Search

This paper describes the cutting process of a 0.5 mm thickness polycarbonate (PC) worksheet stacked on another 0.5 mm thickness PC underlay by an adhesive tape layer. As the first investigation, the cutting line force of the two-line blade (90/30deg.) on the laminated PC sheet was measured with a load cell and the sheared profile of the worksheet was observed by a CCD camera in order to reveal the effect of the time delay of the ultrasonic blade excitation on the deformation flow of the worksheet. The profile parameters such as the inclination angle, elevation angle and necked height of sheared section were observed with respect to the blade excitation patterns by varying the delayed time. Secondarily, in order to investigate the effect of the excitation-duty rate on the profile parameters of the sheared zone, two kinds of ultrasonic-excitement patterns were applied to the laminated PC sheet by varying the duty rate. Through the two experiments, it was revealed that both, the positioning control in the case of the time delay and the power density control in the case of the duty ratio were superior for varying the profile parameters of sheared zone.

Surface discharge on dielectric materials seriously restricts the performance of many electrical and electronic systems. Based on Pockels electro-optic effect, the surface discharge on polymeric insulating materials has been investigated under HVAC in Atmospheric Air. Different polymer materials such as polyimide (PI), polyethylene terephthalate (PET) and polyvinylidene fluoride (PVDF) films are employed. One single cycle of sinusoidal HVAC with peak value of 5 kV and duration time of 50ms is applied on a needle-plate electrode configuration and the applied voltage starts from the positive or negative half-cycle, respectively. During the development process of surface discharge on different materials, the dynamic observation of surface charge distribution is recorded in a cycle by a high speed camera. The experimental results show that there is significant voltage polarity effect observed, and the radius of negative charge distribution is smaller than that of positive charges. The positive charge density on PET and PI is lower than that on PVDF, which are closely related to the enhancement of electric field by permittivity and thickness of polymer films. Moreover, PI, PET and PVDF samples behave different surface charge distribution. Positive streamer channels on PI and PET are gradually growing into a snake-like pattern with voltage applying, and positive streamers on PVDF develop instantaneously and exhibit as a regular spoke-like pattern. The influence factors on different discharge patterns are discussed in the paper.

We present imaging data and photometry for the COSMOS survey in 15 photometric bands between 0.3um and 2.4um. These include data taken on the Subaru 8.3m telescope, the KPNO and CTIO 4m telescopes, and the CFHT 3.6m telescope. Special techniques are used to ensure that the relative photometric calibration is better than 1% across the field of view. The absolute photometric accuracy from standard star measurements is found to be 6%. The absolute calibration is corrected using galaxy spectra, providing colors accurate to 2% or better. Stellar and galaxy colors and counts agree well with the expected values. Finally, as the first step in the scientific analysis of these data we construct panchromatic number counts which confirm that both the geometry of the universe and the galaxy population are evolving., Comment: 19 pages, 13 figures, 14 tables, Accepted to ApJS for COSMOS speciall issue

We present results from a narrow-band optical survey of a contiguous area of 1.95 deg^2, covered by the Cosmic Evolution Survey (COSMOS). Both optical narrow-band (lambda_c = 8150 AA and Delta_lambda = 120 AA) and broad-band (B, V, g', r', i', and z') imaging observations were performed with the Subaru prime-focus camera, Suprime-Cam on the Subaru Telescope. We provide the largest contiguous narrow-band survey, targetting Ly alpha emitters (LAEs) at z~5.7. We find a total of 119 LAE candidates at z~5.7. Over the wide-area covered by this survey, we find no strong evidence for large scale clustering of LAEs. We estimate a star formation rate (SFR) density of ~7*10^-4 M_sun yr^-1 Mpc^-3 for LAEs at z~5.7, and compare it with previous measurements., 26 pages, 19 figures. to appear in the ApJ Supplement COSMOS Special Issue

At present, the photometric data for clusters at z ≲ 0.2 mainly come from photographic photometry. The lack of CCD data for such clusters is simply due to the fact that no CCD camera had been available until recently that covers the wide extension of clusters within a reasonable amount of observing time. We have developed a large mosaic CCD camera and conducted multicolor imaging observations of z ≲ 0.2 clusters using the 40-inch Swope telescope at Las Campanas Observatory.

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)

This paper describes the development of a small robot using a novel mechanism. The robot performs bipedal locomotion using a pendulum unlike existing bipedal robots. The number of servomotors is also considered, and a mechanism that minimizes the number of axes required to raise and walk a leg, and a novel walk theory, are proposed. This walking is named ldquoteko teko walking.rdquo CAE is used to perform simulation and design verification by a computer, and a low cost, efficient CAE-based development method is proposed.

IL-1 receptor antagonist (IL-1Ra)-deficient mice spontaneously develop several inflammatory diseases, resembling rheumatoid arthritis, aortitis, and psoriasis in humans. As adoptive T cell transplantation could induce arthritis and aortitis in recipient mice, it was suggested that an autoimmune process is involved in the development of diseases. In contrast, as dermatitis developed in scid/scid-IL-IRa-deficient mice and could not be induced by T cell transfer, a T cell-independent mechanism was suggested. The expression of proinflammatory cytokines was augmented at the inflammatory sites. The development of arthritis and aortitis was significantly suppressed by the deficiency of TNFalpha or IL-17. The development of dermatitis was also inhibited by the deficiency of TNFalpha. These observations suggest that TNFalpha and IL-17 play a crucial role in the development of autoimmunity downstream of IL-1 signaling, and excess IL-1 signaling-induced TNFalpha also induces skin inflammation in a T cell-independent manner.

We have developed a 64-channnel superconducting quantum interference (SQUID) system that can analyze the vector components of a cardiac magnetic field, and that is more compact and requires fewer SQUID sensors and measuring circuits than existing systems. To adjust the position of the dewar, containing sensor array and liquid He relative to the chest, the bed is capable of three-dimensional movement. The magneto-cardiogram (MCG) data from the 64 channels are stored and analyzed using a personal computer. Each measurement site contains first-order gradiometers to detect the Bz component on the torso. The typical noise of the system in a magnetically shielded room (MSR) is less than 20 fT//spl radic/Hz. Tangential components at each measuring site can be calculated from the first-order gradient of Bz in the x- and y-directions. Two types of magnetic-field patterns of the Bz component and the Bxy tangential components are obtained at the same time. The position and distribution of a current source in the heart can be visualized through a two-dimensional projection using the analyzed tangential magnetic field.

The hemorrhagic disorders associated with circulating anticoagulant are rare but well known as the disease derived from inhibitors(alloantibody or autoantibody), which arise against specific coagulation factors. We encountered a patient with hemorrhage at the lower limbs originating from an unknown coagulation pathway inhibitor. Activated partial thromboplastin time(APTT) prolonged and cross mixing test indicated a coagulation pathway inhibitor. Levels of coagulation factors VIII, IX, XI, and XII were extremely low. Predonine therapy was effective to stop the hemorrhage. To clarify the inhibitor, we treated the patient's plasma with protein A Sepharose, and the factor VIII activity was recovered to a normal level after passing through the column. Gel filtration of the patient's plasma and protein A Sepharose adsorbed fraction of the patient's plasma with Superdex 200 revealed that the inhibitor activity against blood coagulation eluted at the position of IgG, which was detected by the prolongation of APTT of normal pooled plasma. These data indicated that the inhibitor was IgG and its inhibitory spectrum was non-specific and broad. The results of laboratory tests and the clinical features in our patient were similar to those in a previous study by Dahlback(Blood 62, 218-225, 1983). The hemorrhagic abnormality of that patient was derived from the autoantibody to phospholipid, which inhibits the activation of prothrombin. An investigation into the effect of anti-phospholipid antibody in our patient is currently being performed in our laboratory.

We studied a 29-year-old Japanese male patient with factor XI deficiency; we also studied his parents and one sibling. Factor XI coagulation activity and antigen levels were extremely low (less than 1% of normal level) in both the patient and his brother, and they were half the normal levels in both parents. Sequence analysis of all 15 exons and the exon-intron boundaries of the factor XI gene amplified by polymerase chain reaction revealed a nonsense mutation in exon 8 (Gln263--Stop). Although the parents are first cousins, the mutation was unexpectedly heterozygous in all the family members except the father, who showed the homozygous wild type, indicating that this mutation alone was not sufficient to account for the factor XI deficiency. To explore the genetic abnormality in the father, we analyzed allele-specific expression of the platelet factor XI gene using reverse transcription-polymerase chain reaction and subsequent restriction enzyme digestion. As a result, gene expression from only one allele was severely impaired in the father. This result implies an additional mutation in some regulatory element of the factor XI gene from paternal inheritance. We concluded that the factor XI deficiency of the patient was caused by compound heterozygous genetic abnormalities.

Kininogens are multifunctional plasma glycoproteins. There are two forms of human kininogen: low molecular weight kininogen (LK) and high molecular weight kininogen (HK). Both are derived from the same gene by alternative splicing. Some patients with kininogen deficiency have been reported to be deficient only in HK while others are deficient in both HK and LK (total kininogen deficiency). We analyzed three Japanese patients with total kininogen deficiency by the Csp45I digestion study of exon 5 as previously reported in Williams trait and found that two had the same point mutation of C to T at base 22 of exon 5, resulting in a transition of CGA (Arg) codon to TGA (Stop) codon. This is the first report of molecular characterization of total kininogen deficiency in the Japanese population.

It is widely accepted that atherosclerosis and thrombus formation are closely associated. Recent progress in the development of molecular markers of blood coagulation and platelet enabled us to estimate even the slight activation of blood coagulation and platelet activation systems. We investigated a contract phase-activate coagulation and factor -inhibitor complex (factor XIa-alpha 1 antitrypsin) and expression of CD62P on platelet surface, a specific antigen on platelet, in patients with coronary arterial diseases. We compared these activation markers after stratifying patients according to the number of significant coronary artery stenoses. Patients with three-vessel disease had significantly increased levels of factor XIa-alpha 1 antitrypsin, fibrinogen, and CD62P expression compared to those in other subgroups. Although it is unknown whether these abnormalities are the cause or the result of the vascular lesion, these findings may be important to understand the pathophysiology underlying atherosclerosis or propagation of the atherosclerotic process itself.

A 22-year-old woman was admitted to our hospital with fever, generalized lymphadenopathy, and pancytopenia in February 1995. She was diagnosed as having systemic lupus erythematosus (SLE) based on positivity for anti-nuclear antibody, and polyarthritis among other findings. A diagnosis of disseminated intravascular coagulation (DIC) was made based on the increase of FDP and other data (DIC score: 7). We also detected an anti-fibrinogen antibody. Lymph node biopsy revealed subacute necrotizing inflammation and there were on signs of the hemophagocytic phenomenon in bone marrow. The DIC score improved and the anti-fibrinogen antibody disappeared in association with the response of SLE to prednisolone therapy. The onset of SLE associated with DIC has never been reported before, as far as we could determine. The mechanism of DIC associated with SLE may be related to endothelial damage caused by immune complexes.

Creatine kinase (CK)-MB subunit has been recognized as a useful marker for acute myocardial infarction (AMI). However, we recently experienced one case of osteopetrosis with moderately high CK-MB and an abnormal (more than 100%) CK-MB/total (T)-CK ratio without evidence of AMI in a medical examination. We have already experienced 17 cases with an abnormal CK-MB/T-CK ratios in addition to the present case. Those cases were patients with malignant tumor with metastasis (n = 13), leukemia (2), liver cirrhosis (1), and cerebral death (1), and thereby the band of macro-CK was found in the electrophoresis. However, we detected neither the band of macro-CK nor the abnormal levels of tumor markers such as CEA, alpha-fetoprotein, CA-19-9 in the present case. Instead of the macro CK, the high level of CK-BB was detected in electrophoresis. In the medical examination, especially in screening tests, the CK-MB was generally assayed with use of the immunoinhibition method in automated analyzers. The method principle was based on the absence of CK-BB in the patient serum. Since the patient had the past history of pathological fracture in his boyhood, this patient was diagnosed as osteopetrosis. These results suggest that we must consider the possibility of osteopetrosis when an abnormal CK-MB and CK-MB/T-CK ratio without evidence of serious diseases were found. This is simply because of the assay method of immunoinhibition for CK-MB activity.

One of the responses of activated platelets to certain stimuli is the shedding of microparticles. Many studies have attempted to characterize the role of microparticles under various clinical situations or experimental conditions. Pathological levels of fluid shear stress may occur in diseased small arteries and arterioles partially obstructed by atherosclerosis or vasospasm and such shear stress may induce the activation and aggregation of circulating platelets. We investigated whether high shear stress could cause both platelet aggregation and shedding of microparticles from the platelet plasma membrane. A cone-plate viscometer was used to apply shear stress and microparticle formation was measured by flow cytometry. It was found that microparticle formation increased as the duration of shear stress increased. Both microparticles and remnant platelets showed procoagulant activity on their surfaces. Investigation of the mechanisms involved in shear-dependent microparticle generation showed that binding of von Willebrand factor to platelet glycoprotein Ib, influx of extracellular calcium, and activation of platelet calpain were required to generate microparticles under high shear stress conditions. Activation of protein kinase C promoted shear-dependent microparticle formation. These findings suggest that local generation of microparticles in atherosclerotic arteries, the site at which pathological levels of shear stress could occur, contributes to arterial thrombosis by providing and expanding a catalytic surface for the coagulation cascade.

We investigated whether platelet activation could be quantitatively detected in patients with coronary artery disease using flow cytometric analysis of the expression of CD62P and CD63, which are activation-specific antigens on the platelet surface. Platelet samples were obtained from 16 healthy control subjects and 65 patients, of whom 25 had angiographically normal coronary arteries and 40 had at least one major coronary artery stenosis (> or = 50% narrowing). In both patient groups, CD62P expression was significantly higher than in the control group, but the difference between the two patient groups was not significant. In contrast, CD63 expression did not differ among the three groups. We also compared expression of these antigens after stratifying the patients according to the number of significant coronary artery stenoses. Patients with three-vessel disease had significantly increased CD62P and CD63 expression compared with the other subgroups. Our findings indicate that platelet activation occurs in patients with severe coronary artery stenosis.

The assay for activated coagulation factors has recently been considered to be a useful tool for detecting hypercoagulable state. Measurement of activated factor XI and alpha 1 antitrypsin complex developed by us is the only one measure for detecting contact phase hypercoagulation. The ACTIVATED FACTOR XII kit (Shield Diagnostics) has recently been commercially available, and the reference range was reported in the instruction manual. However, because the recovery rate of purified factor XIIa (XIIa) in normal pooled plasma by this assay kit was strikingly decreased, the effects of inhibitors in plasma, such as low molecular weight serine proteinase inhibitor (diisopropyl fluoro-phosphate, DFP), the specific inhibitor for XIIa (corn trypsin inhibitor, CTI), the main inhibitor for XIIa (C1 inhibitor, C1I) were examined. These three inhibitors and XIIa were incubated for 18 hours, then the XIIa levels of these complexes and the recovery rate in the normal pooled plasma were assayed by this assay kit. The recovery ratios in the normal pooled plasma were; DFP-XIIa 82%, CTI-XIIa 63%, and C1I-XIIa 0%, XIIa without inhibitor 7%. These results suggest that this XIIa kit does not reflect the levels of complexed form of XIIa and inhibitors in plasma samples.

We investigated the role of alpha IIb beta 3 in microparticle generation by normal and thrombasthenic platelets stimulated with collagen plus thrombin. Microparticle generation by normal platelets was scarcely inhibited by monoclonal antibodies for glycoprotein Ib and glycoprotein IX. Although one monoclonal anti-alpha IIb beta 3 antibody (NNKY1-32) partly inhibited microparticle generation, 3 other monoclonal anti-alpha IIb beta 3 antibodies had little effect. However, the combination of 4 monoclonal anti-alpha IIb beta 3 antibodies or treatment with a polyclonal anti-alpha IIb beta 3 antibody significantly inhibited microparticle generation (p0.05). Microparticle generation by thrombasthenic platelets also occurred after stimulation with collagen plus thrombin, although at a significantly lower level compared with normal platelets. Monoclonal antibodies for resting alpha IIb beta 3, P-selectin, activated alpha IIb beta 3 and beta 2-glycoprotein I bound to microparticles from healthy platelets. In contrast, only a monoclonal antibody for beta2-glycoprotein I bound to thrombasthenic microparticles. These results suggest that microparticle generation by collagen plus thrombin occurs via two different mechanisms which are dependent and independent of alpha IIb beta 3, respectively. The alpha IIb beta 3-dependent mechanism appears to require activation of alpha IIb beta 3.

Various hemostatic abnormalities have been reported and excess activation of coagulation factors, such as prothrombin, factor VII, factor IX, and factor XI, have been detected in thrombotic diseases states by various assay systems. We recently developed the enzyme-linked differential immunoassay for activated factor XI-alpha 1 antitrypsin complex (FXIa-alpha 1 AT) and applied it with other assays for activated factors such as thrombin-antithrombin III complex (TAT) to detect the hypercoagulable state in clinical samples. In patients with DIC, the FXIa-alpha 1 AT level in plasma increased before onset of DIC. In patients with non-insulin-dependent diabetes mellitus, FXIa-alpha 1AT and TAT levels were increased in the patient plasma. FXIa-alpha 1AT was related to the severity of urinary albumin excretion, whereas TAT was not. Plasma FXIa-alpha 1AT levels were significantly increased in patients with angiographically proven coronary artery disease, and showed a positive correlation with TAT, fibrinogen, and Lp(a). Evaluation of activated coagulation factor provides useful information on the diagnosis of thrombotic disease.

We investigated the association of amyloid beta-protein precursor (APP) and platelet derived microparticles in 20 normal controls and 91 patients with various diseases causing a thrombotic tendency. Compared with the controls, the mean percentage of APP-positive microparticles was significantly greater in the patients with cerebral infarction (39.1 +/- 17.7%, p0.001), diabetes (31.1 +/- 12.6%, p0.001), and uremia (30.1 +/- 14.7%, p0.01), but not in those with hypertension (8.2 +/- 6.3%, p = NS). Sixteen patients with cerebral infarction, 20 with diabetes, and 11 with uremia had microparticles with very high APP levels. In normal controls, 7.2 +/- 3.7% of the microparticles were positive for P-selectin, while the percentage in cerebral infarction, diabetes, uremia, and hypertension was respectively 43.5 +/- 15.1%, 40.0 +/- 12.8%, 31.8 +/- 12.2%, and 11.6 +/- 7.3%. There was a significant correlation between P-selectin and APP positivity of microparticles. Our results suggest that microparticle APP may have a regulatory influence on coagulation abnormalities.

A simple and reliable analytical procedure was developed for determination of platelet-activating factor (PAF) in human plasma using radioimmunoassay (RIA). The assay system consisted of lipid extraction with 2-propanol, lipid separation by Amprep octadecyl minicolumn chromatography and thin-layer chromatography and RIA (charcoal method), and was suitable for quantitation of 30-1000 pg of PAF. The sensitivity of RIA for PAF was notably higher than that for sn-2-short-chain PAF-like phosphatidylcholines. This assay system was then applied for measurement of PAF in human plasma. The normal level of plasma PAF was 54 +/- 40 pg/ml (n = 35), whereas plasma PAF levels in patients with liver cirrhosis (LC) and disseminated intravascular coagulation (DIC) were significantly elevated to 238 +/- 314 pg/ml (n = 14) and 591 +/- 328 pg/ml (n = 14), respectively. The values obtained using this assay system were comparable to those obtained by gas chromatography/mass spectrometry analysis and bioassay. These results indicate that our new assay system is useful for determining changes in the level of plasma PAF associated with diseases such as LC and DIC.

Osseointegrated implant bridges (OIB) have shown excellent progressing in the majority of cases. Since 1983, OIBs has been used to treat about 110 cases in Tokyo Dental College Hospital with more than 500 fixtures. The survival rate of OIB, especially the functioning survival rate of these fixtures, is described, herein. The survival rate of the superstructure of OIB has been almost 100% in both maxilla and mandible. However, the functioning survival rate of OIB fixtures was 88% in maxillary cases and 96% in mandibular cases at less than 1 year. Very few fixtures had to be removed after more than 1 year in either maxilla or the mandible. It appears that the functioning survival rate of OIB fixture is generally decided during the period just between connecting these components and 1 year. A few fixtures may need to be removed later.

We used flow cytometry to investigate surface membrane protein expression by platelets and platelet-derived microparticles from normal individuals and a patient with Glanzmann's thrombasthenia. Microparticles were detected by both forward scatter and side scatter using FACScan. The binding of coagulation factors on microparticles was investigated by using monoclonal anti-Factor IX (IXa) and anti-Factor X (Xa) antibodies. Furthermore, the procoagulant activity of microparticles was measured with a chromogenic substrate (S-2222) using a microtiter enzyme-linked immunosorbent assay. Both types of platelets showed similar release of microparticles. Microparticles released from platelets after activation with the calcium ionophore A23187 did not bind factors IXa and Xa, but when purified factors Va and Xa were added to the incubation buffer, factor Xa binding increased markedly in both normal and thrombasthenic platelets. Both normal and thrombasthenic platelets showed a similar time-dependent release of microparticles when activated with A23187. However, the binding of an antibody to granule membrane protein-140 also increased time-dependently in normal microparticles, but was little increased in thrombasthenic microparticles. These findings suggest that glycoprotein IIb/IIIa does not participate in the expression of prothrombinase activity on the surface of activated platelets and microparticles, whereas this glycoprotein appears to have an important role in the movement of granule membrane protein-140 from platelets to microparticles.

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.

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)

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)

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)