Release of Bβ peptides and F (g) DP from fibrin (ogen) was studied after the activation of Glu-plasminogen (Glu-plg) by urokinase (UK) in the plasma or clot and fibrinogen or fibrin. Bβ peptides or FDP were released faster from the clot than the plasma. In a purified system, FDP was released faster than FgDP after the activation of Glu-plg by UK in the presence of fibrin or fibrinogen. Release of Bβ 15-42 from purified fibrin was slower than the release of Bβ 1-42 from fibrinogen when Glu-plg was activated by UK. The presence of α₂ antiplasmin (α₂AP) slowed the release of Bβ 1-42 from fibrinogen, thus resulted in faster release of Bβ 15-42 in comparison to Bβ 1-42. These results indicated that Glu-plg was activated better by UK in the presence of fibrin than fibrinogen, but the release of Bβ peptides from purified fibrin and fibrinogen depended upon the presence of α₂AP. Since fibrin prevented inactivation of plasmin by α₂AP, the presence of α₂AP inactivated plasmin in the fluid phase.

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Automated assay method for purified tissue type plasminogen activator (tPA) was established which used a flexible centrifugal auto-analyzer COBAS FARA (F. Hoffmann-La Roche & CO.) and a chromogenic substrate S-2288. A release of the product, p-nitroaniline was continuously monitored by the absorbance at 405nm and the data obtained were analyzed by a computer-aided processor. When compared with the conventional assay methods reported, this method has several advantages. The time required for the measuring and data-analysis is very short (10min). The sensitivity is high; a small quantity of sample can be measured (10μl) with less amount of the expensive substrate, S-2288 (26μM).

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Concentrations of fibrinogen, plasminogen, antithrombin III and α₂-macroglobulin in plasma were determined in subjects with age 80 who were apparent healthy and lived in urban in 1978. The similar coagulation analysis were carried out again in 17 subjects among these subjects in 1983. There were statistically significant correlations between all parameters at the interval of 5 years in the same subjects, although the levels of fibrinogen and α₂-macroglobulin tended to increase whereas the concentration of plasminogen and antithrombin III decreased with ageing. The differences of all these parameters determined in 1978 and in 1983 were statistically significant. However, there were no statistically significant differences in the levels of these parameters measured in 1978 between in the subjects who survived in 1983 and ones who expired before 1983. From these results, it is concluded that apparently healthy subjects aged 80 become more thrombotic after 5 years, and that there is some “individuality” in patterns of the parameters related to blood coagulation. However, there were no evidences to suggest that hypercoagulability affected disadvantageously the prognosis of apparently healty subjects with age 80.

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Platelet function, prostaglandins, coagulation and fibrinolytic states influenced by exercise stress were evaluated in 7 patients with effort angina. Upon exercise all patients exhibited positive tests for ischemia and platelet specific protein β-thromboglobulin (β-TG) increased significantly (p<0.01) suggesting intravascular platelet activation but thromboxane B₂ (TXB₂), 6-keto-PGF_1α, fibrinopeptide A (FpA) and fibrinopeptide Bβ 15-42 (FpBβ 15-42) stayed unchanged (Fig. 2). At rest, plasma β-TG, TXB₂, 6-keto-PGF_1α and FpA were in normal or near normal ranges but FpBβ 15-42 was increased suggesting an increased fibrinolytic state. Oral administration of oxygen radical scavenger co-enzyme Q₁₀ 120mg/day for 2-4 weeks prevented the exercise induced elevation of β-TG but exerted no effects on prostaglandins and fibrinopeptides.

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Spontaneously hypertensive rats (SHR) were discovered among Wistar Kyoto rats (WKY). In this study, prothrombin time, activated partial thromboplastin time, levels of factors II, V, VII, VIII, IX, X, XI, XII and XIII, plasma fibrinogen content, heparin cofactor activity (levels of antithrombin III) and activity of α₂-plasmin inhibitor in plasma were determined using an automatic analyzer in 30 WKY (15 males and 15 females) and in 30 SHR (14 males and 16 females) with 10 weeks of age. Mean systolic pressure (±SD) measured using programmable sphygmomanometer in the tails of the rats elevated in SHR. Activities of factors VII and X and heparin cofactor activity in plasma were lower in SHR than in WKY. Levels of factors XII, VIII and II also fell slightly in SHR. Whereas, concentrations of fibrinogen and factor XIII in plasma were slightly higher in SHR than in WKY.

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The process of the fibrinogen-fibrin conversion under a velocity gradient was observed by using a flow birefringence apparatus. This was done in order to investigate the role of the release of fibrinopeptides A and B (FPA and FPB) in the formation of the fibrin structure. Fibrinogen, whose FPA and/or FPB are released by thrombin or reptilase, forms fine clots at high pH and/or high ionic strength, while the hydrolyzed fibrinogen forms coarse clots at lower pH and/or low ionic strength. Under experimental conditions, when forming fine clots, a clear chromatic polarization was observed by the flow birefringence measurement only in the fibrinogen and thrombin systems at a given velocity gradient, e. g., 1, 670s^-1. On the other hand, no flow birefringence was observed in the fibrinogen and reptilase systems under experimental conditions when forming fine or coarse clots. The occurrence of flow birefringence in the fibrinogen-thrombin systems indicates the formation of optically anisotropic particles at a given range of velocity gradient. Thus, the anisotropic particles, formed from fibrin monomer catalyzed by thrombin, could be distinguished from isotropic particles formed by reptilase, by using the flow birefringence observation. The release of FPB appears to be a major contributing factor in disturbing the formation of isotropic particles, caused by aggregation of fibrin protofibrils.

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An acid stable trypsin-plasmin inhibitor (ASTI) was found to be produced when several SH-proteases were incubated with human plasma. The maximal activity produced with bromelain was approximately 40U/ml plasma, which represented about a 10-fold increase compared with that of the normal human plasma (less than 5U/ml). On the other hand, lesser activity was observed with several serine proteases, trypsin, elastase, followed by chymotrypsin. Neither the plasma fibrinolytic enzymes including plasmin and urokinase, nor plasma kallikrein had any effect on the ASTI production in the same condition.
An increase in the ASTI acativity was also demonstrated in plasma derived from patients with acute pancreatitis (21U/ml), and the attivity was almost parallel to that of tosyl-L-arginine methylester (TAMe) hydrolyzing activity in plasma. In an experimental anaphylaxis of the rabbit, the ASTI activity in plasma also increased more than 5 times after antigen (bovine serum albumin) was injected.

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The effects of local vibration on blood fibrinolytic activity were studied. (I) The palms of 10 healthy males were exposed to vibration (35Hz, 5G, 5mm p-p) for 5 minutes keeping the normal blood flow in the forearms. The euglobulin fractions were separated from the pre- and post-vibration blood samples. The fibrinolytic activity was measured with the fibrin plate method. It increased after vibration in spite of no hemoconcentration as follows; fibrinolytic areas (pre/post)=4.9±3.9/7.2±5.2mm². (II) The fibrinolytic activity was kept at high level even 30 minutes after vibration. In case of rabbits with hypercholesterolemia, the activity did not increase. They suggest that the vascular plasminogen activator from the endothelial cells functionally takes part in the increased fibrinolytic activity. (III) In patients with arteriosclerosis (myocardial infarction and cerebrovascular accident, n=36), the fibrinolytic activity (pre/post) were 19.2±8.1/20.4±10.5mm², though 22.2±18.2/28.2±18.2mm² in controls (n=30). A possibility of vibration load test is anticipated as a method for the physiological diagnosis of arteriosclerosis.

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The purpose of the present study is to clarify the effect of taurine on platelet release reaction and platelet ATP metabolism in 13 patients with ischemic heart disease and 10 patients with cerebral infarction.
In vitro, amounts of ATP in platelet rich plasma (PRP-ATP) and platelet ATP (PLT-ATP) were measured by a luciferin-luciferase method before and after the incubation with or without 10mM taurine and also after the stimulation by 4.6μM ADP. In the study using taurine, PRP-ATP was not significantly changed and PLT-ATP was significantly decreased by the addition of 4.6μM ADP. On the other hand, in the control study, PRP-ATP was significantly increased and PLT-ATP was significantly decreased by the addition of 4.6μM ADP. Before and after oral administration of taurine 6g/day for 4 weeks, platelet aggregation was measured by Born's method and plasma β-thromboglobulin (β-TG) and platelet factor 4 (PF4) were measured by the radioimmunoassay. The platelet secondary aggregation induced by 4.6μM ADP, and plasma PF4 were significantly decreased after taurine treatment.
The present results indicate that the effects of taurine on platelet function may be the primary inhibition of release reaction and that taurine may inhibit secondly platelet aggregation. It remains to be investigated wether the inhibition of taurine to platelet release reaction is due to ATP-dependent mechanism or ATP-independent one.

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Platelet aggregation induced by platelet-activating factor (PAF) was inhibited by the prior incubation of PAF solution with citrated human plasma. Such neutralization by plasma of PAF was found to be dependent on the amount of plasma preincubated with PAF and on the period of the incubation. Preincubation of ADP solution with plasma did not affect its platelet aggregating activity. PAF-neutralizing activity was abolished by the pretreatment of plasma with diisopropylfluorophosphate (DFP) in a dose-dependent manner, and a complete inhibition was observed with 5mM DFP. Plasma lipoprotein fraction, prepared by ultracentrifugation, was found to account for the inactivation of PAF by plasma, and lipoprotein-deficient plasma was virtually inactive.
PAF, identified as 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, is known to be metabolized by a specific enzyme called acetylhydrolase which hydrolyzes the acetyl moiety at the sn-2 position of the molecule. This enzyme has been found in various tissues as well as in plasma. The dose-dependent inhibition by DFP implies that acetylhydrolase is solely responsible for the inactivation of PAF by plasma and that the hydroxy group of a serine residue plays an important role as the active site of this enzyme. The result showing the association of PAF-neutralizing activity with plasma lipoproteins may suggest the existence of a unknown mechanism involved in the interaction between platelets and lipoproteins.

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The effects of tumor promoter (PMA, phorbol-12-myristate-13-acetate) on thrombin-stimulated human platelet responses and membrane phospholipid metabolism were investigated. Thrombin (0.1U/ml)-induced serotonin release was inhibited by pretreatment with PMA (50μg/ml) for 5min. This inhibition was associated with a suppression of polyphosphoinositide breakdown and increase of cytoplasmic free Ca²⁺ ([Ca²⁺] i). PMA was observed to increase the incorporation of [³²P]phosphate into phosphatidylinositol 4, 5-bisphosphate (PIP₂) and phosphatidylinositol 4-phosphate (PIP). This indicates that treatment with PMA suppresses the hydrolysis of PIP₂ by phospholipase C, resulting in the insufficient supply of a Ca²⁺ releaser, IP₃. A considerably high radioactivity in PIP would be due to the enhanced conversion of phosphatidylinositol (PI) to PIP via PI kinase rather than the inhibited PIP breakdown, since there was no indication of PIP hydrolysis during stimulation with thrombin alone. These observations provide evidence that protein kinase C subserves a negative feedback role in a receptor-mediated cell activation.

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In human platelets both membrane phospholipid metabolism and Ca²⁺ mobilization are known to be closely associated with signal transduction. Phosphoinositide metabolism and changes in cytoplasmic Ca²⁺ level were investigated in [³H] inositol-labeled and quin 2-loaded platelets respectively.
In the presence of 1mM CaCl₂ thrombin (0.1-1.0U/ml) caused a transient decrease in [³H] phosphatidylinositol 4, 5-bisphosphate (PIP₂) and the rapid formation of [³H] inositol trisphosphate (IP₃) within 10 seconds. [³H] Inositol bisphosphate was also accumulated in the early phase but [³H] inositol mono-phosphate was accumulated later. The formation of [³H] inositol phosphates was not affected by 1mM EGTA. PIP₂ was resynthesized above the initial level and [³H] phosphatidylinositol 4-phosphate (PIP) increased. [³H] Phosphatidylinositol was found to be a lipid pool for PIP and PIP₂. Cytoplasmic free Ca²⁺ concentration ([Ca²⁺] i) increased rapidly coinciding with the process of IP₃ formation and thereafter declined gradually toward the resting value. Under conditions that IP₃ formation was inhibited a small increase in [Ca²⁺] i was observed. Moreover IP₃ was observed to be a Ca²⁺ releaser from Ca²⁺-accumulating human platelet membranes.
These results indicate that a series of phosphoinositides metabolism may regulate the formation of second messengers to be contributory to human platelet activation.

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In twenty patients with ischemic cerebrovascular disease fibrinopeptide A (FPA) and fibrinopeptide Bβ 15-42 (Bβ 15-42) were serially assayed in cerebrospinal fluid (CSF) and in plasma.
After plasma FPA levels began to be elevated, it took a peak from 5th to 14th day after stroke (p<0.05), and gradually returned to control level. Bβ 15-42 levels in plasma were constantly elevated at least during first three weeks (Fig. 1). On the other hand, values of FPA in CSF were initially elevated and then reached to a peak on 15th to 21st day after stroke (p<0.01). However values of Bβ 15-42 in CSF remained at control level (Fig. 2).
Suggestions of these results are as follows. Coagulation activity was accelerated following the onset of stroke with a short time lag. This delay of coagulation activity was considered to be due to the stroke progression, that is, continuous activation of thromdin in the ischemic regions. And it caused the elevation of FPA that released into the subarachnoid space crossing the broken blood-brain barrier. While fibrinolytic activity in plasma was continuously elevated, that in CSF was not influenced by stroke. The reason of this difference was not proven in our series.

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Two cases of cardiac tamponade associated with anticoagulant treatment in the early period after cardiac valve replacement are reported. The first case, a 48-year-old female with mitral valve re-stenosis underwent mitral valve replacement and received anticoagulant treatment with warfarin after surgery. Cardiac tamponade occurred on day 5 postoperatively when the thombotest value was 9%.
The second case, a 49-year-old male with hypertension underwent aortic valve replacement and thoreafter received warfarin. Cardiac tamponade occurred on day 10 postoperatively when the thrombotest value was 13%. Both were treated successfully by surgical drainage.
Postoperative anticoagulant treatment with warfarin appeared to the development of cardiac tamponade in both cases.

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Changes in platelet counts, prothrombin time, partial thromboplastin time, thrombo test, normo test (hepaplastin test), fibrinogen, euglobulin lysis time (ELT) and Bβ 15-42 peptide were investigated on 16 patients with benign disease and 27 with malignant disease before and after operation in general surgical patients. The same investigation was performed on 6 patients with benign disease and 29 with malignant disease who had intermittent sequential compression of the legs for 48 hours postoperatively.
The ¹²⁵I-fibrinogen uptake test was performed on 45 patients with malignant disease who had intermittent sequential compression of the legs for 48 hours postoperatively.
Following results were obtained.
1) It suggests that influence on activity of plasminogen activator was produced by leg compression but no influence on activity of plasmin and plasminogen was produced by leg compression.
2) Incidence of deep vein thrombosis (DVT) in intermittent sequential leg compression group reduced to 8.9 per cent compared with 18.0 per cent in control group.

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The influence of DDAVP on factor XI (F. XI), factor XII (F. XII), prekallikrein (PKK) and High Molecular Weight Kininogen (HMWK) in normal subjects was studied to clarify the hemostatic mechanism. A 12-16μg of DDAVP was injected intravenously to ten normal aubjects and aPTT, VIII: C, VIIIR: Ag, XI: C, XI: Ag, XII: C, XII: Ag, PKK: C, PKK: Ag, HMWK: C and HMWK: Ag were measured serially before and after the injection. And in vitro serial changes of aPTT, F. XI, F. XII and HMWK in mormal plasma after interaction with urokinase (UK) was studied. XI: C, XII: C and HMWK: C increased after DDAVP injection. But antigens of these did not increase. XII: C and HMWK: C after injection of DDAVP may be induced by fibrinolytic activity activated by the release of plasminogen activator. The increase of XI: C may be secondary activation due to the increase of XII: C and HMWK: C.

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