We contrived new method of the adjudication of platelets spreadability (Die Ausbreitung-sfähigkeit der Thrombozyten) which had been developed by Marx (1960) and Breddin (1965). Using this method, we examined platelets spreadability of blood diseases and uremia, and investigated mechanism of platelets spread.
Methed: Object-slide was made according to Breddin method and we used the slide made of plastic. Our method is to classify each one by platelets Diameter and Form. Classification of diameter; Platelets is divided into five groups by its diameter and figure out real dimension of its dimeter. Normal mean diameter was 7.88±0.34u. (Fig. 1) Classification form; Platelets is divided five types by its form and figure out ratio of abnormal form. Ratio of abnormal form was under 5per cent. (Fig. 2) Adjudication of platelets spreadability; We judge by mean diameter and ratio of abnormal form, besides attach importance to its distribution pattern.
Clinical cases: In uremia (6 cases), platelets spreadability decreased, but increased remarkably after P. D. in all cases. (to 7.05±0.40u from 5.22±1.13u) We tried next studies. Using washing normal platelets to add uremic plasma, its spreadability inhibited remarkably, and case of added normal plasma for washing uremic platelet's, spreadability was inhibited slightly. Consequently we concieve in uremia drop of spreadability is mainly because of plasma and partly because of platelets abnormality. Result of several blood diseases is showen Fig. 3.
We investigated spreadability of platelets under the influence of several factor: a) Adhesion; Platelets did not adhere to slide was not spreaded. b) Plasma factor; Used washing normal platelets. In absence of plasma, platelets did not spreaded. In case of added plasma, platelets spreaded almost normaly. In case of added afibrinogen-plasma (used trombin), spreadability inhibited remarkably. In case of added serum, platelets did not spreaded. c) Glycolysis; Added 1mM A. T. P. increased spreadability. In added 1mM A. T. P, and 100mg/dl glucose to washing platelets, platelets spreaded. Added antiglycolysis (monoiodo acetic acid and 2-deoxy d-glucose) decreased spreadability. d) Low temperature; In 4°C, spreadability was inhibited.
We conceive that platelets spread after adhesion to slide, plasma factor is required and spreadability relates to the energy of glycolysis metabolism.

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The ⁵¹Cr-labeling method for measuring platelet life span has been introduced by R. H. Aster in 1965. But his method has not been widely adopted as a routine examination, because a largevolume of blood had to be drawn to label the platelets.
The author tried to improve the method by using only 200ml of blood and carried out the test clinically in 11 cases with various disorders. The ¹⁴C-serotonin release test was done simultaneously to detect the platelet antibody. Methods:
(1) To a double bag containing 30ml of ACD (A), 0.15M citric acid was added in a volume determined by a donor's hematocrit (Aster 1965).
(2) Collection of 200ml of blood.
(3) Separation of PRP and PPP by centrifugation.
(4) Platelets were suspended in 3-5ml of plasma and 300-500μCi of isotonic sodium ⁵¹Cr-chromate was added.
(5) Addition of 50mg of ascorbic acid in 20ml of plasma after 20min. incubation.
(6) Removal of the supernatant after centrifugation.
(7) Re-suspension of platelets into 20ml of plasma and infusion into a recipient.
Pesults:
Ir took 2.5-3 hours to perform the entire procedure.
The radioactivity infused into recipients ranged from 11 to 41μCi (mean 19μCi).
Platelet recovery ranged from 30 to 68.3%.
Platelet survival was as follows:
ITP: 3 cases, 1-1.5days (Platelet Half Life Time 0.1-0.5day)
ITP in remission: 7.5 (2.4)
reactive thrombocytosis: 2 casis 9, 9.5 (3.0, 3.5)
liver cirrhosis: 3 cases, 7-8.5 (3.0-4.0)
chronic hepatitis: 6 (3.3)
sideroblastic anemia: 7.5 (2.2)
Surface scanning: High spleen/liver ratio was observed in 3 cases.
Conclusion:
(1) Platelet survival studies were performed successfully with 200ml of blood. This method seemed to give accurate information clinically, when donor's platelet count was more than 40, 000/μl.
(2) Clinically it was much appropriate to express the platelet survival by half life time rather than whole survival time.
(3) For interpretation of the platelet survival curve, it was informative to have checked the anti-platelet antibodies.

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A number of factors such as vascular wall, blood stream, and plasma composition are involved in thrombus formation. To analyze these complicated mechanism and predict thrombogenic episodes, metabolic and kinetic study of platelets has been conducted on the cases with increased platelet count and vascular damages. This study deals with the evaluation of enormous increase of peripheral platelet count as a risk factor in thrombogenesis, by means of thrombokinetic study.
A total of 11 cases were studied as subject, composed of 4 cases of thrombocythemia, 4 cases of chronic myelogenous leukemia (CML) with more than 1, 000, 000 platelets, 1 case of myelofibrosis with myeloid metaplasia (MMM), and 2 cases of vascular disease.
To predit hypercoagulable state in vitro, aggregation and adhesiveness have been generally studied. We measured platelet adhesivenss by Salzmann's method. Platelet aggregation was measured by aggregometor (Bryston's company). Many workers reported that platelet adhesiveness and aggregation were increased in thrombogenesis. Among our cases thrombosis was observed in 1 case of CML and 1 case of thrombocythemia.
In patient with CML, platelet adhesiveness and aggregation were increased, the latter being induced by low concentration of epinephrine. On the other hand, a patient with primary thrombocythemia had absence of epinephrine induced platelet aggregation, and decreased adhesiveness. We did not find a good correlation between platelet function test and episodes of thromboembolism in patients with thrombocytosis.
In this respect, platelet kinetics study seems to be useful to detect early stage of platelet plug formation; adhesion in vivo. We performed platelet kinetics study of all cases with thrombogenic state, using ⁵¹Cr-labeled platelet.
The results of this study showed that the platelet survival time was significantly shortened in 2 cases of thrombocythemia, 3 cases of CML, 1 case of MMM, and 2 cases of vascular disease. The means of platelet survival (T1/2) in cases with thrombogenic risk factor was 2.85 days, shorter than that of normal subject, 3.5-4.5 days.
Platelet turnover was also increased; almost twice as that of control. Among these cases, thrombosis like symptoms were observed in two cases.
The shortening of platelet survival time implied accelerated platelet consumption and suggested a condition prone to thrombus formation and an indication for prophylatic antithrombotic therapy.

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The hemodynamic benefits of replacing defective heart valves with prosthetic valves are compromised by thromboembolic complications. After replacement of artificial heart valves, platelets are destroyed by prosthetic heart valves, and consumed by interaction with prosthetic valves.
It has been shown that patients with substitute heart valves have short platelet survival time, which can be lengthened after treatment with platelet inhibitor drugs as Dipyridamole. For this purpose we have given 450mg daily dosage of Dipyridamole in all patients with prosthetic valves.
To determine the relation thromboembolism to the presence of substitute heart valves, kinetic studies of platelet were performed in 14 patients. Six patients had received mitral valve prostheses, 4 had aortic valve prostheses, and 4 had double artificial valves. In accordance with platelet survival half time, platelet survival was determined that it was normal or not. The normal platelet survival half time was 4.0±0.5 days (S. E. M.). Average platelet survival time in 4 patients with aortic artificial valves was 3.85±0.75 days (survival half-time±S. E. M.), in 6 patients with mitral artificial valves, 3.73±0.67 days, and in 4 patients with double prostheses, 3.71±0.74 days. In these patients average platelet survival was almost normal, because they were given Dipyridamole. Thromboembolism occurred in 3 of the 14 patients (21 percent). In this group, platelet survival was shortened in 2 of 3 patients and was normal in one. In these patients, kind of prostheses was Kay-Shiley valve. In spite of treatment with Dipyridamole, it was seen that platelet survival was shortened in 6 patients. In the case of non-thromboembolism episode, it was thought that platelet was destroyed by prothetic heart valves, or was reduced by platelet aggregation and adhesiveness. Therefore there was confirmed that platelet behavior changing factor needed furthermore. In the case of thromboembolism episode, it was thought that platelet was reduced by formation of clot at artificial heart valve and its surrounding. Therefore anticoagulant and fibrinolysin therapy, or artificial valve re-replacement needed in this case.

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Thromboembolism continues to be a major complication occurring after prosthetic heart valve replacement. It is thought that thrombus formation on prosthetic surfaces may be primarily related to the adherence of platelets with subsequent platelet aggregates and the deposition of fibrin.
Platelet adhesiveness was examined in 40 patients with Björk-Shiley prosthetic heart valves; 28 of them were receiving 1500-2250mg of clofibrate, 5 receiving 300mg of dipyridamole, and the remaining 7 receiving warfarin alone. None of the patients had a history of thromboembolism. Platelet adhesiveness in a glass bead column was determined by a modified technique of Salzman. It is known that by this method not only platelet adhesiveness was detected but also platelet aggregation induced by ADP which was released from erythrocytes hemolysing on the glass surface owing to the blood flow. Student's t-test was used to compare the means for statistical significance.
The mean platelet adhesiveness in patients with prostheses was decreased, and differed significantly (p<0.005) from the normal control value. It seemed likely that a platelet suppressant drug such as clofibrate or dipyridamole play a role in decreased adhesiveness. The mean platelet adhesiveness in 13 patients with an aortic prosthesis receiving clofibrate alone was not statistically different from that in 9 patients with a mitral prosthesis receiving clofibrate in combination with warfarin. Four of 7 patients receiving warfarin alone showed a relatively decreased platelet adhesiveness of below 15%, but there was no obvious correlation between platelet adhesiveness and Thrombotest value in these patients.
The mean platelet count in patients with double prostheses was relatively low, and differed significantly from that in patients with an aortic or with a mitral prosthesis (p<0.0005 and p<0.01 respectively). The mean platelet adhesiveness in patients with an aortic prothesis was slightly decreased, and differed significantly (p<0.05) from that in patients with a mitral prothesis. The mean platelet count in patients with atrial fibrillation was signficantly (p<0.0005) different from the count in those with normal sinus rhythm, but no significant difference was found in platelet adhesiveness between the two groups.
No correlation was found between platelet count and adhesiveness, and also between hematocrit (ranging 24 to 47%) and platelet adhesiveness in the patients with prostheses.
The measurement of platelet adhesiveness by this method is easy to do and thought to be useful. However, further studies are expected on correlations between the platelet adhesivness and other platelet functions including platelet aggregation, platelet survival time and also the clinical incidence of thromboembolism.

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The thromboembolism following the artificial valve replacement in patients with acquired heart valve diseases is one of the most important complications. This study has been aimed to clarify the specific abnormalities of platelet function and coagulofibrinolytic system after the operation for valve replacement.
Methods:
Thirteen patients with heart valve diseases were divided into 2 groups as follows. In 7 of them the valve repair has been performed (group I), and in 6 the valve replacement has been done (group II).
Laboratory data have been chronologically checked just before the operation, and for 6 months after the operation.
Platelet aggregation was studied with an aggregometer (Bryston Co.), with the modified method of O'Brien's technique. ADP and collagen were used as aggregating agents.
Platelet aggregation was estimated by indices of maximum aggregation rate (%), T1/2 (sec) and “R”. “R” is a new index postulated by us which means the changing rate of T1/2 (Y) obtained by change of platelet concentration (X) in collagen aggregation, that is R=d log(Y)/d log(X), We believe by clinical and experimental data that when “R” is small, the platelet aggregability is large and when “R” is large, the platelet aggregability is small.
Fibrinogen and plasmin activity in plasma were measured by ordinary methods. FDP in plasma was estimated by the HIT method.
Results:
The mean values of T1/2 induced by both ADP and collagen were shorter in group II than group I during the course observed. However, the mean value maximum aggregation rate induced by ADP was smaller in group II than in group I. This might be resulted from the effect of clofibrate which was given in group II.
Though “R”s remained in normal range in the majority of group I, they were out of normal range in about half of group II; sometimes smaller, and sometimes larger within a observation period of postoperative days. This variation of “R”s means that in patients of group II the platelet function is in a very unstable state, sometmes in hyperaggregability state by the stimulation with artificial valve, and occasionaly in hypoaggregability state by the secondary damages due to microthrombi.
Specific changes were not seen in fibrinogen amount and plasmin activity but high values of FDP were noticed more frequently in group II than in group I.
These results indicate that in patients with artificial heart valve, microthrombi may be made and thereafter FDP may be produced as a result of hyperaggregability of platelets.
It is also important problems that such a hyperaggregability state was induced even in these patients given clofibrate and warfarin.
Further studies will be needed to establish the pathogenesis and prophylactic methods for thrombosis in the patients with artificial heart valve.

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The effect of dipyridamole on human platelet adhesivenss was studied by the author's modification of Hellem's II method and its effect on platelet aggregation was observed by the turbidmetric method of Born using aggregometer (Bryston).
The adhesivenss, in vitro, was inhibited by dipyridamole at 100-500μg/ml blood, and the inhibited degree of the the adhesiveness was slighter than that of ADP-induced aggregation. In vivo, by intravenous injection of dipyridamole, the adhesiveness was inhibited obviously during 5-15 minutes, and recovered after 30 minutes. It was also lowered during the course of oral abministration of dipyridamole (75-250mg/day), but was rised by the nterruption of its administration.
ADP-aggregation (0.4μg) was also inhibited by dipyridamole at 50-500μg/ml PRP. Only secondary aggregation was inhibited by its low concentrations, whereas primary aggregation was also inhibited by its high concentrations. Lag phases of adrenalin (1μg)-and collagen (50%)-aggregation were prolonged by this inhibitor at 50-200μg/ml, and such aggregation was completely inhibited at the concentration, over 200μg/ml.
The inhibitory effect of dipyridamole upon ADP-induced aggregation was not influenced by the period of its pre-incubation with PRP and by HCl used in these experiments. Platelet aggregation induced by ADP (0.4-40μg/ml of PRP) which had been incubated with 100μg of dipyridamole was similar to that induced by the same concentrations of ADP without the preincubation.
Fifty μg of dipyridamole/ml PRP slightly inhibited ADP release induced by Kaolin, and its concentration over 200μg/ml inhibited the release so remarkably that ADP thus released was not enough to aggregate platelets.
Bovine fibrinogen-aggregation was not influenved by dipyridamole at the concentration up to 500μg/ml.

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Inhibitory activity of Flurbiprofen [2-(2 fluoro-4-biphenylyly) propionic acid], antiinframmatory compound, on normal human platelet aggregation was tested in vitro.
At final concentration of 10^-5M flurbiprofen inhibited completely the platelet aggregation which was induced by 10 and 3μg/ml (final concentration) collagen. Flurbiprofen of 10^-3M (final concentration) inhibited the 2nd phase of platelet aggregation induced by 5×10^-6M (final concentration) ADP, but did not inhibit the 1st phase of 2×10^-6M (final concentration) ADP-induced platelet aggregation. When flurbiprofen was added after collagen-induced platelet aggregation or 2nd phase of ADP-induced platelet aggregation actually began, they were not inhibited by flurbiprofen.
It seems that flurbiprofen has no influence on ADP itself, but inhibits the release reaction of platelets, induced by collagen or ADP.
In scanning electronmicroscopic studies, many small balloon-like structures were found on the surface of ADP-or collagen-induced platelet aggregation mass, and these structures were seen to be round shape pseudo podes of platelets in transelucent electronmicroscopic studies. When ADP-or collagen-induced platelet aggregation was inhibited by flurbiprofen, gathering platelets atached each other with only slim pseudo pode. No particular changes were observed in the internal structure of platelet affected by flurbiprofen.

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The effects of structural changes of collagenplatelet reaction were investigated. The collagen used was derived from bovine skin collagen, of which telopeptide was removed with proctase. It was dissolved in 0.05% solution of acetic acid and then dialyzed against deionized water to adjust to about pH 5.5. This collagen solution was very stable compared with one containing salt. The digestion with collagenase from Clostridium hystoliticum was followed as a decrease in the specific optical rotatiod at 405nm. A, B and C-fractions wereisolated by fractional ammonium sulphate precipitation when the values of [α]⁴⁰⁵ dropped from -1000° to -900°, -700° and -590°, respectively. D-fraction was prepared by fractionation using hydroxyatite column when it dropped to -800°. Each fraction was dialyzed against deionized water and especially the solutin of D-fraction was concentrated with Diaflo of 0.2-0.3%. Platelet aggregation was measured with Aggregometer.
The collagen stored at 4°C for 5 months showed increased ability of platelet aggregation compared with the freshly prepared one and the same effects on it with native collagen. The sedimentation pattern of ultracentrifugation of stored collagen showed the presence of gel-like aggregates in the solution. These results suggest that telopeptide of native collagen is not essential to platelet aggregation, but accelerates the association of collagen which induces platelet aggregation. Although D-fraction showed dose dependence for induction of platelet aggregation in both lag time and maximum aggregation rate, increased lag time and platelet aggregation ability at only higher concentration were observed compared with the collagen untreated with collagenase. It suggests that the length of collagen molecules plays a part but is not a critical factor in platelet aggregation. A, B and C-fractions did not aggregate platelet at 37°C. D-fraction of low concentration altered platelet shape as shown in the transient increase of optical density of the mixture of D-fraction and PRP. D-fraction of low concentration added previously to PRP showed inhibitory effect on the aggregation ability of D-fraction of higher concentration which was added succesively, while the addition of A-fraction showed no inhibitory effect on it. It could be cosidered. that the molecule chain of A-fraction could not adhere to platelet because it was denatured at 37°C and lost its triple helical structure, but higher dose of D-fraction could adhere to and aggregate platelet because the molecule chain of it held the triple helical structure. A-fraction showed thebaility of platelet aggregation when it was measued at 22°C, because its triple helical structure was well preserved at this low temperature. The collagen denatured by heating at 60°C for 20min. showed also transient increase of optical density in the pattern of platelet aggregation which indicated shape change platelets in PRP, but no inhibitory effect on the ability of platelet aggregation of the collagen. These results suggest that there are different mechanisms in platelet-collagen reaction and the heat denatured collagen.

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The role of c-GMP in platelets has been studied by investigating the influence of c-GMP derivatives, such as N⁶-2′-0-dibutyryl c-GMP and 8-bromo c-GMP, on platelet function.
Dibutyryl c-GMP (500μM-5μM) and bromo c-GMP (1mM-10μM) did not cause platelet aggregation and serotonin release. At high concentrations these agents inhibited platelet aggregation and serotonin release induced by ADP and epinephrine. At lower concentrations the effect of these agents on epinephrine-induced aggregation was sometimes stimulatory, but was significant.
Furthermore, the possibility has been invesstigated that aggregation induced by arachidonate might be mediated by increase of c-GMP levels in platleets.
Addition of 1mM arachidonate to platelet rich plasma rapidly caused aggregation and serotonin release, but incubation of platelets with arachidonate did not alter the basal level of c-GMP in platelets.
These results do not support the hypothesis that c-GMP might play a regulatory role in inducing platelet aggregation and release reaction.

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Recently, roles of cyclic AMP and cyclic GMP in platelets have been getting important on mechanisms of platelet aggregation. However, behaviours of c-AMP and c-GMP phosphodiesterase (PDE) in platelet aggregation are still unknown. So relationships between activities of c-AMPPDE and c-GMPPDE in platelets and platelet aggregability induced by ADP and adrenaline were observed using 57 human materials. Activities of c-AMPPDE and c-GMPPDE were measured by Hidaka and Shibuya method (Biochem. Med. 10: 301, 1974). Using the same samples, platelet aggregabilities induced 3 and 10μM ADP and 0.1 and 1μg/ml of adrenaline were measured by an optical density method. In a platelet aggregation curve, slope of the curve and intensities of primary aggregation and aggregation at 5 minutes after the addition of reagent were measured. In 16 cases, screen filtration pressures (SFP) induced by 3μM ADP were also measured.
Activities of c-AMPPDE and c-GMPPDE of platelets were 1.17-8.36 and 4.16-24.39pmol/min/10⁷ platelets respectively. These means and standard errors were 3.92±0.26 and 11.97±0.89pmol/min/10⁷ platelets respectively. There were statistically significant correlations between slope induced by 10μM ADP and c-AMPPDE or c-GMPPDE activities respectively (p<0.001). These correlation coefficients (r) were 0.31 on c-AMPPDE and 0.48 on c-GMPPDE activities. Also a significant correlation between an activity of c-GMPPDE and an intensity of primary aggregation induced by 10μM ADP (r=0.28, p<0.05). Using 1μg/ml of adrenaline, there was a significant negative correlation between an activity of c-GMPPDE and an ntensity of 5 minutes aggregation (r=-0.38, p<0.01). SFP showod a significant negative correlation to a c-GMPPDE activity (r=-0.520, p<0.05). These results suggest important roles of c-AMPPDE and c-GMPPDE activities of platelets on platelet aggregation.

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The effects of sustained isometric handgrip exercise on platelet aggregability and the left ventricular function were assessed in 29 patients with ischemic heart disease (Table 1) on placebo and EG626, a newly synthesized cyclic AMP phosphodiesterase inhibitor. The platelet aggregability was assessed by using a new device (Sano, 1974) which avoids centrifugation and needs only a small amount of citrated blood (Fig. 1). The left ventricular performance was estimated by a non-invasive technique, systolic time intervals. Parameters of systolic time intervals (LVET, PEP, ICT and PEP/ET) were expressed as indices following Motomiya's multiple regression equations (Table 2).
RESULTS (1) Platelet Aggregability: On placebo, an enhancement of platelet aggregability was observed after isometric handgrip exercise in patients with ischemic heart disease but not in healthy control subjects. The enhancement of platelet aggregability was prevented by pretreatment of EG626, administered orally 90 minutes prior to the exercise (Fig. 1).
(2) Left Ventricular Function (Fig. 3): a) At Rest: Systolic blood pressure was significantly lower on EG626 pretreatment than on placebo (p<0.01). Heart rate, diastolic blood pressure and all indices of STI's were not different between two regimens.
b) During Handgrip Exercise: The increase in heart rate by exercise was significantly smaller on EG626 pretreatment. The increase in systolic and diastolic blood pressures was not different between two regimens. Handgrip exercise produced significant decrease in LVETi on both placebo and EG626 regimens (p<0.05 and 0.01 respectively). However these decrements were not statisitically different between two regimens. Handgrip exercise produced significant increase in PEPi, ICTi and PEP/ETi both on placebo and EG626 (p<0.01 each). The increment was smaller on EG626 in PEPi and PEP/ETi (p<0.05 and 0.01 respectively) and not different in ICTi between two regimens.
The foregoing results indicate the followings: Handgrip isometric exercise produced significant deterioration in systolic time intervals indicating decrease in stroke volume, cardiac output and also probable impairment in myocardial contractility in patients with ischemic heart disease. EG626 pretreatment improved above deterioration in the left ventricular function induced by the exercise.
These effects of EG626 on platelet aggregability and the left ventricular performance could be attributed to the cyclic AMP level as this compound reportedly increases c-AMP level in platelet and myocardium.

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When human platelets in plasma were exposed to a small amount (nonaggregating concentration) of collagen, epinephrine or arachidonic acid, their responsiveness to aggregating agents were potentiated and they were aggregated by a subsequent addition of nonaggregating concentration of the stimulants. Otherwise the nonaggregating concentrations of the stimulants were uncapable to induce platelet aggregation. The potentiation of platelet responsiveness to aggregating agents was also caused by collagen- or epinephrine-treated platelet menbranes, but not by arachidonic acid-treated membranes. Furthermore, the soluble fraction of collagen- or epinephrine-treated membranes contained some material responsible for platelet potentiation, indicating that the responsible material was released from platelet membranes by collagen or epinephrine. It is suggested that the material may be arachidonic acid since the soluble fraction of collage- or epinephrine-treated membranes contained a larger amount of the precursor of prostaglandin F₂α than the untreated membranes and arachidonic acid is the precursor of Prostaglandin F₂α. The potentiation of platelet aggregability by the small amounts of the stimulants was increased in nephrotic syndrome whose albumin concentration in plasma is low, and the increased potentiation was abolished by the addition of albumin. The abolishment of the potentiation of platelets by albumin could be explained by postulating that some part of arachidonic acid released from membranes immediately binds to albumin and becomes unavailable to platelets.

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Peroxidation of arachidonic acid by human platelets was studied by malonaldehyde measurement and oxygen uptake. Platelets were prepared by collecting blood into 3.8% trisodium citrate, isolation in the presence of EDTA, washing in 0.154M NaCl (saline) and finally suspending in Tris-buffered saline (pH 7.4) for malonaldehyde determination or in saline for measurement of oxygen uptake. Malonaledhyde was measured by reaction with thiobarbituric acid and oxygen uptake by the platinum electrode method. Peroxidation of arachidonic acid was induced by incubation of this fatty acid with platelets and the optimal incubation condition for formation of malonaldehyde was examined. The maximum value of malonaldehyde was produced approximately 1min after initiation of the reaction at 37°C. The activity of platelets was almost completely inhibited by heating them at 100°C for 5min. The highest specific activity was found in the particulate fraction that sedimented between 12, 000g and 105, 000g. A burst in oxygen uptake occurred concomitantly with peroxidation of arachidonic acid by platelets. Aspirin inhibited both malonaldehyde production and the burst in oxygen uptake induced by platelets. These results are in accord with the concept that endoperoxide intermediates in prostaglandin biosynthesis (prostaglandin G₂ and prostaglandin H₂) are formed by human platelets in response to arachidonic acid. Methods used in the present investigation may be employed as convenient procedures for an assay of prostaglandin synthetase activity of platelets in the evaluation of new anti-platelet drugs as well as for elucidation of pathogenesis of platelet dysfunction.

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Studies on the sialic acid of platelets in adults, newborns, and cases with various kinds of diseases were carried out by the method of Madoff and Warren. Additionaly the sialyltransferase of platelets was asssyed on the platelets of adults and newborns by the Bosman's method which was partially modified by us. The sialic acid of platelets in newborns was markedly low (Fig. 1) and the sialic acid of a case with Bernard-Soulier syndrome was low in the meaning of per protein of platelet, though rather high by the case of single one platelet (Table 1). The changes of sialic acid in the MLNS (acute febrile mucocutaneous lymph node syndrome) during the course of illness were interesting. The sialic acid of platelets decreased at 10 to 20 days from the onset of the illness and then increased slowly up to the normal range in accordance with improvement (Fig. 2). By the administration of flurbiprofen, the sialic acid increased rapidly, being decreased by cessation of the administration.
The sialyltransferase of platelets in newborns and cord blood were seemed to be slightly low in our present results. In order to get final conclusion on the sialyltransferase of platelets in newborns, further studies would be necessary.

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Thromboembolic and hemorrhagic episodes were encountered not infrequently in the period of thrombocytosis of myeloproliferative disorders; chronic myelogenous leukemia (CML), primary thrombocythemia (P. Th.), myelofibrosis with myeloid metaplasia (MMM) and polycythemia vera (P. V.). Establishment of the early detection and the regimen for prevention of these episodes are mandatory and prompted us to study the biochemical background of abnormalities in platelet functions; absence of second aggregation by epinephrine and of collagen induced aggregation, decreased platelet retention.
To analyse glucose metabolism of platelets, to which release reaction and aggregation are dependent as the energy source, we observed the activity of Embden-Meyerhof pathway and TCA cycle by measuring ¹⁴C-lactate and ¹⁴CO₂ production from ¹⁴C-6-glucose. Basal metabolism of platelets in three cases of CML, three of P. Th. and one case of each MMM and P. V., were as active as control. Epinephrine stimulated control platelets in ¹⁴CO₂ and lactate production twice as much of basal level, but could not stimulate platelets of CML. Preincubation of these platelets with PGE₂, which alone could not induce metabolic response, brought about enhanced metabolic activity in two of three cases but to the level of control. Platelets in MMM and P. V. behaved similar to those of CML. In primary thrombocythemia, platelets responded to epinephrine in two of three cases as well as contol and exhibited enhanced activity by PGE₂.
Since these platelets in myeloproliferative disorders, aggregated normally by exogenous ADP, relese mechanism seemed to be suppressed or regulated by feed-back mechanism to raise threshold for thrombus formation. In CML, α-receptor on the platelet membrane seemed to be not sensitive enough for epinephrine to induce metabolic response and production of mediator (s) for release reaction. In thrombocythemia, production of mediator (s) for release reaction might be not enough, in spite of fairly good increse of glycolysis by platelets.
With the results obtained by this study, we concluded that metabolic abnormalities exist in the platelets of myeloproliferative disorders, being accompanied with release abnormality. The mechanism how those mediators; cyclic nucleotides, prostaglandins and their metabolites are participating to give rise these abnormalities, is under investigation. Metabolic study as well as kinetic study are promising to detect and evaluate initiation of platelet consumption and hypercoagulable state, and to institute fit therapy to prevent hemorrhagic thrombosis.

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Case 1. 60-year-old female with history of three times' hematemesis. Received esohageal transection and splenectomy for Banti's syndrome. Postoperatively developed thrombophlebitis of right leg and was administered heparin. Two months later admitted for posttransfusion hepatitis. Main laboratory findings on admission are T. Bilirubin 4.1mg/dl, GOT 156u., GPT 126u. Characteristic finding is hypercoagulability. Reduction in r and k and increase in ma on thromboelastogram were conspicuous. Spontaneous platelet aggregation of platelet rich plasma (PRP) containing 30×10⁴ platelets/ml without an addition of ADP by Evans' aggregometer was most characteristic.
Electronmicroscopic examination of the platelets showed unremarkable change of internal structure but formation of chracteristic pseudopodia which may explain morphological alterations suggesting an abnormality of platelet release reaction. ADP in patient's platelets was definitely increased. High activity of ATPase in platelets was also demonstrated. This spontaneous aggregation disappeared 4 hours after administration of 0.5g aspirin.
From the above results it can be said that main causes of spontaneous aggregation of this particular case are sought in an acceleration of energy generation system in metabolic pool of the platelets as well as an increase in ADP content of the storage pool.
Case 2. 25-year-old female with liver cirrhosis. Also showed spontaneous platelet aggregation. Same mechanism as Case 1 was considered.

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Platelet fatty acid composition (wt%) of human, rat, and rabbit were studied. The results were as follows:
(Fatty acids whose wt% higher than 10% were listed.)
In rabbit platelet fatty acid composition, the arachidonic acid (20:4) was lowest and the linoleic acid (18:2) was highest among those of the three species. The results of previous study on the platelet fatty acid composition of rabbit fed cholesterol-supplemented diet for six months were also reported. In the study the basic diet (RC-5) and the cholesterol-supplemented diet were obtained from Oriental Kobo Kogyo Co., Tokyo. Ingredients of RC-5 were as follows: Crude fat 4.8g, crude protein 22.8g, and water soluble non-nitrogenous substance 46.2g in 100g. Fatty acid composition of the fat was 16:0, 16.8%, 16:1, 2.3%, 18:0, 2.8%, 18:1, 12.1%, 18:2, 39.3%, and 18:3, 26.7%. The body weight, the serum total cholesterol, and the serum triglycerides of the two groups (the rabbits fed basic diet only and the rabbits fed cholesetrol-supplemented diet) before and six months of experiment were as follows:
The platelet fatty acid composition of the two groups at six months experiment were as follows:
(Figures indicate mean±SEM.)
The platelet fatty acid composition of the cholesterol-supplemented diet group was not significantly different from that of the basic diet group.

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Since Born introduced the turbidometric method observing platelet aggregation, there have been many evaluations of platelet activity through the aggregogram.
We have recently found that the platelet aggregogram will yield a straight line following a log·log transformation of transmission, i. e.,
-log·log(T_∝/T)=at+c…(1)
The direction coefficient “a” of the regression equation (1) is considered the rate constant of the platelet aggregation, according to the following relationships,
-logT=mx+k…(2)
dx/dt=-a(x-x_∞)…(3)
and -log·log(T_∞/T₀)=at_r+c…(4)
where T, T_∞ and T₀ are the transmisson of at a given time t, of limiting value and of platelet rich plasma respectively, x and x_∞ are the number of platelets and its limiting value and a, c, m and k are constant. The t_r is the time interval of lag phase. Based on these relations, the aggregation in the aggregometer is felt the first order reaction and it was supported by the photomicroscopical observation through the sampling method. From this standpoint, authors used “a” and “t_r” to evaluate the platelet activity in the collagen induced reaction, while only “a” was used when ADP-induced aggregation was studied. In this paper, the effects of glycolytic metabolism and the effects of the nucleic acid relating substances are estimated with this method. High concentration of glucose, from 500mg/dl up to 2000mg/dl, acted as an inhibitor to collagen-induced aggregation, as shown in Fig. 1. Sodium fluoride, NaF, is known as an inhibitor of anerobic glycolytic metabolism as well as an inducer of relese reaction, as shown in Fig. 2. High concentration of glucose did not act as an inhibitor to the NaF-induced aggregation, while DBc-AMP and theophylline showed inhibitory action to the NaF aggregation, as shown in Fig. 3 and Fig. 4. DBc-AMP showed a remarkable inhibition to the collagen-induced aggregation, as shown in Fig. 5. This inhibition was more remarkable than that of c-AMP. AMP, Adenosine, DBc-GMP, and GMP showed an inhibitory action as well as theophylline. Inosine, Cytidine and Uridine showed on effect on “a”, but they showed a slight tendency to prolong “t_r”.

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Platelet aggregation by ADP (first phase) can be supposed as being a consecutive reaction:
a(Platelets)+b(ADP)→k₁(Aggregates)→ka₂(Platelets)+b′(Products)
Then the initial rate equation of platelet aggregation is
v₀=k₁·[Platelets]^a·[ADP]^b
where “v₀” is the initial rate of this reaction. In this expression, “a=2, 0<b≤2” was ascertained experimentally and the rate constant “k₁”, which partially shows the character of the reaction, was calculated in each case. Thus, the dimension of “k₁” is influenced by the value of “b”, and only when “b” is equal in each case, “k₁” can be used to compare the degree of platelet aggregation.
When [Platelets] is contant in the initial rate equation, “v₀” in relation to [ADP] is obtained from the expression
v₀=C·[ADP]^b
where “C” is k₁·[Platelets]² and constant. Whe “b” is “b₁” in Case I and “b₂” in Case II, and “b₁” is smaller than “b₂”, Curve: v₀=C·
[ADP]^b₁ and Curve: v₀=C·[ADP]^b₂ cross each other when graphed. Therefore, if [ADP]_I=II indicates the ADP concentration at the intersecting point, it equalizes “v₀”, in both case. When [ADP] is smaller than [ADP]_I=II, “v₀”in Case I is larger than that in Case II. On the other hand, when [ADP] is larger than [ADP]_I=II, “v₀” in Case I is smaller than that in Case II. This means that the intuitive aggregometer indexes, such as the initial rate “v₀”, the half life period “t1/2” and the proportion of maximum aggregation, are not very useful for expressing the degree of platelet aggregation.
These experimental facts hold good under the supposition that there is one relationship between the dispersion system of particles of cellular dimension and spectrophotometer attenuance. Although a satisfactory answer for this problem has not yet been found, we are convinced that this is a suitable supposition because experimentally, “a” is equal to “2” in any case.
We suggest future work on the basis of these experimental facts.

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Platelet adhesiveness and aggregability were studied comparatively using human citrated blood.
1) Adhesiveness in glass beads (Hellem I) was compared to the intensity of platelet aggregation as measured concurrently by the optical density (O. D.) method using ADP, adrenaline and collagen in 25 patients in convalescence of various diseases. No significant correlation between two test results was found (Fig. 1).
2) Citrated blood (0.38%) was filtrated through glass filter (glass beads column or glass-wool braid) repeatedly (up to 4 times) with the filter renewed each time. Platelet count (by Coulter Counter) and platelet aggregability were assessed before and after filtrations. Platelet aggregability was measured by O. D. method. the screen filtration pressure (SFP) method and a method for assessment of platelet sensitivity to ADP-aggregation.
i) Platelet count decreased significantly (P<0.01-0.05) after each filtration.
ii) The intensity of platelet aggregation measured by O. D. method 5 minutes after addition of ADP (10μM, final concentration) or SFP method using 3μM ADP did not change sigificantly after filtrations when the platelet numbere of the pre-filtration and post-filtration samples were equalized using platelet-poor plasma (PPP) (Fig. 2 and Fig. 3).
iii) The SFP value decreased significantly (P<0.01) after filtration when the platelet numbers of the pre-filtration and post-filtration samples were not equalized (Fig. 4).
iv) Platelet sensitivity to ADP-aggregation of the platelets in the pre-filtration and post-filtration samples was assessed by a new device (Sano et. al. 1974). Platelet-rich plasma (PRP) was separated without centrifugation. Then the PRP and serially two-fold diluted ADP solution (2^-5-2^-15mg/ml) were mixed. The minimum effective ADP concentration to give platelet aggregation was detected microscopically. The sensitivity increased after each filtration in both of the observations with and without platelet number equalization (Fig. 5).
v) It was suggested that the degree of platelet injury may be reflected more on the platelet sensitivity to ADP-aggregation than on the intensity of platelet aggregation.

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(1) Platelet factor 4 (PF-4) wich shows antiheparin activity was purified from the human platelets. After homogenization by sonifier and Al(OH)₃ gel absorption, crude PF4 was further purified by the Heparin-Sepharose affinity column chromatography and G-75 sephadex gel filtration. Final PF-4 activity was 186% and purification fold was 500. The purified PF4 neutralized 200u/ml of heparin. The molecular weight of final PF4 was found to be about 20, 000.
(2) In normal rabbits a single injection of 2000r endotoxin, extracts of gastric cancer tissue, anti-rabbit platelet immune guinea pig serum and a subsequent infusion of normal saline induced disseminated intravascular coagulation (DIC). The remarkable changes of platelet count and PF4 levels were observed. In contrast to these facts, in the thrombocytopenic rabbits treated with Busulfan DIC was not induced by the infusion of the same trigger substances and PF4 levels did not change dominantly.
(3) The relationship of PF4 to haemorrhagic tendency in the patients with DIC, hypoplastic anemia and ITP was investigated. PF4 levels increased markedly in the haemorrhagic patients with DIC and hypoplastic anemia, but it decreased in the patients with ITP. In all cases it was observed that when platelets counts increased and haemorrhage was relieved, PF4 levels became to normal. PF4 would be a good parameter to reflect the platelet function in haemorrhagic disorders and thromboembloic diseases.

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The clinical syndrome secondary to the elevated blood viscosity, called as “serum hyperviscosity syndrome”, “blood high viscosity syndrome”, or “syndrome of hyperviscosity” has been paid attention recently. In clinical practice we frequently see hyperviscosity syndrome associated with paraproteinemia. We present hemorheological studies on eleven cases of multiple myelomas (IgG 6 and IgA 5) and two cases of macroglobulinemia. Among eleven cases multiple myelomas two patients manifested epistaxis and gingival bleeding presumably due to hyperviscosity of the blood.
We measured blood viscosity by using capillary viscometer and Brookfield microviscometer at 37°C and 20°C respectively. The plasma relative viscosities of thirteen cases at 37°C ranged between 1.84 and 4.47 with the avarage 2.69 (normal 1.72±0.06). The whole blood viscosities at 37°C 115sec^-1 fell between 3.25 and 6.09cp with the average 4.88cp (normal 4.2±0.3). The only 500% of cases revealed abnormal viscosity of whole blood. The plasma containing a large amount of paraprotein showed nonnewtonian behavior. The clear relation was noted between paraprotein concentration and plasma relative viscosity. The shear dependency (ηWB11.5sec^-1/ηWB230sec^-1) was follow: IgG<IgA<IgM.
The rheological properties of blood containing a large amount of paraprotein and cryoglobulin are extremely complicated and further investigation on cases of hyperviscosity are required in varing temperature.

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The many coagulation factors increase during pregnancy, accordingly pregnant women often complicate thromboembolic episodes. The side effect of oral contraceptives such as thromboembolism and disorders of liver function have long been called attention, but the suitable and simple test to predict these side effects has not been known.
Prothrombin time (PT) and Thrombotest (TT) are sensitive only at low coagulation activities. On the other hand, Hepaplastintest (HT) gives reliable results in the assay of hypercoagulable state, still more HT can be used as a liver function test.
It is the purpose of this paper to clarify wether HT is available to assay the hypercoagulability during pregnancy and to check the side effects during the use of oral contraceptives.
HT was studied in 41 non-pregnant, 40 pregnant (pregnancy VII th-X th month), and 90 sex-hormones administrated women. TT, PT, GOT and GPT were also examined. The results were following.
1) HT mean value in normal non-pregnant women was 101.26±16.08%.
2) HT mean value in pregnancy VII th month women was 131.40±14.8%, in VIIIth 142.50±13.87%, in IXth 143.66±7.10%, in Xth 158.07±4.15%. These high values could be measured by using non-diluted samples. None of these pregnant women complicated thromboembolic disease. Therefore the relation between these high values and an increasing tendency to thrombosis was not determined.
3) HT, GOT and GPT was studied in 3 groups taking sex-hormones.: 7 women taking Mestranol (60γ/day×11 days), 41 women taking Norethisterone (15mg/day×5 days) and 42 women taking oral contraceptives (Anovlar 1 Tab/day×21 days). HT values elevated moderately during the use of sex-hormones and reduced after the ceasing. 10% women in each groups had high HT values above the normal non-pregnant women. None of them had thromboembolic disease.
No correlation between the HT values and the GOT and GPT values could be seen. The GOT value elevated above 40 units in 4 cases and GPT in 8 cases, whose HT values were in normal range. HT could not be available to check the slight liver disorders during oral contraceptives.
4) It was concluded that HT was useful for the assay of hypercoagulable state. The possible correlation between high HT values and an increased risk of thrombosis should be further investigated.

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Usefulness of normotest (NT) in management of oral anticoagulant therapy was studied in comparison with Quick's prothrombin time (PT) or thrombotest (TT).
Materials:
Twentysix male and seven female patients with thromboembolism on long-term warfarin treatment (mean maintenance dose; 3.9mg per-day) were subjected. Control samples were obtained from youg doctors and laboratory technicians.
Results:
1. Effect of temperature on PT, NT and TT activities of oxalated plasma samples stocked for 0, 24 or 72 hours at -20, 4 or 22°C was investigated.
NT was most stable among three tests.
2. Reproducibility of the results in normal subjects or patients by three different technicians (A: beginner, B: more expert, C: the most expert technician) was investigated. Both reproducibility and reliability were highest in NT. Procedures of both NT and TT were equally simple as compared with PT.
3. The following correlations among activities of PT, NT and TT were observed; between PT and NT: Y=0.806X+9.29, PT and TT: Y=0.590X+3.43, NT and TT: Y=0.719X-3.30.
Correlation coefficients were 0.813, 0.793 and 0.929, respectivelly. (p<0.001).
4. In 8 normal subjects, PIVKA inhibitor ranged -0.15 to 0.25 (mean±sd; 0.094±0.148), while 33 patients under warfarin therapy, the index ranged 0.13 to 0.59.
There was significant negative correlation between the inhibitor and TT, although no correlation of NT to the inhibitor was observed. In addition, the coincidence rate of therapeutic range was higher between NT and PT than that of TT and PT.
Discussion:
Although prothrombin time has long been used for anticoagulant therapy, there still remains problem to be solved. That the therapeutic range of clotting activity of 15-30% was determined by short time difference of 7 seconds is one of them.
In addition, there are a certain technical difficulty and troublesome procedures such as centrifugation and standarization of tissue thromboplastin used.
While thrombotest is expected to be sensitive to all vitamin K dependent clotting factors including IX and endogenous inhibitors.
The problem is that sensitivity of this test is not superior to PT.
Determination of therapeutic range in NT is easier than in PT because of longer time difference of 60 seconds.
Thus, the NT is useful, convinient and reliable method for management of anticoagulant therapy in spite of insensitivity to endogenous inhibitor and factor IX.

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Hepaplastintest (HPT) was studied on 96 patients with hepato-biliary discorders at Hirosaki University Hospital and its affiliated hospitals for recent four months.
As healthy controls HPT were carried out in 50 males and 50 females of healthy hospital employee. HPT was determined on 3.8% citrated venous blood. Coagulation activity was reported in per cent derived from correlation curve of batch number 163.
The mean HPT value±SD for healthy controls was 77±12.0 (male 78.5±13.2, female 75.7±10.7).
There was no significant difference in mean value between male and female. The mean HPT value±SD showed 34.7±16.8 in 9 cases with extreme stage of acute hepatitis. In 6 of these 9 cases the author observed precipitous fall of the HPT value to bellow 30%. And severe 4 out of these 6 cases were complicated with disturbed consiousness.
The mean HPT values±SD for other various hepatobiliary diseases were as follows: 13 cases on recovery stage of acute hepatitis, 60±16.1; 17 cases with chronic hepatitis, 64.4±13.1; decompensated hepatic cirrhosis, 43.5±8.7; 9 cases with compensated hepatic cirrhosis, 55.3±11.7; 4 cases with primary hepatoma, 55.3±12.0; 11 cases with metastatic tumor of the liver, 65±12.8; and 5 cases with constitutional hyperbilirubinemia including 4 Gilbert diseases, 88±13.1. The mean HPT value for each disease mentioned above, except constitutional hyperbilirubinemia, was significantly lower than that of the control group.
HPT was reflected slightly on total protein, S-GOT, S-GPT, fibrinogen, cholesterol and partial thromboplastin time. HPT was correlative apparently with γ-globulin, platelet, prothrombin time with Quick's method (sec), Thrombotest (%) and ICG.
Further more HPT was correlative with factor II, VII and X but not with fator IX.
If HPT was remarkably impoved in cases with acute hepatitis in spite of the very low initial value less than 30% in the beginning stage of the disease they showed clinical improvement followed by desirable prognosis. It could be suggested the HPT well reflected the synthesis of coagulation factors or the protein synthesis in liver cells.
So we could conclude that HPT reflected well the degree of hepatic cellular disfunction and was enable one to suspect the degree in severity and prognosis of the hepatobiliary disorders, especially of acute hepatitis.

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The liver has close relation with blood coagulation. All the factors of blood coagulation except factor VIII are synthesized by the liver. Hepaplastintest (HPT) is a method to assay the activity of three coagulation factors (II, VII, X) which are synthesized by the hepatocyte. As the level of coagulation factors depends both on hepatic synthesis and on the effect of inhibitors, it is suspected that HPT (an inhibitor-insensitive method) might be available for liver function test.
HPT was compared with other biochemical tests to asess its diagnostic and prognostic value in hepatocellar disease. Fourty five patients with acute hepatitis, chronic hepatitis, liver cirrhosis and primary liver carcinoma were studied. Still, HPT in rabbits with acute liver injury induced by carbon tetrachloride is assayed, and our results are summarized below.
1) The value of HPT decreased in the early stage of acute hepatitis, but increased following the recovery. HPT often became restored to normal before the transaminase.
2) In the patients with chronic inactive hepatitis, HPT values varied within normal range, but in most cases with chronic active hepatitis and especially with liver cirrhosis ramained decreased for long. However, in the cases with primary hepatoma the values of HPT deviated almost only within the normal range or decreased slightly.
3) We found highly significant correlation of HPT with serum albumin, cholinesterase, ICG and prothrombin time.
4) In rabbits with acute liver cell damage induced by CCl₄, HPT values most decreased one day after CCl₄ administration, while SGOT and SGPT most elevated one day and three days respectively after the administration of CCl₄. HPT values were improved and became normal during seven days, however SGPT remained at a high level.
In the clinical and experimental study we observed that HPT often normalized earlier than the SGPT activity of serum in acute liver injury, but it was not clear whether transaminases became elevated before the HPT values became reduced.
HPT, which reflects the synthesis of three clotting factors in the hepatocyte, is usef ull as diagnostic and prognostic tools. HPT also has advantage of easy technical practicability.

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In an attempt to evaluate the clinical significance of “Hepaplastintest”, comparative studies have been undertaken by simultaneous observation on Hepaplastintest, Owren's Thrombotest and Quick's one step prothrombintest respectively.
159 cases with liver diseases, including acute, chronic hepatitis, cirrohsis of the liver, hepatoma and fatty liver.
Significantly lower hepaplastintest values were obtained in acute stage of acute hepatitis, chronic hepatitis active form and cirrohsis as compared with other types of liver diseases.
Earlier recovery of hepaplastintest value as within two weeks was impressed in acute hepatitis, contrasting to definite delay of recovery in subacute hepatitis as long as more than one month.
In chronic hepatitis active form with sublobular zonal necrosis, concomitant lower values both in choline esterase arid hepaplastintest were impressive.
In cirrohsis, preceeding depression of hepaplastintest value was noted shortly before the onset of hepatic failure.

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