we investigated platelet-activating factor (PAF)-induced changes in cytosolic free Ca²⁺ concentration ([Ca²⁺] cyt) using three Ca²⁺-indicators, quin 2, fura-2 and aequorin, and effects of ONO-6240, a specific PAF-antagonist, on platelet aggregation and changes in [Ca²⁺] cyt stimulated with PAF.
In the presence of 1mM extracellular Ca²⁺, PAF induced increase in [Ca²⁺] cyt as determined by three indicators. In the absence of extracellular Ca²⁺, however, there was little or no increase in [Ca²⁺] cyt stimulated with PAF. These results suggest that PAF increases [Ca²⁺] cyt mainly by influx of extracellular Ca²⁺.
ONO-6240 inhibited PAF-induced platelet aggregation at a concentration being from 1μM to 100μM dose-dependently, whereas it didn't inhibit collagen-induced platelet aggregation at a concentration of 100μM. ONO-6240 inhibited PAF-induced increase in [Ca²⁺] cyt in a dose-dependent manner as determined by these indicators, as well as platelet aggregation. These findings suggest that the increase in [Ca²⁺] cyt is closely related to platelet activation evoked by PAF.

抄録全体を表示

Indium-111-labeled platelet scintigraphy was performed before and 4 weeks after the administration of ticlopidine hydrochloride in 3 thrombotic patients with positive studies on initial imaging. Although the suppression of platelet aggregability was mild, the second imaging showed decreased platelet accumulation on the surface of thrombi and platelet survival times prolonged after the administration of ticlopidine in two patients; one with giant leg varix and one with dacron patch in the left ventricle. Although Indium-111-labeled platelet scintigraphy was equivocal before and after ticlopidine in another patient with mural thrombi in the abdominal aorta, abdominal CT scan revealed a moderate decrease in the size of the mural thrombi after ticlopidine therapy. Thus, we conclude that Indium-111-labeled platelet scintigraphy is useful in the evaluation of the effect of antithrombotic drugs in some patients, and that ticlopidine hydrochloride, in a dosage which mildly suppresses the platelet aggregability, inhibits platelet deposition on thrombi. These antithrombotic treatment may facilitate the resolution of thrombi.

抄録全体を表示

A 85-year-old female, who had neither easy bruising nor family history of bleeding, was referred to our clinic because of prolonged activated partial thromboplastin time (105.1sec) and prothrombin time (62.2sec). Clotting factors other than factor X were normal. Circulating anticoagulant was not detected. Factor X activity (F. X: C) was 0.4%, 0.8%, and 5.2% by the determination using prothrombin time method, activated partial thromboplastin time method and Russell's viper venom time method, respectively. F. X: C was also below 5.0% by synthetic chromogenic substrate method. Factor X antigen (F. X: Ag), on the other hand, was 72% by the method of enzyme immunoassay. Thus the patient was diagnosed as cross reacting material (CRM) positive.
Because her daughters' levels were 31% and 39% in F. X: C and 72% and 89% in F. X: Ag respectively, they should be heterozygous. Although her parents' F. X: C can not be determined, the patient is probably homozygous.
Factor X deficiency is rare disease. Only 13 families have been reported in Japan. This is the first report in Japan that showed low factor X activity determined by 4 different methods and positive CRM.

抄録全体を表示

Organic disturbances induced by endotoxin shock and the effects of anticoagulants and antiplasmins on disseminated intravascular coagulation (DIC) occuring as a complication, were studied experimentally in dogs. Endotoxin was intravenously infused in dogs for 90 minutes at a dose of 2mg/kg to induce endotoxin shock. For therapy, heparin at a total dose of 1000units/kg and EACA at a total dose of 100mg/kg were given to experimental dogs. Half of the dose was given both before and simultaneously with the infusion of endotoxin. The parameters used to evaluate the organic disturbances and effects of treatment for DIC were WBC, platelet counts, levels of plasminogen, antiplasmin, antithrombin III, prothrombin time, blood pressure, microcirculatory status in the liver and kidney, S-GOT, S-GPT, S-BUN, S-Creatinine, respiration rate, and level of CH₅₀ over a period of four hours.
Immediately after the onset of endotoxin shock, DIC occurred and the symptoms of multiple organ failure (MOF) were observed. In the untreated cases, MOF progressed steadily in a short period of time after the onset of endotoxin shock. Administration of heparin improved the microcirculatoon of major organs, while that of EACA aggravated the microcirculatory status of major organs.
These findings demonstrated that treatment for MOF should be started as soon as endotoxin shock is diagnosed and that anticoagulants such as heparin are useful in preventing the occurrence of DIC and MOF, while a single administration of antiplasmins is useless in improving DIC and MOF.

抄録全体を表示

A spleen fibrinolytic proteinase (non-plasmin) activity was measured in normal pregnant and puerperal mice. The weight of the spleen and blood parameters (APTT, PT, DBCL and fibrinogen level) were also examined.
The normal pregnant mice were divided into two groups in accordance with their fetus length; group I: ≤1.5cm and group II: >1.5cm by the growing of the fetus. The changes of leukocyte count and PT were not significant compared with the control (non-pregnant). However, APTT was shortened, DBCL supressed and fibrinogen content slightly increased in group II. The spleen weight was significantly increased in the group I, while it recovered in group II. In the latter group, a apecial spleen fibrinolytic activity was remarkably enhanced by nearly three times compared with the control, in spite of the recovery of the spleen weight.
The same examinations were made in the puerperal mice, either continuing lactation or non-lactation ones. The enhanced spleen fibrinolytic activity was slightly remained in both lactation and non-lactation groups 1-3 weeks after delivery. Results of other parameters were the same as those of pregnant mice.
The inhibitor studies on the spleen extract indicated that the spleen fibrinolytic activity was not derived from plasmin but chymotrypsin like protease.

抄録全体を表示

To avoid infectious risk of viruses such as hepatitis B, nonAnonB, and AIDS upon using fibrinogen preparations, we heve attempted to inactivate viruses by heat-treatment employng model viruses such as AIDS, Mumps, Polio I, Sindbis, Cytomegalo and Parvo. We heve previously demonstrated that heat-treatment of lyophilized factor VIII concentrates at 65°C for 96hrs is more effective to inactivate the viruses than the case of albumin products in solution at 60°C for 10hrs, which is known to be no risk of hepatitis B, nonAnonB virus infection.
In the case of fibrinogen, we have proved that AIDS virus was easily inactivated by heat-treatment of the dried sample at 65°C within 4 hrs. Our results obtained for the heat-inactivation of Parvo virus at 65°C have also suggested the possibility to reduce infectious risk of hepatitis viruses. Clotting activities of fibrinogen with regard to clotting time, clottability and clot opacity were not significantly influenced by the heat-treatment up to 144hrs at 65°C. Furthermore, there has been no difference in the physicochemical as well as in the immunochemical properties of fibrinogen between heated and unheated samples.

抄録全体を表示

Cell lysate obtained from cultured vascular endothelial cells contained a substance which induced platelet aggregation. This substance was identified as a phospholipid, platelet-activating factor (PAF), by thin-layer chromatography, phospholipase A₂ digestion, inhibition by a specific antagonist CV-3988, and agonist-specific refractory state. It was further found that PAF and collagen together induced extensive aggregation of platelets even with the concentrations by which each agonist alone could not induce aggregation of platelets at all. Once endothelial cells are damaged they not only let collagen fibers be exposed, but also might release intracellular PAF. These two compounds cooperatively ensure thrombus formation.

抄録全体を表示

The role of platelet-activating factor (PAF) in human platelet function was studied by examining the possible involvement of PAF in agonist-induced aggregation and the mechanism by which PAF exerts its aggregating activity.
Platelet aggregation induced by PAF was inhibited by aspirin or 5′-p-fluorosulfonylbenzoyl adenosine (FSBA), an inactive ADP analogue. The combined use of these two agents (each at 5μM) completely abolished PAF-induced second-wave aggregation in all platelet samples tested. We next examined the effect of a PAF-antagonist, CV-3988, on platelet aggregation. Pretreatment of platelets with 1-50μM CV-3988 slightly, but in a dose-dependent manner, inhibited the aggregation in response to 0.2U/ml thrombin. This antagonist had no effect on ADP- and collagen-induced aggregation.
PAF production by washed human platelets was studied by measuring [³H] acetate incorporation into [³H] PAF. [³H] PAF production in response to 10μM ADP, 5μg/ml collagen or 2U/ml thrombin were 263±136.7, 1, 021±763.6 and 1, 112±628.9dpm/10⁸ platelets, respectively (Mean±SD, n=3). The production by unstimulated controls was 412±207.6dpm/10⁸ platelets.
These results suggest that most part of the activity of PAF towards platelets may depend on thromboxane A₂ and ADP. Platelets synthesize PAF in response to collagen and thrombin, and thrombin-induced platelet aggregation may be partly dependent on the generation of PAF.

抄録全体を表示

We have prepared murine monoclonal antibodies to porcine platelet glycoprotein (GP) Ib and GPIIb/IIIa for further study of the porcine hemostatic mechanism. One monoclonal antibody, designated PP3-4C, blocked ristocetin-induced platelet agglutination and caused 80% inhibition of ristocetin-induced ¹²⁵I von Willebrand factor (vWF) binding to platelets at a concentration of 12μg IgG/ml. Another monclonal antobody, designated PP3-3A, completely inhibited ADP- or collagen-induced platelet aggregation at 6μg IgG/ml. At a concentration of 10μg IgG/ml PP3-3A completely inhibited binding either of ¹²⁵I-fibrinogen or of ¹²⁵I-vWF to ADP-stimulated platelets. Immunoaffinity chromatography of solubilized surface-radiolabeled porcine platelets and subsequent SDS-polyacrylamide gel electrophoresis demonstrated that PP3-4C recognized a glycoprotein with an apparent molecular weight of 160, 000 (non-reduced), and 140, 000 (reduced). PP3-3A recognized glycoproteins with apparent molecular weights of 130, 000 and 80, 000 (non-reduced), and 115, 000 and 100, 000 (reduced). These monoclonal antibodies to porcine platelet membrane glycoproteins which are structural and functional analogues of human GPIb and GPIIb/IIIa will be useful for in vitro and in vivo studies of the mammalian hemostatic mechanism.

抄録全体を表示

In saponin-permeabilized platelets, GTPγS (1-100μM) induced serotonin secretion and enhanced thrombin-induced secretion. In [³H] arachidonic acid (AA)-labeled platelets, GTPγS stimulated [³H] AA release and [³H] diacylglycerol (DG) formation. RHC 80267, an inhibitor of DG lipase, was not effective to reduce [³H] AA release, suggesting hydrolysis of phospholipids by phospholipase A₂. NaF, an activator of GTP-binding proteins, also stimulated platelet responses. Thrombin-induced AA liberation was almost completely inhibited with pretreatment of pertussis toxin (PT) (10μg/ml), whereas DG formation was decreased by only 20-40% in the toxin-treated platelets. Although GDPβS suppressed AA release and DG formation in a dose-dependent manner, the half maximal inhibition required less than 10μM for AA release but more than 100μM for DG production. These results indicate that GTP-binding protein (s) plays an important role in signal transduction during platelet activation. Since AA release and DG formation are differently modulated by PT and GDPβS, the distinct GTP-binding proteins may regulate the activity of phospholipase C and phospholipase A₂.

抄録全体を表示

Actin binding proteins in nonactivated or thrombin activated platelet were analyzed by SDS-PAGE after actin Sepharose chromatography of platelet homogenate.
90K protein, which had been thought to be actin gelation inhibitory protein, and actin were contained in nonactivated platelet samples or activated ones.
A protein of about 45K was much more detected in nonactivated platelet sample than in activated one. This protein thought to have a characteristics of binding to actin in nonactivated state and dissociation to actin in activated state.
The role of this protein in platelet activation must be futher investigated.

抄録全体を表示

Phospholipase C activity in membranes obtained from untreated platelets was stimulated by GTP and its analogs in a dose-dependent manner. On the other hand, membranes from dibutyryl-cyclic AMP (dbcAMP)-pretreated cells did not cause enhancement of the enzyme activity. The basal phospholipase C activity without GTPγS was not changed but the GTPγS-stimulated phospholipase C activity was markedly suppressed. However, in the the presence of a protein kinase inhibitor (H-8), this inhibitory action of cyclic AMP was not observed. The pretreatment of platelets with dbcAMP did not prevent the thrombin-induced increase in GTPγS-binding and GTPase activities. Thus, these results suggest that G-protein itself and its coupling with thrombin-receptor may not be influenced by dbcAMP pretreatment. Experiments of photoaffinity labelling with 8-N₃-[γ-³²P] GTP demonstrated that the membranes from dbcAMP-pretreated cells exhibited much more labelling of 240-kDa band when compared to control membranes. The 240-kDa photolabelled protein showed identifical mobility with the protein phosphorylated by cyclic AMP-dependent protein kinase.

抄録全体を表示

We investigated the inhibitory effect of aspirin on collagen induced platelet aggregation using rat PRP (platelet rich plasma) and GFP (gel filtered platelet). In the case of PRP, low concentration of collagen induced aggregation was inhibited by aspirin but when collagen concentration was increased, aggregation was observed in spite of the presence of aspirin. On the contrary, in the case of GFP, collagen induced aggregation was always inhibited by aspirin regardless of the collagen concentration. But when rat plasma was added to GFP, the inhibitory effect of aspirin on collagen induced aggregation decreased in a plasma dose dependent manner. These studies suggest that plasma factor(s) are essential for the aspirin noninhibited aggregation which is induced by high concentration of collagen.

抄録全体を表示

To investigate the role of platelet in ischemic heart disease, plasma levels of β-thromboglobulin (βTG) and thromboxane B₂ (TXB₂) were measured in 12 normal subjects and 18 patients with effort angina pectoris (EAP), 14 with vasospastic angina pectoris (VSA), 31 with old myocardial infarction (OMI). Levels of βTG and TXB₂ were monitored before, during and after an ergometer exercise.
1) Plasma βTG levels at rest were significantly elevated in the patients with EAP and with OMI, compared to that in normal subjects (p<0.05).
2) During ergometer exercise, plasma, βTG levels were significantly (p<0.05) increased in the cases of EAP, especially in the cases having significant ST depression, and chest pain at maximal exercise.
3) TXB₂ levels changed during exercise neither in the normal nor in the patients with ischemic heart disease.
4) At rest, platelet aggregation by collagen were decreased in the cases with EAP, VSA and with OMI.
5) There were no significant changes in TXB₂ production by arachidonate and thrombin, at rest and during exercise in 4 groups.
These results indicate that platelets are activated in the patients with ischemic heart disease. Measurement of plasma, βTG level may be useful to detect the presense of myocardial ischemia.

抄録全体を表示

A new inhibitor of blood coagulation, calphobindin (CPB; MW 34000) isolated from human placental tissue by EDTA extraotion was pharmacologically studied as an anticoagulant. CPB in concentrations ranging 3 to 30μg/ml gave only a slight inhibitory effect on human intact whole blood clotting time (WBCT; r+k=28min) estimated with thromboelastograph. A significant prolonging effect of CPB, however, was observed against tissue factor (TF)-added WBCT in a concentration of 1μg/ml, resulting in a complete neutralization of added TF (2μg/ml) by 3 to 10μg/ml of CPB. Heparin gave a significant prolonging effect not only on intact WBCT but on TF-added WBCT in a concentration of 0.03U/ml and a severe blood coagulation defect in a concentration of 0.3U/ml.
In a rat DIC model induced by TF iv infusion (20mg/kg) for 60min, reduction of plasma fibrinogen level (50 to 60%) was protected by simultaneous administration of 20mg/kg of CPB. Heparin also gave a prophylactic effect on fibrinogen reduction in a dose of 200U/kg.
These results suggest that CPB seems to be able to medicate the tissue factor poison caused by inflow of TF into blood stream.

抄録全体を表示

Six monoclonal antibodies against a new placental coagulant inhibitor (Calphobindin: CPB) were obtained. Two kinds of ELISA systems for CPB were developed using three monoclonal antibodies. Calcium ion interfer with CPB assay on one system. On the other system calcium ion did not interfer with CPB assay, but phospholipids, phosphatidylserine, phosphatidylethanolamine and phosphatidylglycerol, did in the presence of calcium ion. Both ELISA systems could detect CPB between 0.4-25ng/ml in buffer. CPB added to human plasma was recovered almost quantitatively by both systems in the presence of EDTA.

抄録全体を表示

We examined the behavior of rabbit factor XI (rabbit F. XI) in Watanabe-hereditable hyperlipidemic rabbit (WHHL rabbit), to study the participation of F. XI in atherosclerosis. Rabbit F. XI was isolated with use of anti-human F. XI monoclonal antibody-Sepharose. Purified rabbit F. XI was single band in sodium dodecylsulfate polyacrylamide gel electrophoresis (M. W.: 80, 000), and the specific coagulant activity was 75unit/mg protein. ¹²⁵I-rabbit F. XI, iodinated with Bolton-Hunter reagent, was administered intravenously to WHHL rabbit and Japanese white rabbit (normal). Then blood was taken from ear vein and the trichloroacetic acid precipitate of plasma was counted. Turnover of ¹²⁵I-rabbit F. XI was significantly faster in WHHL rabbit (T1/2=2.84±0.44 days) than in normal (T1/2=4.44±0.42 days). By en-face autoradiography, specific accumulation of ¹²⁵I-rabbit F. XI was observed in thoracic aorta of WHHL rabbit. In contrast, no radioactivity was observed in normal. These results suggest that activation of F. XI proceeds on the atherosclerotic lesion of WHHL rabbit.

抄録全体を表示

Basic study on normal and abnormal tracings of sonoclot coagulation analyzer (SCA) was performed. Beginning of fibrinogenesis can be detected earlier by SCA than by thromboelastgram. Recalcified citrated whole blood or plasma can be used. Reproductibility of the tracing was good. Not only the concentrations of hemostatic factors and platelet counts but also Ca²⁺ concentration, the concentrations of F XII activators, hematocrit value affect the tracing. Normal value of the onset time of Japanese adults, which is said to show the interval between the initiation and the beginning of fibrinogenesis was male: X±2SD=124.5±44.2sec, female: X±2SD=96.6±37sec. The sexual difference is statistically significant (p<0.001). SCA is considered to be applicable to clinical investigation of hemostasis.

抄録全体を表示

In eighty patients with thrombotic conditions plasma cross-linked fibrin derivatives (XDP) were measured by ELISA, and the relationships between XDP and clinical conditions, extent of anticoagulation and other parameters of coagulation and fibrinolysis were analized.
Following results were obtained;
1) Plasma levels of XDP were high in 3 of 10 patients with prosthetic heart valves, in 7 of 18 patients with venous thrombosis and in 37 of 52 patients with arterial thrombotic disorders.
2) High XDP value was ferquently noted in patients with inadequate anticoagulation or without anticoagulation.
3) Higher XDP values were obtained in patients with old myocardial infarction or old cerebral infarction than in arteriosclerotic patients without such episodes.
4) High XDP was noted in 6 of 15 patients with negative results of FDP test and in 9 of 20 patients with negative results of fibrinopeptide A.
5) XDP values showead weak positive correlation with vWf: Ag and weak negative correlation with antithrombin III, α₂-plasmin inhibitor or plasminogen.
These results indicate that plasma XDP may be one of useful parameters in patients with chronic thrombotic conditions, especially in patients under anticoagulation.

抄録全体を表示

Plasma levels of crosslinked fibrin degradation products (XDP) in some plasma samples from patients with disseminated intravascular coagulation (DIC) associated with acute promyelocytic leukemia (APL) were significantly lower than those from non APL patients, while the plasma levels of D-fragment of fibrin/fibrinogen degradation product (FDP (D)) were markedly increased. The dissociation of FDP (D) and XDP was observed only when α2 plasmin inhibitor (α2PI) levels fell below 60% of the control level in APL patients. Plasma levels of α2PI-plasmin complex in these plasma samples from APL were all more than 10μg/ml. These data suggested that hyperfibrinolytic state would occur in APL patients under the condition that α2PI levels fell markedly.

抄録全体を表示