Ultrastructural localization of alkaline phosphatase activity in human megakaryocytes and platelets was examined. In megakaryocytes, alkaline phosphatase activity was demonstrated in the plasma membrane and cytoplasmic vacuoles as lead phosphate deposits. No reaction product was observed in platelets. In the control samples incubated in a substrate free medium, and in a complete medium containing levamisole, no reaction products were observed. The functional significance of alkaline phosphatase in megakaryocytes has not been clarified; however, this finding indicates that the enzyme composition of the megakaryocyte plasma membrane is different from that of the platelet plasma membrane as well as the megakaryocyte demarcation membrane system. The mechanism whereby platelets are released from megakaryocytes is still debated; however, this finding suggests that the megakaryocyte plasma membrane does not delineate platelet fields.

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A case of 23-years old female patient, who was in the twenty four weeks pregnant, developed thrombotic thrombocytopenic purpura (TTP) and was treated by the infusion of normal plasma with dramatic response in platelet number.
We detected a platelet aggregating factor (PAF; Lian) activity in her plasma, and an inhibitory activity in normal plasma (immunoglobulin IgG). Patient's plasma did not aggregate fixed platelet, but induced the aggregation and the release reaction of washed platelets. This aggregation was slightly inhibited by the preincubation of washed platelets with aspirin, but were not inhibited with dipyridamole or PGE₁. The PAF activity was not inhibited by thrombin or heparin, but inhibited by normal immunoglobulin (IgG) and its Fab portion. The PAF activity was inhibited by normal human IgG, but not by patient IgG. The mechanism of production and release of PAF and its physicochemical characteristics are still remained to be identified.

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An effect of plasma fibronectin (FN) on the adhesion of human fixed washed platelets (FWP) to collagen was evaluated. FWP did not adhere to denatured collagen (heated collagen, gelatin agarose). The percent adhesion to collagen column (1300μg) was 51% at 5ml/min flow rate. The addition of purified fibronectin (FN) decreased the degree of adhesion to collagen dose dependently; 34% (FN 31μg/ml), 29% (62μg/ml), 16% (125μg/ml) and 3% (250μg/ml). The inhibitory effect of FN on the platelet adhesion was also observed by pretreatment of the collagen beads with purified FN. The addition of purified von Willebrand factor (vWF), however, increased the adhesion and the enhancing effect of vWF on the adhesion was observed at higher flow rate conditions. Pretreatment of FWP with anti-FN antibody or anti vWF: Ag antibody did not change the degree of adhesion to collagen.
Plasma FN bound to collagen in suspension and the affinity of FN to collagen was greater than that of vWF: Ag; 97% FN and 60% vWF: Ag bound to 250μg/ml collagen. The binding of ritocetin cofactor (vWF: RCo) to collagen (12.5-100μg/ml) was partially inhibited by FN, but both proteins bound completely to the collagen at 200-500μg/ml.
These data suggested that the adhesion of FWP to collagen is not mediated through plasma FN, while plasma vWF controls the adhesion at high flow rates.

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Inhibition constants of heparin and heparinoids, which formed soluble complexes with fibrinogen, for the fibrinogen and thrombin system were determined by the turbidimetrical method, derived from a kinetic study on the initial stage of the fibrinogen-fibrin conversion. A commercial heparin (molecular weight, 9, 000; 152units/mg) and dextran sulfate (DSC), low molecular weight dextran sulfate (DSA), carboxy methyl cellulose (CMC), chondroitin sulfate (CHS), and sulfated methyl cellulose (SMC) were investigated. Among heparinoids, DSC inhibited more effectively the reaction of fibrinogen and thrombin than heparin. At high concentrations (>0.08mg/ml), heparin and heparinoids acted as noncompetitive inhibitors, and inhibition constants for those substances were smaller in the following order of DSV<heparin<DSA<CMC<CHS<SMC.
The apparent inhibition of heparin and DSC for the reaction of fibrinogen and thrombin increased with decreasing concentrations. At low concentrations (<10^-4mg/ml), both heparin and DSC acted as hyperbolic competitive inhibitors of thrombin. The inhibition constant of heparin was 1.07×10^-8M, which agrees with values estimated from a thrombin and synthetic substrate system. The scarce inhibition effect of DSC in the presence of antithrombin III for the fibrinogen-thrombin system suggests that DSC can not induce the conformational change of antithrombin III, different from heparin.

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The fibrinopeptide A (FPA) as a indicator of thrombin and the fibrinopeptide Bβ15-42 (FPBβ115-42) as a indicator of plasmin of CSF in 55 patients with SAH were measured serially by RIA. FPA was extremely high on the day 0-1 (1230.0±429.3ng/ml) and decreased rapidly (control: 1.2±0.9ng/ml). FPBβ15-42 was high on the day 0-1 (13.4±1.7ng/ml) and decreased slowly (control: 0.88±0.4ng/ml). Bradykinin (BK) of CSF in 27 patients with SAH were also measured serially by RIA. BK levels were 122.7±22.7pg/ml on day 0, 38.6±6.1pg/ml on day 1, 22.7±6.3pg/ml on day 2, and decreased slowly (control: 8.0±3.3pg/ml).
BK is produced by the activation of Hageman factor that is considered to be activated by trabeculae as collagen bundles in the subarachnoid space. The coagulation system in the subarachnoid space might be activated by Hageman factor in the case of SAH. Trabecuia could be considered to play a role to activate Hageman factor of the coagulation system in case of bleeding as well as support the central nervous system.

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In 40 patients with prosthetic heart valves and 3 patients who underwent open mitral commissurotomy, fibrinopeptide A (FPA) and soluble fibrin monomer complex (SFMC) were measured by enzyme immunoassay and fibrin monomer test (FM test), respectively. The relationship between these two parameters and either von Willebrand factor antigen (vWF:Ag), antithrombin III (ATIII) or thrombo-test (TTO) were analyzed.
FPA was elevated in 41.9% and FM test was positive in 25.6% of the patients. High FPA values were obtained frequently in patients with positive FM test. TTO, vWF:Ag and ATIII were not correlated with plasma concentration of FPA. FM test was not correlated with TTO or vWF:Ag, but positive results in FM test were frequently observed in potients with low plasma level of ATIII.
These results indicate that low TTO does not mean suppressed thrombin generation in vivo, and that FPA and FM test would be valuable parameters of hypercoagulability in these patients.

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A characterization of the platelets treated with sodium citrate has been made by isoelectric equilibrium analysis. Isoelectric focusing was carried out in a 50-ml column fitted with platinum electrodes. An aliquot of 1-5×10⁸ platelets were focused at 0-1°C for 4 hours by post-pH equilibrium loading of the platelets in a sucrose linear gradients (0-50%) containing 1% ampholine at pH 3-10. After focusing, the column was drained. The pH and the OD at 420nm were determined in 1.0ml fraction.
Unmodified platelets of healthy subject show a surface isoelectric point (pI) of 4.22. Those of hepatic cirrhosis show pI between 3.80 and 4.50. Platelets from thrombasthenia show a pI of 4.20. When amino groups are blocked by formaldehyde, the platelet surface of healthy subject has a pI of 3.91. Platelets treated with neuraminidase are isoelectric at pH 4.54.

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The effects of 9, 11-epihio-11, 12-methano-thromboxane A₂ (STA₂), a stable analogue to thromboxane A₂, on platelet aggregation and its interaction with α blockers (phentolamine, prazosin and yohimbine) were studied in both mongrel dogs and healthy male volunteers in vitro.
The results were as follows; (1) In dogs, though STA₂ did not have a potency of inducing platelet aggregation, STA₂ revealed enhancing effect on platelet aggregation induced by epinephrine, and this effect of STA₂ was inhibited by α blockers, especially α₂ blockers, (2) In healthy volunteers, STA₂ itself induced platelet aggregation, and this effect of STA₂ was also inhibited by α blockers, but dominancy of α₁ or α₂ blocker was unclear.
It was concluded from these data that STA₂ was a potent platelet aggregatory agent in human and this activity was inhibited by α blockers.

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Endothelial cell growth factor from platelets, designated as Vascular Endothelial cell Proliferation Factor (VEPF), was purified to an apparent homogeneity from fresh human platelets using eight steps, e. g.: QAE-Sephadex chromatography, ammonium sulfate precipitation, DEAE-Sepharose chromatography, chromatofocusing separation, Mono P chromatography and HPLC gel chromatography using Superose 12 and TSK 4000 columns. The half maximal stimulation was obtained at a concentration of less than 10ng/ml (less than 200pM). The highly purified material stimulated the growth of porcine aortic endothelial cells but was inactive against human foreskin fibroblasts.

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We studied the effects of cytochalasin B and phalloidin on platelet secretion and protein phosphorylation induced by ionophore A23187.
(1) Preincubation of platelets with cytochalasin B or phalloidin suppressed the ATP release induced by A23187 in a dose dependent manner.
(2) In the absence of these reagents, exposure of ³²P-labelled platelets to A23187 resulted in increased phosphorylation of the 20, 000-dalton protein (P20) and the 47, 000-dalton protein (P47).
(3) Treatment of ³²P-labelled platelets with cytochalasin B or phalloidin suppressed the increased phosphorylation of both P20 and P47 caused by A23187.
In conclusion, it was considered that intact network configuration of actin filaments would support full phosphorylation of P20 and P47 during platelet activation.

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In this study, the effectiveness of various drugs affecting the arachidonate cascade such as OP-4148, a derivative of PGI₂, aspirin (ASA), an inhibitor of cyclooxygenase, OKY-046, an inhibitor of thromboxane (TX) synthetase and CV-3988, an antagonist against Platelet Activating Factor (PAF) were determined in animal experiments using rabbits along with a study on the relationship between changes in platelet and the renal function after burn injuries. A third degree burn of 35% of the total body surface area was prepared by immersing the back of rabbits in 80°C hot water, and the effectiveness of OP-41483, ASA, OKY-046 and CV-3988 was studied. In no therapy group, all rabbits died after 8 hours or more. Renal function tests in this group showed a decrease in creatinine clearance and an increase in F_ENa and CH₂O. Such changes were improved slightly with infusion therapy, With OP-41483, OKY-046 and ASA, distinct improvement was observed (in order of extent of improvement), but with CV-3988 improvement was rather small. Resultantly, those drugs may also effective to prevent the organ failure after burn injury clinically.

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Factor VIII related antigen (VIIIR: Ag) has been shown to be localized in the endothelial cells of capillaries in oral mucosa, using the peroxidaselabeled antibody immunoelectron-microscopy. We have clearly demonstrated that endothelial cells secrete VIIIR: Ag not only to the lumen of capillaries but also to the subendothelium by epinephrine or DDAVP. The findings suggest that the secretion of VIIIR: Ag from endothelial cells to the subendothelial areas by preoperative administration of epinephrine or DDAVP may share in the hemostatic effect of these drugs by promoting the adhesion of platelets to basement membrane following endothelial injury.

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We developed an assay for activated factor XI-α₁antitrypsin complex (F. XIa-α₁AT) in plasma. Purified F. XI was activated with β-XIIa and treated with α₁AT. Thus we prepared the standard F. XIa-α₁AT. Assay for F. XIa-α₁AT was carried out essentially according to the method of Harpel. F. XIa-α₁AT in sample plasma (10-fold dilution, 200μl) was adsorbed on anti-F. XI monoclonal antibody beads and developed with peroxidase-labeled anti-α₁AT Fab'.
To eliminate the effect of plasma on this assay, standard F. XIa-α₁AT was diluted with 10% F. XI-deficient plasma which did not contain the complex. The absorbance of the standard complex containing F. XI (0.016-0.12μg/assay, i. e. 20-150%) was a little lower than that of the complex alone, because F. XI compete the binding of F. XIa-α₁AT to the beads. Either F. XI or α₁AT alone did not increase the color development. Therefore we diluted the standard F. XIa-α₁AT with 10% F. XI-deficient plasma containing F. XI (0.08μg/assay, i. e. 100%). Recovery of added F. XIa-α₁AT was satisfactory (over 90%).
25 normal individuals had low level of F. XIa-α₁AT (below 0.18ng/assay), whereas 30 patients with disseminated intravascular coagulation (DIC) demonstrated high level of this complex (0.18-4.2ng/assay). These data indicated that this assay was useful to the diagnosis of DIC.

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Disseminated intravascular Coagulation (DIC) syndrome is not only a coagulopathy but also a microrheological disorder. In this study 20 cases of DIC were evaluated whose basal diseases were acute leukemia (n=7) (group A), malignant lymphoma (n=3), solid tumors (n=7), and others (n=3) (group B, n=13). 1) influence of acute circulatory and/or respiratory failures, 2) complicated organ failures, 3) manifestations of bleeding, 4) abnormalities of coagulation studies, and 5) effect of treatment were observed.
In group A only one case of respiratory disturbance was observed as a cause of DIC. but in group B 8 cases of circulatory or respiratory disturbance were thought to be cause of DIC. 8 cases of organ failure were detected in total 20 cases (6 cases of respiratory failure, one case of CNS disturbance and one case of multiple organ failures). In group A bleeding was severe due to thrombocytopenia, but in group B their manifestations were not so severe (mainly oozing). Heparin or combination of heparin and gabexate mesilate was administered to the patients. Combination therapy appeared to be more effective for amelioration of DIC, especially for the clinical manifestations.
In conclusion, abnormalities of blood flow in microcirculation caused by respiratory or circulatory disturbance influence on the onset and course of DIC in group B, that is, in pre-DIC or chronic DIC state.

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Polyclonal and monoclonal anti-tissue plasminogen activator (t-PA) antibodies were characterized by using enzyme immunoassay (EIA) in which β-D-galactosidase was coupled to anti-t-PA antibody (Fab'). 2:2 B10 and 1:3 G5 antibodies, specific for both one-chain and two-chain t-PA, strongly bound with one-chain t-PA purified from cultured melanoma cell lines, but 1:3 C5 antibody bound weakly with such t-PA. When polyclonal t-PA antibody was used as the first reaction antibody immobilized on silicone pieces, anti-t-PA polyclonal antibody mainly reacted with 2:2 B10 or 1:3 C5 antigenic determinant. When t-PA levels in the plasma were determined, the presence of EDTA enhanced the sensitivity of t-PA determination by the present EIA technic. 2:2 B10 monoclonal antibody detected a part of t-PA molecules in the plasma that polyclonal antibody detected. T-PA was mainly detected in the endothelial cells, but not in the muscular layer of inferior mesenteric artery when immunochemical technic was used where polyclonal t-PA antibody was applied.

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The presence of two types of plasminogen activators (PA's) was detected in the human early milk.
1) Two types of PA's were separated by lysine-Sepharose chromatography. The bound fraction (HMW-HMPA) was further purified by hydroxylapatite chromatography and Sephacryl S-200 gel filtration. The unbound fraction (LMW-HMPA) was purified by benzamidine-Sepharose chromatography and Sephacryl S-200 gel filtration.
2) By the gel filtration method, the molecular weight of HMW-HMPA was determined to be 69kDa and that of LMW-HMPA 49kDa.
3) By the chromatofocusing method, four kinds of isoelectric points were detected from HMW-HMPA between pH 5.3 and 5.9, and two from LMW-HMPA at about pH 8.1 and 8.4.
4) By the parabolic rate assay, it was observed that the activity of HMW-HMPA was remarkably enhanced in the presence of fibrin monomer, but the LMW-HMPA only a little.
5) The examination of the quenching effects of anti P-HPA (porcine heart plasminogen activator) IgG and anti H-UK (human urokinase) IgG on the activity of human milk PA's revealed that the nature of HMW-HMPA was tissue type and that of LMW-HMPA UK type.

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Glu-plasminogen (Glu-Plg) and Lys-Plg were specifically separated with high-performance affinity chromatography, but it was almost impossible to analyze them in human plasma in a short time because of the existence of a large amount of other components. This problem was solved by introducing specific detection system. Plasminogens were specifically detected by plasmin activity which was produced by the on-line activation using urokinase. A small amount of human plasma (40μl) without pretreatment could be directly applied to the column packed with an adsorbent coupled with p-aminobenzamidine (Asahipak GS520-AHA-ABA; Asahi Chemical Industry Co.). The existence of Lys-Plg was observed in most plasmas. When urokinase was added to the normal plasma, Lys-Plg disappeared faster than Glu-Plg and active plasmin was not detected at all. These findings directly indicate that Lys-Plg is more susceptible to activation by urokinase than Glu-Plg and that the active plasmin in plasma is immediately trapped by inhibitors. Differences between normal and hereditary abnormal plasma was also clearly observed. This new system should be widely applicable in many fields, e. g., in clinical diagnosis and a therapeutic monitor for thrombosis.

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Orthotopic liver transplantation was done using the heparinized hydrophilic catheter-bypass method in mongrel dogs. Four dogs survived for 6-8 days without the use of immunosuppressive drugs and 7 dogs survived for 4-67 days with the use of Ciclosporin. Postoperative changes in blood coagulation and fibrinolysis were measured in both the control group and the Ciclosporin group. Antithrombin III, α₂-plasmin inhibitor, fibrinogen and plasminogen decreased after liver transplantation and reached the lowest point in 6-24 hours, then gradually increased. The activities of prothrombin time, Thrombotest and Hepaplastintest also decreased, but the activities of long survival group increased after 2-3 days. It was found that the measurement of blood coagulation and fibrinolysis was good parameters for hepatic function or rejection. Hepatotoxicity of Ciclosporin was suggested by the decrease of Hepaplastintest in the Ciclosporin group.

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We have developed highly sensitive enzyme immunoassay or radio-immunoassay of Clq, C3 and FDP in body fluids. For examining relationship between excreted uriniary rare proteins and renal disorders, Clq, C3, IgG, fibrin degradation products (FDP), FDP-E and urokinase (UK) have been measured in daily urine samples from 10 normal volunteers, 23 patients with primary nephritis and 7 patients with secondary nephritis.
Both Clq and C3 excretion were high in the presence of immune complexes in glomeruli. In IgA nephropathy, pericapillary immune deposition rather than mesangial deposition may result in increased in Clq and C3 excretion. In membranous nephropathy, results indicating the activation of alternative pathway of complements were observed. In lupus nephritis, urinary complements excretions were increased at the active phase of the disease. These excretions of complements in urine were not related to the total protein leakage.
Urinary FDP and FDP-E were detected in high levels in cases of impaired renal function. Mesangial proliferation was observed in these patients. In such cases, FDP and FDP-E excretions were not related to the total protein leakage, which suggests that FDP and FDP-E excretions were observed in increase in prolif erative change.
In conclusion, the measurements of urinary rare protein components may be useful for screening of renal disorders.

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