Reference values of hemostasis related factors of healthy Japanese adults. II. Protein C.
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Adult↗
Biomedical subjects
M Kazama
Publications and source records attributed to M Kazama.
Soluble thrombomodulin antigen in conditioned medium is increased by damage of endothelial cells.
Thrombomodulin (TM) exists not only in endothelial cells but also in circulating plasma as soluble heterogeneous fragments. A release mechanism of soluble TM antigen from endothelial cells was investigated. Cultured human umbilical vein endothelial cells released about 0.6% of total cellular TM antigen into conditioned medium during 24 h. The release of TM antigen was not influenced by addition of various concentrations (0.01-5.0 microM) of monensin, which inhibits intracellular transport of secretory proteins, though the secretion of plasminogen activator inhibitor-1 from the cells was inhibited. The release of TM antigen was not increased when total cellular TM level increased 1.3- or 1.4-fold relative to control cells after stimulation with 0.1-1.0 U/ml thrombin or 3 mM dibutyryl cAMP, respectively. Exposure of endothelial cells for 6 h to mixture of 1 microM N-formyl-methionyl-leucyl-phenylalanine (FMLP) and 100 ng/ml lipopolysaccharide (LPS) decreased cellular TM level by 30% relative to control cells without increase in the TM release. The FMLP and LPS-stimulated leukocyte treatment of the cells increased the release of TM antigens into the medium in a time-dependent manner and the increased release of TM antigen paralleled the extent of cell damage as measured by 51Cr release. Hydrogen peroxide treatment of the cells increased the release of TM antigens into the medium in a time- and concentration-dependent manner. The increased release of TM antigen by hydrogen peroxide also paralleled the extent of cell damage.(ABSTRACT TRUNCATED AT 250 WORDS)
Blotting, Western↗
Immobilized urokinase column as part of a specific detection system for plasminogen species separated by high-performance affinity chromatography.
Immobilized urokinase was used as part of a post-column reactor for the specific detection of human plasminogen species which were fractionated using a high-performance affinity column. After on-line activation of each peak, plasmin activity was measured by mixing the eluate with a specific fluorogenic substrate and the product was detected by a fluorescence monitor. This detection system gave linear calibration graphs for both purified plasminogens (0.1-50 micrograms) and plasminogens contained in plasma (25-100 microliters). Relative standard deviations for the determination of plasminogens in plasma were 6.1-6.6% (n = 12), showing good reproducibility. The detection limit was as low as 0.1 micrograms of plasminogen. Immobilized urokinase was very stable and no appreciable decrease in activity was found after 100 cycles of operation. In combination with an immobilized benzamidine column, this system made it possible to separate and detect Glu-plasminogen and Lys-plasminogen contained in human plasma samples as small as 100 microliters without any pretreatment.
Chromatography, Affinity↗
[Analysis of hemostatic abnormality in various disease using molecular-I. Liver disease].
We examined the hemostatic abnormality of liver disease using hemostatic molecular markers, i.e. TAT, FPA and SFMC for coagulation, B beta 15-42, FDP, D dimer and PIC for fibrinolysis, t-PA and TM for vessel wall. The molecular markers for coagulation were generally increased in cases of liver disease, which was most sensitively reflected by FPA. On the other hand, it was postulated that SFMC was a marker reflecting the complication of DIC in these cases. Hyperfibrinolysis of liver disease was sensitively reflected by the increase of B beta 15-42, and an occasional increase of SFMC or FDP was thought to indicate the complication of DIC in these cases. A high correlation was found between t-PA and TM. It was postulated that the increase of the both markers in liver disease was due to deteriorated clearance by liver dysfunction, although TM is regarded as a marker reflecting endothelial injury. It was expected that visualization of hemostatic disorder of liver disease was made practical with the use of radar chart of these molecular markers.
Antifibrinolytic Agents↗
Increased thrombomodulin values in plasma of diabetic men with microangiopathy.
To study the significance of plasma thrombomodulin (TM) values in diabetes mellitus, we determined plasma TM in 34 patients with non-insulin-dependent diabetes mellitus (NIDDM) men, mean age 54 (SE 2) years. Plasma TM was determined by an enzyme immunoassay with anti-TM monoclonal antibodies. The plasma TM values were significantly greater in NIDDM patients with nephropathy than in patients without nephropathy (P less than 0.001). Also, a significant positive correlation was noted between the concentration of plasma TM and serum creatinine (r = 0.55, P less than 0.001). The plasma TM values of the patients with retinopathy were significantly greater than the values of those without it (P less than 0.002). Furthermore, we noted a significant positive correlation (r = 0.78, P less than 0.001) between plasma TM and the severity of diabetic retinopathy as graded by Scott's classification. These results suggest a close relationship between TM and diabetic microangiopathy.
Adult↗
[Soluble thrombomodulin: a specific parameter of endothelial injury].
Thrombomodulin (TM) is a constituent glycoprotein of endothelial cell membrane, and soluble TM is present also in plasma and urine. It was revealed by experiments using cultured HUVEC in vitro that TM is released from endothelial cell membrane not with monensin, thrombin, fibroblast growth factor, interleukin-1 or endotoxin, but with H2O2 or endotoxin-treated granulocytes. And the release was suppressed by the coexistence of gabexate mesilate or superoxide dismutase. It was suggested that soluble TM was released from endothelial cell membrane by its injury and digested to multiple molecular forms by endogenous and granulocytic protease(s). TM level in circulation is increased in cases of SLE, MCLS, diabetic angiopathy. It was increased in cases of overt DIC and decreased to the normal level when the patient was recovered from DIC. TM level in circulation was also increased in cases of decompensated liver cirrhosis and markedly in cases of renal insufficiency. It was concluded that plasma TM is a parameter reflecting endothelial injury due to inflammation or metabolic disorders of vascular system. But the interpretation of increased plasma TM was difficult when renal insufficiency was complicated.
Cells, Cultured↗
Evaluation of international normalized ratios by a controlled field survey with 4 different thromboplastin reagents.
A nationwide survey has been performed in Japan involving 75 laboratories to assess the relative reliability of different methods of reporting prothrombin time results in anticoagulant control. The interchangeability of results using prothrombin time, prothrombin activity percentage, prothrombin ratio and international normalized ratios (INR) were compared with four different thromboplastin reagents and a range of coagulometers. A secondary batch of reference thromboplastin of human brain origin (BCT/454) was used to calibrate the local thromboplastins and for comparison of methods of reporting. The study revealed the closest agreement of the results between BCT and the other reagents, and the regression lines of these reagents were almost identical, when the results were reported as INR. Box-Whisker plot analysis showed that the distribution of the results was large with the more deficient plasmas with all methods of reporting. It was found by this analysis that the interchangeability of the results was greatest when the results were expressed by INR, because the mean values obtained of each plasma using different thromboplastin reagents gave the lowest CV and the frequency of the far-out data was least, compared with the other methods of expression. On the other hand, the type of coagulometer had almost as much effect as the thromboplastin reagent on the prothrombin time, even if INR was used. Interchangeability of INR would be further improved by providing ISI values for each reagent/instrument combination.
Humans↗
Reference values of hemostasis related factors of healthy Japanese adults. I: Circadian fluctuation.
The circadian fluctuation of hemostasis related parameters was examined on 16 healthy Japanese adults (male 9, female 7). Twenty one parameters were measured in this study, i.e. fibrinogen, the activity of F.II, F.V., F.VII, F.VIII, F.IX, F.X., F.XI, F.XII, antithrombin III, plasminogen, alpha 2-antiplasmin, as well as the antigen level of F.IX, von Willebrand Factor, protein C, tissue-type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), beta-thromboglobulin, platelet factor 4, fibrinopeptide A, plasmin-alpha 2-antiplasmin complex and FDP. Fluctuation was not significant in almost all of the parameters except F.VIII, F.IX, beta-thromboglobulin, platelet factor 4, tPA and PAI-1. Although the fluctuations of F.VIII, F.IX, beta-thromboglobulin and platelet factor 4 were statistically significant, they remained within the normal ranges. On the other hand, tPA and free PAI-1 showed significant circadian fluctuation, of which levels were highest at 9:00. It was postulated that the significant circadian fluctuation of fibrinolytic activity will be regulated by the balance between tPA and PAI-1 in plasma.
Adult↗
Heparin-like glycosaminoglycan is a receptor for antithrombin III-dependent but not for thrombin-dependent prostacyclin production in human endothelial cells.
Antithrombin III (ATIII) induced a marked increase in prostacyclin (PGI2) release from cultured human umbilical vein endothelial cells (HUVEC) after incubation for more than 2 hr and the induction continued for 8 hr, while thrombin induced the increase within 10 min. ATIII-dependent production of PGI2 was abolished by addition of heparin, but pretreatment of HUVEC with polyclonal antibody against thrombomodulin could not prevent the PGI2 productions by ATIII and thrombin. ATIII-dependent PGI2 production was significantly inhibited by pretreatment of HUVEC with beta-D-xylosides or heparitinase, though neither pretreatment affected thrombin-induced PGI2 production. After treatment of HUVEC with 1 micrograms/ml cycloheximide. ATIII-dependent PGI2 production was completely abolished. These results indicate that the mechanism of the induction of PGI2 production by ATIII involves heparin-like glycosaminoglycans on HUVEC and the stimulation of synthesis of a protein related to PGI2 production. The ATIII-induced PGI2 production is very different from that induced by thrombin.
Antibodies, Monoclonal↗
Cyclic AMP increases thrombomodulin expression on membrane surface of cultured human umbilical vein endothelial cells.
Dibutyryl-cyclic AMP (Bt2cAMP; final concentration 1-5 mM) or beraprost sodium (synthetic prostacyclin, 100 nM) enhanced the expression of thrombomodulin (TM; an anticoagulant factor of endothelial cells) on the membrane surface of cultured human umbilical vein endothelial cells up to 1.4 times over the control within 9 hrs after the treatment, while the expression fell below the control level at 12 hrs and thereafter. 8-Bromo-cAMP (final concentration 1-5 mM) or 3-isobutyl-1-methylxanthine (IBMX; an inhibitor of phosphodiesterase; final concentration 10-1000 microM) enhanced the expression of TM on the cell surface at 12 hrs after the treatment. The enhancement of TM expression caused by Bt2cAMP was inhibited by incubation with phorbol 12-myristate 13-acetate. These results suggest that cAMP stimulates expression of TM in the endothelial cells.
1-Methyl-3-isobutylxanthine↗
Establishment of enzyme immunoassay of human thrombomodulin in plasma and urine using monoclonal antibodies.
Thrombomodulin, TM, is an endothelial cell membrane protein acting as a cofactor for the activation of plasma protein C. Soluble TM is present in plasma and urine of normal subjects. Enzyme immunoassay, EIA, for human TM was developed using mouse monoclonal antibodies against human placental TM in this paper. We obtained four types of the monoclonal antibodies against human placental TM. EIA sandwich method using three types of the monoclonal antibodies enabled us to measure almost all of 6 and 7 TM subspecies in plasma and urine, respectively, except 1 subspecies, 31 kDa TM. There was no interference from other components of plasma and urine in the assay conditions. Titration curves of purified TM in buffer or in normal plasma were linear within the range from 0.08 to 10 ng/ml. The coefficient of variation at 0.08 ng/ml TM was 4.7%. TM titer with buffer, assayed by this method, was reduced by the addition of thrombin at the final concentration of 20 U/ml, but the titer with plasma was not reduced even at 100 U/ml. These concentrations of thrombin are far larger than those which would be formed in circulation. TM levels in plasma and urine of normal subjects collected in the morning were 35.2 +/- 8.32 ng/ml (n = 346) and 111 +/- 31.6 ng/ml (n = 33), respectively. TM level in plasma did not differ from the level in serum. Circadian fluctuation of plasma TM was not significant in 10 normal adults, although a tendency of increase in TM excretion to urine was found rather in the day time than the other times.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Thiolprotease inhibitor, EST, can inhibit thrombin-induced platelet activation.
Participation of thiolprotease in platelet activation was investigated. When platelets were treated with EST (L-trans-epoxysuccinyl-leucylamide (3-methyl)butane-ethyl ester, a membrane-permeable thiolprotease inhibitor) for 30 min, thrombin-induced aggregation and secretion were inhibited, and remained so even when the platelets were washed and resuspended in EST-free medium after the pretreatment. The inhibitory action of EST on thrombin-induced platelet aggregation and secretion was both dose-dependent and incubation-time-dependent. The inhibitory action of EST on platelet aggregation and secretion was shown not only in response to thrombin but also to platelet activating factor (PAF). Pretreatment of platelets with 1 mM EST for 30 min inhibited the 65% of calpain (thiolprotease) activity in platelets. The phosphorylation of 40 kDa and 20 kDa proteins of platelets caused by stimulation with thrombin was blocked by the pretreatment with 1 mM EST. Phosphatidylinositol hydrolysis and inositol-1-phosphate production, which appear after stimulation of platelets with thrombin, were also inhibited by the pretreatment with 1 mM EST. The results suggest that EST was incorporated to inside of platelets, and inhibited activation of platelet through inhibition of thiolprotease.
Adult↗
Plasma thrombomodulin in liver diseases.
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Humans↗
Clinical effects and basic studies of thrombolytic therapy on cerebral thrombosis.
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Animals↗
Characterization of calpain I-binding proteins in human erythrocyte plasma membrane.
The calpain-binding components on the plasma membrane were characterized using calpain I. 125I-labeled calpain was bound to inside-out membrane vesicles from human erythrocyte in a Ca2(+)-dependent manner, but not to right-side-out membrane vesicles. The maximum binding was observed at more than 5 microM Ca2+. The binding amount of calpain to the inside-out membrane vesicles was decreased when the vesicles were pretreated with 100 micrograms/ml of trypsin, chymotrypsin, elastase, or pronase P for 30 min at 37 degrees C, although the binding to the vesicles pretreated with 200 micrograms/ml of phospholipase A2 or C was not affected. Calpain-binding proteins in the membrane were analyzed by using a modified immunoblotting for calpain. Immunostained bands of 240, 220, 89, 72, 52, and 36 kDa were detected as the calpain-binding proteins in the native membrane. All of these bands had disappeared in trypsin-treated membrane. The disappearance of bands was dose-dependent with respect to trypsin and paralleled the reduction of binding amount of calpain to the trypsinized membrane. In calpain-treated membrane, the 240 and 36 kDa bands were retained in the blotting, though the other bands disappeared dose-dependently with respect to calpain. These results suggested that the significant component in the inner surface of plasma membrane for binding of calpain was proteinaceous and the calpain-binding proteins could be classified into two species, i.e. substrates of calpain (220, 89, 72, and 52 kDa protein) and non-substrates (240 and 36 kDa protein).
Calpain↗
Alpha-tocopherol-induced hexagonal HII phase formation in egg yolk phosphatidylcholine membranes.
The effect of alpha-tocopherol and its acetate on the membrane structure of egg yolk phosphatidylcholine (egg PC) dispersions was investigated using phosphate-31 nuclear magnetic resonance (31P-NMR) and small-angle X-ray diffraction. The incorporation of alpha-tocopherol into egg PC dispersions induced a change in the 31P-NMR spectrum from a multilamellar bilayer line shape to a hexagonal HII one. The phase transition by alpha-tocopherol was also confirmed by small-angle X-ray diffraction analysis. The amount of hexagonal HII phase increased with increase in concentration of alpha-tocopherol. Egg PC dispersions containing a molar ratio of 0.8 of alpha-tocopherol gave a 31P-NMR spectrum of an approximately hexagonal HII type at 37 degrees C. The amount of hexagonal HII phase increased with increasing temperature, indicating that the alpha-tocopherol-induced phase transition is thermotropic and that the transition temperature of egg PC membranes from the lamellar to the hexagonal HII phase is lowered by alpha-tocopherol. The incorporation of alpha-tocopherol acetate did not induce any phase transition. This fact indicates that the hydroxyl group of alpha-tocopherol may play an important role in the hexagonal HII phase formation of egg PC dispersions.
Egg Yolk↗
Elevation of plasma thrombomodulin level in diabetic patients with early diabetic nephropathy.
Thrombomodulin (TM) is a membrane protein in the vascular endothelium, and it plays an important role as a cofactor in the thrombin-catalyzed activation of protein C. It has also been found in human plasma; however, its clinical significance is not known. In this study, fasting plasma TM concentrations in 67 diabetic patients with different degrees of albuminuria (39 men aged 57 +/- 8 yr, 28 women aged 57 +/- 11 yr; means +/- SD) and 34 age- and sex-matched healthy subjects were investigated by use of a one-step sandwich enzyme immunoassay, a new method developed by H.I. and others. As a screening, the patients were divided into three groups according to the first morning urinary concentrations of albumin: group 1, less than 30 micrograms/ml (normoalbuminuria); group 2, 30-140 micrograms/ml (microalbuminuria); group 3, greater than 140 micrograms/ml (clinical nephropathy). There was no significant difference in plasma TM level between the control group (17.7 +/- 3.7 ng/ml, n = 34) and group 1 (16.9 +/- 3.4 ng/ml, n = 30); however, plasma TM concentrations in group 2 (22.8 +/- 3.4 ng/ml, n = 22) and group 3 (29.6 +/- 6.1 ng/ml, n = 15) increased significantly compared with those in the control group and group 1, respectively. As a further investigation, three timed overnight urine collections were made. The patients were allocated to three groups according to their rates of albumin excretion: group I, less than 20 micrograms/min (normoalbuminuria); group II, 20-200 micrograms/min (microalbuminuria); group III greater than 200 micrograms/min (clinical nephropathy).(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Plasma thrombomodulin levels as an indicator of vascular injury caused by cyclosporine nephrotoxicity.
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Adult↗