To clarify the effect of blood sampling through the indwelling catheter on indices of platelet function, we measured the levels of plasma β-thromboglobulin (βTG) and platelet factor 4 (PF4). In 12 patients with heart disease, direct sample was obtained by two-syringe technique with a 21-gauge needle inserted into the antecubital vein and catheter sample was also obtained immediately after advancing a 16-gauge Teflon catheter of 50cm length into the superior vena cava. In 4 patients (33%), catheter sample showed extremely high levels (more than 50%) of both βTG and PF4 as compared with those of direct sample. In 7 normal subjects, direct samples were obtained from the right and left antecubital veins immediately before and 45 minutes after catheterization, respectively. Catheter sample was also obtained 45 minutes after catheterization., βTG and PF4 levels of direct sample obtained 45 minutes after catheterization (21±6 and 5±1ng/ml, respectively) were similar to those before catheterization (22±8 and 5±1ng/ml), while their levels of catheter sample (66±36 and 18±10ng/ml) were significantly higher than those of direct samples. In another 4 normal subjects, catheter samples were repetitively withdrawn through a Teflon needle of 6cm length immediately after insertion to the antecubital vein and 10, 15 and 30 minutes thereafter. βTG and PF4 levels increased progressively (βTG: from 19±7 to 106±49, PF4: from 6±1 to 44±28ng/ml). Microscopy of the inner surface of the indwelling catheter disclosed massive platelet aggregates and fibrin strands. This study demonstrated that collection of blood through the catheter of any length caused an artificial elevation of plasma βTG and PF4 levels, which was derived from catheter-induced platelet α-granule release, and that catheter insertion itself had no effect of artificial increase in βTG and PF4 in vivo.

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Intravenous bolus injection of 2.5% fluorescein sodium, 2ml/kg, constantly produced platelet thrombi at the site of microvasculature where the filtered light, 20.7mW/mm² in intensity, had been radiated. The phenomena were observed with an intravital microscopy and recorded on VTR which allowed better analysis in playback mode. Using this model developed by Sato and Ohshima, the inhibitory effect of Trapidil on thrombus formation in the venules, 40-60μm in diameter, was studied in the rat mesentery. Trapidil was administered either intravenously (group A) or orally (group B). Two mg/kg/min of intravenous infusion of Trapidil induced significant decrease in blood pressure. The initiation time of thrombus formation, ti, was significantly prolonged compared with that of control. Marked retardation of the time to stop flow was also observed. In the case of intravenous infusion of Trapidil, 0.2mg/kg/min, blood pressure remained unchanged. Although no difference in ti was found between Trapidil loaded and control rats, is was significantly prolonged in Trapidil group. In group B, 30mg/kg/day of Trapidil were given orally for 6 days. Although no difference in ti was observed between Trapidil loaded and control rats, is was significantly prolonged in Trapidil group. The results indicated that Trapidil inhibited both initiation and growth of thrombus formation in this model. In group A, no significant difference in is was found between high and low dose of Trapidil. Decrease in flow velocity secondary to significant drop in blood pressure might be responsible for this difference.

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Platelet function and factor VIII were studied in seventeen patients with CML. The clinical symptom of bleeding tendency was seen in two out of 17 cases.
Prolonged Duke bleeding time was seen in only two out of 14 patients; on the other hand, the Simplate bleeding time was prolonged in five of ten cases. The platelet retention decreased in 13 of 17 patients.
The maximal extent of aggregation induced by ADP, collagen and epinephrine was reduced in 24%, 24% and 41% of patients examined, respectively. The second wave of ADP- and epinephrine-induced aggregation was absent and the lag preceding the onset of collagen induced aggregation was prolonged in platelets with reduced maximal extent of aggregation induced by each aggregating agent. Abnormal ristocetin induced aggregation was seen in nine out of 17 patients. The platelets of all patients were normally aggregated with arachidonic acid.
The intracellular concentrations of ATP and ADP were significantly reduced, and the ratio of ATP/ADP was greater than normal. ATP released from platelets was reduced, as determined by Lumi-aggregometer.
In patients with abnormal ristocetin induced platelet aggregation, FVIIIR: Ag, FVIIIR: CoF and FVIII: C were all decreased. No significant inactivation of factor VIII was induced in normal plasma by its incubation with patient's plasma.
The present study shows that in some patients with CML, acquired von Willebrand's disease may occur.

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Autoradiography using ¹¹¹In-oxine labeled autologous platelets was performed to observe the behavior of platelets in induced vascular injury by activated platelets in rabbit's brain and lung.
Cerebrovascular injuries were induced by injection of arachidonic acid (AA) (0.7mg/kg) into right internal carotid artery. Fourteen animals were pretreated with antiplatelet drug, ticlopidine (200mg/kg) and 10 were controls. Before the AA injection, ¹¹¹In-oxine (300μCi) labeled platelets were injected intravenously. Evans blue was given as a marker of disturbances of blood brain barrier. Sixty min after the AA injection, brains were removed and autoradiographic and electron microscopic studies were done. In the nontreated animals and some of the treated animals whose platelet aggregability was not suppressed, blue staining were seen in the cerebral hemisphere of injection side and hot radioactivity in autoradiogram were revealed in corresponding area. In the treated animals whose platelet aggregability was remarkably suppressed, no or slight blue staining or radioactivity were recognized. Only in hot radioactive area, platelet thrombi and vascular injuries were seen.
Vascular injuries of lung were produced by decompression after keeping animals under hyperbalic condition (6 atomosphere absolute for 40min). Before this procedure, ¹¹¹In-oxine labeled platelets were injected. Lungs of both 4 control and 4 decompression sickness animals were removed and autoradiographic and lightmicroscopic observations were performed. In lungs of decompression sickness animals remarkable spotty high radioactivity and prominent platelet aggregates in the vessels were seen. These findings were not seen in control animals.
Our results suggested important roles of platelets in induced vascular injuries. And this autoradiographic approach seemed to be quite useful for observation of platelet's behavior in injured vessels and evaluation of antiplatelet drugs.

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Tissue plasminogen activator (t-PA), which has high affinity to fibrin, is secreted into culture medium from a human melanoma cell line (Bowes). The mechamism of t-PA secretion remains still unclear. Since t-PA is glycoprotein, effect of monensin on the secretion of t-PA was investigated using two series of experiment. In both series, monensin was added to the medium 4 hours before the experiment. In A series, monensin was further added during the experimantal (sampling) period (6 hours). In B series, monensin was not added during the sampling period in order to observe withdrawal effect of monensin. The results showed that, in A series, the secretion of t-PA was markedly depressed in 0.1, 1 and 10μM monensin and the intracellular t-PA was accumulated as twice as control. In B series, the secretion of t-PA was suppressed in a dose-dependent manner, and the intracellular t-PA was accumulated further in 10μM, but decreased in 1μM. Monensin inhibited DNA, RNA, and pretein syntheses in a dose-dependent manner in both A and B series. The effects of monensin on the release of previously produced protein showed that monensin increased protein release. The results obtained in the present study indicated that monensin disturbed intracellular transport system of t-PA in melanoma cell, as well as other glycosylated proteins.

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Hemocoagulase (Reptilase-S, Zeria Pharm. Co., Ltd., Tokyo) is a ferment from the venom of Bothrops genus, and a well-known hemostatic agent. Recently, a clinical study reported the possible influence of Reptilase-S on disseminated intravascular coagulation (DIC). In this study, we experimentally examined the effects of this agent on DIC.
Female Wistar strain rats (200-220g) were used. Coagulation parameters, such as fibrinogen levels, fibrin and fibrinogen degradation products (FDP), platelet counts, prothrombin time (PT), partial thromboplastin time (PTT), and the number of glomeruli with fibrin thrombi were determined 0.5, 1, 2 or 4hr after i. p. injection of Reptilase-S at a dose of 1, 10 or 100 Klobusitzky unit (KU)/kg. Experimental DIC was induced by a 4-hr i. v. infusion of bacterial endotoxin (lipopolysaccharide B, E. coli 055, B5, Difco Lab., Detroit, Mich.). Five min or 1hr after i. p. injection of Reptilase-S at a dose of 0.0001, 0.001, 0.01, 0.1, 1, 10 or 100KU/kg, endotoxin (100mg/kg) was infused for 4hr.
Although the injection of 100KU/kg of Reptilase-S resulted in an increase in FDP, and decreases in platelet counts and fibrinogen levels with a prolongation of PTT, the administration of 1 or 10KU/kg of this agent did not have any significant effect on the coagulation parameters (FDP, fibrinogen levels, PT, PTT and platelet counts) or formation of glomerular fibrin thrombi. Pretreatment with Reptilase-S at a dose of 0.1 or 1KU/kg significantly inhibited the increases in FDP and the number of glomeruli with fibrin thrombi. The prolongation of the PT and PTT, and the reduction of the fibrinogen levels and platelet counts were also inhibited by the pretreatment.
These results showed that Reptilase-S failed to induce DIC. Although the exact mechanism was unclear, Reptilase-S seems to protect against the aggravation of DIC induced by endotoxin in rats.

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Recent studies have shown that high density lipoprotein (HDL) contributes to the pathogenesis of atherosclerosis and attention has been directed to HDL cholesterol (HDL-C) level as a negative risk factor against ischemic heart and cerebrovascular disease. The purpose of this study is to elucidate the influence of decreased plasma HDL-C on the content of red cell membrane cholesterol.
Red cell membrane cholesterol (RBC-C) was analysed in 24 survivors of cortical artery area infarction with decreased plasma HDL-C levels, whose plasma total and lipoprotein cholesterol were measured concurrently. The results were compared with findings in 9 controls with normal plasma HDL-C levels. Each cholesterol level in two groups was summarized in table 1. The mean value of RBC-C in patients was significantly increased, compared to that in the control group (Fig. 1). The content of RBC-C correlated negatively with that of plasma HDL-C (Fig. 2), and positively with the ratio of low density and very low density lipoprotein cholesterol (LDL+VLDL-C) to HDL-C (Fig. 3). Weak correlation was seen between the concentration of RBC-C and that of LDL+VLDL-C or total cholesterol (Fig. 4, 5).
The results suggest that the decreased content of plasma HDL-C will affect the pathogenesis of atherosclerotic disease by the increase of red cell membrane cholesterol.

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Protein kinase C was purified to the apparent homogeneity from a 100, 000×g supernatant fluid of human platelets homogenate, employing the steps of DEAE cellulose, Ultrogel AcA-34 and phenyl-Sepharose CL-4B chromatographies. The purified enzyme appeared as a single protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, having a Mr 82, 000. With histone H-1 as a substrate its activity was 20-fold higher in the presence of phospholipid and Ca²⁺ than in the presence of phospholipid and EGTA, EGTA or Ca²⁺ alone. The enzyme has a K_m of 8.3μM and a V_max of 1.5μmol/mg/min for histone H-1. Platelet protein kinase C undergoes an apparent autophosphorylation. The enzyme was most active at 10-20mM mg²⁺ with histone H-1 as a substrate, wherease with mixed myosin light chains from turkey gizzard the maximum activity was observed at 1mM mg²⁺. Of several myosin light chains isolated from a variety of sources, the enzyme phosphorylates the 20, 000-dalton light chain of myosin from human platelets and turkey gizzard smooth muscle at a significant rate. The enzyme also phosphorylates the two phosphorylatable light chains of myosin from Limulus. These results suggest that platelet protein kinase C can phosphorylate the regulatory light chain of myosin non-muscle and smooth muscle, in which myosin phosphorylation catalyzed by myosin light chain kinase is thought to control contraction and cytoskeletal related processes.

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Calmodulin (CaM), an intracellular calcium binding protein, is widely distributed in human tissues and cells. A. sensitive radioimmunoassay for CaM has been applicated to tissue extracts and cells since it was developed recently. In the present study, the contents of platelet CaM in normal subjects and several diseases were determined using Calmodulin RIA Kit.
The results of this study were as follows: (1) The contents of CaM in platelet from patients with ITP, SLE, DVT. DIC, aplastic anemia, acute leukemia in recovery phase and MPD were significantly increased than that from normal subjects. (2) The contents of CaM in platelet from patients with ITP which responded to therapy tended to be decreased, comparing with those at initial diagnosis or those did not respond to therapy. (3) there was no correlation between the contents of CaM in platelet and platelet aggragability. (4) There was a negative correlation between the content of platelet CaM and platelet lifespan, indicating that the CaM level was inversely related to pletelet age.

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We have developed two assay systems (amidolytic assay and platelet aggregation assay) for platelet factor X activator activity (Pl. FXAA) with purified coagulation factors and gel filtered platelets (GFP), and investigated the properties of Pl. FXAA. The present study shows the correlation of human factor VIII to Pl. FXAA. In platelet aggregation assay, GFP aggregation behavior was observed after addition of factor X, factor II and CaCl₂. In amidolytic assay, the rate of factor Xa generation from factor X by GFP was determined with chromogenic substrate S-2222.
GFP from hemophiliacs were tested to Pl. FXAA assay. GFP from hemophilia B patients showed the similar aggregation behavior and amidolytic activity as control. On the other hand, GFP from hemophilia A patients showed low FXAA compared to control GFP, and that was significant in platelet aggregation assay. Furthermore, GFP prepared from factor VIII inhibitor patient or pretreated with factor VIII inhibitor plasma in vitro showed low FXAA, and the reduced extent was very marked in platelet aggregation assay and insignificant in amidolytic assay. While, both inhibitor-GFP and control-GFP showed the similar GFP aggregation after addition of factor Xa, factor II and CaCl₂ in platelet aggregation assay. Furthermore, purified human factor VIII added in vitro accelerated dose-dependently Pl. FXAA in both assays.
These results suggest that factor VIII enhances factor Xa generation by GFP, and reduced Pl. FXAA in hemophilia A patients is due to the reduced membranebound factor VIII.

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Despite of accumulated information of the LBS (lysine binding site) of the plasmin, little is known of characteristic stereogeometry at the active center. The present studies were kinetically made by using selective synthetic substrates or inhibitors, with expectations that tri-pod structure of highly selective substrates and inhibitors would indicate the replica structure of the active center. From the results obtained, we conclude that the plasmin active center assumes the following three pockts; (1) the first pocket is characterized just to fit the positively charged head of the lysine residue, (2) the second is equipped with very fine structure just to fit (2R, 4R) structure of 4-metyle-2-piperidine-carboxylic acid, but not other stereoisomers of the above mentioned radical (Table 1), and (3) when examined by D-Ile-L-Phe-L-Lys-pNA, 3-methyl-valeric acid-L-Phe-L-Lys-pNA and L-Ile-L-Phe-L-Lys-pNA, the third pocket is to fit (a) D-Ile-, (b) 3-methyl-valeric acid-, and (c) L-Ile-, with differend Km values (a) 20μM, (b) 180μM, and (c) 330μM respectively, the highest affinity of the third pocket for the terminal D-Ile derivative being indicated (Table 2).

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Serum FDP was found to be elevated in patients with liver cirrhosis, but we are still unable to clearly differentiate between primary fibrinolysis and fibrinolysis secondary to disseminated intravascular coagulation (DIC)
Whether elevated FDP shows secondary fibrinolysis or not was investigated in 31 patients with decompensated liver cirrhosis.
The following tests were performed: hepaplastin test, prothrombin time, anti-thrombin III, plasminogen activator, fibrinogen, plasminogen, α₂ plasmin inhibitor and FDP.
The following results were obtained.
1) Decompensated liver cirrhosis is associated with hypercoagulable and hyper-fibrinolytic states.
2) The patients with 20μg/ml or less of FDP (25 of 31 cases (84%) of decompensated liver cirrhosis), were not in hypercoagulable state but were in hyper-fibrinolytic state . Therefore, we thought that these cases do not have a tendency of developing DIC.
3) On the other hand, in the patients with 40μg/ml or more of FDP (5 of 31 cases (16%) of decompensated liver cirrhosis), hypercoagulable and hypofibrinolytic states were in front. Therefore, these cases have secondary fibrinolysis.

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A family of congenital dysfibrinogenemia, in which the propositus comlicated acute occlusion in the left side internal carotid artery and four out of nine tested family members had abnormal fibrinogen with no history of thrombosis is reported. The abnormal fibrinogen was characterized by a defective polymerization in the formation of fibrin.
The occlusive thrombus that had been formed in the carotid artery shown by angiogram, could be removed by the fibrinolytic therapy with the local infusion of urokinase. It is suggested that the dysfibrinogenemia might be easily dissolved by fibrinolysis because of the poor polymerization.

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Nine leukemic cell lines, KML-1 (APL), PL-21 (APL), HL-60 (APL), ML-1 (AML), KCL-22 (CML), KEN-L-1 (common ALL), NALM-18 (pre-B cell ALL), TALL-1 (T-cell ALL) and RC-K8 (histiocytic lymphoma), were investigated about their thromboplastin (TP) and plasminogen activator (PA) activities by the method based on factor Xa generation in the presence of factors VII and X using chromogenic substrate S₂₂₂₂ and fibrin-agar method, respectively. The granulocytic cell lines such as ML-1, HL-60, and PL-21 were found to have significant TP activity in their cell lysates. The cell lines including RC-K8, HL-60, NALM-18 and KML-1 had significant PA activity in their cell lysates. A large amount of PA activity was found in the culture medium from RC-K8. The PA from RC-K8 was immunologically identical to urokinase and showed two different fibrinolytic bands, molecular weights of 58, 000 and 53, 000 on SDS-PAGE followed by zymography. Therefore, RC-K8 seems to be useful as one of the PA-producing cell lines.

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Features of blood coagulation system and postoperative anticoagulation therapy with Warfarin on 22 patients in which cardiac valve replacement was performed, were investigated by compared to 18 patients from peripheral vascular disease.
Coagulation factor VIII was increased in both group of valvular disease and vascular disease before operation. There was no tendency of increase in F. VIII during Warfarin therapy continued for 36 months after operation. Prothrombin time was not correlated well with Thrombotest nor Hepaplastintest during Warfarin therapy. Thrombotest was affected by protein induced by vitamin K abscence (PIVKA) increased above 6-8μg/ml. It was noteworthy that % F. VIII (AHF/RAG) was reduced after valve replacement surgery, as compared with vascular disease in which reconstructive surgery was performed. These results suggest that postoperative anticoagulation therapy with Warfarin should be controlled by combination of thrombotest and Hepaplastintest. Decrease in % F. VIII (AHF/RAG) might indicate the hypercoagulability induced by mechanical valve prosthesis implantation.

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