It has been reported that the GPIIb and GPIIIa exist predominantly as the Ca²⁺-dependent, heterodimer complex, and incubation of intact platelets with EDTA at 37°C causes progressive dissociation of GPIIb-IIIa complexes. We used two monoclonal antibodies against GPIIb-IIIa complex (NNKY 1-32, NNKY 2-18) to study the effects of cations that influence the reassociation of dissociated GPIIb and GPIIIa, and studied the change of platelet aggregation under those conditions. Ca²⁺, Mg²⁺ and Mn²⁺ promoted the reassociation of dissociated GPIIb and GPIIIa in intact cells. But the promotive effect of Mn²⁺ on the reassociation of GPIIb and GPIIIa was less than that of Ca²⁺ and Mg²⁺. Ca²⁺ and Mg²⁺ also restored the aggregabilities of platelets, whereas Mn²⁺ did not. Differences were found in aggregability between samples. These results suggested that some different cations participate in the formation and maintenance of GPIIb-IIIa complex, and especially Ca²⁺or Mg²⁺ need for those conditions.

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PAIgG from patients with ITP was evaluated by a competitive solid phase microenzyme immunoassay, and its specificity for anti-platelet antibody in patients with ITP was investigated by Western blotting. PAIgG values were elevated in most ITP patients whose platelet count was under 50, 000/μl, and within or close to normal range whose platelet count was over 50, 000/μl. Likewise PAIgG values elevated in ITP patients whose megakaryocyte count was over 200/μl, and within or close to normal range in most patients whose megakaryocyte count was normal. Following SDS-PAGE of whole platelet lysates or platelet membrane lysates, platelet fractions were transferred onto nitrocellulose strips by Western blotting. The binding immunoglobulin was detected with an avidin-biotin peroxidase system. Several bands of bound immunoglobulins were detect in the whole platelets lysates of all ITP patients, but most of the detectable bound immunoglobulins disappeared when platelet membrane lysate was used. This finding suggests that some immunoglobulins from ITP patients may bind to cytoplasmic proteins in the whole platelet lysate.

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The interacion between influenza virus and platelets was observed under a transmission electron microscope by mixing virus (PR-8 strain) with PRP of human or rabbits. The adsorption of the virus onto the surface of the platelet was immediately observed. Some of the viruses started to invade into the platelets and were seen in the open canalicular systems. After two minutes, the platelets deformed significantly, marked decreased number of the granules and vacularization were seen. After 5 minutes, the mass of platelet aggergates can be seen. In addition, 2ml of virus suspension was intravenously administered to rabbits to see coagulation and pathological changes. The platelet counts decreased singnficantly, fibrinogen level decreased only after 2 hours, but fibrin monomer test showed negative. The platelet thrombi were observed in pulmonary capillaries and splenic tissues.
From the above results, it is suspected that platelet aggregation is partly responsible for the development of thrombocytopenia in the inital stage of viral infection.

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We describe a new murine monoclonal antibody, OP-G2, against platelet glycoprotein (GP) IIb/IIIa that induces platelet aggregation and release of ATP from platelets. OP-G2 was an IgG₁ antibody, which did not react with platelets from two patients with Glanzmann's thrombasthenia and immunoprecipitated GP IIb and GP IIIa from solubilized radiolabeled normal platelets. In the concentration range of 2-80μg/ml of IgG, OP-G2 induced platelet aggregation with a lag phase in PRP from 11 of 20 normal subjects. The lag phase decreased with the increasing concentration of OP-G2 in 9 of 11 normal subjects. It is noteworthy, however, that the intensity of maximal aggregation paradoxically decreased at higher concentration than 32μg/ml. In contrast to intact OP-G2 IgG, no aggregation was observed by Fab of OP-G2. The increasing amounts of OP-G2 (Fab) prolonged the lag phase of platelet aggregation induced by OP-G2 (IgG, 20μg/ml). Finaly, OP-G2 (Fab, 40μg/ml) completely inhibited OP-G2-induced platelet aggregation including the shape change. These data indicated that the platelet aggregation was mediated by Fab region of OP-G2. Furthermore, OP-G2 (Fab) inhibited dose-dependently ADP-, epinephrine-, and collagen-induced platelet aggregation. Therefore, we conclude that OP-G2 has both effects on platelet function: inducing and inhibiting platelet aggregation.

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We used four monoclonal antibodies against platelet membrane and flow cytometry to study the conformational change on the platelet surface in ADP or collagen stimulation. If ADP or collagen was added to normal PRP or to washed platelets that had been incubated with various buffers, the binding of fibrinogen and monoclonal anti-GPIIb/IIIa complex antibodies changed. On the other hand, when ADP was added to PRP preincubated with monoclonal anti-GPIb antibody, the increase of light transmission continued and the fixed binding of fibrinogen was observed. The results obtained from this study following possibilities.
1. The binding sites fibrinogen are newly expressed on GPIIb/IIIa complex by the stimulation of agonist or the extracellular Ca²⁺ concentration, and the GPIIb/IIIa complex increases quantitatively in the case.
2. The binding GPIIb/IIIa complex is stable at Ca²⁺ concentration near the in vivo level and the structure hardly changes on stimulation by a weak agonist such as ADP.
3. The binding of vWF to GPIb after ADP stimulation inhibits the dissociation of the first aggregation or enhances the aggregation.

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Inositol 1, 4, 5-trisphosphate (IP₃) is formed in response to receptor stimulation by phospholipase C-mediated hydrolysis of phosphatidylinositol 4, 5-bisphosphate, and is thought to be responsible for the release of Ca²⁺ from intracellular stores. Cyclic AMP (cAMP) also serves as a second messenger that mediates the inhibition of platelet functions but the mechanism underlying the inhibitory action is not clear. The effect of cAMP on IP₃-induced Ca²⁺ release in human platelets permeabilized with saponin were studied. Ca²⁺ was sequestered by intracellular organelles in the presence of ATP (2mM) and IP₃ (2μM) released 40 to 50% of the sequestered Ca²⁺ The addition of cAMP (20μM) to permeabilized platelets did not alter Ca²⁺ uptake but depressed IP₃-induced Ca²⁺ release. When permeabilized platelets were incubated with [γ-³²P] ATP, cAMP stimulated phosphorylation of 20k-, 22k-, 24k- and 50k-Da proteins. These results indicate that cAMP may modulate IP₃-induced Ca²⁺ release in human platelets through protein phosphorylation.

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We have investigated the changes in intracellular pH (pHi) and cytosolic free Ca²⁺ concentration ([Ca²⁺]cyt) in platelet activation evoked by thrombin and thromboxane A₂ analogue, STA₂. pH was measured with the new fluorescent dye, 2′, 7′-bis (carboxyethyl) 5, 6-carboxyfluorescein (BCECF), and [Ca²⁺]cyt was determined by the fluorescent Ca²⁺ indicator, fura-2. Resting pHi of human platelets averaged 7.03±0.08 pH unit, which is similar to the values reported by others. There was an initial fall of 0.01-0.02 pH unit immediately after addition of thrombin. This was followed by an increase in pHi to between 0.07-0.18pH unit above the resting level. The resting [Ca²⁺]cyt level of around 100nM was increased abruptly to 1-3μM after stimulation with thrombin. Thrombin-induced platelet aggregation, increase in [Ca²⁺]cyt and intracellular alkalinization were inhibited by removal of Na⁺ or by treatment of platelet with Na⁺/H⁺ exchange inhibitor, amiloride.
STA₂ induced a transient increase in [Ca²⁺]cyt, however, it failed to cause a distinct alkalinization. Increase in [Ca²⁺]cyt and platelet aggregation evoked by STA₂ were observed even after removal of extracellular Na⁺. These results suggest that there may be another pathway in relation to platelet activation and Ca²⁺ mobilization independent of intracellular alkalinization.

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An occurrence of phosphatidylinositol 4, 5-bisphosphate (PIP₂) phosphomonoesterase in human platelets was demonstrated by analyzing phosphoinositides metabolism. The activity of the enzyme was maximum at pH7.0. It was active even in the absence of Ca²⁺ or Mg²⁺ but it was enhanced in the presence of Mg²⁺ or NaF. The activity was inhibited by pyrophosphate. The activity was not altered in the presence of Ca²⁺ Thereby, besides phosphodiesteric cleavage by phospholipase C, the amount of PIP₂ in activated platelets may be reduced by the combined effect of PIP₂-phosphomonoesterase and suppressed activity of PI-kinase by Ca²⁺.

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Nine phosphatase activities were separated from human platelets by DEAE-Sepharose CL-6B and AcA 44 column chromatography. Two of which were pNPP phosphatases and had little activity toward ³²P-histone IIA. The other seven phosphatase fractions were inactive toward pNPP in the presence of EDTA, but could be activated by Mn⁺⁺ or Mg⁺⁺. Histone IIA phosphatase activities of these fractions were significantly increased by Mn⁺⁺ or Mg⁺⁺ (Fr D, F), even though a considerable amount of the activity was observed in the presence of EDTA. All of these protein phosphatase activities were inhibited by NaF, a protein phosphatase inhibitor. Among phosphoproteins in Triton X-100 lysate of thrombin stimulated platelets, 47k phosphoprotein was remarkably dephosphorylated in the presence of EDTA. Dephosphorylation of the other six phosphoproteins (250k, 180k, 120k, 95k, 47k, 28k) was enhanced and became apparent in the presence of Mn⁺⁺ or Mg⁺⁺, which was also inhibited by NaF. All histone IIA phosphatase fractions dephosphorylated eight cytoskeletal phosphoproteins (250k, 150k, 110k, 95k, 80k, 47k, 28k, 20k) without strict substrate specificities. Thus, it was suggested that protein phosphatases in platelets might play a role in the negative feed back mechanisms by dephosphorylating phosphoproteins involved in platelet reaction.

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Platelets in whole blood from 7 healthy subjects were studied serially under scanning electron microscopy (SEM) and light microscopy (LM) to evaluate their morphological changes during their activation as exactly as possible. No drugs (anticoagulants) were used in sampling blood in this study.
1. Needle-shaped particles (0.7×0.2μm) immediately appeared on OCS and around the platelets in all subjects, but disappeared shortly. These particles were coloured by May-Giemsa stain, and they were conidered to be protein. The serial changes in their morphology and other properties suggested that they were derived from alphagranules. From these findings, the rapid release from alphagranules was considered to occur during activation of unstimulated platelets.
2. After the appearance of the needle-shaped particles, spherical particles 0.1-0.3μm in diameter were observed on platelets and fibrin fibers in experiment (2). Moreover, marked accumulation of microparticles was observed around platelets experiment (1) after the appearance of the fibrin net.

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We reported previously the establishment and platelet aggregating activity of human lung adenocarcinoma cell line KUM·LK-2 which produce spontaneous metastasis in nude mouse. Platelet aggregating potential in vivo and vitro, and metastatic potential of KUM·LK-2 were performed under using anti-platelet agent DN 9693 which owned phosphodiesterase blocking activity and not owned anti-cancer activity. DN 9693 blocked platelet aggregating potential and fatal acute thromboembolism of nude mouse induced by KUM·LK-2 cell. KUM·LK-2 metastases were produced subcutaneous implantation group only. But in the case of naturall killer remission using anti-asialo GM1, KUM·LK-2 produce metastases intravenous injection group too. DN 9693 blocked the metastasis of KUM·LK-2 in the case of natural killer reduced nude mouse completely. From these results DN 9693 blocked platelet aggregating activity of KUM·LK-2 cell in vivo and vitro, and blocked metastasis. It was concluded that platelet aggregating activity of KUM·LK-2 cell is necessery to produce metastasis. It was first experiment of human cancer metastasis blocking using anti-platelet agent and proof that anti-platelet agent may usefull for anti-metastasis of human cancer.

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It has been suggested in experimental animal cancer system that platelet aggregating potential of cancer cell and platelet aggregating material (PAM) is one of the most important factor in cancer metastasis. We established a human lung cancer cell line (KUM·LK-2) which produce spontaneous lung metastasis in nude mouse. PAM was extracted from KUM·LK-2 cell and PAM owned aggregating potential in human heparinized-PRP. PAM was differ from well known aggregating materials like ADP, collagen and others. PAM didn't contain fibrinogen and sialic acid, and was not concerned witht hromboxan composition. PAM was considered high molecular weight protein itself containing phospholipid and aggregating potential was concerned with ATP composition of platelet. This was first confirmation of platelet aggregating potential in human cancer cell line which own metastatic potential. Platelet aggregating potential of KUM·LK-2 cell may play a important role in blood borne metastasis.

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Enhanced erythrocyte aggregation and decreased eryhrocyte deformability are thrombogenetic factors, especially in microcirculation where shear rate is low. The aim of this report is to investigate the relationships between erythrocyte aggregation, erythrocyte deformability, hematocrit, plasma viscosity and intracellular free calcium of erythrocyte. Venous blood samples were taken from resting subjects into Na-citrate. Erythrocytes were suspended in calcium free HEPES buffer. Erythrocyte aggregation was measured at high and low shear rate by using the technique which is based on the increase of light transmission through whole blood that occurs when individual cells aggregate into rouleaux or rouleaux-rouleaux complexes; gaps in whole blood between the cell aggregates produce the increased light transmission. The levels of [Ca²⁺] i of erythrocytes were measured by using fluorescent Ca²⁺ indicator, fura-2 acetoxymethyl ester. Plasma viscosity was measured via capillary viscometry and erythrocyte deformability was measured by the method of Reid et al. and whole blood hematocrit by microhematocrit method. Both erythrocyte aggregation and erythrocyte deformability were decreased with increasing plasma viscosity and [Ca²⁺] i of erythrocyte. Erythrocyte aggregation was enhanced with increasing hematocrit. Thus these behaviors of erythrocytes in microcirculation contribute to formation of microthrombi especially at low shear rates.

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In is well known that the fluid cadaveric blood is a characteristic of sudden death.
However, a number of cases of sudden death after alcohol drinking have been reported where soft blood clots have been observed.
We, also, have observed such a tendency in 240 autopsies of strangulation and 57 autopsies of cerebral crushing.
In our present study, we have tested in the effects of ethanol on blood coagulation in vivo and in vitro.
No distinct difference was observed after ingestion of whisky by human subjects, but platelet aggregation was inhibited and blood clots were strengthened in vitro by direct addition of ethanol.
Animal experiments were also made by oral administration of ethanol to rabbits and mice. (10ml/kg of 40% ethanol)
The blood flow in femoral muscule and body temperature in mice significantly decreased about 1 hour after administration of ethanol.
Factor XIII of blood in rabbits was reduced 2 hours after administration of ethanol.
These results would suggest the formation of soft blood clot in sudden death after alcohol drinking.

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A role of histidine-rich glycoprotein in controlling heparin-like compounds on the endothelial cell surface is yet unclear. Incubation of purified porcine ¹²⁵I-histidine-rich glycoprotein retaining heparin neutralizing activity with cultured porcine aortic endothelial cells resulted in only unsaturable binding that is not inhibited by excess unlabeled histidine-rich glycoprotein. This binding was not inhibited by excess unlabeled purified porcine antithrombin III or protamine sulfate and by prior treatment of endothelial cells with heparitinase. On the other hand, displacement of ¹²⁵I-antithrombin III specifically bound to endothelial cells by unlabeled histidine-rich glycoprotein was much less potent than that by unlabeled antithrombin III. The endothelial cell-mediated acceleration of thrombin inactivation by antithrombin III was diminished by protamine sulfate, but was not affected by histidine-rich glycoprotein even at a histidine-rich glycoprotein/antithrombin III molar of -7:1. These data indicate that histidine-rich glycoprotein neither binds to endothelial cell surface heparan sulfate nor interferes with the interaction of this compound with antithrombin III, suggesting that it may not play an important role in the modulation of anticoagulant activity of endothelial cells.

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Effect of cepharanthin on blood coagulation system and possible usefuness for the treating animals with experimental disseminated intravascular coagulation (DIC) were investigaed. Prothrombin time (PT), activated partial thromboplastin time (APTT) and kaolin-activated clotting time were markedly prolonged by the presence of cepharanthin, whereas recalcification clotting time remained unaffected. The activity of each coagulation factor was not decreased after treating with this agent. Cepharanthin inhibited both platelet aggrgation and release of platelet factor 4 (PF 4). In treating the experimental DIC induced by endotoxin, cepharanthin alone prevented both the decrease of platelet count and antithrombin III (AT III) activity. Plasma heparin activity was maintained higher in the group which was received with both heparin and cepharanthin than that with heparin alone. These data suggested that cepharanthin might be useful for treating patients with DIC.

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By using the methop of enzyme immunoassay, plasma abnormal prothrombin (protein induced by vitamin K absence or antagonist-II: PIVKA-II) was measured in patients with hepatobiliary diseases. In 21 (60%) of 35 patients with hepatocellular carcinoma (HCC), plasma PIVKA-II levels were increased (>0.10 arbitary unit/ml). After curative resection of HCC, plasma PIVKA-II levels decreased to within normal ranges. Therefore, plasma PIVKA-II determination was very useful in the detection and monitoring of therapy for HCC patients.
Plasma PIVKA-II levels were also increased in both postoperative patients receiving antibiotics containing N-methltetrazolethiol without vitamin K, and patients with external biliary drainage.

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The changes of protein C was studied in the patients with cardiac valve replacement at the beginning of warfarin therapy. Protein C and thrombotest were measured during a period from the first day to the eighth day postoperatively in the 15 cases under going cardiac valve replacement.
Warfarin therapy was started on second postoperative day in initial dosis of 2.0±1.3mg per day. After then, warfarin was administered in dosis of 2-3mg per day continuously.
According to the medication of warfarin, protein C and thrombotest decreased from 106.0±14.0% (control) to 68.5±14.7% (4 days postoperatively) and from 109.6±13.9% (control) to 39.4±19.8 (4 days postoperatively) respectively. There were three cases who had decreased protein C under 50% in 6 days postoperatively. Nevertheless thrombotest was decreased under 30% at the same time in these cases.
These results suggest protein C is not decreased significantly by the medication of small dosis warfarin.

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The present study was conducted to analyse the predisposed conditions developing multiple organ failure (MOF) in sepsis. Primarily a sepsis model was established to confirm the hypothesis that DIC predisposes MOP.
Fecal suspension was injected fined into ligated biliary tree in Japan male rabbits. After the insult, serial blood sampling disclosed the onset of DIC with decrease of pletelet counts, leukocyte counts, and fibrinogen concentrations and with prolongation of PT and APTT values. Simultaneously serum bilirubin and creatinine levels elevated. Plasma endotoxin concentrations also gradually elevated. In the histological examinations of the liver, multiple focal necrosis and neutrophil infiltration into sinusoid were observed.
Heparin (50u/kg/h) and MD 805 (a specific trombin inhibitor; 300μg/kg/h) treatments significantly prevented decrease of platelet counts and elevation of bilirubin and creatinine levels in sepsis. The histological changes of the liver were minimized by both heparin and MD 805. Thus, these results indicate that hypercoagulable state undoubtedly contributes to the progression of MOF, which can be controlled by anticoagulant therapy.

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Plasma fibrinopeptide A (FPA), fibrinopeptide Bβ_1-42 (Bβ_1-42), fibrinopeptide Bβ_15-42 (Bβ_15-42), tissue-type plasminogen activator (t-PA), D-dimer and serum FDP-E levels were determined in 16 patients (AML 4, AMoL 1, ALL 6, CML-blastic crisis 1, CLL 1, Malignant Lymphoma 3) with hematological malignancy without DIC. FPA levels at the time of before and during therapy were significantly higher than normal control levels. And in clinical remission state, FPA levels decreased to almost normal range. This tendency was especially remarkable in 5 patients with acute leukemia and the sequential FPA levels were related to the disease activity. Bβ_1-42, Bβ_15-42, t-PA levels were significantly higher than normal control, but were not related to the disease activity. D-dimer, FDP-E levels were also significantly higher than normal control, and had a marked relationship with the grade of DIC.

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Various sclerosants for esophageal varices (polidocanol; PO, ethanolamine oleate; EO, human thrombin; TH, absolute ethanol; ET, sodium tetradecyl sulfate; STS) were injected into mongrel dogs to clarify the effects on blood coagulation, fibrinolysis and histological changes. Hemolysis and thrombocytopenia were observed in all dogs treated with sclerosants. Prolongation of prothrombin time and activated partial thromboplastin time were observed in dogs of EO, TH and STS groups. Plasma fibrinogen decreased significantly in dogs with TH. No remarkable changes of plasminogen and antithrombin-III were observed in any dogs. α₂-plasmin inhibitor decreased to 50-70% in ET, TH and STS groups. Degree of tissue damage caused by sclerosant was most remarkable in ET group. It caused necrosis around the vessel. STS and EO groups, however, showed mild damage of the vessels than ET group. They caused thrombi for more than a week, and after a month, organization of the vessel space. So at the sclerotherapy for esophageal varices, we should use sclerosants considering these effects.

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Increased plasminogen activator (PA) secrection has been observed in transformed and malignant cells and PA is thought to be involved in the processes of invasion and metastasis. Recently both types of plasminogen activator such as tissue type PA (tPA) and urokinase type PA (uPA), have been detected in human plasma.
In this study, to investigate the relationship between circulating PA and malignant state, we have measured plasma PA levels (PA activity, tPA and uPA antigen) in 69 women with gynecologic malignancies. These levels were compared to levels in a control group of 33 women with benign gynecologic tumors.
In the case of uterine tumors, significantly higher levels of tPA antigen were measured in patients of stage IV with metastasis (p<0.05). In the case of ovarian tumors, significant higher levels of tPA antigen were measured in patients of stage III and stage IV who showed dissemenation or metastasis (p<0.01). There was no correlation between PA activity or uPA antigen levels and malignant state.
These retults suggest that increased synthesis of tPA may play a role in cancer invasion and metastasis.

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