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Biomedical subjects

Y Komiyama

Publications and source records attributed to Y Komiyama.

At least 19 recordsLinked to original sources
PubMed1992

The role of phospholipids and the factor VII Gla-domain in the interaction of factor VII with tissue factor.

Whether or not the factor VII Gla-domain is involved in the high-affinity interaction of factor VII and tissue factor via calcium-dependent interactions with surrounding phospholipids is unknown. To investigate this, we have purified the factor VII Gla-peptide (FVII-GP) from digested recombinant human factor VIIa and assessed its effect on factor VII:tissue factor interactions. FVII-GP inhibited the activation of factor X by factor VIIa in the presence of either soluble or cell surface tissue factor half-maximally at 0.5 microM and 2.7 microM, respectively. However, FVII-GP failed to inhibit the specific binding of factor VIIa to cell-surface tissue factor, and did not inhibit the ability of tissue factor to stimulate the amidolytic activity of factor VIIa. Unrelipidated tissue factor apoprotein stimulated the amidolytic activity of factor VIIa to the same extent as relipidated tissue factor apoprotein. These findings suggest that the factor VII Gla-domain does not directly interact with tissue factor, but rather is important for calcium binding and concomitant expression of other factor VII epitopes necessary for tissue factor recognition and binding. To test this hypothesis, we have prepared a monoclonal antibody against a putative factor VII epitope that participates in the interaction of factor VII with cell-surface tissue factor (peptide 195-206) and assessed its ability to bind to factor VII in the presence and absence of calcium. Binding of this monoclonal antibody (PW-4) to intact factor VIIa was calcium-dependent and could be inhibited in a dose-dependent manner by peptide 195-206.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1992

Purification of factor XIa inhibitor from human platelets.

An inhibitor of activated factor XI (FXIa) in human platelets was recently identified as an amyloid beta-protein precursor (APP). We purified an FXIa inhibitor (XIaI) from the supernatant of activated human platelets, and assessed its inhibitor activity toward FXIa amidolytic activity. Approximately 90 micrograms of XIaI that cross-reacted with anti-APP antibody was obtained from two hundred units of platelet suspension by employing a six-step column chromatography procedure. The molecular weight of the purified XIaI was 94,000. The Ki value of XIaI to factor XIa was 526 +/- 120 pM, and the inhibition was enhanced by the addition of ZnCl2. The amino-terminal sequence of XIaI was L-E-V-P-T-D-G-N-A-, which is identical to that for the leucine (N18) to alanine (N26) sequence of APP751 and the amino-terminal sequence of protease nexin-2.

Amino Acid Sequence↗
PubMed1992

Polyarteritis nodosa with atrophy of the left hepatic lobe.

A 73-year-old Japanese man with a history of partial gastrectomy due to gastric cancer 4 years previously was admitted because of intermittent fever. The patient developed abdominal pain, erythema, and myalgia in addition to the fever during the final clinical course, and died of acute heart failure. Autopsy disclosed atrophy of the left lobe of the liver and acute myocardial infarction. Neither metastasis nor recurrence of the cancer was observed. Small- and medium-sized arteries of the visceral organs showed various stages of necrotizing vasculitis with narrowing of the lumina. The vasculitis was most prominent in the left lobe of the liver and in the heart. Narrowing of the portal vein due to portal tract inflammation in addition to vasculitis of the hepatic arteries may have induced ischemia and infarction, which had resulted in atrophy of the left hepatic lobe.

Aged↗
PubMed1991

Human plasma and recombinant factor VII. Characterization of O-glycosylations at serine residues 52 and 60 and effects of site-directed mutagenesis of serine 52 to alanine.

Factor VII is a multidomain, vitamin K-dependent plasma glycoprotein that participates in the extrinsic pathway of blood coagulation. Earlier studies demonstrated a novel disaccharide (Xyl-Glc) or trisaccharide (Xyl2-Glc) O-glycosidically linked to serine 52 in human plasma factor VII (Nishimura, H., Kawabata, S., Kisiel, W., Hase, S., Ikenaka, T., Shimonishi, Y., and Iwanaga, S. (1989) J. Biol. Chem. 264, 20320-20325). In the present study, human plasma and recombinant factor VII were isolated and subjected to enzymatic fragmentation. Peptides comprising residues 48-62 of the first epidermal growth factor-like domain of each factor VII preparation were isolated for comparative analysis. Using a combined strategy of amino acid sequencing, carbohydrate and amino acid composition analysis, and mass spectrometry, three different glycan structures consisting of either glucose, glucose-xylose, or glucose-(xylose)2 were detected O-glycosidically linked to serine 52 in plasma and recombinant factor VII. Approximately equal amounts of the three glycan structures were observed in plasma factor VII, whereas in recombinant factor VII the glucose and the glucose-(xylose)2 structures predominated. In addition to the O-linked glycan structures observed at serine 52, a single fucose was found to be covalently linked at serine 60 in both human plasma and recombinant factor VII. Carbohydrate and mass spectrometry analyses indicated that the fucosylation of serine 60 was virtually quantitative. Metabolic labeling studies using [14C]fucose confirmed the presence of O-linked fucose at serine 60. In order to assess whether the carbohydrate moiety at serine 52 contributes to the biological activity of factor VII, we have constructed a site-specific mutant of recombinant factor VII in which serine 52 has been replaced with an alanine residue. Mutant factor VIIa exhibited approximately 60% of the coagulant activity of wild-type factor VIIa in a clotting assay. The amidolytic activity of mutant factor VIIa was indistinguishable from that observed for recombinant wild-type factor VIIa. In addition, the ability of mutant factor VIIa in complex with either purified relipidated tissue factor apoprotein or tissue factor on the surface of a human bladder carcinoma cell line (J82) to activate either factor X or factor IX was virtually identical to that observed for wild-type factor VIIa. These results indicate that the carbohydrate moiety O-glycosidically linked to serine 52 does not appear to be involved either in the interaction of factor VIIa with tissue factor, or the expression of its proteolytic activity toward factor X or factor IX following complex formation with tissue factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Alanine↗
PubMed1991

Inhibition of recombinant human blood coagulation factor VIIa amidolytic and proteolytic activity by zinc ions.

Although it is well established that calcium is an essential cofactor in blood coagulation, recent experimental evidence suggests that zinc may also play an important role in hemostasis. In the present study, we have examined the effect of zinc ions on the amidolytic and proteolytic activity of recombinant factor VIIa in the presence of physiological levels of calcium ions. The amidolytic activity of factor VIIa was inhibited half-maximally by 20 microM zinc. The amidolytic activity of a derivative of factor VIIa lacking the gamma-carboxyglutamic acid domain was also inhibited half-maximally by 20 microM zinc, suggesting that the mechanism of zinc inhibition of factor VIIa amidolytic activity did not involve its gamma-carboxyglutamic acid residues. The amidolytic activity of a complex of recombinant tissue factor and factor VIIa was inhibited half-maximally by 70 microM zinc. In contrast to the results obtained with factor VIIa, the amidolytic activities of other human vitamin K-dependent coagulation proteases including factor Xa, thrombin and activated protein C were not appreciably affected by 50-100 microM zinc. The proteolytic activation of factor X by a complex of factor VIIa and relipidated tissue factor apoprotein was inhibited half-maximally by 40 microM zinc, whereas activation of factor IX in this system was inhibited half-maximally by 70 microM zinc ions. Considerably higher levels of zinc (approximately 100 microM) were required to inhibit half-maximally the rate of factor X activation by a complex of factor VIIa and functional tissue factor on the surface of either a human bladder carcinoma cell line, J82, or stimulated human umbilical vein endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1991

[Factor XIa-alpha 1 antitrypsin complex].

We developed a new rapid assay for the factor XIa-alpha 1 antitrypsin complex (FXIa-alpha 1AT) in plasma using an anti FXI monoclonal antibody (KMXI-1). In 20-fold diluted plasma samples, this assay was not affected by co-existing FXI or non specific color development of the plasma. Normal level of FXIa-alpha 1AT (11 +/- 4.1 ng/ml plasma) increased with the aging of healthy adults. The FXIa-alpha 1AT levels of patients with disseminated intravascular coagulation (DIC) rose along with the progression of the disease, and the appearance of high levels and the peak of FXIa-alpha 1AT developed faster than FDP-E or alpha 2plasmin inhibitor-plasmin complex (alpha 2PI-PmC) in most patients. These results indicate that, in addition of FDP and alpha 2PI-PmC, FXIa-alpha 1AT is a useful molecular marker for DIC.

Adult↗
PubMed1990

Proteolytic activation of human factors IX and X by recombinant human factor VIIa: effects of calcium, phospholipids, and tissue factor.

Previous studies indicated that factor VIIa, in complex with tissue factor, readily activates either factor X or factor IX in the presence of calcium ions. In order to assess the relative physiological importance of the activation of factor IX versus the activation of factor X by recombinant factor VIIa, we have obtained steady-state kinetic parameters for the factor VIIa catalyzed activation of factor IX and factor X under a variety of cofactor conditions that include calcium alone, calcium and phospholipids, calcium, phospholipids, and tissue factor apoprotein, and calcium and cell-surface tissue factor. Calcium alone stimulated the activation of factors IX and X by factor VIIa maximally at 1 and 2.5 mM, respectively. In the presence of 25 microM phospholipids, maximal rates of factor IX and factor X activation were achieved at 2.5-5 mM calcium. With calcium alone, or with phospholipid and calcium, the initial rates of factor IX activation by factor VIIa were significantly higher than that observed for factor X. Kinetic studies revealed that the Km for the factor VIIa catalyzed activation of factor IX was essentially constant in the presence of 5 mM calcium and 1-500 microM phospholipid, whereas the Km for factor X activation varied with phospholipid concentration, reaching a minimum at 7-20 microM phospholipid. At all concentrations of added phospholipid, the kcat/Km ratio for the activation of factor IX by factor VIIa appeared to be considerably greater than that observed for the activation of factor X.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1990

Increased accumulation of nonenzymatically glycated fibrinogen in the renal cortex in rats.

We have determined that the nonenzymatic glycation of fibrinogen altered its biological functions in vitro. Thrombin clottability of rat fibrinogen incubated with glucose decreased with increasing incubation time, but was not affected by the glucose concentration. Fibrin prepared from glycated fibrinogen showed a significant resistance in susceptibility to plasmin degradation. We also examined the in vivo distribution of glycated fibrinogen in renal cortex. Iodine labeled rat glycated or unglycated fibrinogen was injected into streptozocin-induced diabetic and control rats. No appreciable difference in the plasma disappearance rate in control rats was observed (half-lives in hours for glycated, 25.6 +/- 0.37; unglycated, 26.1 +/- 0.74). The radioactivity of fibrinogen retained to the renal cortex was calculated 24-hours after injection. In both control and diabetic rats, the retention rate of glycated fibrinogen in renal cortex was significantly higher than that of the unglycated. These results suggest that glycated fibrinogen may occur in a more resistant form to plasmin digestion with fibrin deposition as confirmed in in vitro studies. Therefore, we suggest that glycated fibrinogen may partly contribute to the development of diabetic microangiopathic lesions such as glomerulosclerosis.

Animals↗
PubMed1990

Determination of the minimal concentrations of contact activation factors in deficient substrate plasmas required to assess accurately factor XII, factor XI, factor IX, and high molecular weight kininogen.

Using combined deficient plasmas prepared by passage of a deficient plasma over an anti-factor XI-monoclonal antibody column, we have determined the threshold concentrations of each coagulation factor of contact phase in factor-deficient substrate plasmas required to determine accurately the functional activities of factor XII, factor XI, factor IX and high molecular weight kininogen (HMWK). In order to reliably quantitate factor XI and factor IX activity levels, at least 20% factor XII and 20% factor XI, respectively, were required in the deficient substrate plasmas. In the assessment of factor XII activity, approximately 40% factor XI was required in the factor XII-deficient substrate plasma. On the other hand, only 11-12% factor XI or HMWK was required in the deficient substrate plasmas in the assessment of these two clotting factors. Our data emphasize that deficiencies of other clotting factors may reduce the apparent activity of the clotting factor in question if their concentration is rate-limiting in the clotting assay.

Blood Coagulation Disorders↗
PubMed1990

Difference in changes of membrane fluidity of polymorphonuclear leukocytes stimulated with phorbol myristate acetate and formyl-methionyl-leucyl-phenylalanine: role of excited oxygen species.

Polymorphonuclear leukocytes (PMN) were stimulated with phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl phenylalanine (FMLP) to clarify the role of excited oxygen species in inducing changes of membrane fluidity. Membrane fluidity was assessed by the excimer-forming lipid technique using pyrenedecanoic acid and flow cytometry. Membrane fluidity of PMN decreased following stimulation with PMA, and the extent of decrease was both time- and dose-dependent. FMLP at 10(-5) M induced a decrease, while FMLP at 10(-7) M induced a rapid increase. On stimulation with 10(-7) M FMLP as well as in a resting condition, the change of membrane fluidity of PMN from patients with chronic granulomatous disease (CGD) was similar to that of normal PMN. However, on stimulation with PMA or 10(-5) M FMLP, CGD PMN did not show a significant decrease. In addition, normal PMN incubated with catalase inhibited the decrease. These findings suggest that the generation of excited oxygen species, particularly of H2O2, is important in inducing a decrease of PMN membrane fluidity.

Adult↗
PubMed1990

Flow cytometric study of injuries in cultured endothelial cells by neutrophils of the inherited cataract rats.

We examined the mechanism of endothelial injuries in the inherited cataract rats (ICR), which have a number of age-associated spontaneous injuries in the aortic endothelium. Cell cycle traverse rate of endothelial cells of ICR was shorter than that of Wistar rats. The rate was estimated from bromodeoxyuridine (BrdU) incorporation into cell nuclei measured periodically after BrdU pulse labeling. Next we established the method for measurement of cultured endothelial cell injury by neutrophils with flow cytometry by assessing the regeneration of injured endothelial cells. By the use of the gate analysis method, contaminated neutrophils were excluded from the analysis. Endothelial cell injury by neutrophils of Wistar rats was detectable at 1 x 10(5) neutrophils (1 neutrophil to 1 endothelial cell) when stimulated with 10 ng/ml phorbol myristate acetate (PMA). Extent of injury increased with an increasing number of neutrophils and the concentration of a stimulator, PMA. We detected endothelial cell injury by ICR neutrophils not only when they were stimulated but also in a resting condition, and ICR neutrophils yielded more injury than Wistar rat neutrophils. Number of adhered neutrophils to endothelial cells and effects of plasma or lymphocytes were the same between two strain rats. Scavengers of hydrogen peroxide and singlet oxygen inhibited the ICR neutrophil-induced endothelial cell injury. These findings indicate that an increase of generation of excited oxygen species from neutrophils, particularly of singlet oxygen, may cause injury of endothelial cells in this specific strain of rats.

Animals↗
PubMed1990

Bovine plasma protein C inhibitor with structural and functional homologous properties to human plasma protein C inhibitor.

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Decrease of polymorphonuclear leukocyte membrane fluidity in uremic patients on hemodialysis.

We measured membrane fluidity of polymorphonuclear leukocytes (PMN) from 14 uremic patients on hemodialysis by the excimer-forming lipid technique with pyrenedecanoic acid using flow cytometry. Membrane fluidity of PMN was significantly lower in the uremic patients during the predialysis period. During hemodialysis, progressive normalization in membrane fluidity was observed. Cross-incubation studies indicated that this observation is ascribed to factors in the patients' serum and that they can be removed by hemodialysis. When sera of these patients were fractionated by Sephadex G-25 column chromatography, the specific fraction responsible for a decreased fluidity was found in the low-molecular-weight fraction. Uremic patients have an increased risk of infection, which may be partly due to altered membrane fluidity of their PMN.

Adult↗
PubMed1989

Accumulation of 125I-factor XI in atheroma of rabbit with hereditary hyperlipidemia (WHHL-rabbit).

We have studied the turnover and accumulation of rabbit factor XI (F.XI) in atherosclerotic lesion in Watanabe-hereditable hyperlipidemic rabbit (WHHL rabbit) to reveal the participation of blood coagulation in atherosclerotic lesion. Rabbit F.XI was iodinated and administered intravenously to WHHL rabbits and Japanese white rabbits. The turnover of 125I-rabbit F.XI was significantly faster in WHHL rabbits (T1/2 = 2.84 +/- 0.44 days) than in normal rabbits (T1/2 = 4.44 +/- 0.42 days). The thoracic aorta of WHHL rabbit was strongly labelled with 125I-rabbit F.XI, in sections obtained after 5 days by en-face autoradiography, whereas no radioactivity was detected in normal aorta. By an immunohistochemical study of WHHL rabbit aorta, we confirmed that many F.XI- and fibrin-related compounds existed in the atheroma, whereas albumin did not in these area. These results suggest that the activation of F.XI proceeds on the atherosclerotic lesions of WHHL rabbits.

Animals↗
PubMed1989

New rapid assay for factor XIa-alpha 1 antitrypsin complex--application to DIC.

We developed a new rapid assay for the factor XIa-alpha 1 antitrypsin in complex (F.XIa-alpha 1 AT) in plasma with the use of a newly produced anti-F.XI monoclonal antibody (KMXI-1). This assay was completed within about 5 hours, and the minimum assay range extended sensitivity about 5-fold over the former assay (Thromb. Res. 44, 489-501, 1986). In 20-fold diluted plasma samples, this assay was not affected by co-existing F.XI or nonspecific color development of the plasma. Normal levels of F.XIa-alpha 1 AT (11 +/- 4.1 ng/ml plasma [n = 96]) increased with the aging of healthy adults. The F.XIa-alpha 1 AT levels of patients with disseminated intravascular coagulation (DIC) rose along with the progression of the disease, and the appearance of high levels and the peak of F.XIa-alpha 1 AT developed faster than FDP-E or alpha 2 plasmin inhibitor-plasmin-complex in most patients. These results indicate that, in addition to FDP, F.XIa-alpha 1 AT is a useful molecular marker for DIC.

Adult↗
PubMed1989

Change of membrane fluidity of rat neutrophils accompanying Escherichia coli inoculation.

Membrane fluidity of rat neutrophils was studied following Escherichia coli inoculation, and characteristic changes were observed. Membrane fluidity was assessed by the excimer-forming lipid technique using pyrenedecanoic acid and flow cytometry and expressed as the fluorescence intensity ratios of excimer and monomer pyrenedecanoic acid (IE/IM ratio). High IE/IM ratios indicated high membrane fluidity. The IE/IM ratio of rat neutrophils (0.50 +/- 0.048) increased after E. coli inoculation, reaching a maximum of almost 1.00 after 10-20 min and then returning to its starting value. Intravenous injection of heat-killed E. coli or E. coli-conditioned culture supernatants into rats induced a rapid increase of IE/IM ratios, which returned to initial levels after 20 min. The effect on membrane fluidity of in vitro neutrophil incubation with E. coli, heat-killed E. coli, or E. coli-conditioned culture supernatants was similar to that observed in vivo. Addition of 5 mM ethylenediaminetetraacetic acid (EDTA) did not affect neutrophil membrane fluidity. Addition of either 5 micrograms/ml cytochalasin B or 10(-5) M colchicine did not directly affect neutrophil membrane fluidity but did block the change observed following incubation with bacteria.

Actin Cytoskeleton↗
PubMed1989

Spontaneous injuries in the aortic endothelium of the inherited cataract rats and their prevention by tocopherol. A study by scanning electron microscopy.

The aortic endothelium of inherited cataract rats (ICR), which spontaneously develop cataracts and neutrophilia, was examined by scanning electron microscopy using silver nitrate staining and pressure fixation. In ICR aged 4 weeks, the luminal surface of the aorta was similar to that in Wistar rats from which they had been derived. However, 8 weeks after birth, endothelial cells were upraised and partially detached from an underlying tissue. At 16 weeks, morphological changes exhibited by such detaching cells were more evident than at 8 weeks and fibrin was seen to be adhering to the surface of these cells; no platelet involvement was noted, however. Oral administration of DL-alpha-tocopheryl acetate for 2 weeks resulted in a reduction in the extent of endothelial injury and the luminal surface of the aorta became similar to that seen in 4- or 8-week-old animals. We found that the number of age-associated spontaneous injuries occurring in the aortic endothelium of ICR could be reduced by tocopherol administration.

Aging↗