Haemorrhagic diathesis often manifest in liver diseases. It has been recently found that the activation of Kallikrein-Kinin (K-K) system causes acceleration of coagulation, fibrinolysis and compliment system.
We have studied K-K system in 30 normal subjects, and 56 cases of liver diseases; acute hepatitis (8), chronic hepatitis (8), liver cirrhosis (19) and hepato-billiary carcinoma (21). Plasma levels of prekallikrein (PK) and high molecular weight (HMW)-kininogen were reduced in patients with chronic hepatitis and liver cirrhosis, but plasma levels of kallikrein-like activity, kallikrein-inhibitor activity and serum levels of kininase II activity were elevated in the most of patients with liver diseases.
Accordingly, a correlation was shown between the elevated plasma kallikrein-like activity and the reduced PK in the patients with liver diseases.
In severe stages of liver damages, such as in cases with endotoxin shock accompanied with liver diseases, plasma levels of HMW-kininogen reduced primarily and followed by reduced plasma levels of low molecular weight (LMW)-kininogen. We concluded therefore that, in contrast to conventional opinions concerning the decreased state of plasma factor of K-K system in liver damages, plasma K-K system is activated in the patients with liver damages.

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The authers examined the effect of IgG₁ (λ) on the ristocetin-induced platelet aggregation in a patient with multiple myeloma associated with acquired von Willebrand's disease.
We isolated monomeric IgG with the combined procedure of ammonium sulfate fractionation and column chromatography on DEAE-cellulose, as aggregated IgG inhibited the ristocetin-induced platelet aggregation. We found that IgG₁ (λ) from the patient's plasma inhibited ristocetin-induced platelet aggregation but IgG from normal human plasma hardly inhibited. Molecular weights of heavy chain and light chain of IgG₁ (λ) from the patient's plasma were estimated to be approximately 58, 000 and 35, 000, respectively. On the other hand, molecular weights of heavy chain and light chain of IgG from normal human plasma were estimated to be approximately 58, 000 and 30, 000, respectively. Therefore, the difference in molecular weight of light chains might be correlated with the inhibitory activity of IgG₁ (λ) on the ristocetin-induced platelet aggregation. Furthermore, the inhibitory activity of IgG₁ (λ) molecules might be derived from interfering in the interaction of ristocetin, von Willebrand factor and blood platelets.

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The aim of this report is to investigate the effects of eicosapentaenoic acid (EPA) and docosahexenoic acid (DHA) on hemostasis, blood lipids and fatty acid composition of plasma and platelet. Twelve volunteers were subjected to the cross-over study comparing the effect of EPA with that of DHA. They were divided into 2 groups: group A (mean age, 64.5 years) in which EPA 2g/day was given for 4 weeks and after 4 weeks off treatment, DHA was given for the subsequent 4 weeks and group B (mean age, 62.5 years) in which DHA was given first followed by EPA treatment. Significant decrease in platelet aggregation was observed following the treatment with 2g of DHA, but platelet aggregation tended to increase after the treatment with 2g of EPA. Platelet aggregation tended to decrease following the treatment with 2g of EPA, but it was increased after the treatment with 2g of DHA. The ratio, (EPA+DHA)/arachidonic acid, in both plasma and platelets was increased after EPA treatment, but there was no consistent change of the ratio after DHA treatment. DHA thus appears to be more potent than EPA in inhibiting platelet aggregation. However, this inhibitory effect was blunted by the prior administration of EPA or DHA.

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Platelet aggregation induced by Polybrene was investigated in normal subjects and patients with various disorders. Polybrene aggregated normal washed platelets in buffer, and the aggregation became more extensive in the presence of plasma von Willebrand factor. Polybrene-induced platelet aggregation (PIPA) and ATP release in citrated platelet-rich plasma (PRP) from normal subjects were inhibited by prostacyclin and indomethacin in a dose-dependent manner. PIPA in PRP and ATP release were markedly decreased in thrombasthenia and decreased in congenital afibrinogenemia, platelet release mechanism abnormality due to defective intracellular calcium mobilization, and platelet-type von Willebrand's disease with increased ristocetin-induced platelet aggregation. However, a patient with platelet cyclo-oxygenese deficiency showed nearly normal response to Polybrene. These results provided us additional informations about the mechanism of platelet aggregation and release reaction induced by these cations, and the interaction between platelets (platelet receptors) and von Willebrand factor.

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In this paper, results of the one year follow-up on the effects of ticlopidine on platelet aggregation (PA), other blood parameters and peripheral hemodynamics in patients with arteriosclerosis are reported. We measured PA, plasma β-thromboglobulin (β-TG), plasma euglobulin lysis time (ELT), plasma lipids and peripheral hemodynamics in 9 patients (mean age 64.4 years) including chronic state of ischemic heart disease, cerebral infarction and arteriosclerosis obliterans before treatment with oral administration of 300mg of ticlopidine per day, at 1, 3, 6 and 12 months during treatment. Blood flow (BF) and maximum venous outflow of the forearm and calf were measured by venous occlusion plethysmography using strain gauge to investigate peripheral hemodynamics.
PA, induced by ADP, collagen, adrenaline and arachidonic acid, decreased significantly at 1 and 12 months following ticlopidine treatment. No significant change in other blood parameters was found following the treatment except the increase of plasma phospholipid at 12 months following the treatment. Significant increase in BF of the bilateral forearm and calf was observed at 12 months following the treatment.
These results show that an anti-platelet drug, ticlopidine may have beneficial effects on peripheral hemodynamics in the treatment of patients with arteriosclerosis.

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A thromboxane A₂ (TXA₂) synthetizing activity of platelets collected from myocardial infarction (MI) was measured and compared with that of healthy subject. Platelets were collected from 13 healthy volunteers and 30 patients with MI after 3 hours to 21 days from the onset. Platelets were separated from plasma and suspended in Ca⁺⁺ and Mg⁺⁺ free Tyrode solution. Thirty seconds and 1 and 5 minutes after the incubation with arachidonic acid (25uM) or thrombin (0.1unit/ml) in aggregometer, the amount of TXB₂. Which was synthetized in platelets, were measured by RIA. The amount of TXB₂ was decreased in MI after 12 hours to 4 days in comparison with that of healthy group with a statistical significance. The amount was increased in MI after 5 to 10 days. On the contrary, the platelet aggregation was enhanced in MI after 12 hours to 4 days. A negative correlation was observed between the synthetized amount of TXB₂ and intensity of aggregation of platelets. In the case stimulated with thrombin, the same tendency was observed in the TXA₂ synthetizing activity of platelets. However, a significant difference was not found. These findings suggest a presence of selective consumption of platelets with higher synthetizing activity of TXA₂ in acute stage of MI.

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Endothelial cells were isolated from human umbilical cord and cultured by the modified method of Jaff e et al. 6-keto PGF_1α content in supernatant over the monolayer cells was employed as the parameter of PGI₂ generation. PGI₂ generation was increased by addition of Nifedipine (×2.10)>Diltiazem (×1.78)>Verapamil (×1.59). This effect was diminished when the cells were pretreated with each of Mepacrine or ASA, even when Arachidonic acid or PGH₂ was added. The increase of PGI₂ generation by Ca antagonist was considered to be due to activation of phospholipase A₂. The PGI₂ generation induced by A-23187, Arachidonic acid, or PGH₂ was remarkably decreased by preincubating the cells with TMB-8. The dependency of PGI₂ generation upon the intracellular Ca⁺⁺ was considered to be dominant in the early step of AA cascade. The enhanced PGI₂ generation by A-23187 or Diltiazem was almost diminished in Ca⁺⁺ free medium. While preincubation with MIX caused increase in intracellular c-AMP concentration, and decrease in PGI₂ generation, addition of Diltiazem had no effect on c-AMP concentration. Through these experimental results, it is concluded that the enhanced PGI₂ generation via phospholipase A₂ induced by Diltiazem may be originated from intra- and extra- cellular Ca⁺⁺ mobilization and may not be dependent on c-AMP process.

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The interaction between platelets and fibrin during clotting was examined using a rheological technique.
At an early stage of clotting, a wave form of the shear stress was distorted for all samples including PRP, PPP and fibrinogen solution, indicating a non-linearity in the stress-strain relation in the sample. When the clotting approaches completion, the distortion of the wave form becomes more marked in the clot of PPP, fibrinogen solution and reptilase catalyzed PRP, but completely disappeared in the clot of PRP and fairly disappeared in that for fibrinogen solution containing washed platelets and calcium ion.
The non-linearity for the clot under a large strain amplitude appears to be caused by the high stretching of fibrin forming network chain in the clot structure. The disappearance of non-linearity in PRP clot would be due to clot retraction, that is, platelets adhere to polymerizing fibrin and contractile protein in platelets exerts contractile force to fibrin network. The rigidity of the clot is thus increased and the stress-strain relation becomes linear.
It is expected that the observation of the wave form of the shear stress in clot would be used as monitoring technique that gives information about the interaction between platelets and fibrin.

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The effects of multistage treadmill exercise testing (TMT) on blood coagulability and platelet function were evaluated in 11 male patients with ischemic heart disease (IHD) and 11 age-matched healthy male controls. Near-maximal TMT was performed according to the modified Bruce's protocol. The blood for the measurements was sampled from the antecubital vein at rest, and immediately and 6min after exercise.
No significant changes in the platelet sensitivity to ADP-aggregation and the plasma β-TG levels were observed after exercise in IHD and control groups. Plasma cAMP and catecholamines (epinephrine and norepinephrine) increased after exercise in both groups. However, the degree of increase in catecholamines was greater in the IHD group than healthy controls. Plasma fibrinogen increased after exercise in the IHD group, and antithrombin III increased in both groups. Prothrombin time and partial thromboplastin time were shortened after exercise in both groups. The biological activities of factors VIII and XII were enhanced after exercise in the IHD group but remained unchanged after exercise in healthy controls.
In conclusion, there was a tendency for blood coagulability to be enhanced after exercise in IHD, which seemed to be caused by the marked increase in plasma catecholamines.

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The plasma coagulation system was converted to a mathematical model which was described using differential equations. The calculated output patterns of the mathematical model against various input stimulations were compared with results of in vitro assay.
The simulated results of (1) Hemophilia A, (2) anticoagulation effect of antithrombin III and (3) anticoagulation effect of heparin corresponded to the results of in vitro assay and clinical reports. However, the simulated result of (4) anticoagulation effect of synthesized arginine derivative No. 805 (MD-805) did not correspond to the results of in vitro assay. Therefore, a new series simulation of MD-805 was done, supposing that MD-805 had an inhibitory activity not only on coagulation factor ha but also VIIa. The new simulation pattern closely resembled the results of in vitro assay. From these facts, it was theoretically indicated that MD-805 also has an inhibitory activity on VIIa.

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Concentrations of fibrinogen, plasminogen, antithrombin-III, α₁-antitrypsin, α₂-macroglobulin, C1-inhibitor and α₂-antiplasmin in plasma of arteriosclerosis obliterans, thromboangitis obliterans, deep venous thrombosis and Raynaud's syndrome were measured by radial immunodiffusion except α₂-antiplasmin assayed using amidolytic substrate S-2251 (Table 1).
Concentrations of each factor in each disease were compared with that of other disease. The correlation between antigen level or activity of each factor and prognosis in arterial occlusive diseases was investigated (Fig. 1). The average diseased years were 2.9±1.7 (SD) yrs in ASO and 7.2±4.3 (SD) yrs in TAO. The patients aggravated in the course of treatment were designated as poor cases.
On ASO was there correlation between prognosis and concentrations of fibrinogen or C1-inhibitor (Fig. 1). On TAO, significant correlation was identified between prognosis and antigen levels of fibrinogen, α₁-antitrypsin or C1-inhibitor (Fig. 1).
It has been reported that C1-INH interacts with C1s, plasmin, kallikrein, factor XIIa and XIa. The role of C1-INH on thrombogenesis and thrombolysis remains uncertain.
In a group with raised levels of C1-INH antigen, a large percent of patients with ASO underwent limb amputation (P<0.05), and patients with TAO had suffered from aggravating ischemic toe ulcers (P<0.05).
On both ASO and TAO, the patients with raised levels of C1-INH antigen resulted in unfavorable prognosis (P<0.01-0.02).

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Thirteen monoclonal antibodies to human protein C were prepared according to the hybridoma technique of Köhler and Milstein using BALB/c mice and murine myeloma cells (P3Ul). The antibodies were characterized where they recognize on the protein C molecule and whether they effect on the biological functions of protein C. Immunoblotting technique clarified that six antibodies bound to the L-chain of protein C and five antibodies to the H-chain. Two antibodies neither bound to the L-chain nor the H-chain, though they bound to the nonreduced protein C. Two of the antibodies directing toward the H-chain (MFC-1, 13) inhibited the amidolytic activity of activated protein C. In addition to these two, another one toward the H-chain (MFC-10) and two toward the L-chain (MFC-9, 11) inhibited the Factor Va inactivation of activated protein C. One of the antibodies which inhibited the amidolytic activity of the enzyme (MFC-1) blocked the inhibition of the enzyme by protein C inhibitor. Another one toward the H-chain (MFC-5) inhibited the activation of protein C by thrombin regardless of the presence or absence of thrombomodulin. These results would further inform us the relationship between the structure and function of protein C.

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The initial stage of the fibrinogen-fibrin conversion catalyzed by thrombin was studied kinetically by the measurement of the turbidity change of the system. The value of the induction period, obtained by extrapolating the initial and maximum slope portion to the zero turbidity in turbidity-time curves, was related to an equation similar to the equation expressed in the enzyme kinetics. At that time, the fibrin monomers at the end of the induction period was assumed to be at constant concentration, and then to form instantly such smallest polymers of fibrin monomers as detectable by turbidimetry.
The constant derived from our equation, corresponding to the Michaelis-Menten constant was estimated to be 1.79×10^-6M, which is somewhat smaller than the Michaelis-Menten constant in the hydrolysis reaction of fibrinogen catalyzed by thrombin. The inhibition constants of various thrombin competitive inhibitors were estimated in the systems of fibrinogen, thrombin, and inhibitors. The inhibition constants estimated by our turbidimetrical procedure were agreed well with or a little smaller than those measured in the system of synthetic substrate, thrombin, and inhibitors. Thus it was concluded that a practical and handy assay could be carried for a study on the initial stage of the fibrinogen-fibrin conversion.

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This report will deal with the evidence of the interaction in vivo as well as in vitro between thrombin and factor VIII, and with its clinical application. Formation of intrinsic factor X activator activity with thrombin (Xa)-activated factor VIII have also been studied. To elucidate the influence of thrombin (Xa) on the analytical procedures, thrombin (Xa) employed was coupled to Sepharose. The insolubilized thrombin (Xa) complex was readily eliminated from the test samples by filtration prior to their analysis. Thrombin caused an increase in factor VIII coagulant activity (VIII:C), which was followed by a decay of VIII:C to below baseline levels. Factor VIII was also activated by Xa especially in the presence of calcium and phospholipid. With a constant level of factor IXa, factor X activation was directly proportional to the phase of the activation and inactivation of VIII:C by thrombin (Xa). Sequential activation of factor VIII by thrombin and Xa showed that after peak activation, no further enhancement of factor VIII activity occurred, whether thrombin was the initial activation followed by Xa or vice versa. Crossed immno-affinity electrophoresis demonstrated that plasmas with normal and thrombin-activated factor VIII had identical electrophoretic patterns. VIII: C in the DIC plasma, after incubating in vitro with thrombin, was lower compaired with that in normal plasma, thus indicating a low activation in vitro in DIC. The rabbit DIC plasma also showed no further activation resembling to the human DIC plasma.

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Factor VIII-related antigen (VIII R:AG) in four normal persons and twenty patients with liver diseases were analized by 4.35% large pore polyacrylamide gel-two-dimensional crossed immunoelectrophoresis (PAGE-CIE). At the same time, plasma levels of VIII:C, VIII R:AG and VIII R:WF were measured.
The electrophoretic pattern of VIII R:AG in four normal persons showed two precipitin peaks near the cathode, whereas that of VIII R:AG in patients with liver diseases showed various patterns. Abnormal patterns like three or four precipitin peaks were seen frequently in cases of advanced liver diseases as fulminant hepatitis, decompensated cirrhosis and hepatocellular carcinoma.
There was found discrepancy between VIII R:AG and VIII R:WF in eight out of fourteen cases with abnormal patterns.
PAGE-CIE reflects more clearly the qualitative defect of VIII R:AG in cases of advanced liver diseases than customary CIE, however the reason of the qualitative defect remains to be elucidated even though some disturbance of synthesis and catabolism including plasmin interaction may be involved.

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Hypercoagulability, hypofibrinolysis and tendency of kinin increasing during normal pregnancy and labor have been reported, however systemic informations of plasmin activity based on stage-dependent analysis have not been reported yet. In this study, we tried to find out the changes of fibrinopeptide A (FPA) and fibrinopeptide Bβ15-42 (FPBβ15-42) in normal pregnancy and delivery. These factors of FPA and FPBβ 15-42 in 30 non-pregnant women who were chosen as control and 170 cases of normal gravida from early stage to term pregnanacies togather with 18 cases of transvaginal delivery and 12 cases of cesarean section delivery were investigated. Levels of FPA and FPBβ15-42 during pregnancy gradually increased from midterm of pregnancy and increased markedly in late state of pregnancy (A=2.7±1.2, Bβ9.9±3.6ng/ml), Levels of FPA and FPBβ15-42 in the 2nd stage of labor (A=5.0±1.6, Bβ15-42=13.8±5.3) were significantly increased when compared to term pregnancy or during false labor. These findings suggested that chronic DIC conditions secondary to hyperfibrinolytic activity appeared during pregnancy and labor. In puerperium or after operation, levels of FPA were seen with its peak at 15mins after transvaginal delivery and 20-30mins after placental separation in cesarean section. FPBβ 15-42 was noted with its peak at 3 hours in puerperium and post-operation. These patterns closely resembled those of acute DIC with hyperfibrinolytic activities of the blood.

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This study was conducted to elucidate the influence of the diabetic status and hemostatic factors which might participate in the develoment of microangiopathy (retinopathy, nephropathy and neuropathy).
The results obtained from 52 patients with diabetes (52±12 years of age, male 18, female 34) were as follows:
The development of the microangiopathy, observed in thirty seven among fifty two patients, correlated well with the suffering duration (1-25 years) of diabetes mellitus (DM). Increased level of fibrinogen and von Willebrand factor, hyperfunction of platelet demonstrated by ADP-induced aggregation as well as increase in number, decreased AT III, α₂-PI and plasminogen level were observed with thier higher grades as their severity of grade of microangiopathy advanced. In DM of poor controlled groups (28 cases; FBS>120mg/dl, 2hrs blood sugar level after a meal>200mg/dl), platelet counts, fibrinogen and von Willebrand factor level were elevated and ADP-induced platelet aggregation was more hyperactive significantly than that of good controlled groups (22 cases; FBS=120mg/dl, 2hr blood sugar level after a meal≤200mg/dl, for at least on month). These findings were more remarkable in brittle type of DM. These results support the hypercoagulable state exist in DM and suggested that these factors in plasma and platelets promote the injury of the vessel wall directly or indirectly to induce the miroangiopathy.

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In the presence of 10mM triethanolamine, the fibrin-binding ability of urokinase (UK) in freshly obtained human urine was compared to that of commercially obtained urinary UK and two different sources of human malignat melanoma tissue plasminogen activator (TPA). In contrast to the non-binding property to a fibrin/Celite column of commercially obtained UK, which had molecular weights (MW) of approximately 50, 000 and 30, 000 by a zymographic method, fresh urine contained at least three different molecular forms of fibrin-binding UK, all of which were adsorbed on the fibrin/Celite column at neutral pH, and could be eluted with 0.3-1.0M NaCl in phosphate buffer, 0.2M Arg, 2M KSCN, and 2M urea, respectively. The main fibrin-binding UK was a high molecular form of MW approximately 100, 000, and the minor ones were of MW 150, 000-200, 000 and 45, 000.
On the other hand, most of the TPA derived from human cell cultured melanoma (Bowes) was found to be fibrin-non-binding, although TPA extracted from human melanoma tissue with 2M KSCN showed a fibrin-binding property. In the latter, PA activity was completely adsorbed and could be eluted with 6M urea buffer.
These findings strongly indicate that the fibrin-binding property of PA is related to the molecular forms of PA, and is not directly dependent on their sources.

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A Phase II study (early stage) was carried out to investigate clinical effects of Lysyl-Plasminogen (Lys-PLg) prepared from human placenta on cerebral thrombosis.
Sixty mg of Lys-PLg was administered to 71 cases with cerebral thrombosis, once a day for successive 7 days.
Overall improvement at remarkable to moderate grades was evaluated to be 26.8% on the 7th day from the initiation of administration and to be 57.6% at 4th week.
Percentage for clinical effects was relatively higher in younger thrombosis within 14 days from the onset of clinical episode, but even in older thrombosis than 1 month still good clinical results were obtained.
The side effects of this therapy were observed with only in 7 cases, i. e. a case with fever, another with eruption and 5 with slight elevation of transaminase levels in serum.
No cases of hemorrhagic infarction were encountered with Lys-PLg administration in this study.

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We have demonstrated that placental protein (PP 5) first detected by Bohn (1972) shows the same inhibitory effect on platelet aggregation as found in the isolation process of human placental extracts by our previous report. In this report, coagulation and fibrinolytic activities on placental platelet aggregation inhibitor (PPAI) and placental protein (PP 5) were examined. Materials and Methods: The microsomal fraction with an inhibitory effect of platelet aggregation was obteined from human placental extracts. The supernatant fraction concentrated by a column of Bio-beads SM-2 was isolated by gelfiltration on Sephadex G-150 or G-100. PP 5 was measured by RIA using a double antibody method. Antiplasmin and placental UK inhibitor (Kawano) activities were assayed by the chromogenic substrates, S-2251 and S-2444, respectively. Results: 1) PPAI consists of two types of proteins with molecular weights of 78, 000 and 112, 000. 2) Although PPAI appeared relatively the shortened clotting time, it showed neither antiplasmin activity nor UK inhibitory effect. 3) PP 5 had relatively prolonged APTT and thrombin time, but it was found to inhibit the activity of plasmin. 4) Although PP 5 showed no UK inhibitory activity, it was detectable that PP5 is mixed into the placental UK inhibitor (Kawano) by SDS-PAGE method.
In conclusion, it is suggested that PPAI and PP 5 might contribute the homeostasis of fluidity of blood in the placenta.

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The euglobulin lysis time (ELT) in antecubital vein and femoral vein was investigated in 35 patients before and after bilateral intermittent sequential leg compression for one hour. After compression, ELT in antecubital vein was shortened, and that in femoral vein was significantly shortened (P<0.05).
The mean and peak blood velocity of the femoral vein before and during compression was measured using Doppler ultrasound in 20 patients. The mean blood velocity increased about 183% (P<0.001), and the peak blood velocity also increased 178% (P<0.001).
ELT was measured pre- and post-operatively in 9 patients who had intermittent sequential compression during operation and succeeding two days and in 9 controls who had no compression. Both in antecubital vein and in femoral vein, ELT in the compression group was significantly shorter than that of the control group in the first postoperative day.
This study demonstrates that intermittent sequential compression of the legs increased fibrinolytic activity, and this effect may contribute to the prevention of deep venous thrombosis.

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Levels of fibrinogen, plasminogen, antithrombin III, α₂-plasmin inhibitor and α₂-macroglobulin in plasma were determined using single radial immunodiff usion method in an old people's home in 1979, 1981 and 1982. Comparison of results of these parameters in the same subjects between in 1981 and 1982 was possible in 67 subjects. Comparison between in 1979 and 1981 was done in 61 subjects. Results of coagulation analysis in 1979 and 1982 were compared in 49 subjects.
Generally, concentrations of plasminogen, antithrombin III and α₂-plasmin inhibitor in plasma were higher in women than in men. There were statistically significant correlations between all the parameters determined at intervals of 1, 2 and 3 years in the same subjects. The coefficients of correlation in each parameter at intervals of 1 to 3 years were as follows: +0.45-+0.57 in fibrinogen, +0.69-+0.81 in plasminogen, +0.55-+0.81 in antithrombin III, +0.46-+0.64 in α₂-plasmin inhibitor and +0.91-+0.93 in alpha;₂-macroglobulin. From these results, it is concluded that old women are generally less thrombotic than old men and that there is some “individuality” in patterns of the parameters affecting blood coagulation and fibrinolysis in the elderly. The significance of the “individuality” upon prognosis of the elderly was discussed.

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