
In four patients with cerebral thrombosis and one patient with myocardial infarction, the life span of platelets measured by method of Stuart, M. J., et al. were shorter than that in healthy persons. (Fig. 3)
From these findings, it is suggested that platelets are consumed more rapidly in these patients than in healthy persons, and that the peroral administration of 300mg of aspirin every three days is sufficient to inhibit the collagen-induced aggregation of platelets in most cases.

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Much evidence suggests an influence of estrogen on platelet functions. In the present study, effects of physiological change in estrogen concentration on platelet aggregability were examined in 20 healthy young men (23-29 years), 42 healthy young women with normal menstrual cycle (17-22 years) and 38 women at the 7th to 40th week of pregnancy (18-35 years). Citrated platelet rich plasma was obtained and platelet aggregation induced by 10 and 3μM ADP (Sigma), 1 and 0.1μg/ml of adrenaline and 3 and 1μg/ml of collagen (Hormon Chemie) was measured using Sienco aggregation meter. In 42 non-pregnant women, blood was collected at their menstruation (mestrual phase), 12 to 16 days (follicular phase) and 3 to 7 days (luteal phase) before the next comming menstruation. Parameters measured in the aggregation curve were slope of initial aggregation, intensities of primary, secondary and 5 minutes aggregation and appearance rate of secondary aggregation induced by high and low doses of ADP and adrenaline. Lag time was measured in collagen-induced aggregation curve also.
During menstrual cycle and pregnancy, primary aggregation did not change in intensity or speed. Also secondary aggregation did not change in intensity. However, secondary aggregation induced by ADP and adrenaline frequently observed in follicular phase and less in menstruation and luteal phase with a statistical significance. In pregnant women intensities of primary and secondary aggregation were not different with those of non-pregnant women. However the appearance rate of secondary aggregation markedly increased during pregnancy. Collagen aggregation did not show any difference among any groups.
The result suggests that estrogen, which increases in the blood both in pregnant and follicular phase, might have a role in the appearance of secondary aggregation which is related to platelet release reaction.

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Although the question whether oral contraceptives are casually involved in thromboembolic disorders remains a very contested issue, a growing number of studies show that a state of hypercoagulability does exist in users of oral contraceptives, and there seems to be general agreement that factors II, VII, X and fibrinogen are increased and antithrombin III (AT III) decreased. Reduction in the level of this inhibitor in this case has been repeatedly confirmed both by coagulant and immunological methods. Many familial cases of thromboembolic disorders due to AT III deficiency have been reported and one of authers showed hyper sensitivity of platelet to thrombin. The recent observation by Fukutake and Nagasawa et al. showed the reduction of AT III measured by coagulant and immunological methods after the addition of estrogen to the plasma in vitro.
Our test system was consisted with platelet, AT III and thrombin. It was set up as next; Platelet was suspended in Ca-free Tyrode buffer and platelet aggregation was induced by vovine thrombin (0.01 NIH unit) containing lowest activity which can produce 100% aggregation. And this 100% aggregability of platelet was supressed to 0-5% by the addition of AT III with lowest concentration (0.06u) 2min prior to thrombin. Platelet aggregation was measured as optical transmission based on Born's method, and purified in highest degree materials were used. This aggregation curve as a control for the next study almost nothing in aggregability.
When 17β estradiol desolved in Tyrode buffer containg 0.05% ethanol was added to platelet suspension previously, platelet aggregation was increased up to 100% in a dose dependent manner. This effect was observed by the addition of estrogen 1.25×10^-7-1.25×10^-10M in final concentration.
The same effect was also observed by the addition of estradiol, progesteron, testosterone, cortisone and cholesterol which are all clasified as a steroid group. There seemed to be no difference in the effect between these steroides. Not only estrogen but also all other samples could not produce any platelet aggregation in another system consisted with only platelet nor platelet and thrombin.
Our in vitro test system is similar to the mechanism of hemostasis on the blood coagulation in the body. The results interprete the mechanism of thromboembolic disorders due to the administration of oral contraceptives through the inhibition of neutralizing activity in AT-III against thrombin, and this phenomenon produce predominance of thrombin resulting platelet aggregation.

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We experienced 8 cases (10 pregnancies) complicated with aplastic anemia during pregnancy and labor for last 10 years and analysed haemorrhagic, obstetrical problems of these cases.
Three cases occurred during pregnancy, 1 case after abortion, 4 cases independent of pregnancy. Anemia became worse in all cases during pregnancy and fresh blood transfusions were required. According to our experiences, minimum RBC counts in patients who had livebirths was 120×10⁴/mm³ (Hb; 3.5g/dl). Platelets counts was also decreased during pregnancy and haemorrhagic tedency appeared, so some cases received platelets transfusions. WBC counts was not almost influenced by pregnancy, but 1 case had fever in puerperium. At delivery the amount of bleeding was various, but in all cases either fresh blood or platelets transfusions were required.
Aplastic anemia becomes more serious during pregnancy but becomes better after delivery. Problems of aplastic anemia with pregnancy are severe anemia, haemorrhagic tendency, danger of infection. So, pregnancy must be interrupted during the early stage of pregnancy, and during the last stage fresh blood or platelets transfusions are required. Vaginal delivery without laceration is the absolute principle, because uterine bleeding can be easily ceased by the biological ligation of uterine muscle. Prudent follow-up and contraception are necessary after delivery.

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This experiment was designed and carried out to isolate and identify the membrane receptor proteins and antigens in association with platelet functions.
The platelet specific membrane proteins against rabbit anti-human platelet antibody, rabbit anti-β₂ microglobulin, platelet antibodies 4 SLE and ITP patients were examind. The O type healthy human ACD blood (200-600ml) was collected and the platelet suspension was prepared. The washed platelets were labelled with ¹²⁵I-Na by lactoperoxydase method, and then washed 3 times with the same buffer. The ¹²⁵I-labelled platelets were sonicated or disrupted by glycerol loading method, and this lysed platelet solution was layered on the top of a solution of 27% sucrose and centrifuged at 4°C at 23, 000rpm for 4 hours. The membrane fraction remained as a narrow band above the sucrose interface, and this band was isolated, suspended with phosphate buffer for washing, and centrifuged at 4°C at 23, 000rpm for 2 hours. These isolated ¹²⁵I-labelled membrane was solubilized with 0.1% Emalgen 911 (HLB 13.7) for 12-16 hours at 4°C, and centrifuged at 78, 000G for 2 hours. The supernatant was used for double antibody immunoprecipitation method. This method was performed as follows: solubilized, iodinated platelet membrane solution was incubated with IgG fraction of antiserum (rabbit anti-human platelet serum, SLE patient's serum, ITP patient's serum, normal rabbit and human serum or rabbit anti-β₂ microglobulin) for 1 hour at 37°C and 18 hours at 4°C. The IgG fraction of rabbit anti-human IgG or goat anti-rabbit IgG was added and the mixtures were again incubated in the same way. The immunoprecipitation was prepared by centrifugation, washed 3 times with PBS, then solubilized in 2% SDS solution and analyzed by 5% SDS polyacrylamide gel electrophoresis. The gel was cut into 1mm slices and their radioactivities were counted.
The ¹²⁵I-labelled solubilized platelet membrane was precipitated as five radioactive peaks by rabbit anti-human platelet antibody and their molecular weights (M. W.) were 18×10⁴, 16.5-15×10⁴, 10.5-9×10⁴, 6.8-6.0×10⁴ and 5.2-5.0×10⁴, respectively. Two of five peaks corresponded to glycoprotein I and III. When SLE serum was used, the radioactivities of glycoprotein III and M. W. 6×10⁴ membrane protein were greater than those obtained by using normal human serum, but ITP serum did not show remarkable changes compared to normal. The anti-β₂ microglobulin bound to the glycoprotein ha remarkably.
These results may suggest that the platelet has various specific antigen determinant sites against anti-human platelet antibody, platelet antibody-positive SLE serum and anti-β₂ microglobulin.
But to decide if these membrane proteins bind to each serum specifically or not, F (ab)₂ fragment of IgG from each serum must be used instead of IgG.

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1) The fatty acid composition (16:0, 18:0, 18:1, 18:2, 20:4 in wt%) of platelet total lipids and platelet aggregation (PAG) in citrated PRP induced by ADP (1 and 0.1mM) were studied in 20 normal subjects. PAG was expressed as percent aggregation measured by light transmission method following aggregation to ADP such as maximum, at 1min. and 3min. after the addition of ADP. The results of Student's t test of the correlation coefficients between PAG and individual fatty acid concentration (wt%) revealed that only those of linoleic acid (18:2) were significant (0.05<p<0.1).
2) The effect of ADP-induced aggregation on the platelet fatty acid composition was studied as follows. PRP was divided into two parts and usual ADP aggregation (1mM) was done on the one PRP and the other PRP was treated in the same manner with the addition of saline in place of ADP. The platelets obtained from the two PRP were then analyzed in the same manner so as to compare the fatty acid composition of platelet total lipids under the two conditions. The correlation coefficients between the difference in individual fatty acid concentration under the two conditions and the concentration of the fatty acid without the addition of ADP were significant in 18:0 and 18:1 (p<0.01) and in 18:2 and 20:4 (p<0.05). From the results it was concluded that fatty acid composition of platelet converged to a certain level when it was aggregated by the addition of ADP. The same trends were observed following aggregation to epinephrine or collagen.

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A method for the determination of human platelet glycolytic enzymes is described. Suspension of washed human platelets with a count of approximately 1×10⁶per μl was prepared from about 10ml of blood with ACD or EDTA. The degradation of enzymes was prevented by the addition of 0.2mM NADP and 1mM DTT to the above suspending medium before disruption. It was exposed to Kentes micro-ultrasonic disintegrator for 5min. below 5°C. All glycolytic enzyme assays were carried out at 37°C in a final volume of 1.5ml at pH 7.4.
Determinations of various enzymes in platelets as well as in erythrocytes were performed in a variety of cases. Included are the cases with G6PD deficiency (2 cases), pyruvate kinase (PK) deficiency (1 case), thrombasthenia (3 cases), essential thrombocythemia (4 cases), myelofibrosis (2 cases) and CML (4 cases).
Platelet phosphoglyceromutase (PGM) activities were found to be increased in 4 cases with essential thrombocythemia. However, it seems that platelet enzyme assays are of not much help in making precise diagnosis of patients with essential thrombocythemia and thrombasthenia.
G6PD activities in platelets as well as in red cells were low in cases with G6PD deficiency, whereas in all other enzymes, the values were either high or normal. It is concluded that enzyme assays in platelets as well as in red cells are useful diagnostic tools in making precise diagnosis of patients with congenital glycolytic enzyme deficiencies.

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

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