Stimulants-induced changes in platelet membrane fluidity were observed by electron spin resonance (ESR) using spin probe (5SAL). The order parameter of stearate spin probe was markedly decreased when the washed platelets were aggregated by thrombin, collagen or Ca²⁺-ionophore A23187. However, isolated plasma membranes did not show any decrease in the order parameter by the addition of thrombin. After the platelets were treated with aspirin and colchicine which are potenti inhibitors for aggregation, the consequent addition of thrombin caused no significant increase in membrane fluidity. Moreover, any change in order parameter was not detected after treatment with N-ethylmaleimide which inhibited both aggregation and secretion. On the other hand, secretion induced by thimerosal or thrombin in the presence of EDTA (5mM) was accompanied with a decrease in the order parameter.
By the addition of thrombin to the washed platelet suspension, some significant alterations were observed to occur in the phospholipid composition. The content of phosphatidylinositol was decreased after the 30 sec-stimulation by thrombin, with an accompanying increase in the diacylglycerol level. The 60 min-treatment of platelets by the drug caused decreases in phosphatidylcholine and phosphatidylethanolamine, resulting in increases in their corresponding lysophospholipids.
When platelets were activated by thrombin and examined by electron microscopy after freeze-fracture, no change was observed in distribution of intramembranous particles. The distribution of cholesterol-filipin complexes formed in the platelet membranes including intracellular organelles was not different from that of the untreated platelets, thus implying that the cholesterol distribution was not affected by aggregation.
These results suggest that the increase in platelet membrane fluidity might be caused by reorganization of membrane (lipid) constituents rather than their compositional modification.

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For an analysis of relationship between membrane physicochemical properties and functions of platelets, aggregability, electrophoretic mobility, sialic acid amount and membrane glycoproteins were measured for platelets from 21 healthy aged males, 14 prostatic hypertrophy patients, 10 males with stomach or lung cancer, 2 prostatic cancer patients, 16 prostatic cancer patients on estrogen (stilbesterol diphosphate 300mg/day) and 15 similar patients on aspirin 330mg/day. Prostatic cancer patients on estrogen and stomach or lung cancer patients had significantly higher electrophoretic mobility than control. A good linear correlation was found between sialic acid amount and electrophoretic mobility of platelets (r=0.950, p<0.001). Platelet electrophoretic mobility had negative correlations with primary aggregation induced by adrenaline and by ADP, respectively, but it had no correlation either with secondary aggregation or with maximal aggregation. Treatment of rabbit platelets with increasing doses of neuraminidase resulted in increasing aggregation associated with decrease in electrophoretic mobility. Thus, surface negative charge seems to have the role to inhibit primary aggregation. Surface negative charge was influenced with composition of membrane glycoproteins, estrogen and changes in population of platelets. In addition, aspirin increased surface negative charge without the effect on sialic acid. Membrane glycoprotein patterns for prostatic cancer on estrogen indicated a significantly lower relative percentage of GP I and higher GP IV. A positive correlation was present between relative amount of GP II and intensity of primary aggregation induced by ADP. These results suggest that membrane physicochemical properties of platelets may affect their function.

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Human platelets contain a large quantity of Ca²⁺-activated, phospholipiddependent protein kinase originally found in rat brain (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695). The activation of this enzyme appears to be directly coupled to phosphatidylinositol (PI) turnover which is induced by thrombin, since at micromolar concentrations of Ca²⁺ the enzyme absolutely depends on phospholipid as well as on a small amount of unsaturated diacylglycerol which is derived from the PI turnover. Among various phospholipids phosphatidylserine is the most active. The enzyme thus activated phosphorylates preferentially an endogenous protein having a molecular weight of 40, 000 in vitro. This protein is also shown to be phosphorylated rapidly in platelets in vivo which are stimulated by thrombin. The phosphorylation of this protein is generally accepted to be intimately related to the thrombin-induced serotonin release. Such phosphorylation in vitro as well as in vivo is profoundly and selectively inhibited in parallel manners by phospholipid-interacting drugs such as dibucaine and chlorpromazine, both of which are known to inhibit platelet activation. These results suggest that PI turnover plays a role in transmembrane control of protein phosphorylation during the thrombin-induced activation of human platelets.

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We have examined morphological and biochemical changes in membrane surface components of platelets during thrombin induced aggregation. To washed human platelets suspension (0.5×10⁹/ml), bovine thrombin was added at concentration of 0.5 NIH units/ml and the mixture was stirred at 37°C for various period of time. Thrombin treated and control (non-treated) platelets were then subjected to electron microscopic observation, lactoperoxidase iodination and SDS polyacrylamide gel disc electrophoresis for glycoprotein analysis. Electron microscopic observation of platelet-aggregates formed 3min. after addition of thrombin demonstrated that there were irregularly spaced bridges of amorphous and filamentous substances between closely approximated plasma membranes. These interplatelet connection reacted with ruthenium red and was thought to be derived from membrane surface. Ten min. after adding thrombin, there found a distinct contact between outer leaflets of membranes of platelets and partial fusion of the membrane was observed.
Iodine labelling pattern in membrane polypeptides of thrombin treated platelets showed a significant reduction in labelling of GP I, II, III and actin. These changes were initiated 30″ after thrombin treatment and reached maximum after 3min. incubation with thrombin. Two new peptides were identified as having a surface orientation after thrombin treatment. The molecular weight of the two components were 80, 000 and 65, 000 respectively. Similar new peptides were demonstrated in inside labelled platelet membranes. No significant change was observed in membrane glycoprotein profile of thrombin treated platelet aggregates. Surface labelling pattern was never changed when thrombin induced platelet aggregation was inhibited either by the addition of 1mM EDTA or by no stirring. These results suggest that interaction of surface components of platelet membranes, particularly glycoproteins may involve the mechanism of early phase of platelet aggregation by thrombin.

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We have demonstrated that thrombin-induced platelet release reaction is closely related to the specific hydrolysis of glycoprotein V (GPV) which is one of the surface membrane glycoproteins and whose molecular weight is approximately 80, 000 to 90, 000 daltons.
In this study, we have further studied the influence of the other serineproteases on GP V, using trypsin and α-chymotrypsin.
GP V was hydrolyzed not only by thrombin but also by trypsin in a concentration or time-dependent manner, and its disappearance was accompanied with release reaction. Trypsin also hydrolyzed GP I (M. W. 150, 000) and high molecular weight glycoproteins as well, chymotrypsin had no effect on the membrane glycoproteins nor the release reaction.
Platelets pretreated with thrombin showed the decreased release reaction after stimulation with trypsin or thrombin, but there was no remarkable change in platelets pretreated with chymotrypsin, which have the functionally affected GP I.
Heparin had specific inhibitory effect against thromb-ininduced release reaction in a concentration dependent manner and its effect was remarkably increased by using platelets pretreated with chymotrypsin instead of normal washed platelets.
As the results, the most important glycoprotein for the serineproteases to induce release reaction was GP V, which was hydrolyzed specifically by thrombin or non-specifically by trypsin. On the other hand, it was considered that GP I was the receptors for heparin as well as for thrombin, and heparin exhibited its inhibitory effect on thrombininduced release reaction by means of competition for these receptors on GP I.

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When disseminated intravascular coagulation (DIC) persists, heparin becomes inefficacious at times, since activated factors impaired the inhibitory capacity of antithrombin III, while reacting in the presence of heparin. From the point of view, the application of serine proteinase inhibitor has been proposed for the prevention and treatment of DIC. In the experiment, radioactive fibrinogen was given to the rats and while DIC was induced by infusion of thrombin or tissue thromboplastin, deposition of the radioactive fibrin to the kidneys were directly detected by a γ-counter and monitored continuously. Cocomittant with platelet count, whole blood radioactivity and organs radioactivity, the effect of aprotinin on experimental DIC was investigated in comparison with that of FOY. The continuous administration of 120μmol of FOY/kg/h effectively inhibits the microthrombi formation induced by thrombin or tissue thromboplastin. Aprotinin failed even at the dose of 50, 000u/kg/h. On the other hand, as for fibrinolysis, FOY at the above dose not show any alteration on the naturally occered secondary fibrinolysis when thrombin-induced microthrombi were firstly provoked. Aprotinin was potent in antiplasmin that at 5, 000u/kg/h or 2, 000u/kg the fibrinolynosis was completely blocked. From these results, it may be concluded that without combination with other anticoagulant agents, aprotinin alone in management the patient of DIC was underserved.

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Aggregability, sialic acid content and electrophoretic mobility were measured for platelets from 13 gynecological patients 1 day before, 1 day, 1 week and 1 month after laparotomy. Platelet electrophoretic mobility (EPM) was measured by a fully automatic instrument, Laser Zee 3000 system, which determined mean EPM of about 700 platelets with good reproducibility. For all patients, platelet membranes were isolated and their glycoprotein patterns were determined by electrophoresis on SDS-polyacrylamide gels. A significant increase in platelet EPM was observed 1 day after a laparotomy associated with a decrease in platelet count and aggregability. This suggests that a selective consumption of platelets with smaller surface negative charge may occur during postoperative hemostatic plug formation or under surgical stress. In that time, a decrease in sialic acid amount in platelets was observed. A good linear correlation was observed between EPM and sialic acid amount (r=0.856, p<0.001). It suggests that variations in sialic acid content reflect differences of residues which have a direct bearing on the effective surface charge. One day after the laparotomy, a decrease in relative amount of glycoprotein I and increases in glycoprotein II and III (p<0.001-0.005) were observed. There might be an agent which affects the platelet membrane itself or causes a change in the platelet population.

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Aggregability, sialic acid content, and electrophoretic mobility were measured for platelets from 2 prostatic cancer patients, 20 prostatic cancer patients on estrogen (Stilbesterol diphosphate 300mg/day), 14 prostatic hypertrophy patients, 10 stomach or lung cancer patients, 21 healthy aged male controls, 13 healthy young females and 16 healthy young males. Healthy young females and aged male controls had significantly high electrophoretic mobility compared with healthy young males (p<0.01 and p<0.001, respectively). Prostatic cancer patients on estrogen and stomach or lung cancer patients had significantly higher electrophoretic mobility than controls (p<0.001). A good linear correlation was found between whole cell sialic acid and platelet electrophoretic mobility (r=0.95, p<0.001). The primary aggregations induced by adrenaline and by ADP had negative correlations with platelet electrophoretic mobility respectively, but there was no correlation between secondary aggregation and platelet electrophoretic mobility. This surface negative charge seems to have the role to inhibit platelet primary aggregation. Treatment of washed rabbit platelets with increasing doses of neuraminidase resulted in increasing aggregation by ADP and by collagen associated with decrease in electrophoretic mobility. When the prostatic cancer patients were given aspirin, the platelet electrophoretic mobility was found to increase and the linear relationship between electrophoretic mobility and sialic acid was lost.

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1) Types of epinephrine-induced aggregation in chronic myeloproliferative disorders were classified into three types according to a concentration of epinephrine added in the PRP. The first type of aggregation curve revealed normal responsiveness in response to a usual concentration of l-epinephrine (5μM). The second type revealed defective or decreased responsiveness to 5μM l-epinephrine, but a normal responsiveness appeared at a higher concentration of l-epinephrine. The third type showed no responsiveness to any concentration of l-epinephrine (10μM, 100M and 1mM). We called above those types of aggregation curves Type I, Type II and Type III, respectively.
2) ³H-DHEC (³H-Dihydroergocryptine) binding assay in washed intact platelets was studied according to the method of Newman et al. with a minor modification. Platelets in Type I revealed normal values in ³H-DHEC binding sites. In Type II, ³H-DHEC binding sites were slightly fewer than in normal controls or reduced to half numbers of normal controls. In Type III, ³H-DHEC binding sites revealed half numbers of normal controls.
3) The cAMP level in the PRP in Type I showed normal enhancement by PGE₁ and a sufficient decrease in response to epinephrine. In Type III, the cAMP level in the PRP demonstrated only 10 to 20% of normal enhancement by PGE₁ and no responsiveness to epinephrine. Whereas, in Type II, various attitudes were observed in response to PGE₁ and epinephrine.
4) Platelets in Type III revealed sufficient potentiation of ADP aggregation due to a low-dose epinephrine. Therefore, the mechanism of epinephrine aggregation might be different from that of potentiation of ADP aggregation due to a low-dose epinephrine.

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The reaction of platelets to bilirubin and its photodegradation products was investigated. Based on the results showing in Figure 1, various concentrations of bilirubin were added to gel filtration platelets solution (GFP). The most intence aggregation was induced by 5-10mg/dl of bilirubin at final concentrations (f. c.) in GFP from adult blood, whereas it was induced by 0.5-1.0mg/dl of bilirubin (f. c.) in GFP from umbilical blood. The less intence aggregation was induced by photodegradation products of bilirubin, and no aggregation wasobserved after exposure to light for 80 minutes or longer.
Bilirubin induced GFP aggregation was inhibited perfectly or partially by addition of albumin, ethanol, prostaglandin E₁, prostaglandin I₂, aspirin and flurbiprofen. Heparin enhanced the aggregation at low concentration (≤20units/ml), but inhibited it at high concentration (≥40units/ml). However, dl-α-tocopherol (≤300mg/dl) had no inhibitory effect on the aggregation.
¹⁴C-labelled serotonin was released after exposure to light in platelet rich plasma from umbilical blood, but not in platelet rich plasma from adult blood.
Those results indicate that phototherapy may induce DIC by activation of platelets, especially in neonates.

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A possibility of association between the platelet aggregation and active oxygens was investigated. The ADP-, collagen-, thrombin- or epinephrin-induced platelet aggregations in PRP were inhibited by addition of Tiron, 1, 4-Diazabicyclooctane (DABCO) and 2, 5-Dimethylfuran (DMF), the scavengers on superoxide radical (O₂^-) and singlet oxygen (¹O₂). The arachidonic acid-induced platelet aggregation was not inhibited by the scavengers. The scavengers on hydroxyradial (·OH) and hydrogen peroxide (H₂O₂), mannose and catalase, respectively, did not affect on the all platelet aggregations examined in this work. The results of platelet aggregometry, release reaction of ³H-serotonin and electron microscopy demonstrated that human platelets were aggregated by incubation with xanthine-xanthine oxidase system (active oxygens producing system). Furthermore, the aggregation caused by this system was inhibited by addition of superoxide dismutase, DABCO and DMF. These results suggested that these aggregations were induced by O₂^- and ¹O₂. It was postulated that active oxygens would participate in the process of platelet aggregation, as this was induced by the active oxygens generating system and ADP-, collagen-, thrombin- or epinephrine-induced aggregations were inhibited by addition of the scavengers of O₂^- and ¹O₂.

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Lysine-naphthalene-sulfonamide derivatives activated TAME (tosyl-L-arginine methyl ester) esterase activity of thrombin. These compounds stimulated also thrombin-induced aggregation, ATP release and serotonin secretion from human platelet. On the other hand, analogues of these compounds which contain arginine residue in stead of lysine residue inhibited not only TAME esterase of thrombin but also thrombin-induced platelet aggregation. Lysine-naphthalene-sulfonamide and arginine-naphthalene-sulfonamide were the opposite actions each other against thrombin. The structure and activity relationship in these naphthalene-sulfonamide suggest that lysine and arginin residues play a very important role in the function of thrombin.

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A group of 30 rabbits (22 with no pretreatment, 8 thrombocytopenic) were given 0.7mg/kg of sodium arachidonate (AA) within 4-5sec. into the right carotid artery. Thirty minutes later, Evans blue was injected. Another 12 rabbits were given 20mg/kg of ADP within 1 minute. One hour after injection, brains were observed histologically. Brains of 77% (17/22) of AA-injected rabbits showed irregular blue staining in the ipsilateral cerebral hemisphere. Extravasation of plasma substance was histologically observed in this area. Frequent platelet adhesion on the luminal wall was seen. Derangement of endothelial lining was apparent around the area to where platelets adhered. The changes were not observed in the opposite hemisphere or in the thrombocytopenic group. Sixty six percent (8/12) of ADP injected rabbits showed marked blue staining and edematous changes which were confined to the right cortex. Ipsilateral hemisphere was stained in 2 cases and 2 cases showed no changes. The ADP-injected group showed similar, but less pronounced histological changes. Platelet count was more marked and of longer duration in the AA group as compared to the ADP group. These vascular injuries may be related to the consumption of intra-vascular platelet aggregates. Activated platelets might play an important role in the genesis of vascular injury.

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Aspirin is known as an agent which inhibits platelet malondialdehyde (MDA) production and its aggregation by blocking cyclooxygenase.
Recently, Stuart et al described a new method for measuring platelet survival by using this principle. It is very useful because its technique is very simple and nonradioisotopic, however there are some problems must be clarified further on it.
In this paper, observation of platelet kinetics by a single low-dose aspirin administrated orally, or intravenously as a pulse labeling in normal volunteers were investigated.
The mean platelet survival time is calculated with linear recovery of daily MDA formation using a computer programme.
In normal subjects administrated single oral dose of 500mg or 1.0g aspirin, platelet survival curve revealed a lag phase for the first two days, followed by linear recovery, and the mean platelet survival time was 9.7±1.4 (M±SD) days. It has been suggested that high-dose aspirin inactivates the cyclooxygenase not only in peripheral platelets, but megakaryocytes in bone marrow.
The mean platelet survival time after oral administration of low-dose aspirin (63mg, 125mg) was 8.9±1.4 days and that after intravenous injection (50mg, 100mg) was 8.7±0.3 days respectively, which are shorter than that after oral high-dose aspirin.
MDA production was inhibited completely and immediately after the intravenous injection of aspirin (50mg or 100mg). Platelet survival curve showed almost linear without a lag phase during the first two days in this case.
It is concluded that platelet survival time should be measured by using low-dose intravenous pulse labeling technique.

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Isolated rat aortae were incubated at 22°C in 1ml of Tris-buffered saline (pH 7.4). The incubation medium was changed every 10min and the amounts of PGI₂ in the medium were immediately bioassayed as an inhibitory activity against rabbit platelet aggregation induced by ADP.
An addition of arachidonic acid to the medium caused the increased generation of PGI₂, followed by gradual decrease even in spite of the presence of the same amount of arachidonic acid, whereas the generation from PGH₂ was kept constant. The decrease of PGI₂ generation from arachidonic acid was prevented by tryptophan, which was required by PG hydroperoxidase with heme compounds as a cofactor. MK-447, which was reported to exert tryptophan-like action in PG endoperoxide biosynthesis, also prevented the decrease. These results indicate that MK-447 can act as a tryptophan-like cofactor in the PGI₂ generation.
When isolated rat aortae were mechanically stimulated by manual stretch before each 10min incubation, the PGI₂ generation from endogenous arachidonic acid was kept fairly constant after initial transient decrease. The addition of MK-447 to the medium obviously accelerated the PGI₂ generation. The present results suggest that MK-447 may be a good candidate of drugs which prevent the thrombus formation.

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Catalase (CAT), glutathione peroxidase (PGx), as well as superoxide dismutase (SOD), which are known to protect membrane from oxidative damage, were examined in 24 patients with myeloproliferative disorders, including twelve patients with chronic myelogenous leukemia (CML), six with essential thrombocythemia (ET) and six with polycythemia vera (PV). The CAT activities were significantly decreased in the platelets of ET and PV as compared with normal subjects, however it was normal in those of CML. The GPx was also decreased in some patients with these disorders, whereas SOD was increased in some patients. The individual data were further evaluated about the interrelationship among these three enzymes. Two of ET (2/6) and one of PV (1/6) didn't show increased SOD activities as to compensate the reduced CAT or GPx and two of them had the symptom of bleeding tendency and decreased platelet aggregability. The rest of the patients exhibited compensatory high SOD activities for the other decreased enzymes and each patient had never experienced bleeding tendency. These findings suggest that the balance of these three enzyme activities might be playing an important role in the hemostatic function of platelet in these thrombocythemic states.

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Detection of thrombi by scintiphotography and measurement of platelet survival with ¹¹¹In-oxide labeled autologous platelets were carried out in patients with thrombosis. Platelets were separated using differential centrifugation from 44ml of venous blood obtained in 6ml ACD solution from a subject. They were suspended in 3ml ACD-saline solution (pH 6.5). Three hundred μCi of ¹¹¹In-oxide were added to the platelet suspension, and then the mixture was incubated for 20 minutes at room temperature. One hundred and fifty μCi of labeled platelets resuspended in autologous plasma were injected to the patient. Imaging by scintillation camera on the body and detection of platelet radioactivity in the blood were done following the course of time.
A normal subject (case 1) and four patients suspected of thrombi (cases 2-5) were studied. Cases 2 and 3 had been implanted Dacron-graft 6 and 4 years before respectively, because of abdominal aortic aneurysm. Case 4 who had old myocardial infarction with left ventricular aneurysm and case 5 who suffered from mitral stenosis have been suspected of intracardiac thrombi by two dimensional echocardiography. In normal case, radioactivity on the spleen and liver and also cardiac blood pool was observed at 1 hour after the injection of ¹¹¹In-labeled platelets. At 5 days after, the radioactivity on the cardiac blood pool was decreased, whereas that of the liver and the spleen were increased. Unusual platelet depositions on the Dacron-graft were observed in case 2 and 3. The accumulation of labeled platelets to thrombus at the left ventricular aneurysm in case 4 and to left atrial thrombus in case 5 was cleary detected as the camera images. Mean platelet survival time was normal in case 1 (8.6 days), shortened in case 2 (3.4 days) and case 3 (3.7 days), and slightly shortened in case 4 (6.9 days) and case 5 (5.6 days).
Imaging of thrombi and measurement of platelet survival using ¹¹¹In-oxine labeled platelets are considered to be an excellent method for the diagnosis and decision of treatment on various disorders with thrombi.

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The platelet aggregating activity of tumor cells was studied in vitro using ten established human cancer cell lines, Lewis lung cancer cell and Chang liver cell. Four out of ten human cancer cell lines induced the aggregation of human platelet. The aggregating reaction was irreversible and was preceded by a lag period. The platelet aggregating activity was shed from the cells growing in culture and associated with cell membrane fragments.
Growth promoting activity of human platelet lysate on human tumor cells was also studied. Three cell lines examined, were stimulated to proliferate by addition of platelet lysate.
These results suggest that human tumor cells aggregate the platelet and the aggregated platelet successibly liberate growth promoting factor which activate the growth of tumor cells and thus formation of the tumor thrombus in peripheral blood vessels may be accelerated.

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1) 培養細胞を発育様式, 細胞形態およびECに特徴的な Weibel-Palade bodies の確認とを合わせてECと同定した.
2) Rubber spatula による機械的操作で得た約2×105個の初代培養EC浮游液は既知の合成PGI₂, 10ng/mlに相当する血小板凝集抑制効果を示し, その効果は5μMのASAにより完全に阻害された.

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The effects of dexamethasone and prednisolone on the prostacyclin synthesis, protein synthesis and cell size of cultured human endothelial cells were studied.
Inhibitory activity of endothelial cells treated with dexamethasone on ADP induced platelet aggregation was reduced, when compared with the non-treated endothelial cells. This observation showed that dexamethasone blocked prostacyclin synthesis in cultured endothelial cells.
Cell surface area after culturing the cells with dexamethasone or prednisolone for 48h were calculated by using image analysing system. The mean surface area of the endothelial cell treated with 10μM dexamethasone was 1.63 times greater than that of the untreated endothelial cell. The morphological change of endothelial cells was associated with an increase in protein synthesis without an increase in DNA synthesis or cellular replication. These effect of dexamethasone on morphological alteration and protein synthesis of the endothelial cells were completely inhibited by a protein inhibitor, cycloheximide.
Our results indicate that dexamethasone inhibits the prostacyclin formation in the cultured endothelial cell and can increase endothelial cell size. These effects of glucocorticoids are compatible with hemostatic effect in the thrombocytopenic state.

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The intimal damage was induced by the injection of saturated saline solution into the femoral artery of rabbit. The endothelium was disappeared on 2 days after the irritation. The subintimal hyperplasia was shown on 9 days after the irritation. In the experimental group, the artery was cut on 2 days after the irritation and was anastomosed immediately under the operative-microscope. In the control group, the artery was anastomosed without the irritation. Anticoagulants were not used systemically, but the arterial lumina was washed with the heparinized saline solution.
All arteries in both groups were patent on 7 days after anastomoses, but many of them in the experimental group were stenosed due to thrombus.
Histologically, subintimal hyperplasia of the artery was shown in the experimental group, but it was not in the control group. It was thought that the blood flow prohibits the anastomosis from occlusion even in the region with thrombus formation.

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The arterial thrombus was made experimentally in rabbit carotid arteries by the methods of both denudation of the endothelium and stenosis of the lumen.
A Fogarty balloon catheter (2F or 3F) was inserted in the common carotid artery and balloon was inflated by air at the bifurcation, that was pulled back gently to the proximal portion. Then inserted portion was by-passed by asiliconized polyethylene-tube. Immediately, the carotid artery with the de-endothelialization was ligated with a steel wire (0.5mm) at the distal portion, and was stenosed by pull out of the wire.
Another carotid artery treated only de-endothelialization or luminal stenosis by the same methods. The artery with only endothelial denudation showed marked platelet adhesion and aggregation at the subendothelium, but never formed any thrombus at 30 minutes. The artery with only luminal stenosis formed platelet thrombus in 80% at the site of the ligation, but the normal endothelium did not showed either platelet adhesion, thrombus formation, nor the luminal obstruction.
The combined treatment of the de-endothelialization and the luminal stenosis, a thrombus was formed in 100% and luminal obstruction was observed in 78% of the studied arteries at 30 minutes. The thrombus was formed at the distal portion and protruded the head to down stream over the stenosed portion, and it was accompanied by a coagulation thrombus.
These results suggested that platelet adhesion and aggregation occured by the endothelial injury, and combination of the arterial luminal stenosis might be necessary to arterial obstructive throinbus formation.

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We examined the 87 autopsy cases for the thrombus induced by inserting various catheters for diagnosis or treatment in the veins or arteries. Organization of the thrombus is classified into fresh (I), organizing (II-a, II-b, II-c) and organized (III-a, III-b). Dating of the thrombus was determined by measuring the interval between the day of the catheterization and the day of the patient's death. The thrombi of the elder patients tend to be delayed in organization. It is speculated that the intimal thickening, poor in cellularity may be correlated to the delay of the organization. The process of the organization of the thrombus is mediated by covering by newly formed endothelial cells, and infiltration and proliferation of smooth muscle cells from the underlying intima or media into the thrombus. Cellularity is very low in the intima showing fibrous thickening in the aged. Therefore, the number of the smooth muscle cells and other cells concerned in the organization of the thrombus is small. Other factors, such as senescence of cells and smooth muscle cell proliferating factors may have influence on the delay of the organization also.
Fibrinolytic therapy for thrombosis has been widely used nowadays. We should take account of the stage of organization of the thrombus when we treat the thrombosis.

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Formation of an insoluble fibrin network plays an important role in the process of wound healing. To form such an insoluble fibrin, thrombin, the blood clotting factor XIII (FXIII) and calcium ion are required. For the enhancement of local fibrin accumulation, we immobilized thrombin and F XIII on absorbable gelatin sponge and also on some suture materials.
Basic studies on the degree of fibrin formation on the materials both in vitro and in vivo showed that a large amount of fiibrin had formed and adapted with tissues.
Clinically, the immobilized silk suture materials were used for skin suture and the extraction force to take out the sutures was measured. The extraction force was almost 2 times as large as that of control silk sutures.
As a model of embolus formation in vessels, we applied Chandler's method. A large embolus was formed on the immobilized sponge in a moment. So, we utilized the sponge as a thrombogenic material for transcatheter embolization. Embolization was performed for a patient with inoperable hepatoma. The tumor stain disappeared and no recanalization could be seen.
We are going to utilize these materials for promotion of wound healing protection of anastomoses, transcatheter embolization and so on.

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Hemostatic and healing mechanism at synthetic vascular graft implanted in the canine inferior vena cava was studied by light and scanning electron microscopy.
Six of 7 grafts in the control group and all the 6 grafts in the batroxobin-defibrinogenated group were patent, and provided for the investigation.
Blood loss from the anastmotic suture lines appeared to be slightly greater in the defibrinogenated group, in which the fibrinogen concentration was reduced below 0.4g/l than in the controls. Hemostatic clot formed at the anastomosis revealed no significant differences in platelet morphology between the two groups. These findings comfirmed that platelets play a key role in primary hemostasis during defibrinogenation.
Loose and spongy structure of initial mural thrombus resulting from diminished fibrin formation, might cause slightly delay in pseudointimal organization and endothelial cell invasion within the first 21 days. After 70 days, the graft had a similar appearances of the lining.

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[¹⁴C] arachidonic acid and [³H] myoinositol double labelled platelet membrane vesicles were prepared from human platelet. Upon the stimulation of [¹⁴C] arachidonic acid and [³H] myoinositol labelled platelet membrane vesicles by thrombin, the formation of not free arachidonic acid but diglycerides was observed. Simultaneously, a decrease in radioactivity was observed not in the pool of phosphatydylcholine but in that of phosphatidylinositol. From these observation, it may be considered that platelet membrane vesicle is devoid of diglyceride lipase and that not phospholipase A₂ but phospholipase C is involved in the early step of platelet aggregating reaction.

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The opportunity to carry out oral surgery in patients under anticoagulant therapy has been increasing, although a dilemma exists between the hemostasis in the dental procedures and the contineous anticoagulant management.
It is our purpose to know the safety of oral surgery among the patients using anticoagulant.
The present study was based on forty one oral surgery in 19 patients treated Nagoya University Hospital during 1973 to 1978. All cases had been managed by anticoagulant therapy with Warfarin for the replaced prosthetic valve, or for the management of mitral or aortic disease, etc.. The oral procedures consisted of 30 dental extractions, 5 scaling, two resections of torus mandibularis, two cases of alveoloplasty, removal of an apical cyst and biopsy in each case.
The mean value of thrombotest with the therapeutic dose of Warf arin was 24.3±11.8%. And the value on the data of dental extraction was found to be 49.9±21.2%. Postoperative bleeding, only for one or two days, was observed in 10 cases with dental extraction. A comparison of the value between cases of the bleeding and non-bleeding after dental extraction revealed no significant difference, of which the group with bleeding and non-bleeding were 56.6±23.6% and 46.6±20.2%, respectively.
The present results may indicate that the bleeding at oral surgery is not related to the anticoagulant therapy and that it is not necessary to discontinue the therapy for oral surgery. However, further detailed studies are necessary to establish a comprehensive policy.

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In 34 cases of cancer, plasma levels of prekallikrein (PKK), high and low molecular weight kininogens (HMWK and LMWK) and total kininase activity were studied in relation to coagulation-fibrinolytic system. Prekallikrein levels were measured with the chromogenic substrate Bz-Pro-Phe-Arg-pNA and the others by bioassay methods. Prekallikrein and HMWK showed a marked decrease in 10 cases with DIC (to 34, 33% of normal, respectively) and a moderate decrease in 24 cases without DIC. A moderate reduction in LMWK levels (to 56% of normal) was found in the cases with DIC and a slight reduction, in the cases without DIC. The above three factors had positive linear correlations with each other and with antithrombin III, plasminogen and albumin levels in citrated plasma as to those cases without DIC. Plasma total kininase activity showed no significant difference between the cases with cancer and the normal subjects.
These results suggest that plasma levels of prekallikrein, HMWK, LMWK, antithrombin III and plasminogen may parallel with each other and with plasma albumin levels and that LMWK may have a possible relation to coagulation-fibrinolytic system.

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To clarify the interaction between F. VIII and thrombin, coagulation studies and affinity chromatographies were accomplished.
Incubation of normal plasma or purified F. VIII with thrombin (0.01-1.0U/ml f. c.) resulted in an marked increase and then in rapid decrease in VIII: C. Re-addition of the same dose of thrombin into the exhausted normal plasma or purified F. VIII failed to reincrease VIII: C, accordant to the fact that VIII: C in some of DIC cases did no longer increase by addition in vitro of thrombin.
When purified F. VIII preparation was applied on thrombin- (or Xa-) Sepharose column, approximately one half of F. VIII complex was washed out by starting buffer with the protein front, and the eluting buffer (0.3M in NaCl) also produced the rest F. VIII complex (VIII: C, VIIIR: AG and VIIIR: WF) altogether. The eluting F. VIII was not or less activated by additional trace of thrombin and was more labile as compared to native F. VIII. Its electrophoretic mobility remained unchanged on crossed immunoelectrophoresis.
Conclusively, the results seemed to be an additional evidence of the interaction or activation of F. VIII by thrombin (and/or by Xa) and to explain the discrepancy between one-stage and two-stage VIII: C in some DIC cases.

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Highly concentrated F. VIII materials (HC F. VIII) for replacement therapy to hemophilia A contain less amount of fibrinogen than cryoprecipitates. It has become easy to administer a large amount of F. VIII, but there may be some problems in cases of multitransfusion that the starting material of these HC F. VIII is a large scaled pool plasma.
Seventy-one cases of hemophilia A under the frequent replacement therapy by HC F. VIII were investigated on liver function tests and F. VIII inhibitor through 10.2±2.7 months. The mean of total amount of F. VIII administered for individuals within the investigated term are 21, 700±20, 400 units.
Comparative study of GOT, GPT and γ-GTP before and after following-up term showed no significant changes statistically. Both the titer of HBs antibody and the number of HBs antibody positive patients proved increasing tendency, which might be explained with a high titer of HBs and HBc antibody contained in HC F. VIII. F. VIII inhibitory titer showed mild increasing tendency statistically, it may be too early to conclude that the phenomena caused by the multitransfusion of these materials. Because, the mean of the highest titer of these individuals through the objective term is considerably high, 0.903±0.375 unit and 34 out of 71 cases showed over 1.0 unit once at least through the whole course of the objective term.

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