Antithrombin III (AT III) is not only a most potent thrombin inhibitor but also an inhibitor against coagulation factors Xa, XIa, IXa, and XIIa and heparin accelerates the inhibitory reaction of AT III remarkably. Since Egeberg first described an inherited AT III deficient family, about thirty articles dealing with inherited AT III deficient families have been published as full papers. The present review analyzes the relation between AT III deficiency and thrombotic tendency in the inherited AT deficient families.
The mean age of the propositus was 30.2±14.2y/o (mean±S. D.) and the sex distribution was male dominant (male: female ratio approximately 2:1). The cases where the propositus had been suffering from recurrent thrombosis was 36%, and that of the first episode of thrombosis for the propositus was 48%. The sites of thrombosis were located mostly in the veins of the lower half of the body such as the ilio-femoral vein, the deep veins of the legs, mesenteric vein and vena cava inferior, often complicating with pulmonary embolism.
The total numbers of persons in the inherited AT III deficient families whose AT III was examined, amounted to 434 in 23 articles. Depressed AT III level measured by either the immunological or biological method was found in 189 persons. The average appearance rate of AT III deficient persons in the affected families was 52.1±18.0%. The incidence of persons who had both AT III deficiency and thrombotic episodes was calculated by dividing the numbers of AT III deficient persons by the total number of persons whose AT III was examined in the deficient family. The average value for the affected families was 26.5±12.8%.
These figures thus indicate that about half of the persons in the inherited AT III deficient families had AT III deficiency, and that about half of these AT III deficient persons had thrombotic episodes. Analysis of the reports on the inherited AT III deficiency thus revealed that AT III deficiency did not always induce thrombosis: in younger age group, thrombosis was never apparent. Additional factors which induce thrombosis in AT III deficient persons must therefore be present. Trauma, surgery, or pregnancy may be one of such factors. About one quarter of the persons in the affected families had AT III deficiency without thrombosis. The existence of such “silent” AT III deficiency suggests that the incidence of inherited AT III deficiency may be much higher than anticipated.

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A new method of platelet aggregation using diluted blood was investigated with Screen Filtration Pressure (SFP). Diluted blood passed through the nuclepore membrane with 5μ diameter, and changes in pressure following passage of blood through the filter were indicated as control·SFP. Results obtained using diluted blood added with 3μM ADP were presented as ADP·SFP. Platelet aggregability was presented as (ADP·SFP) minus (control·SFP). In the 31 healthy individuals, mean platelet aggregability was 152±99mmHg. Reproducibility of ADP·SFP was stable within 30 to 60 minutes after venopuncture. There was a statistically significant positive correlation between platelet aggregability assayed using SFP method and that measured using Born's optical density method in 39 neurosurgical prtients. There was no correlationship between tlatelet aggregability and haematocrit values or platelet count in randomized cases. There pesults indicated that platelet aggregability determined with SFP was not affected by changes in haematocrit values and/or platelet counts. In clinical application of this method, platelet aggregability in cases of classical migraine was higher than those of healthy subjects or patients with tention headache. It is pointed out that SFP method is a simple and reliable for measurement of platelet aggregability and is useful for clinical applications.

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The binding sites of concanavalin A (Con A) on the membranes of human platelets were studied by using ferritin-conjugated concanavalin A (fer. -Con A) in the three different conditions.
Suspension of washed platelets (S. W. P.) was prepared by washing P. R. P. in phosphate-buffered saline (0.01M P. B. S., pH 7.2-7.4) two times and resuspended in P. B. S.. Destructed platelets (S. D. P.) were prepared by washing P. R. P. in P. B. S. once and by rinsing with 3.8% sodium citrate (S. C.) followed by suspending in 3.8% S. C..
Experiment (1): Particles of fer. -Con A were observed on the membrane of the washed platelets in the distance of 15-20nm from the outer leaflet of the membrane.
Most of the particles were distributed linearly in parallel with the membrane very densely at the concentration of 0.4mg/ml of fer. -Con A (as ferritin concentration), less densely at 0.2mg/ml and much less densely at 0.1mg/ml respectively.
Experiment (2): Particles of fer. -Con A were arranged in linear on the outside of the disrupted membrane in the distance less than 20nm, but not on the inside.
Experiment (3): Particles of fer. -Con A were distributed in appearance of “mass or branch” at distance of 10-40nm.
In each experiment, control samples were pre-treated with inhibitors of the lectin before mixing with fer. -Con A.
In conclusion, the specific Con A binding site was localized in the surface coat of human platelets and divided into two types in mode of distribution. One type was arranged in appearance of “mass or branch” in the outer layer of the surface coat, whereas the other was linearly arranged in the inner layer.

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Recently a synthetic vasopressin DDAVP is used for hemostatic control of hemophiliacs. The hemostatic mechanism of DDAVP in hemophilia A and von willebrand's disease is understandable because of the increment of factor VIII activity and von Willebrand factor, but in hemophilia B the mechanism is yet unclear. The influence of DDAVP on platelet function in normal subjects was studied to clarify the hemostatic mechanism.
Eight μg of DDAVP was injected intravenously to fifteen normal subjects and VIII: C, VIIIR: WF, aPTT, platelet count, prothrombin consumption, platelet adhesion (Hellem II method), platelet aggregation induced by ADP, adrenaline, collagen and ristocetin, platelet shape and platelet factor 3 were measured before and after (one and two hours) the injection.
VIII: C and VIIIR: WF increased by 2.5 and 1.5 times, respectively (p<0.001). Increase of prothrombin consumption rate (p<0.002) and platelet adhesion rate (p<0.01) was statistically significant. APTT also shortened (p<0.001) and platelet aggregation induced by a low concentration of ristocetin (1mg/ml) increased in 11 out of 15 cases. In platelet shape study only the pseudopod increased significantly (p<0.01).
The accerelation of prothrombin consumption and platelet adhesion may be induced by the increase of VIII: C and VIIIR: WF, and DDAVP seems not to give any direct effects on platelet function in normal subjects.

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In the previous study, urokinase was administered by several kinds of methods to patients with thrombo-embolic disease and its fibrinolytic activity was evaluated by means of euglobulinlysis time and fibrin plate method with euglobulin fraction.
For the purpose of evaluating the effect of an increased dose and prolonged infusion of urokinase on plasma fibrinolytic activity, a dose of 18×10⁴IU of urokinase was infused intravenously for six hours following a bolus of 3.6×10⁴IU in 29 patients with thromboembolic disease. During these procedures plasma fibrinolytic activity was estimated by the same means as those used in the previous study.
The dose of urokinase used in this study seemed insufficient to maintain effective fibrinolytic activity, as far as evaluated by euglobulin lysis time.
Values of the standard fibrin plate method with euglobulin fraction varied so markedly that this method is not suitable for evaluating fibrinolytic therapy in individual cases.
Although several factors which might influence euglobulin lysis time were examined, no determinant one was found.
After starting urokinase infusion, the amount of whole plasmin measuered by SK-activated euglobulin lysis time decreased gradually and slightly but this decrease was significant. This may be explained as metabolic clearing of the plasmin-α₂-plasmin inhibitor complex by reticuloendotherial system.
Since no significant changes were seen in the serum FDP level after infusion of urokinase in all 29 cases, determination of FDP was not an appropriate way of evaluating fibrinolytic activity in our experiment.

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Human factor IX has been purified from a commercial prothrombin complex concentrate (Proplex^®) with a yield of 17%. The purified factor IX preparation had a specific activity of 171 units per mg protein. The purification procedure involves BaSO₄ adsorption-elution, 40-70% saturated (NH₄)₂SO₄ precipitation, DEAE-Sepharose CL-6B chromatography, heparin-Sepharose 4B chromatography, and preparative disc gel electrophoresis. The final product is homogeneous on an analytical polyacrylamide gel electrophoresis, and has an apparent molecular weight (mol. wt.) of 57, 000 which is not changed by chemical reduction.
The monospecific antiserum to factor IX was raised against rabbits, and possessed an inhibitory activity equivalent to 35 Bethesda units per ml.
In the course of factor IX purification, prothrombin (mol. wt. 72, 000) has also been purified (36% yield), and has been shown to have a specific activity of 8.6 units per mg protein.

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MCI-9038 (MD-805, synthetic thrombin inhibitor No. 805), (2R, 4R)-4-methyl-1-[N²-(3-methyl-1, 2, 3, 4-tetrahydro-8-quinolinesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid monohydrate, is the new thrombin inhibitor that shows a potent and highly selective inhibitory effect.
We devised the new experimental arterial thrombosis in rabbits and evaluated the effect of MCI-9038 on the thrombus formation of this thrombosis.
This experimental thrombosis was developed at the carotid artery by injuring the vessel wall by acetic acid, which shows a marked potency of a penetration and stimulation to tissues, and the procedure was as follows; the exposed carotid arteries of a rabbit were surrounded with absorbent cotton in a plastic capsule, and then absorbent cotton was soaked sufficiently with an aqueous 90% acetic acid for three hours.
The size of the thrombus was classified as follows; occlusion, large, middle, small and no thrombus. The thrombus was mainly consisted of platelets. The continuous infusion at rates of 1 and 3μg/kg/min of MCI-9038 showed the obvious inhibitory effects on the thrombus generation, the plasma concentrations being 0.16-0.17μM and 0.41-0.49μM respectively. Heparin was administered intravenously at five minutes before soak of acetic acid and showed the dosedependent inhibitory effects at a dose of 300units/kg or more.
Oral administration of MCI-9038 at doses more than 10mg/kg (plasma concentration; 0.17-0.25μM) showed the inhibitory effect, but not at a dose of 2.5mg/kg.
Aspirin snowed the obvious inhibitory effect at an oral administration of 300mg/kg.
From above results, it is considered that MCI-9038 would be used clinically as an effective antithrombotic agent.

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Purpose; In the serious cases of toxemia of pregnancy, the coagulation pattern and the morphological findings suggest participation of the intra-renal vascular coagulation. Accordingly, it is considered that control of mother and fetus by administration of drugs with anticoagulant and fibrinolytic system activating agent is useful. Therefore, taking women in normal course of gestation (300 cases) as the control and 17 cases of pre-eclampsia (gestosis index, 9.1±1.5), daily dose 3, 000-1, 500mg of DS and 48, 000 IU of UK were administered into the cubital vein by one shot or drip (10-30 minutes) twice daily, i. e. in the morning and evening and the effect of preventing aggravation of pre-eclampsia has been examined and the result will be reported here.
Method; G. I, BP, edema, urine volume, urine protein, Ccr (ml/mm) blood platelet counts, PT, PTT, PRT, TT, factors XII, IX, VIII, X, VII, V, II and XIII, Fbg, Plg-Act, pro-Act, Plg, SFMC, serum and urine FDP, AT-III, C₁-INA, α₂-PI, α₂-M and α₁-AT.
Results; Comparison between pregnant women and the pre-eclampsic women revealed enhancement of the intrinsic coagulating factor activity, marked decrease in the extrinsic coagulating factor activity, depletion of Plg-Act, rise in SFMC, marked increase in blood and urine FDP, and decrease in AT-III and α₂-PI, suggesting a tendency of thrombus formation accompanied with hypercoagulable and low fibrinolytic states. After administration of DS plus UK, the following coagulative patterns were observed, namely, normalization of the intrinsic coagulating factor activity, recovery to normal levels of the extrinsic coagulating factors (FVII. V. XIII) activity, Plg-Act and blood FDP, increasing tendency of pro-Act, Plg, AT-III, α₂-PI, α₂-M, α₁-AT and disappearance of urine FDP at the same time, improvement of clinical findings such as a fall of B·P, disappearance of edema, reduce in urine protein and increase in Ccr (ml/mm). We believe the present therapy is enough effective in treatment of severe pre-eclampsia and the intrarenal vascular coagulation.

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We have alreadey elucidated the administration of Defibrase as a defibrinating agent promoted fibrinolysis. In this study, we carried out animal experiment using hybrid dogs for investigating the mechanism of fibrinolysis induced by the administration of Defibrase, especially on the relationship between fibrinopeptide A, des A fibrinmonomer and Defibrase itself and a release reaction of plasminogen activator from vascular wall.
Fibrinolytic phenomenon was not observed at all on the intravenous administration of 0.18mg/kg body weight of fibrinopeptide A. However, when 7.5, 17.0 and 34mg/Kg body weight of des A fibrinmonomer were administered, the concentration of FDP was increased and fibrinogen and α₂-plasmin inhibitor levels were decreased depending on the dosage administered. Similar findings were also found following Defibrase administration. To observe a release reaction of plasminogen activator from vascular wall, perinfusion experiments were performed using hybrid dog's hind limbs. Among groups of 20, 50 and 100BU of Defibrase administration in a way of one shot injecton, only three out of five dogs in a group received 100BU of Defibrase showed release of 0.1IU/ml of plasminogen activator, although which would not be strong enough to promote fibrinolysis.
The fact that des A fibrinmonomer, which is a secondary byproduct on the administration of Defibrase, may play a major role in fibrinolysis phenomenon induced by a release of plasminogen activator from vascular wall should be considered in the administration of Defibrase.

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It is known that ristocetin, an aminoglycoside antibiotic, induces the platelet agglutination in concert with the ristocetin-cofactor (von Willebrand factor) in normal plasma and also causes the precipitation of fibrinogen and other plasma proteins. The fibrinogen precipitated by ristocetin redissolves if the ristocetin is removed from the solution. The precipitation of fibrinogen is partially inhibited by the presence of urea. In the course of our study for the isolation of fibrinogen from plasma by affinity chromatography on ristocetin-agarose, we utilized the above observations about the relationship between ristocetin and fibrinogen. Ristocetin-agarose adsorbs fibrinogen and other proteins from plasma. After incubation of ristocetin-agarose with plasma, the agarose packed in a column was washed with buffer containing 1M NaCl and treated with 8M urea. Fibrinogen eluted in urea fraction has a clottability of 95-97% and is not contaminated with cold-insoluble globulin, plasminogen, Factor VIII, Factor XIII, immunoglobulin G and other plasma proteins. This fibrinogen has also the native characterization about 1) the release of fibrinopeptides from fibrinogen by thrombin treatment, 2) polymerization of fibrin monomer and 3) the substrate for activated Factor XIII. The present procedure could achieve a high recovery, over 90%, of fibrinogen from plasma. This study showed that ristocetin-agarose is a suitable adsorbent of fibrinogen purification from plasma by affinity chromatography, especially from limitted small amount of valuable plasma from the patient such as dysfibrinogenemia.

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1. Using chromogenic synthetic substrates, S-2222 and S-2238, a new technique to measure the time course of Xa and IIa generation in the plasma defibrinated with Ancrod was designed and applied for analysis of bypassing activity of FEIBA.
2. The generation of Xa and IIa was returded in hemophilic plasma and formed much lesser activity in comparison with normal plasma. However, the generation patterns of Xa and IIa in hemophilia A or B plasma without inhibitor were normalized by the addition of factor VIII concentrates or prothrombin complex concentrates respectively. The addition of FEIBA in the hemophilia A or B plasma with inhibitor brought on the promotion of generation of IIa accompanied with insufficient generation of Xa. In those cases above mentioned, partial thromboplastin time after the drip infusion of FEIBA was shortened.
3. When antiserum of factor IX or factor VII was added to the mixture of FEIBA and phospholipids suspended in calcium cloride solution, the inhibitory effect of antifactor VII serum was much stronger than that of anti-factor IX serum on IIa generation in the mixture. Therefore, it is suggested that factor VII or VIIa in FEIBA might play an important role for the generation of IIa in the bypassing therapy.

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The tissue contents of fibrin (Fb.), fibrinogen (Fbg.) and their derivertives (Denv.), and the autofibrinolytic rate of the tissues were examined to analyse the role of blood coagulation and fibrinolysis in the tissues under the pathologic process.
The tissue contents of Fb., Fbg. and Deriv. were estimated by the measurement of FDP in the digested tissues with latex agglutination method after the tissues were reacted with plasmin at 37°C for 4hrs. The autofibrinolytic rate was also estimated by the measurement of FDP production in the incubation medium after the sonicated tissues were incubated and autofibrinolysed at 37°C for 1hr.
With these methods, in the course of rat Masugi nephritis, glomerular contents of Fb., Fbg., and Deriv. were shown to increase immediately after nephrotoxin injection and be followed by the additional slow decrease. In contrast, autofibrinolytic rate of the glomeruli showed biphasic after nephrotoxin injection: initial rapid increase in the first 4hrs and the following decrease thereafter until 24hrs. At the same time urinary excretion of FDP in the process of the nephritis paralleled with the autofibrinolytic rate but not with the tissue content of Fb., Fbg. and Deriv.
The results suggested that these assay methods are simple, sensitive and useful in the evaluation of blood coagulation and fibrinolysis in the various tissues under the pathologic process.

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Platelet aggregation, plasma β-thromboglobulin (β-TG) and platelet ATP and ADP contents were examined in 20 patients with chronic glomerulonephritis (CGN) and 10 normal controls. Contents of platelet ATP and ADP which were extracted with ethanol were measured by Holmsen's firefly luciferase method. The mean values of platelet aggregation and plasma β-TG in the patients were significantly higher compared with those of normal controls. The mean platelet ATP content in the patients was 7.10±0.20 (SE)μ moles/10¹¹ platelets, significantly (p<0.05) lower than that of normal controls (7.87±0.36μ moles/10¹¹ platelets). The mean platelet ADP content was 3.34±0.18μ moles/10¹¹ platelets in the patients. This value was lower than that of normal controls (3.59±0.22μ moles/10¹¹ platelets) although this did not reach significance. The mean platelet ATP content was significantly (p<0.05) lower in patients with hypoalbuminemia than in those without it.
The results suggest that platelet aggregation and release reaction were increased in patients with CGN. Decreased adenine nucleotides in platelets may be related to the increased platelet energy metabolism in addition to the decreased storage pool in the patients.

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Recently, the vascular injury have been investigated as the most important complications of Diabetes Mellitus (DM). We studied on the correlation between Kallikrein system and haemostasis in DM. The results are as follows.
1) Platelet β-thromboglobulin (β-TG) levels were 31.0±18.0ng/ml in normal subjects and 87.0±61.8ng/ml in DM. In the group with retinopathy β-TG level was 65.8±41.5ng/ml and it was 97.5±53.5ng/ml in the group with nephropathy.
2) There was a positive correlation cetween β-TG and platelet factor 4 levels in DM. (r=0.81)
3) Prekallikrein (PK) level was 94.8±6.0% in normal subjects and 83.9±38.0% in DM. In DM group without complications, PK level was 111.8±24.9%.It was 56.6±34.3% in the group with retinopathy and 76.4±26.3% in the group with nephropathy. In PK level significant decrease was recognized in DM with complications.
4) Factor XII level increased in DM. There was significant correlation between F-XII and PK levels in DM. (r=0.74)
5) There was a positive correlation between β-TG and PK levels in DM.
6) Plasminogen level decreased in DM. There was a correlation between plasminogen and PK levels. (r=0.3)
7) α₂-plasmin inhibitor level increased in DM. There was also a correlation between α₂-Pl and PK levels. (r=0.36)
8) In cholorpropamide alcohol flashing test, skin temperature showed a gradual elevation. But, in comparison with normal subjects group, it was relatively low in DM group. Platelet aggregation and PK levels gradually decreased in normal subjects. But, in DM with retinopathy groups they were widely variable. The changes of conjuctive capillaries by alcohol test was poor in vascular complication groups. The changes of PK levels suggested Kallikrein formation and Kallikrein converted Kininogen to Kinin. Kinin had vasoactive effect and Insulin like activity.
It is concluded that the changes of these factors levels in vascular complications with DM, and showed thrombotic tendency and disturbances of circulation and glucose metabolism. These haemostatic factors are related to the progress and prognosis of vascular complication in DM.

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PGE₁ is one of prostaglandins which inhibit platelet functions and have vasodilating activity as well as PGI₂. Newly developed PGE₁ analogue (ONO-1206) was supplied for the clinical evaluations by Ono Pharmaceutical Company. This research was performed to analyze the effect of orally administered ONO-1206 on platelet functions and to evaluate the usefulness on the thromboembolic disorders.
Comparing with PGI₂, this analogue demonstrated the similar inhibitory activity on the platelet aggregation in the in vitro study. Oral administration of ONO-1206 on the patients with thromboembolic disorders showed the dose-dependent inhibition on platelet aggregation and adhesiveness. This activity continued for 180min. (max. at 120min.). Daily oral administration (20μg or 30μg t. i. d.) was continued for two weeks and its effect on blood pressure, heart rate, platelet aggregation, platelet adhesiveness, and platelet c-AMP level were evaluated. Both of administration doses caused remarkable depression of platelet aggregation and increase of platelet c-AMP level and mild suppression on the platelet adhesiveness. Blood pressure was decreased in almost all cases but heart rate remained unchanged. Clinical improvement of symptoms were observed in the patients with deep vein thrombosis or angina pectoris.
These results suggest the effectiveness and usefulness of this oraly administered PGE₁ analogue against the prevention and treatment of thromboembolic disorders.

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Prostacyclin (PGI₂), the major active metabolite of arachidonic acid in the vascular endothelium, is characterized by antiaggregatory and vasodilator properties. In this report, the significance of PGI₂ on fetal platelets was studied. Platelet aggregation induced by ADP, collagen, adrenalin were found to be augmented in maternal blood but suppressed in the umbilical cord blood.
Plasma β-thromboglobulin level is higher in maternal and umbilical cord blood, compared with control. Plasma 6-keto-PGF_1α and TxB₂ values were significantly higher in umbilical cord blood than in maternal blood (p<0.05). Plasma cyclic AMP in the umbilical cord blood was significantly (p<0.001) higher than in maternal blood, but PDE activity showed no difference between mother and fetus.
A much larger amount of PGI₂-like substance was released from the umbilical cord artery and vein than from the intraplacental vein and chorionic tissue. But release of that was decreased from the umbilical cord artery (4.4±2.7nmoles/mg tissue/hour) and vein (2.9±2.1) of pre-eclampsia complicated by IUGR.
These results indicate that various factors, such as PGI₂, TxA₂ and cyclic nucleotides, may co-exsist in high levels in fetus and the balance is needed for the maintenance of physical interaction between platelet and vascular endothelium in the fetal blood vessels.
Especially PGI₂ may play an important role in this balance and in the regulation of fetoplacental circulation.

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The effects of a novel antianginal agent, molsidomine (N-ethoxycarbonyl-3-morpholinosydnonimine (SIN-10)) and its metabolites (3-morpholinosydnonimine (SIN-1), N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A), cyanomethylenaminomorpholine (SIN-1C) on human platelet aggregation and guanylate cyclase were investigated. SIN-10 and SIN-1C were weak inhibitors of platelet aggregtion induced by collagen (1μg/ml) and ADP (4μM). SIN-1 and SIN-1A were 1, 000 times more potent inhibitors of platelet aggregation than were SIN-10 and SIN-1C. The concentration of SIN-1 and SIN-1A producing 50% inhibition of the platelet aggregation induced by 1μg/ml of collagen were 0.6μM and 0.08μM respectively. SIN-1A was about 10 times more potent inhibitor than SIN-1. Human platelet guanylate cyclase was markedly stimulated by SIN-1 and SIN-1A and half maximal stimulation by these agents were produced by 2μM and 0.2μM, respectively. SIN-10 and SIN-1C did not stimulate the guanylate cyclase activity at concentrations up to 10mM.
We now report that the metabolites of SIN-10, such as SIN-1 and SIN-1 A inhibit human platelet aggregation in vitro and also stimulate guanylate cyclase purified from human platelets. Inhibition of platelet aggregation by SIN-10 metabolites may account for a part of their beneficial effects in coronary artery disease.

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Inhibitory activity of microvessel for ADP induced platelet aggregation was investigated using the glomeruli isolated from rat kiney. The activity recovered in the incubation medium was increased during the incubation with A. A. for 10-20min. And this activity was highly correlated with the number of incubated glomeruli. When indomethacin was added to the incubation medium, this activity was decreased, but was not inhibited by 15-HPAA addition. Half time of this activity in the medium was approximately 20min at room temperature. ¹⁴C-A. A. incubated with isolated glomeruli was converted to several PGs which were identified as PGD₂, E₂ and TxB₂ (A₂) by thin-layer chromatography. They are the major products synthesized by rat isolated glomeruli, although the main PGs synthesized in large vessel as aorta was identified as PGI₂. And these products could not be detected when indomethacin was added. From these results it was concluded that the prostaglandin synthesis in the vessel wall might be different from each other in the localization and size of vessels in various many organs, and might be related to the functional aspect of vessel wall.

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The positional distribution of fatty acids in phospholipids was analyzed for human platelets activated by thrombin. At 30 sec after thrombin-activation, when the content of phosphatidylinositol (PI) fell by 40%, the 2-position of PI underwent a significant decrease in arachidonate (77.6%→61.5%) with a compensating increase in oleate and stearate. However, following PI resynthesis (10min after thrombin-activation), its positional distribution tended to revert to that of non-activated platelets. No significant change was detected in the fatty acyl positioning in other phospholipids. In addition, evidence is presented that human platelet lysates acylate 1-acyl-sn-lycero-3-phosphorylinositol and the acylation rate for arachidonate was 2.5 times higher than for any other unsaturated fatty acids tested. These findings indicate that thrombin-induced alteration in fatty acid paring of PI is subsequently rearranged by the deacylation-reacylation after PI resynthesis.

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Metabolism of arachidonic acid (AA) by human platelets was studied by TLC. Gelfiltered platelets (GFP) were eluted both with Ca²⁺-free Tyrode albumin solution (pH 7.3) and with albumin-free Haslam solution (pH 7.4). GFP was stimulated by different concentrations of thrombin (f. c.: 0.06-5u/ml). Platelet lipids were extracted by the method of Lapetina and Cuatrecasas, followed by TLC in the two kinds of slovent systems: I; Tou's solvent system; chloroform/methanol/acetic acid/H₂O (81: 10: 45: 5, v/v) and II: the solvent system of Hong and Levine; the upper layer of ethylacetate/glacial acetic acid/iso-octane/H₂O (90: 20: 50: 100, v/v). ¹⁴C-AA was incorporated into platelet phospholipids: PC 60.7%, PI 16.9%, PE 10.8% and PS 10.2%. On the other hand, free AA was released mainly from PC and PI. Radioactivity of 13% of free ¹⁴C-AA was liberated from phospholipids after stimulation of GFP with 5u/ml of thrombin for 5min. AA metabolites, such as PGD₂, PGE₂, TXB₂, HETE and HHT were also detected by TLC. No spots and radioactivities corresponding to HHT were detected in aspirin (1mM)-treated normal platelets and in platelets from the patient with congenital cyclooxygenase deficiency after stimulation by thrombin. Free AA was not liberated from phospholipids in platelets from the patient with congenital phospholipase A₂ deficiency.

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