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1.
Anal Chem ; 92(3): 2388-2392, 2020 02 04.
Article in English | MEDLINE | ID: mdl-31899617

ABSTRACT

A novel multilayer modulator chip offering a robust miniaturized interface for multidimensional liquid chromatography has been developed. The thermoplastic microfluidic device comprises five tailor-made functional layers, and the chip is compatible with commercially available switching-valve technology. The modulator chip allows for robust ultrahigh-pressure operation up to 65 MPa. Peak-dispersion characteristics of system peaks were assessed directly at the valve outlet by monitoring fluorescein injection profiles with laser-induced fluorescence detection. Integration of a microporous monolithic mixing entity in the microchannels significantly narrows the resulting peak profile. Proof-of-concept of the applicability of the microfluidic modulator chip is demonstrated in a heart-cut multidimensional strong-cation-exchange-reversed-phase liquid chromatography proteomics analysis workflow coupled to nanoelectrospray mass spectrometry for the target analysis of Glu-1-Fibrinopeptide B spiked in a protein digest mixture of bovine serum albumin.


Subject(s)
Fibrinopeptide B/analysis , Glutens/analysis , Lab-On-A-Chip Devices , Nanotechnology , Proteomics , Animals , Cations/chemistry , Cattle , Chromatography, Liquid , Chromatography, Reverse-Phase , Mass Spectrometry , Serum Albumin, Bovine/chemistry
2.
Chin J Nat Med ; 16(4): 241-251, 2018 Apr.
Article in English | MEDLINE | ID: mdl-29703324

ABSTRACT

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections. Qingfei oral liquid (QFOL), a traditional Chinese medicine, is widely used in clinical treatment for RSV-induced pneumonia. The present study was designed to reveal the potential targets and mechanism of action for QFOL by exploring its influence on the host cellular network following RSV infection. We investigated the serum proteomic changes and potential biomarkers in an RSV-infected mouse pneumonia model treated with QFOL. Eighteen BALB/c mice were randomly divided into three groups: RSV pneumonia model group (M), QFOL-treated group (Q) and the control group (C). Serum proteomes were analyzed and compared using a label-free quantitative LC-MS/MS approach. A total of 172 protein groups, 1009 proteins, and 1073 unique peptides were successfully identified. 51 differentially expressed proteins (DEPs) were identified (15 DEPs when M/C and 43 DEPs when Q/M; 7 DEPs in common). Classification and interaction network showed that these proteins participated in various biological processes including immune response, blood coagulation, complement activation, and so forth. Particularly, fibrinopeptide B (FpB) and heparin cofactor II (HCII) were evaluated as important nodes in the interaction network, which was closely involved in coagulation and inflammation. Further, the FpB level was increased in Group M but decreased in Group Q, while the HCII level exhibited the opposite trend. These findings not only indicated FpB and HCII as potential biomarkers and targets of QFOL in the treatment of RSV pneumonia, but also suggested a regulatory role of QFOL in the RSV-induced disturbance of coagulation and inflammation-coagulation interactions.


Subject(s)
Biomarkers/blood , Drugs, Chinese Herbal/pharmacology , Fibrinopeptide B/analysis , Heparin Cofactor II/analysis , Proteome/drug effects , Proteomics , Respiratory Syncytial Virus Infections/blood , Respiratory Syncytial Viruses/drug effects , Animals , Chromatography, Liquid , Disease Models, Animal , Drugs, Chinese Herbal/therapeutic use , Fibrinopeptide B/genetics , Gene Expression Regulation/drug effects , Heparin Cofactor II/genetics , Lung/pathology , Mice, Inbred BALB C , Respiratory Syncytial Virus Infections/drug therapy , Tandem Mass Spectrometry
3.
PLoS One ; 12(12): e0189972, 2017.
Article in English | MEDLINE | ID: mdl-29253022

ABSTRACT

Biologically uncommon d-aspartate (d-Asp) residues have been shown to accumulate in proteins associated with age-related human disorders, such as cataract and Alzheimer disease. Such d-Asp-containing proteins are unlikely to be broken down completely because metabolic enzymes recognize only proteins or peptides composed exclusively of l-amino acids. Therefore, undigested d-Asp-containing peptides may exist in blood and, if detectable, may be a useful biomarker for associated diseases. In this study, we investigated d-amino acid-containing peptides in adult human serum by a qualitative d-amino acid analysis based on a diastereomer method and LC-MS/MS method. As a result, two d-Asp-containing peptides were detected in serum, both derived from the fibrinogen ß-chain, a glycoprotein that helps in the formation of blood clots. One of the peptides was fibrinopeptide B, which prevents fibrinogen from forming polymers of fibrin, and the other was same peptide with C-terminal Arginine missing. To our knowledge, this is the first report of the presence of d-amino acid-containing peptides in serum and the approach described will provide a new direction on the serum proteome and fragmentome.


Subject(s)
Amino Acids/blood , Peptides/blood , Adult , Alzheimer Disease/blood , Amino Acids/chemistry , Aspartic Acid/metabolism , Biomarkers/blood , Cataract/blood , Chromatography, Liquid , Crystallins/metabolism , Fibrinogen/metabolism , Fibrinopeptide B/analysis , Humans , Middle Aged , Proteomics , Tandem Mass Spectrometry , Young Adult
4.
Anal Biochem ; 417(2): 174-81, 2011 Oct 15.
Article in English | MEDLINE | ID: mdl-21756868

ABSTRACT

Although most time-of-flight (TOF) mass spectrometers come equipped with vacuum matrix-assisted laser desorption/ionization (MALDI) sources, the atmospheric pressure MALDI (API-MALDI) source is an attractive option because of its ability to be coupled to a wide range of analyzers. This article describes the use of an API-MALDI source coupled to a TOF mass spectrometer for evaluation of the effects of medium- and long-term storage on peptidomic profiles of cryopreserved serum samples from healthy women. Peptides were purified using superparamagnetic beads either from fresh sera or after serum storage at -80°C for 18 months or at -20°C for 8 years. Data were preprocessed using newly developed bioinformatic tools and then were subjected to statistical analysis and class prediction. The analyses showed a dramatic effect of storage on the abundance of several peptides such as fibrinopeptides A and B, complement fractions, bradykinin, and clusterin, indicated by other authors as disease biomarkers. Most of these results were confirmed by shadow clustering analysis, able to classify each sample in the correct group. In addition to demonstrating the suitability of the API-MALDI technique for peptidome profiling studies, our data are of relevance for retrospective studies that involve frozen sera stored for many years in biobanks.


Subject(s)
Atmospheric Pressure , Cryopreservation , Neoplasms/blood , Peptides/blood , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Bradykinin/blood , Clusterin/blood , Complement System Proteins/analysis , Female , Fibrinopeptide A/analysis , Fibrinopeptide B/analysis , Humans , Specimen Handling
5.
Kidney Int ; 79(5): 546-554, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21150872

ABSTRACT

Biomarkers that evaluate the response to erythropoietic-stimulating agents largely measure inflammation and iron availability. While these are important factors in modifying an individual's response to these agents, they do not address all aspects of a poor response. To clarify this, we isolated peptides in the serum of good and poor responders to erythropoietin in order to identify biomarkers of stimulating agent response. Ninety-one candidate biomarker targets were identified and characterized using mass spectrometry, of which tandem mass spectroscopy provided partial amino-acid sequence information of 17 different peptides for 16 peptide masses whose abundance significantly differed between poor and good responders. The analysis concluded that three peptides associated with a poor response were derived from oncostatin M receptor ß (OSMRß). The 13 serum peptides associated with a good response were derived from fibrinogen α and ß, coagulation factor XIII, complement C3, and cysteine/histidine rich 1(CYHR1). Poor response was most strongly associated with the OSMRß fragment with the largest molecular weight, while a good response was most strongly associated with CYHR1. Immunoblots found the abundance of intact OSMRß and CYHR1 significantly differed between good and poor responders. Thus, two measurable biomarkers of the response to erythropoietic-stimulating agents are present in the serum of treated patients.


Subject(s)
Erythropoietin/therapeutic use , Oncostatin M Receptor beta Subunit/blood , Proteins/analysis , Renal Dialysis , Biomarkers/blood , Cytokines/blood , Female , Fibrinopeptide B/analysis , Humans , Male , Peptide Fragments/blood , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
6.
Anal Chem ; 80(19): 7467-72, 2008 Oct 01.
Article in English | MEDLINE | ID: mdl-18729472

ABSTRACT

A nanostructured diamond-like carbon (DLC) coated digital versatile disk (DVD) target is presented as a matrix-free sample support for application in laser desorption/ionization mass spectrometry (LDI-MS). A large number of vacancies, defects, relative sp(2) carbon content, and nanogrooves of DLC films support the LDI phenomenon. The observed absorptivity of DLC is in the range of 305-330 nm (nitrogen laser, 337 nm). The universal applicability is demonstrated through different analytes like amino acids, carbohydrates, lipids, peptides, and other metabolites. Carbohydrates and amino acids are analyzed as sodium and potassium adducts. Peptides are detectable in their protonated forms, which avoid the extra need of additives for ionization. A bovine serum albumin (BSA) digest is analyzed to demonstrate the performance for peptide mixtures, coupled with the material-enhanced laser desorption/ionization (MELDI) approach. The detection limit of the described matrix-free target is investigated to be 10 fmol/microL for [Glu(1)]-fibrinopeptide B (m/z 1570.6) and 1 fmol/microL for L-sorbose (Na(+) adduct). The device does not require any chemical functionalization in contrast to other matrix-free systems. The inertness of DLC provides longer lifetimes without any deterioration in the detection sensitivity. Broad applicability allows high performance analysis in metabolomics and peptidomics. Furthermore the DLC coated DVD (1.4 GB) sample support is used as a storage device for measured and processed data together with sampling on a single device.


Subject(s)
Carbohydrates/analysis , Compact Disks , Diamond/chemistry , Nanostructures/chemistry , Peptides/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Bradykinin/analysis , Bradykinin/chemistry , Carbohydrates/chemistry , Fibrinopeptide B/analysis , Fibrinopeptide B/chemistry , Molybdenum/chemistry , Peptide Fragments/analysis , Peptide Fragments/chemistry , Peptides/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/instrumentation
7.
Anal Chem ; 79(15): 5927-34, 2007 Aug 01.
Article in English | MEDLINE | ID: mdl-17605467

ABSTRACT

An automated off-line liquid chromatography-matrix-assisted laser desorption ionization (LC-MALDI) interface capable of coupling both capillary and microbore LC separations with MALDI mass spectrometry (MS) and tandem mass spectrometry (MS/MS) has been developed. The interface is a combination of two concepts: analyte concentration from heated hanging droplets and impulse-driven droplet deposition of LC fractions onto a MALDI sample plate. At room temperature the interface allows the coupling of capillary LC separations (i.e., flow rate of <5 microL/min) with MALDI MS. With heating, it can be used to combine microbore LC operated at a relatively high flow rate of up to 50 microL/min with MALDI MS. The collected fractions can be analyzed by MALDI MS and MS/MS instruments, such as time-of-flight (TOF) and quadrupole-TOF MS. Performance of the interface was examined using several peptide and protein standards. It was shown that, using MALDI-TOF MS, [GLU1]-fibrinopeptide B could be detected with a total injection amount of 5 fmol to microbore LC. Chromatographic performance was also monitored. A peak width of 12 s at half-height for [GLU1]-fibrinopeptide B showed no evidence of band broadening due to the interface. The ability of the interface to mitigate ion suppression was studied using a mixture of 100 fmol of [GLU1]-fibrinopeptide B and 10 pmol of cytochrome c tryptic digest. Although fully suppressed under direct MALDI conditions, LC-MALDI analysis was able to detect the 100 fmol peptide with 10 s fraction collection. Finally, the ability to inject relatively large sample amounts to improve detectability of low-abundance peptides was illustrated in the analysis of phosphopeptides from alpha-casein tryptic digests. A digest loaded on column to 2.4 microg and analyzed by LC-MALDI MS/MS resulted in 82% sequence coverage and detection of all nine phosphoserine residues. It is concluded that, being able to handle both high- and low-flow LC separations, the impulse-driven heated-droplet interface provides the flexibility to carry out MALDI analysis of peptides and proteins depending on the information sought after, analysis speed, and sample size.


Subject(s)
Chromatography, Liquid/methods , Cytochromes c/analysis , Fibrinopeptide B/analysis , Phosphopeptides/analysis , Phosphoserine/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Amino Acid Sequence , Animals , Caseins/metabolism , Chromatography, Liquid/instrumentation , Cytochromes c/metabolism , Fibrinopeptide B/metabolism , Hot Temperature , Humans , Molecular Sequence Data , Phosphopeptides/metabolism , Phosphoserine/metabolism , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/instrumentation , Trypsin/metabolism
8.
J Am Soc Mass Spectrom ; 17(11): 1605-1615, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16904337

ABSTRACT

The relative abundances of fragment ions in electron capture dissociation (ECD) are often greatly affected by the secondary and tertiary structures of the precursor ion, and have been used to derive the gas-phase conformations of the protein ions. In this study, it is found that resonance ejection of the charge reduced molecular ion during ECD resulted in significant changes in many fragment ion populations. The ratio of the ion peak intensities in the double resonance (DR)-ECD to that in the normal ECD experiment can be used to calculate the lifetime of the radical intermediates from which these fragments are derived. These lifetimes are often in the ms range, a time sufficiently long to facilitate multiple free radical rearrangements. These ratios correlate with the intramolecular noncovalent interactions in the precursor ion, and can be used to deduce information about the gas-phase conformation of peptide ions. DR-ECD experiments can also provide valuable information on ECD mechanisms, such as the importance of secondary electron capture and the origin of c./z ions.


Subject(s)
Cyclotrons , Peptides/chemistry , Spectrometry, Mass, Electrospray Ionization , Spectroscopy, Fourier Transform Infrared , Cyclosporine/analysis , Cyclosporine/chemistry , Electrons , Fibrinopeptide B/analysis , Fibrinopeptide B/chemistry , Melitten/analysis , Melitten/chemistry , Peptides/analysis , Tandem Mass Spectrometry
9.
Anal Chem ; 78(6): 1995-2000, 2006 Mar 15.
Article in English | MEDLINE | ID: mdl-16536438

ABSTRACT

Electron capture dissociation was implemented in a digital ion trap without using any magnetic field to focus the electrons. Since rectangular waveforms are employed in the DIT for both trapping and dipole excitation, electrons can be injected into the trap when the electric field is constant. Following deceleration, electrons reach the precursor ion cloud. The fragment ions produced by interactions with the electron beam are subsequently analyzed by resonant ejection. [Glu(1)]-Fibrinopeptide B and substance P were used to evaluate the performance of the current design. Fragmentation efficiency of 5.5% was observed for substance P peptide ions. Additionally, analysis of the monophosphorylated peptide FQ[pS]EEQQQTEDELQDK shows that in the resulting c- and z-type ions, the phosphate group is retained on the phophoserine residue, providing information on which amino acid residue the modification is located.


Subject(s)
Fibrinopeptide B/analysis , Substance P/analysis , Tandem Mass Spectrometry/methods , Electrons , Peptide Fragments/analysis , Phosphorylation , Sensitivity and Specificity
10.
Electrophoresis ; 26(24): 4575-89, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16358250

ABSTRACT

The present work describes a dual-column and dual-sprayer LC-MS system for high-throughput proteomic analyses. This system consists of two precolumns for sample desalting and two analytical columns. Each column is terminated by a nanoelectrospray emitter mounted on a robotic arm enabling their sequential positioning in front of the sampling cone of the mass spectrometer. The effluent from each emitter is recorded in separate acquisition channels without detectable crosstalk. Gradient elution to both nanoLC columns is delivered by a single HPLC system via a flow splitter. The reproducibility of retention time and peak intensity of the present multiplex system were comparable to those obtainable using a single emitter configuration. Replicate injections of complex tryptic digests (n = 10) indicated that this system provided good reproducibility of retention time and peak intensity on both columns with RSD values of less than 0.9 and 18.6%, respectively. The application of this system is demonstrated for the monitoring of protein expression changes in U937 human monocyte cells with and without phorbol ester administration. Furthermore, we also demonstrated the use of this multiplex system in a 2-D LC configuration to increase sample loading and throughput for the analysis of biomarker samples of higher complexity. Variations in peptide abundance down to two-fold change were identified across salt fractions for spiked tryptic digests present at a level of 50 fmol in 1.5 microg of plasma samples.


Subject(s)
Chromatography, Liquid/methods , Mass Spectrometry/methods , Proteomics/methods , Animals , Cell Differentiation/drug effects , Chromatography, Liquid/instrumentation , Enkephalin, Leucine/analysis , Fibrinopeptide B/analysis , Humans , Mass Spectrometry/instrumentation , Nanotechnology/methods , Nanotechnology/trends , Rats , Reproducibility of Results , Tetradecanoylphorbol Acetate/pharmacology , U937 Cells
11.
Blood Coagul Fibrinolysis ; 15(6): 451-61, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15311153

ABSTRACT

In order to characterize tissue plasminogen activator (t-PA) binding to gamma-chain residues in fibrinogen, we generated variant fibrinogens substituting alanine for gamma D316, gamma D318, gamma D320, and gamma K321. We measured thrombin-catalyzed polymerization and found normal polymerization with gamma K321A, no polymerization with gamma D316A, and, as reported by Lounes et al. in 2002, impaired polymerization with gamma D318A and gamma D320A. We measured t-PA binding in a solid-phase assay, and t-PA activity by the generation of plasmin. Comparing normal fibrin with fibrinogen, we found a seven-fold increase in binding and a two-fold increase in activity. Binding to all variant fibrinogens was the same as normal. In contrast, t-PA binding to all variant fibrins was weaker than binding to normal fibrin, 2.5-fold for gamma K321A, seven-fold for gamma D320A and 10-fold for gamma D316A and gamma D318A. Plasmin generation in the presence of variant fibrinogens was similar, although not identical, to normal, and plasmin generation in the presence of variant fibrins was impaired for the Asp to Ala variants. As the three variants with the weakest t-PA binding and least activity also showed impaired polymerization, our results support previous findings demonstrating the DD:E complex, found in the normal fibrin polymer, is necessary for the fibrin enhanced binding of t-PA and activation of plasminogen.


Subject(s)
Amino Acid Substitution , Fibrinogen/metabolism , Tissue Plasminogen Activator/metabolism , Fibrinogen/chemistry , Fibrinolysin/biosynthesis , Fibrinopeptide A/analysis , Fibrinopeptide B/analysis , Humans , Mutagenesis, Site-Directed , Protein Binding , Protein Interaction Mapping
13.
J Mass Spectrom ; 38(5): 523-5, 2003 May.
Article in English | MEDLINE | ID: mdl-12794873

ABSTRACT

Polydimethylcyclosiloxanes, an almost ubiquitous air contaminant, can interfere with nanoelectrospray analysis. The sensitivity of nanoelectrospray to these volatile air contaminants was demonstrated in this study. The intensity of the interfering ion signals caused by these compounds can be decreased by changing the position of the nanoESI needle and almost completely suppressed by applying a flow of pure nitrogen around the needle and the sample cone. The nitrogen flow causes a slight shift in charge distribution, but does not influence the sensitivity for peptide detection.


Subject(s)
Air , Artifacts , Fibrinopeptide B/analysis , Laboratories , Siloxanes/analysis , Spectrometry, Mass, Electrospray Ionization/standards , Air Pollutants/analysis , Air Pollutants/chemistry , Dimethylpolysiloxanes/analysis , Dimethylpolysiloxanes/chemistry , Nitrogen , Sensitivity and Specificity , Siloxanes/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Volatilization
14.
Anal Chem ; 75(7): 1615-9, 2003 Apr 01.
Article in English | MEDLINE | ID: mdl-12705593

ABSTRACT

A novel, rugged capillary electrophoresis-electrospray ionization (CE-ESI) interface where the separation column, an electrical porous junction, and the spray tip are integrated on a single piece of a fused-silica capillary is described. ESI is accomplished by applying an electrical potential through an easily prepared porous junction across a 3-4-mm length of fused silica. A stable electrospray is produced at nanoflow rates generated in the capillary by electrophoretic and electroosmotic forces. The interface is particularly well suited for the detection of low-femtomole levels of proteins and peptides. The ruggedness of this interface was evident by the continuous operation of the same column for over a 2-week period with no detectable deterioration in separation or electrospray performance. The new interface was used for the LC-ESI-MS separation and analysis of peptides and proteins. Injection of 25 fmol of [Glu1]-fibrinopeptide B using the new device produced a CE-ESI-MS electropherogram with a signal-to-noise ratio of over 100 for this peptide.


Subject(s)
Electrophoresis, Capillary/methods , Nanotechnology/instrumentation , Proteins/analysis , Proteins/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Amino Acid Sequence , Animals , Apoproteins/analysis , Apoproteins/chemistry , Chromatography, Liquid , Electrophoresis, Capillary/instrumentation , Fibrinopeptide B/analysis , Fibrinopeptide B/chemistry , Horses , Molecular Sequence Data , Myoglobin/analysis , Myoglobin/chemistry , Nanotechnology/methods , Spectrometry, Mass, Electrospray Ionization/instrumentation , Time Factors
15.
Blood Coagul Fibrinolysis ; 13(2): 149-53, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11914657

ABSTRACT

A fibrinogen variant was suspected based on the results of routine coagulation tests in a 2-year-old asymptomatic child. Coagulation studies showed marked prolongation of both the thrombin and reptilase times, and discrepancy was noted between the level of plasma fibrinogen as measured by a kinetic versus immunological determination. Family studies revealed that the father beared the same abnormality. Studies of purified fibrinogen revealed an impaired release of both fibrinopeptides by thrombin. Fibrin monomer polymerization and fibrin stabilization were normal. DNA sequencing revealed a heterozygous G --> T point mutation in exon 2 of the gene coding for the Aalpha chain, which substituted a Gly for Val at position 12. Although the mutation is the same as in fibrinogen Rouen, fibrinogen Saint-Germain I shows a different fibrinopeptide release pattern and a mild factor V deficiency.


Subject(s)
Coagulation Protein Disorders/diagnosis , Fibrinogens, Abnormal/genetics , Point Mutation , Amino Acid Substitution , Child, Preschool , Coagulation Protein Disorders/genetics , DNA Mutational Analysis , Diagnosis, Differential , Factor V Deficiency , Family Health , Female , Fibrinopeptide A/analysis , Fibrinopeptide B/analysis , Genetic Variation , Heterozygote , Humans
16.
Surg Today ; 30(9): 778-84, 2000.
Article in English | MEDLINE | ID: mdl-11039704

ABSTRACT

When a large volume of coagulum remains in the body cavity after trauma or surgery, secondary fibrinolysis occurs, which disturbs the hemostatic balance and results in rebleeding. To better understand this condition, we conducted a clinical study on patients with and without coagula and an experimental study on fibrinolytic activity in a rat model. The results of the clinical study showed that when coagula existed in the body cavity, the blood levels of the fibrin degradation products D-dimer and fibrinopeptide Bbeta15-42 remained high compared with when subjects were under similar stress but without the presence of coagula. In the experimental studies, fibrinolytic activity of the omentum, measured by the fibrin plate method, was higher in rats with hemoperitoneum. This suggests that increased fibrinolytic activity may lead to rebleeding from the area of transient hemostasis when coagulum is present in the body cavity.


Subject(s)
Blood Coagulation/physiology , Fibrinolysis/physiology , Wounds, Nonpenetrating/diagnosis , Adolescent , Adult , Aged , Animals , Child , Female , Fibrin Fibrinogen Degradation Products/analysis , Fibrinopeptide B/analysis , Hemoperitoneum/blood , Hemostasis/physiology , Humans , Male , Middle Aged , Rats , Rats, Wistar
17.
Nihon Rinsho ; 57 Suppl: 573-4, 1999 Sep.
Article in Japanese | MEDLINE | ID: mdl-10543181
18.
Thromb Haemost ; 78(6): 1484-7, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9423799

ABSTRACT

We report the first direct detection of a fibrinogen mutation by electrospray ionisation mass spectrometry. The propositus, from a family with a history of thrombosis, came to attention after a pulmonary embolism subsequent to a spontaneous abortion. Prolonged thrombin (41 s) and reptilase times (26 s) together with an impairment of fibrinopeptide B release suggested a mutation at the thrombin cleavage site of the Bbeta chain. Direct mass analysis of purified fibrin chains from a thrombin induced clot showed that 50% of the Bbeta chains remained uncleaved. The measured mass of the mono sialo isoform of this uncleaved chain was 54150 Da, compared to a value of 54198 Da for normal Bbeta chains. This decrease of 48 Da in the intact protein is indicative of either a Bbeta 14 Arg to Cys, or Arg to Leu substitution. Heterozygosity for the Bbeta 14 Arg --> Cys mutation was verified by PCR amplification and DNA sequence analysis.


Subject(s)
Amino Acid Substitution , Fibrinopeptide B/analysis , Fibrinopeptide B/genetics , Mass Spectrometry/methods , Thrombosis/genetics , Adult , Arginine/genetics , Catalysis , Cysteine/genetics , Family Health , Female , Fibrin/analysis , Fibrin/chemistry , Fibrinogen/analysis , Fibrinogen/chemistry , Fibrinopeptide A/metabolism , Fibrinopeptide B/chemistry , Glycoproteins/analysis , Heterozygote , Humans , Molecular Weight , Mutation/genetics , Mutation/physiology , Point Mutation/genetics , Point Mutation/physiology , Polymerase Chain Reaction , Sequence Analysis , Thrombin/chemistry
19.
Am J Hematol ; 49(2): 143-8, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7771466

ABSTRACT

Small cell carcinoma of the lung (SCCL) responds commonly to combination chemotherapy but resistance to therapy follows. Prior reports have suggested that a relationship may exist between plasma fibrinogen levels and response to therapy in SCCL. This study was designed to determine the possible predictive value of the fibrinogen level for tumor response (chemoresistance) in SCCL. Pretreatment fibrinogen levels were correlated with outcome and response to therapy in a cohort of 119 previously untreated patients with SCCL who were admitted to VA Cooperative Study 188. Higher pretreatment fibrinogen levels at diagnosis correlated significantly with more advanced stage of disease at entry (P < 0.001) and with reduced overall survival (P = 0.030). In addition, higher pretreatment fibrinogen levels were correlated significantly with a reduced likelihood of achieving subsequent disease regression with combination chemotherapy (P = 0.005). Because several clinical trials have shown that anticoagulant therapy improves tumor response rates and survival of SCCL, we postulate that tumor cell thrombin generation not only promotes SCCL growth but may also be primarily responsible for both increased fibrinogen levels and for resistance to chemotherapy. These findings provide incentive for studies of thrombin effects on the development of multidrug resistance, and for new clinical trials of more potent and specific inhibitors of thrombin that may further improve tumor response and survival in SCCL.


Subject(s)
Carcinoma, Small Cell/blood , Fibrinogen/analysis , Lung Neoplasms/blood , Carcinoma, Small Cell/drug therapy , Carcinoma, Small Cell/physiopathology , Cohort Studies , Drug Resistance , Fibrinolysin/analysis , Fibrinopeptide A/analysis , Fibrinopeptide B/analysis , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/physiopathology , Mopidamol/therapeutic use , Randomized Controlled Trials as Topic , Regression Analysis , Thrombin/analysis
20.
J Cell Physiol ; 162(1): 9-14, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7814454

ABSTRACT

We have investigated human neonatal fibroblast synthetic activity in response to fibrin substrates and components of fibrin formation and degradation. Greater than threefold downregulation of procollagen mRNA levels was seen 24 hours after fibroblasts were grown on fibrin gels as compared to tissue culture plastic. This downregulation occurred in both reptilase-generated fibrin (retention of fibrinopeptide B) and thrombin-generated fibrin (loss of both fibrinopeptide A and B). However, fibroblasts grown on fibrin retained their capacity to respond to the stimulatory action of transforming growth factor (TGF)-beta 1. Fibroblasts seeded on reptilase-generated fibrin displayed an abnormal morphology manifested by dendritic appearance and cell rounding, while fibroblast attachment was enhanced by 30% on thrombin-generated fibrin substrate (P < 0.02). Fibrinopeptides A and B, which are generated during fibrin formation, increased and decreased procollagen mRNA levels, respectively. Tissue plasminogen activator (t-PA) increased procollagen mRNA and TGF-beta 1 levels as early as 6 hours after cells were grown on tissue culture plastic, but this stimulation did not occur in cells cultured on a fibrin substrate. We conclude that alpha 1(I) procollagen mRNA levels in cultures of human dermal fibroblasts are consistently down-regulated by a fibrin substrate and are directly and profoundly influenced by complex interactions between components involved in the formation and removal of fibrin.


Subject(s)
Fibrin/pharmacology , Fibrinopeptide B/pharmacology , Fibroblasts/chemistry , Procollagen/genetics , RNA, Messenger/analysis , Blotting, Northern , Cells, Cultured , Down-Regulation , Fibrin/analysis , Fibrin/metabolism , Fibrinopeptide A/analysis , Fibrinopeptide A/metabolism , Fibrinopeptide A/pharmacology , Fibrinopeptide B/analysis , Fibrinopeptide B/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gels , Humans , Infant, Newborn , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tissue Plasminogen Activator/analysis , Tissue Plasminogen Activator/metabolism , Tissue Plasminogen Activator/pharmacology , Transforming Growth Factor beta/analysis , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/pharmacology
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