Plasma von Willebrand factor (vWF) has been shown to have an activity (collagen cofactor; CCo) that enhances adhesion of human fixed washed platelets (FWP) to collagen. A new quantitative method was developed for CCo and used for studies on the roles of platelet membrne glycoproteins (GP) in the adhesion of FWP in the presence or absence of vWF. It was found that large multimer of vWF antigen preferentially bound to collagen. When FWP were treated with monoclonal anti-platelet antibodies or Serratia marcescens protease (SP) and subjected to adhesion to collagen in the absence of vWF, the treatments with a monoclonal anti-GP Ib antibody or SP resulted in decreased adhesion, while the treatments with monoclonal anti-GP IIb/IIIa or anti-thrombospondin antibody did not. Adhesion of FWP to collagen in the presence of normal plasma were inhibited by the addition of monoclonal anti-vWF or anti-GP Ib antibodies, but not of three monoclonal antibodies against GP IIb/IIIa. These findings suggest that GP Ib and the binding of vWF to collagen are involved in the adhesion of platelets in the presence of vWF.

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When platelets are stimulated by activating agents, arachidonic acid (AA) is released from phospholipids and rapidly transformed to metabolites including prostaglandins and hydroxy eicosatetraenoic acids (HETEs). Most of AA metabolites are hydrophilic and the accurate recovery of these metabolites was not obtained by usual lipid extraction methods, such as the Bligh-Dyer method. Two methods are used in this study to evaluate the AA liberation in [³H]AA-labeled cells; the centrifugation method and the method employing an AA metabolism inhibitor, BW 755C.
In the centrifugation method, the reaction was terminated by ice-cold buffer containing 10mM EDTA and centrifuged rapidly by microfuge, and the radioactivity in the resulting supernatant was counted. However, the supernatant fraction contained considerable amount of phospholipids as well as AA and its metabolites. Unmetabolized AA was mainly remained in the pellet fraction.
When BW 755C, an inhibitor of cyclooxygenase and lipoxygenase activities, was employed, the released AA was almost recovered in the lipid fraction. However, in the thrombin-stimulated platelets the radioactivity in the AA fraction reached its peak within 1min after stimulation and gradually decreased. Concomitantly, the radioactivity in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol fell within 1min and showed a progressive increase thereafter. Thus, in the presence of metabolic inhibitor, once released AA was considered to be reincorporated into phospholipids.

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Effects of calcium-channel blockers on platelet activation were examined in vitro by using diltiazem and verapamil. They showed inhibitory effects on aggregation, ATP- and platelet factor 4(PF-4)-release reaction and thromboxane A₂ generation by collagen. An inhibition of thromboxane A₂ generation, measured as thromboxane B₂ (TXB₂), was dose-dependent and more sensitive than other parameters examined. Diltiazem was more potent than verapamil in the inhibition of PF-4 release and (TXB₂) generation.
Addition of chelating extracellular calcium by EDTA did not cause ADP- or collagen-induced platelet aggregation, but thrombin-induced aggregation was still remained. In this condition, effects of Ca-channel blockers were greatly reduced. Thrombin-induced phosphorylations of 20Kd and 40Kd proteins were not inhibited by them in EDTA-containing medium, either. These results suggest an importance of extracellular calcium on the mechanism of effects produced by Ca-channel blockers.

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The effects of angiotensin I (AI) converting enzyme (ACE) related substances upon prostacyclin (PGI₂) generation with special reference to the kinetics of Ca⁺⁺ were investigated using cultured human vascular endothelial cells.
Endothelial cells were isolated from human umbilical cord vein and cultured by the modified method of Jaffe et al. PGI₂ generation or Ca⁺⁺ kinetics were measured using 6-keto PGF_1α RIA assay kit or ⁴⁵Ca, respectively.
AI or bradykinin (BK) (10^-6M) increased PGI₂ generation, however captopril decreased it. On the other hand angiotensin II (AII) did not show any effect. The enhanced PGI₂ generation by AI or BK was remarkably decreased by the pretreatment with an intracellular Ca⁺⁺ immobilizer, TMB-8 (10^-4M). AT and BK enhanced ⁴⁵Ca uptake, while AII did not show any effect on it, and captopril decreased it.
These results suggested that PGI₂ generation by ACE related substances were closely linked to the Ca⁺⁺ uptake to the cytosol from extracellular space or to the release of Ca⁺⁺ from the storage site.

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We have purified factor VIII/von Willebrand factor complex from rabbit plasma and prepared a specific guinea-pig antiserum to the von Willebrand factor antigen. It was used for electroimmunodiffusion to determine von Willebrand factor level in rabbit plasma and for crossed immunoelectrophoresis to study its electrophoretic behavior. Rabbit von Willebrand factor had properties similar to human von Willebrand factor with respect to molecular weight, size heterogeneity, mobility and pattern in electroimmunodiffusion and crossed immunoelectrophoresis, and content in plasma. Plasma von Willebrand factor antigen levels in rabbits ranged between 46 and 147% of the standard plasma. Responses of plasma von Willebrand factor antigen and factor VIII to various bioactive agents were investigated with rabbits. When a small dose of adrenalin, a large dose of noradrenalin, and endotoxin were injected, plasma von Willebrand factor antigen level and ratio of von Willebrand factor antigen to factor VIII activity were elevated in different fashions during 6 hours after administration. The findings could deliver some information about the mechanism of changes in plasma von Willebrand factor observed in human diseases.

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Both the lysine binding site (LBS) and the active center of plasmin (Pl) work together and result in the binding of Pl to its own substrate (fibrin) with extremely high selectivity. In the case of tranexamic acid (t-AMCHA), it binds only LBS, leaving the active center almost quite intact. Therefore, the active center is still able to cleave some oligopeptide-substrates as S-series even in the presence of 10^-2M of t-AMCHA. We report here a novel synthetic plasmin inhibitor called OS-153, chemically speaking, N^α-{trans-[4-(aminomethyl)-cyclohexyl] carbonyl}-L-lysine 4-benzoylanilide, which inhibits the function of the active center of Pl even at low concentration. Fact is, OS-153 inhibits not only the fibrinolysis (I₅₀=6.1μM) but amidolysis (Ki=7.4μM), Furthermore, OS-153 inhibits also “bradykinin forming activity” from the human intact plasma, which is activated by large-dose of streptokinase (SK). This enzymatic activity is characterized by potent amidolysis, which is not inhibited by t-AMCHA, but well inhibited by OS-153. According to Ratnoff, “plasmin has almost innumerable substrates”. The authors also support the possibility of the multiplicity of substrates for Pl, so that the active center directed inhibitor inhibits the different activities of Pl for the multiple substrates other than fibrin. The authors assume that Pl activities not inhibited by t-AMCHA can be more significant than generally considered. The active center directed inhibitors may be good tool to reveal such activities of Pl.

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GPV has a very important role of activating platelets in the presence of thrombin. We now have established a new rapid method for the purification of GPV. The purification procedure consists of (1) extraction of GPV (2) ammonium sulfate precipitation (3) gel permeation HPLC (4) anion exchange HPLC (5) WGA-affinity chromatography.
We recently purified GPV which showed a single silver and PAS stained band on the SDS-PAGE and the apparent molecular weight was 80K dalton. The GPV was hydrolysed by thrombin but not by endoglycosidase F. IEP was estimated as 5.6-6.6.
Anti-GPV antibody was raisd by immunizing a rabbit and its reactive protein was analysed on a nitrocellurose membrane by using a Western blotting method.
Anti-GPV IgG reacted only with GPV and its fragment, but it did not inhibit the thrombin-induced platelet aggregation. The anti-GPV antibody reactive material in plasma was quantified by using Dot-ELISA method. Two patients showed relatively high concentrations of the antibody reactive material before their thrombotic attacks.
We conclude that GPV purified by our rapid technique has the same characteristics as reported before and the detection of anti-GPV antibody reactive materials in patient's plasma is useful for the prediction and/or diagnosis of thrombosis.

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The purpose of the present study is to clarify the inhibitory effect of taurine on the elevation of cytoplasmic Ca²⁺ concentration ([Cai²⁺]) in activated platelets. Using aequorinloaded platelets activated with 1μM A 23187 in normal subjects, [Cai²⁺] was measured by a Platelet Ionized Calcium Aggregometer (Chrono-log). The elevation of [Cai²⁺] in platelets activated with A 23187 was significantly inhibited by the incubation with 1mM taurine for 10min. In order to elucidate the mechanism of this inhibitory effect of taurine, the aequorinloaded platelets were pretreated with 5×10^-5M aspirin (ASA) and 10^-4M dibutyryl cAMP (dbcAMP) before the incubation with 1mM taurine. The pretreatment with both of ASA and dbcAMP significantly inhibited [Cai²⁺] in platelet activated with A 23187. The elevation of [Cai²⁺] in ASA-pretreated platelet activated with A 23187 was further inhibited by the incubation with taurine, but in dbcAMP-pretreated platelet was not. These results suggest the possibility that the inhibitory mechanism of taurine on the elevation of [Cai²⁺] in platelets activated with A 23187 is not related to arachidonic metabolism. But it remains to be investigated whether the inhibition of taurine to platelet activation with A 23187 is due to CAMP-related mechanism or no related one.

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We previously reported that in the patients of myeloproliferative disorders (MPD), intracellular calcium level of the platelets after stimulation is usually lower than normal. In this study, calcium ion movement in platelets from MPD and normal subjects was examined by the use of [⁴⁵Ca²⁺].
In the normals, Ca²⁺ uptake by unstimulated platelets rapidly increased within 10 minutes, and calcium influx into the cytosol increased gradually within 4 hours. Ca efflux was observed to be in equilibrium with the Ca influx. Both internal membrane fraction (dense tubular system) and external membrane fraction of platelets had an ability to uptake calcium ion.
In MPD, whereas, Ca uptake by unstimulated platelets was lower than normal, and Ca influx and efflux were in equilibrium at lower levels. Furthermore, Ca uptake by external membrane fraction was lower and uptake by internal membrane fraction was higher than the normal.
These results suggest that the lower intracellular calcium level of MPD platelets seems to be due to the abnormality of calcium ion movement in pasma membrane and dense tubular system.

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Diabetic retinopathy often progresses after rapid blood sugar control in long-standing diabetic patients. We investigated the changes that happened in the diabetic patients who received blood sugar control from the stand point of platelet aggregation and arachidonate cascade. Platelet aggregation induced by ADP, collagen, PAF or epinephrine, the arachidonate cascade [plasma TXB₂ (TX) and 6-keto PGF_1α (PG) ], and blood viscosity were measured before and about 3 weeks and 6 weeks after blood sugar control in 22 diabetic patients.
Platelet aggregation induced by each agonist and plasma TX increased 3 weeks after blood sugar control but returned to previous level 6 weeks after blood sugar control. Plasma PG gradually increased and the TX/PG ratio decreased gradually after blood sugar control. Blood viscosity also decreased gradually after blood sugar control. There existed correlationship between the degree of increase in plasma TX and the degree of decrease in HBA₁ level in 3 weeks after blood sugar control. It is suspected that the increase of plasma TX may have influence on the deterioration of retinopathy after the rapid control of blood sugar.

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As increase of TXA₂ production in pregnancy-induced hypertension (PIH) has been reported, enzymological analysis of increased TXA₂ production by platelets was undertaken to investigate the significance of increased TXA₂ production in PIH, which was as yet unknonw.
The activity of phospholipase A₂ (PLA₂) and TXA₂ producing enzymes (TPE) was investigated by converting from phosphatidylcholine (PC) to arachidonic acid (AA) and from AA to TXA₂, respectively. Both conversion rate in severe PIH (sPIH) was significantly more than in mild PIH (mPIH) and normal pregnancy (NOR). The apparent Vmax values for PLA₂ in sPIH was significantly higher than in mPIH and NOR. The apparent Km values for PLA₂ in sPIH was higher than in mPIH and NOR. The apparent Vmax and Km values for TPE in sPIH was higher than in mPIH and NOR.
This conlcusion suggested that AA release in sPIH increased more than in mPIH and NOR by reason of high Vmax and low Km values for PLA₂, therefore TXA₂ production in sPIH having higher Vmax and Km values for TPE was more than in mPIH and NOR. Furthermore, a different activity of TXA₂ production between mPIH and sPIH suggested that increased TXA₂ production was an aggravating factor of PIH.

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Antiplatelet effects by combination therapy with aspirin and ticlopidine were investigated in comparison with single aspirin or ticlopidine therapy in 62 patients with cerebral thrombosis and transient ischemic attack. The 14, 21 and 27 patients were given orally 300mg aspirin, 200mg ticlopidine and 81mg aspirin with 100mg ticlopidine, respectively. Aspirin inhibited platelet aggregation (PA) to ADP and arachidonic acid (AA) but not to PAF, while ticlopidine inhibited PA to ADP and PAF but not to AA. In contrast, aspirin with ticlopidine inhibited PA to all the 3 agonists. Aspirin decreased plasma thromboxane (TX) B₂ but not plasma β-thromboglobulin (βTG) or platelet factor (PF) 4, while ticlopidine decreased βTG and PF 4 but not TXB₂. In contrast, aspirin with ticlopidine decreased TXB₂ as well as βTG and PF 4. Platelet survival and lysis remained unaltered after aspirin or ticlopidine alone, whereas platelet survival was longer and percent platelet lysis was lower in 4 patients treated with both aspirin and ticlopidine than in 3 non-treated patients. Hemorrhagic complications were observed in 2, 3 and 8 patients treated with aspirin, ticlopidine and both, respectively. The results above indicate that combination therapy with aspirin and ticlopidine is a potential antithrombotic strategy, although further investigaton appeared necessary to determine optimal doses for minimizing hemorrhagic complications.

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Immediate and prolonged microcirculatory effects of varying single doses of the whole body exposure of γ-rays irradiation were studied by using the intravital-microscopy concurrently with macrocirculatory hematological determination in the healthy male rabbit. The intravital-microscopy applied to the subcutaneous microcirculatory system in a transparent round-table chamber, that has been installed in advance into the ear lobe, revealed that markedly increased intravascular adhesiveness of white blood cells (WBCs) and extensive stasis similar to the intravascular coagulation of red blood cells (RBCs) developed as immediate changes soon after the γ-rays irradiation regardless of difference of exposure doses such as 50, 250, 500 and 1000R. Although these changes disappeared within 3-4 weeks in animals exposed to 50R, they persisted throughout the whole experimental period of 4 weeks at higher doses of γ-rays and the extent of the persisting changes appeared to be dose-dependent. Similarly the macrocirculatory changes such as decreases in measurements of RBCs, WBCs, hematocrit values, hemoglobin concentrations, and platelet counts and increase in erythrocyte sedimentation rates were also noticed in a dose-dependent manner.

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The preventive effect of MK-733 (Simvastatin), a potent inhibitor of HMG-CoA reductase, on hypercholesterolemia and atherosclerosis induced by 1% cholesterol feeding in rabbits was examined. MK-803 (Lovastatin) was used as a comparative drug. The cholesterol feeding remarkably increased the serum total-, VLDL- and LDL-cholesterol and phospholipids levels, and slightly increased the HDL-cholesterol ones. MK-733 was found to prevent dose-dependently the increase of serum cholsterol levels, 2, 263mg/dl (treated control), 95mg/dl (10mg/kg), 445mg/dl (5mg/kg) and 982mg/dl (2.5mg/kg) at 12 weeks later. MK-803 (5mg/kg) inhibited them to 1, 130mg/dl. MK-733 inhibited the increase of VLDL-and LDL-cholesterol levels, but slightly affected HDL-cholesterol ones. MK-733 inhibited the increase of serum phospholipids levels. MK-733 preveted the development of atherosclerosis on aortae.
The preventive effect of MK-733 at 2.5mg/kg on hypercholesterolemia and atherosclerosis was thought to be equal to those of 5mg/kg of MK-803.

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Experimental liver cirrhosis was made in Donryu rats by intraperitoneal administration of thioacetamide. Blood coagulation and fibrinolysis factors of the liver cirrhosis group were lower than those of the normal liver group and liver cirrhotic findings were also histopathologically observed. Plasma endotoxin levels were higher in the liver cirrhosis group than in the normal liver group. But, there was no significant difference in endotoxin levels of inferior vena dava blood, portal vein blood and thoracic lymph duct lymph, either in the group with the liver cirrhosis group or in the normal liver group. Plasma endotoxin inactivating activity in the liver cirrhosis group was lower than that of the normal liver group. It is proposed that the decreased plasma endotoxin inactivating activity in the liver cirrhosis group is one important cause of endotoxemia.

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In order to know the pathological significance of elastase (ELP) released from leukocytes in blood circulation, the laboratory findings which may have connection with high ELP level in sera were searched. ELP value was examined using frozen sera obtained from two laboratories (164 and 125 cases). The value determined using Suc-Ala-Tyr-Leu-Val-pNA remained unchanged by repeated freezing and thawing. The results obtained were as follows.
1) ELP level obtained in sera from healthy human blood (control) was 315±165ng/ml.
2) The value increased significantly in the patients sera which showed high value of leucine aminopeptidase (LAP), γ-GTP, GOT, GPT and ALP. ELP level in sera which LAP, γ-GTP, GOT, GPT and ALP showed high level was 1, 370±825ng/ml (n=18), and it was 690±370ng/ml (n=15) in sera showing high level of only LAP and γ-GTP.
3) A correlation coefficient between ELP and LAP was 0.78, and regression line was y=526x+130 (x=ELP, μg/ml). The coefficient between ELP and γ-GTP was also 0.78, but that between ELP and GOT (or GPT) was low (r=0.29, 0.38).
4) ELP level in sera of patients with renal failure (n=30) was 245±70ng/ml, and it was lower than that in control sera.

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Protamine sulfate (Protamine) has been used for the neutralization of over-doses of heparin in order to avoid bleeding.
In the present study, we examined the neutralization of low molecular weight heparin, FR-860, by protamine and compared the results with those of Novo heaprin (heparin).
The prolongation of aPTT and thrombin time induced by FR-860 was immediately neutralized by protamine in in vivo model of rabbits. Whereas, anti-Xa activity and the prolongation of Xa clotting time induced by FR-860 were resistant to the neutralization by protamine. The prolongation of aPTT, thrombin time and Xa clotting time and anti-Xa activity induced by heparin were completely neutralized by protamine.
In vitro study, anti-thrombin activity of heparin was completely neutralized in parallel with the anti-Xa activity by protamine. The anti-thrombin activity of FR-860 was also completely neutralized in a way similar to that of heparin, whereas the anti-Xa activity was resistant to the netralization by protamine.
These results suggest that the bleeding tendency caused by over-doses of low molecular weight haparin can be specifically prevented by protamine.

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We studied blood coagulation and procoagulant activity of leukemic cells in acute promyelocytic leukemia (APL), exhibited monoblast-like transformation at the 3rd relapse. At the onset, hypofibrinogenemia, a high level of FDP, or decreased antiplasmin activity was observed but antithrombin activity was normal. At the 3rd relapse, fibrinogen and FDP levels were increased, antithrombin activity was decreased, but antiplasmin activity became normal. Although both procoagulant activity and fibrinolytic activity in leukemic cell lysate was high at the onset, fibrinolytic activity of transformed cell was decreased at the 3rd relapse. In HL 60 cells, both procoagulant activity and fibrinolytic activity were high, but in transformed cells with TPA fibrinolytic activity was decreased. Therefore, it was speculated that leukemic cells in APL have high levels of procoagulant activity and fibrinolytic activity, which cause hypofibrinogenemia.

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The plasma levels of von Willebrand factor (vWF), factor VIII procoagulant activity (factor VIII) and protin C (PC), were followed in 39 patients with myocardial infarction (MI).
The mean vWF activity in the acute stage of MI was significantly higher (p<0.01) than that in age-matched controls, whereas in the chonic stage there was no statistically significant difference (Fig. 1).
Factor VIII was not significantly higher than in controls at any stage.
The vWF activities in patients with acute myocardial infarction were not correlated at all with peak CPK levels (Fig. 2).
The mean PC values in MI patients were normal, but 17 patients with abnormally high vWF activity in the acut stage showed PC values significantly lower than those of both nomal controls and 8 patients with nomal vWF activity.
It appeared that increased plasma vWF activity was possibly associated with an increased risk of death from acute myocardial infarction.
The low PC antigen level in patients with higher vWF activity was perhaps due to PC consumption in the acute stage of this disease.
The mechanism of this disease in patients with nomal vWF activity at onset is probably due to spasm of the coronary artery.

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The plasma levels of von Willebrand factor (vWF), protein C (PC) and antithrombin III (AT III) were examined in 32 patiens with cerebrovascular disease (CVD).
The mean vWF activity in 17 patients with cerebral infarction of acute phase was significantly higher (p<0.05) than age matched controls and in 6 patients of chronic phase was within normal limits. The mean PC antigen in 15 patients with cerebral infarction of acute phase was significantly (p<0.05) lower than 64 normal controls and in 6 patients of chronic phase was within normal limits. The mean AT III activity in 18 patients with CVD of acute phase was lower than normal controls, but was not statistically significant.
Linear correlations were found between vWF and PC (r=-0.48, p<0.01), and between PC and AT III (r=0.40, p<0.05) in 32 patients with CVD.
These results suggest that the increased level of plasma vWF in patients with CVD of acute phase may reflect endothelial damage, regeneration of the damaged endothelium and overproduction of vWF from the endothelial cells, and that decreased levels of plasma PC in patients with cerebral infarction is due to consumption at acute phase of the disease.

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The variation of spleen plasminogenolytic activity was investigated using a carrageenan-induced inflammation in mice. Immuno-blotting method using antiserum for human plasminogen was adopted for the observation of plasminogenolysis by the spleen extract with 2M KSCN. The plasminogenolytic activity was detected even in the normal spleen extract. The remarkable enhancement of it in the spleen from the 6th to the 10th day after the carrageenan injection followed the increases of plasma fibrinogen and serum FD (g) P levels. The hydrolysis of plasminogen by the spleen extract made fragments of 55kDa, 35kDa and 28kDa, further the 55kDa fragment changed to the small one. These plasminogenolytic activities were completely inhibited with diisopropyl fluorophosphate, soybean trypsin inhibitor or α₁-trypsin inhibitor, but not inhibited with aprotinin. The variation of the spleen plasminogenolytic activity after the carrageenan injection and these effects of proteinase inhibitors for the activity were similar to the fibrinolytic activity in the spleen. These data show that the plasminogenolytic activity in the spleen was derived from a same proteinase as fibrinolytic activity.

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Coagulation and fibrinolysis were studied in normal subjects and patients with Buerger's disease (TAO), arteriosclerosis obliterans (ASO), acute arterial thrombosis (AAT) and acute deep venous thrombosis (ADVT); the mean ages were 32.8, 42, 5, 63.5, 64.8 and 60.2 years respectively. β-TG significantly elevated in all the patient groups compared with the normal subjects. FPA, FPBβ 15-42 and D-dimer significantly elevated in ASO, AAT and ADVT groups compared with the normal subjects and TAO groups. Therefore in ASO, AAT and ADVT groups, there might be platelet activation, hypercoagulability and increased fibrinolysis, whereas TAO group seemed to have only platelet activation. These results might be dependent on difference in the amount of unorganized thrombi among the patient groups. Low activity of α₂-PI showed effective fibrinolysis in thrombolytic therapy (urokinase and t-PA) by means of ultiple regression analysis. Using fibrin plate method, fibrinolysis area appeared when α₂-PI was less than 50% in urokinase therapy. Judging from the measurement of α₂-PI after administration of urokinase, it was expected that α₂-PI was almost maintained below 50% when 600, 000 units of urokinase were intravenously infused for 3 hours on the first day and an intravenous administration of 300, 000 units of urokinase for 1.5 hours were given daily from the following day.

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