30 results on '"Jung, Stephanie"'
Search Results
2. A fluorescent assay for cryptic transcription in Saccharomyces cerevisiaereveals novel insights into factors that stabilize chromatin structure on newly replicated DNA
- Author
-
Gao, Ellia, Brown, Joshua A R, Jung, Stephanie, and Howe, LeAnn J
- Abstract
The disruption of chromatin structure can result in transcription initiation from cryptic promoters within gene bodies. While the passage of RNA polymerase II is a well-characterized chromatin-disrupting force, numerous factors, including histone chaperones, normally stabilize chromatin on transcribed genes, thereby repressing cryptic transcription. DNA replication, which employs a partially overlapping set of histone chaperones, is also inherently disruptive to chromatin, but a role for DNA replication in cryptic transcription has never been examined. In this study, we tested the hypothesis that, in the absence of chromatin-stabilizing factors, DNA replication can promote cryptic transcription in Saccharomyces cerevisiae. Using a novel fluorescent reporter assay, we show that multiple factors, including Asf1, CAF-1, Rtt106, Spt6, and FACT, block transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Collectively, these results demonstrate that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA.
- Published
- 2024
- Full Text
- View/download PDF
3. Dimers of the platelet collagen receptor glycoprotein VI bind specifically to fibrin fibers during clot formation, but not to intact fibrinogen
- Author
-
Moroi, Masaaki, Induruwa, Isuru, Farndale, Richard W., and Jung, Stephanie M.
- Abstract
The platelet collagen receptor glycoprotein VI (GPVI) has an independent role as a receptor for fibrin produced via the coagulation cascade. However, various reports of GPVI binding to immobilized fibrin(ogen) are not consistent. As a collagen receptor, GPVI‐dimer is the functional form, but whether GPVI dimers or monomers bind to fibrin remains controversial. To resolve this, we analyzed GPVI binding to nascent fibrin clots, which more closely approximate physiological conditions. ELISA using biotinyl‐fibrinogen immobilized on streptavidin‐coated wells indicated that GPVI dimers do not bind intact fibrinogen. Clots were formed by adding thrombin to a mixture of near‐plasma level of fibrinogen and recombinant GPVI ectodomain: GPVI dimer (GPVI‐Fc2or Revacept) or monomer (GPVI‐His: single chain of Revacept GPVI domain, with His tag). Clot‐bound proteins were analyzed by SDS‐PAGE/immunoblotting. GPVI‐dimer bound to noncrosslinked fibrin clots with classical one‐site binding kinetics, with µM‐level KD, and to crosslinked clots with higher affinity. Anti‐GPVI‐dimer (mFab‐F) inhibited the binding. However, GPVI‐His binding to either type of clot was nonsaturable and nearly linear, indicating very low affinity or nonspecific binding. In clots formed in the presence of platelets, clot‐bound platelet‐derived proteins were integrin αIIbβ3, present at high levels, and GPVI. We conclude that dimeric GPVI is the receptor for fibrin, exhibiting a similar KDto those obtained for its binding to fibrinogen D‐fragment and D‐dimer, suggesting that fibrin(ogen)'s GPVI‐binding site becomes exposed after fibrin formation or cleavage to fragment D. Analysis of platelets bound to fibrin clots indicates that platelet GPVI binds to fibrin fibers comprising the clot.
- Published
- 2021
- Full Text
- View/download PDF
4. Dimers of the platelet collagen receptor glycoprotein VI bind specifically to fibrin fibers during clot formation, but not to intact fibrinogen
- Author
-
Moroi, Masaaki, Induruwa, Isuru, Farndale, Richard W., and Jung, Stephanie M.
- Abstract
The platelet collagen receptor glycoprotein VI (GPVI) has an independent role as a receptor for fibrin produced via the coagulation cascade. However, various reports of GPVI binding to immobilized fibrin(ogen) are not consistent. As a collagen receptor, GPVI‐dimer is the functional form, but whether GPVI dimers or monomers bind to fibrin remains controversial. To resolve this, we analyzed GPVI binding to nascent fibrin clots, which more closely approximate physiological conditions.
- Published
- 2021
- Full Text
- View/download PDF
5. A Comprehensive UHPLC Ion Mobility Quadrupole Time-of-Flight Method for Profiling and Quantification of Eicosanoids, Other Oxylipins, and Fatty Acids
- Author
-
Hinz, Christine, Liggi, Sonia, Mocciaro, Gabriele, Jung, Stephanie, Induruwa, Isuru, Pereira, Milton, Bryant, Clare E., Meckelmann, Sven W., O’Donnell, Valerie B., Farndale, Richard W., Fjeldsted, John, and Griffin, Julian L.
- Abstract
Analysis of oxylipins by liquid chromatography mass spectrometry (LC/MS) is challenging because of the small mass range occupied by this diverse lipid class, the presence of numerous structural isomers, and their low abundance in biological samples. Although highly sensitive LC/MS/MS methods are commonly used, further separation is achievable by using drift tube ion mobility coupled with high-resolution mass spectrometry (DTIM-MS). Herein, we present a combined analytical and computational method for the identification of oxylipins and fatty acids. We use a reversed-phase LC/DTIM-MS workflow able to profile and quantify (based on chromatographic peak area) the oxylipin and fatty acid content of biological samples while simultaneously acquiring full scan and product ion spectra. The information regarding accurate mass, collision-cross-section values in nitrogen (DTCCSN2), and retention times of the species found are compared to an internal library of lipid standards as well as the LIPID MAPS Structure Database by using specifically developed processing tools. Features detected within the DTCCSN2and m/zranges of the analyzed standards are flagged as oxylipin-like species, which can be further characterized using drift-time alignment of product and precursor ions distinctive of DTIM-MS. This not only helps identification by reducing the number of annotations from LIPID MAPS but also guides discovery studies of potentially novel species. Testing the methodology on Salmonella entericaserovar Typhimurium-infected murine bone-marrow-derived macrophages and thrombin activated human platelets yields results in agreement with literature. This workflow has also annotated features as potentially novel oxylipins, confirming its ability in providing further insights into lipid analysis of biological samples.
- Published
- 2019
- Full Text
- View/download PDF
6. Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators
- Author
-
Flierl, Ulrike, Nero, Tracy L., Lim, Bock, Arthur, Jane F., Yao, Yu, Jung, Stephanie M., Gitz, Eelo, Pollitt, Alice Y., Zaldivia, Maria T.K., Jandrot-Perrus, Martine, Schäfer, Andreas, Nieswandt, Bernhard, Andrews, Robert K., Parker, Michael W., Gardiner, Elizabeth E., and Peter, Karlheinz
- Abstract
Nucleotide-based drug candidates such as antisense oligonucleotides, aptamers, immunoreceptor-activating nucleotides, or (anti)microRNAs hold great therapeutic promise for many human diseases. Phosphorothioate (PS) backbone modification of nucleotide-based drugs is common practice to protect these promising drug candidates from rapid degradation by plasma and intracellular nucleases. Effects of the changes in physicochemical properties associated with PS modification on platelets have not been elucidated so far. Here we report the unexpected binding of PS-modified oligonucleotides to platelets eliciting strong platelet activation, signaling, reactive oxygen species generation, adhesion, spreading, aggregation, and thrombus formation in vitro and in vivo. Mechanistically, the platelet-specific receptor glycoprotein VI (GPVI) mediates these platelet-activating effects. Notably, platelets from GPVI function–deficient patients do not exhibit binding of PS-modified oligonucleotides, and platelet activation is fully abolished. Our data demonstrate a novel, unexpected, PS backbone–dependent, platelet-activating effect of nucleotide-based drug candidates mediated by GPVI. This unforeseen effect should be considered in the ongoing development programs for the broad range of upcoming and promising DNA/RNA therapeutics.
- Published
- 2015
- Full Text
- View/download PDF
7. Investigating the Control of Listeria monocytogeneson a Ready-to-Eat Ham Product Using Natural Antimicrobial Ingredients and Postlethality Interventions
- Author
-
Lavieri, Nicolas A., Sebranek, Joseph G., Brehm-Stecher, Byron F., Cordray, Joseph C., Dickson, James S., Horsch, Ashley M., Jung, Stephanie, Larson, Elaine M., Manu, David K., and Mendonça, Aubrey F.
- Abstract
AbstractReady-to-eat (RTE) meat and poultry products manufactured with natural or organic methods are at greater risk for Listeria monocytogenesgrowth, if contaminated, than their conventional counterparts due to the required absence of preservatives and antimicrobials. Thus, the objective of this study was to investigate the use of commercially available natural antimicrobials and postlethality interventions in the control of L. monocytogenesgrowth and recovery on a RTE ham product. Antimicrobials evaluated were cranberry powder (90MX), vinegar (DV), and vinegar/lemon juice concentrate (LV1X). Postlethality interventions studied were high hydrostatic pressure at 400 (HHP400) or 600 (HHP600) MPa, lauric arginate (LAE), octanoic acid (OA), and postpackaging thermal treatment (PPTT). Parameters evaluated through 98 days of storage at 4±1°C were residual nitrite concentrations, pH, aw, and viable L. monocytogeneson modified Oxford (MOX) media. On day 1, OA, 90MX, DV, and LV1X yielded lower residual nitrite concentrations than the control, whereas HHP400, HHP600, and LAE did not. LAE, HHP400, and OA reduced L. monocytogenespopulation compared to the control after 1 day of storage by 2.38, 2.21, and 1.73 log10colony-forming units per gram, respectively. PPTT did not achieve a significant reduction in L. monocytogenespopulations. L. monocytogenesrecovered and grew in all postlethality intervention treatments except HHP600. 90MX did not inhibit the growth of L. monocytogenes, while DV and LV1X did. Results of this study demonstrate the bactericidal properties of HHP, OA, and LAE and the bacteriostatic potential of natural antimicrobial ingredients such as DV and LV1X against L. monocytogenes.
- Published
- 2014
- Full Text
- View/download PDF
8. Effects of Different Nitrite Concentrations from a Vegetable Source with and without High Hydrostatic Pressure on the Recovery of Listeria monocytogeneson Ready-to-Eat Restructured Ham
- Author
-
Lavieri, Nicolas A., Sebranek, Joseph G., Cordray, Joseph C., Dickson, James S., Horsch, Ashley M., Jung, Stephanie, Manu, David K., Brehm-Stecher, Byron F., and Mendonca, Aubrey F.
- Abstract
Sodium nitrite exerts an inhibitory effect on the growth of Listeria monocytogenes. The objective of this study was to investigate the effects of various nitrite concentrations from a vegetable source with and without high hydrostatic pressure (HHP) on the recovery and growth of L. monocytogeneson ready-to-eat restructured ham. A preconverted celery powder was used as the vegetable source of nitrite. Targeted concentrations of natural nitrite investigated were 0, 50, and 100 mg/kg. HHP treatments evaluated were 400 MPa for 4 min and 600 MPa for 1 or 4 min at 12 ± 2°C (initial temperature of the pressurization fluid). Viable L. monocytogenespopulations were monitored on modified Oxford medium and thin agar layer medium through 98 days of storage at 4 ±1°C. Populations on both media did not differ. The HHP treatment at 600 MPa for 4 min resulted in L. monocytogenespopulations below the detection limit of our sampling protocols throughout the storage period regardless of the natural nitrite concentration. The combination of HHP at 400 MPa for 4 min or 600 MPa for 1 min with natural nitrite resulted in initial inhibition of viable L. monocytogenes. Ham formulations that did not contain natural nitrite allowed faster growth of L. monocytogenesthan did those with nitrite, regardless of whether they were treated with HHP. The results indicate that nitrite from a vegetable source at the concentrations used in this study resulted in slower growth of this microorganism. HHP treatments enhanced the inhibitory effects of natural nitrite on L. monocytogenesgrowth. Thus, the combination of natural nitrite plus HHP appears to have a synergistic inhibitory effect on L. monocytogenesgrowth.
- Published
- 2014
- Full Text
- View/download PDF
9. Evaluation of the Thin Agar Layer Method for the Recovery of Pressure-Injured and Heat-Injured Listeria monocytogenes
- Author
-
Lavieri, Nicolas A., Sebranek, Joseph G., Cordray, Joseph C., Dickson, James S., Jung, Stephanie, Manu, David K., Mendonça, Aubrey F., Brehm-Stecher, Byron F., Stock, Joseph, and Stalder, Kenneth J.
- Abstract
A sublethally injured bacterial cell has been defined as a cell that survives a stress such as heating, freezing, acid treatment, or other antimicrobial intervention but can repair the cellular damage exerted by the stressor and later regain its original ability to grow. Consequently, sublethally injured cells are not likely to be included in conventional enumeration procedures, which could result in unrealistically low counts unless efforts are made to encourage recovery of the injured cells before enumeration. The objective of this study was to evaluate the use of the thin agar layer (TAL) method for the recovery of pressure-injured and heat-injured Listeria monocytogenesin a tryptic soy broth with 0.6% yeast extract system. Pressure injury consisted of treatment of a culture of mixed L. monocytogenesstrains with high hydrostatic pressure at 400 or 600 MPa for 1 s, 2 min, 4 min, or 6 min at a process temperature of 12 ± 2°C. Heat injury consisted of treatment of a culture of mixed L. monocytogenesstrains at 60 ± 1°C for 3, 6, or 9 min. Growth media were tryptic soy agar (TSA) with 0.6% yeast extract, modified Oxford medium (MOX), and TAL, which consisted of a 7-ml layer of TSA overlaid onto solidified MOX. Counts of viable L. monocytogeneson TAL were higher than those on MOX in the heat-injury experiment but not in the pressure-injury experiment. Therefore, the effectiveness of the TAL method may be specific to the type of injury applied to the microorganism and should be investigated in a variety of cellular injury scenarios.
- Published
- 2014
- Full Text
- View/download PDF
10. Fast, accurate, and convenient analysis of bed densities for columns packed with fine reversed‐phase particles
- Author
-
Jung, Stephanie, Stoeckel, Daniela, and Tallarek, Ulrich
- Abstract
We determine the interparticle porosities of commercially available, analytical, reversed‐phase HPLC columns by Donnan exclusion of a small, unretained, co‐ionic tracer (nitrate ions). The columns contained packings of C18‐modified, endcapped, silica particles, which differed in their nominal particle diameters (1.8–5 μm) and construction (fully porous or core‐shell). Experiments were carried out by monitoring the elution volumes of nitrate samples in a mobile phase of acetonitrile/water 80:20 v/v at increasing concentrations of Tris‐HCl buffer (pH 8.1) from 0.01 to 60 mM. At low buffer concentrations, nitrate ions are completely electrostatically excluded from the intraparticle mesopore space, which is reflected by a plateau region in the elution curves. The elution volume in the plateau region equals the interparticle void volume. Clearly defined plateau regions were observed for all columns, even those densely packed with core‐shell and sub‐2 μm particles, enabling the accurate determination of interparticle porosities to three decimal places in a fast and convenient way.
- Published
- 2011
- Full Text
- View/download PDF
11. Relative antithrombotic effect of soluble GPVI dimer compared with anti-GPVI antibodies in mice
- Author
-
Grüner, Sabine, Prostredna, Miroslava, Koch, Martina, Miura, Yoshiki, Schulte, Valerie, Jung, Stephanie M., Moroi, Masaaki, and Nieswandt, Bernhard
- Abstract
Glycoprotein VI (GPVI) is an essential platelet collagen receptor; therefore, the inhibition of GPVI-collagen interactions may be an attractive antithrombotic strategy. We have previously shown that targeting of GPVI with antibodies leads to the depletion of the receptor and to long-term antithrombotic protection in mice. An alternative agent to interfere with GPVI-collagen interactions might be soluble GPVI acting as a competitive inhibitor, thereby averting undesired effects on platelets. To test this, we expressed soluble dimeric human GPVI, comprising the extracellular domain of the receptor fused to the human immunoglobulin Fc domain (GPVI-Fc), and compared its antithrombotic potential with that of anti-GPVI antibodies in mice. In contrast to a recent report, we found by intravital fluorescence microscopy and ultrasonic flow measurements that GPVI-Fc had no effect on platelet adhesion and thrombus formation at the injured arterial wall, whereas anti-GPVI antibodies profoundly inhibited these processes. Similar results were obtained with a fusion protein comprising the extracellular domain of mouse GPVI and human IgG-Fc. This indicates that direct targeting of GPVI provides significantly stronger protection against arterial thrombosis than soluble GPVI dimer.
- Published
- 2005
- Full Text
- View/download PDF
12. Relative antithrombotic effect of soluble GPVI dimer compared with anti-GPVI antibodies in mice
- Author
-
Grüner, Sabine, Prostredna, Miroslava, Koch, Martina, Miura, Yoshiki, Schulte, Valerie, Jung, Stephanie M., Moroi, Masaaki, and Nieswandt, Bernhard
- Abstract
Glycoprotein VI (GPVI) is an essential platelet collagen receptor; therefore, the inhibition of GPVI-collagen interactions may be an attractive antithrombotic strategy. We have previously shown that targeting of GPVI with antibodies leads to the depletion of the receptor and to long-term antithrombotic protection in mice. An alternative agent to interfere with GPVI-collagen interactions might be soluble GPVI acting as a competitive inhibitor, thereby averting undesired effects on platelets. To test this, we expressed soluble dimeric human GPVI, comprising the extracellular domain of the receptor fused to the human immunoglobulin Fc domain (GPVI-Fc), and compared its antithrombotic potential with that of anti-GPVI antibodies in mice. In contrast to a recent report, we found by intravital fluorescence microscopy and ultrasonic flow measurements that GPVI-Fc had no effect on platelet adhesion and thrombus formation at the injured arterial wall, whereas anti-GPVI antibodies profoundly inhibited these processes. Similar results were obtained with a fusion protein comprising the extracellular domain of mouse GPVI and human IgG-Fc. This indicates that direct targeting of GPVI provides significantly stronger protection against arterial thrombosis than soluble GPVI dimer.
- Published
- 2005
- Full Text
- View/download PDF
13. Von Willebrand factor accelerates platelet adhesion and thrombus formation on a collagen surface in platelet-reduced blood under flow conditions
- Author
-
Tomokiyo, Kazuhiko, Kamikubo, Yuichi, Hanada, Takako, Araki, Tatsuya, Nakatomi, Yasushi, Ogata, Yoichi, Jung, Stephanie M., Nakagaki, Tomohiro, and Moroi, Masaaki
- Abstract
Plasma von Willebrand factor (VWF) has been identified as an indispensable factor for platelet adhesion and thrombus formation on a collagen surface under flow conditions. VWF binds to collagen and then tethers platelets to the collagen surface through interaction with platelet glycoprotein Ib and also contributes to the thrombus formation on the collagen surface. In the present study, we demonstrated that the addition of VWF/factor VIII complex or purified VWF (> 2 ristocetin cofactor activity units/mL) increased platelet adhesion to the collagen surface in platelet-reduced blood (∼ 5 × 104platelets/μL) to the normal level. VWF had no stimulatory effect when it was allowed to bind to the collagen surface before blood flow was initiated. Addition of an excess of FITC (fluorescein-5-isothiocyanate)–abeled VWF to platelet-reduced blood under these flow conditions demonstrated that the VWF was mainly incorporated into the platelet aggregates. These results indicated that the supplemented VWF stimulates the platelet adhesion onto the collagen surface by enhancing platelet aggregation in the platelet-reduced condition. This also suggests a possibility that supplementation of VWF to individuals with thrombocytopenia might be effective for increasing their hemostatic potential.
- Published
- 2005
- Full Text
- View/download PDF
14. Von Willebrand factor accelerates platelet adhesion and thrombus formation on a collagen surface in platelet-reduced blood under flow conditions
- Author
-
Tomokiyo, Kazuhiko, Kamikubo, Yuichi, Hanada, Takako, Araki, Tatsuya, Nakatomi, Yasushi, Ogata, Yoichi, Jung, Stephanie M., Nakagaki, Tomohiro, and Moroi, Masaaki
- Abstract
Plasma von Willebrand factor (VWF) has been identified as an indispensable factor for platelet adhesion and thrombus formation on a collagen surface under flow conditions. VWF binds to collagen and then tethers platelets to the collagen surface through interaction with platelet glycoprotein Ib and also contributes to the thrombus formation on the collagen surface. In the present study, we demonstrated that the addition of VWF/factor VIII complex or purified VWF (> 2 ristocetin cofactor activity units/mL) increased platelet adhesion to the collagen surface in platelet-reduced blood (∼ 5 × 104 platelets/μL) to the normal level. VWF had no stimulatory effect when it was allowed to bind to the collagen surface before blood flow was initiated. Addition of an excess of FITC (fluorescein-5-isothiocyanate)–abeled VWF to platelet-reduced blood under these flow conditions demonstrated that the VWF was mainly incorporated into the platelet aggregates. These results indicated that the supplemented VWF stimulates the platelet adhesion onto the collagen surface by enhancing platelet aggregation in the platelet-reduced condition. This also suggests a possibility that supplementation of VWF to individuals with thrombocytopenia might be effective for increasing their hemostatic potential.
- Published
- 2005
- Full Text
- View/download PDF
15. GPVI levels in platelets: relationship to platelet function at high shear
- Author
-
Best, Denise, Senis, Yotis A., Jarvis, Gavin E., Eagleton, Helen J., Roberts, David J., Saito, Takashi, Jung, Stephanie M., Moroi, Masaaki, Harrison, Paul, Green, Fiona R., and Watson, Steve P.
- Abstract
We have investigated the density of the collagen receptors glycoprotein VI (GPVI) and α2β1on human platelets and their relationship to polymorphisms within the GPVIgene. GPVI levels varied 1.5-fold and showed a weak correlation (r = 0.35) with the levels of α2β1, which varied 3-fold. GPVIgenotype had a significant effect on receptor levels with carriers of the proline 219 allele (approximately 22% of the population) having 10% lower GPVI levels than the more common serine homozygotes. GPVI and α2β1levels were found to be significantly decreased on platelets from patients with myeloproliferative disorders (MPDs). In both the MPD and the control group, GPVI levels were found not to affect platelet function under high shear in whole blood. Similarly murine platelets that express up to 5-fold lower levels of GPVI showed no significant difference than controls in thrombus formation on a high-density collagen-coated surface. However platelets lacking the GPVI/Fc receptor γ-chain (FcR γ-chain) complex or a functional FcR γ-chain (immunoreceptor tyrosine-based activation motif [ITAM] point mutant) exhibited severely abrogated thrombus formation at 800 s–1and 1500 s–1. These results demonstrate that GPVI levels are tightly controlled and play a critical role in thrombus formation on collagen; nevertheless, a range of receptor densities can support platelet function under high shear.
- Published
- 2003
- Full Text
- View/download PDF
16. GPVI levels in platelets: relationship to platelet function at high shear
- Author
-
Best, Denise, Senis, Yotis A., Jarvis, Gavin E., Eagleton, Helen J., Roberts, David J., Saito, Takashi, Jung, Stephanie M., Moroi, Masaaki, Harrison, Paul, Green, Fiona R., and Watson, Steve P.
- Abstract
We have investigated the density of the collagen receptors glycoprotein VI (GPVI) and α2β1 on human platelets and their relationship to polymorphisms within the GPVI gene. GPVI levels varied 1.5-fold and showed a weak correlation (r = 0.35) with the levels of α2β1, which varied 3-fold. GPVI genotype had a significant effect on receptor levels with carriers of the proline 219 allele (approximately 22% of the population) having 10% lower GPVI levels than the more common serine homozygotes. GPVI and α2β1 levels were found to be significantly decreased on platelets from patients with myeloproliferative disorders (MPDs). In both the MPD and the control group, GPVI levels were found not to affect platelet function under high shear in whole blood. Similarly murine platelets that express up to 5-fold lower levels of GPVI showed no significant difference than controls in thrombus formation on a high-density collagen-coated surface. However platelets lacking the GPVI/Fc receptor γ-chain (FcR γ-chain) complex or a functional FcR γ-chain (immunoreceptor tyrosine-based activation motif [ITAM] point mutant) exhibited severely abrogated thrombus formation at 800 s–1 and 1500 s–1. These results demonstrate that GPVI levels are tightly controlled and play a critical role in thrombus formation on collagen; nevertheless, a range of receptor densities can support platelet function under high shear.
- Published
- 2003
- Full Text
- View/download PDF
17. A new monoclonal antibody, mAb 204-11, that influences the binding of platelet GPVI to fibrous collagen
- Author
-
Moroi, Masaaki, Mizuguchi, Jun, Kawashima, Sachiko, Nagamatsu, Michiko, Miura, Yoshiki, Nakagaki, Tomohiro, Ito, Katsuaki, and Jung, Stephanie M.
- Published
- 2003
- Full Text
- View/download PDF
18. Analysis of the interaction of platelet collagen receptor glycoprotein VI (GPVI) with collagen. A dimeric form of GPVI, but not the monomeric form, shows affinity to fibrous collagen.
- Author
-
Miura, Yoshiki, Takahashi, Tsuyoshi, Jung, Stephanie M, and Moroi, Masaaki
- Abstract
Glycoprotein VI (GPVI) is a platelet-specific glycoprotein that has been indicated to react with collagen and activate platelets. Its structure was recently identified by cDNA cloning (Clemetson, J. M., Polgar, J., Magnenat, E., Wells, T. N., and Clemetson, K. J. (1999) J. Biol. Chem. 274, 29019-29024). However, the mechanism of the interaction between collagen and GPVI has not been analyzed in detail because both collagen and GPVI are insoluble molecules. In this study, we expressed the extracellular domain of GPVI as soluble forms as follows: the monomeric form (GPVIex) and the dimeric form of GPVI fused with the human immunoglobulin Fc domain (GPVI-Fc(2)). Purified GPVIex strongly inhibited convulxin (Cvx)-induced platelet aggregation but only weakly inhibited that induced by collagen-related peptide. However, only GPVI-Fc(2), and not GPVIex, inhibited collagen-induced platelet aggregation. The dimeric form of GPVI exhibits high affinity for collagen, as concluded from measurements of GPVI binding to immobilized collagen by both the enzyme-linked immunosorbent assay and surface plasmon resonance methods. GPVI-Fc(2) bound to the surface of immobilized collagen with a dissociation constant (K(D)) of 5.76 x 10(-7) m, but the binding of GPVIex was too weak to allow estimation of this parameter. Cvx did not inhibit the binding of dimeric GPVI to collagen, indicating that the binding site of GPVI to collagen was different from that to Cvx. Taken together, our data indicate that the high affinity binding site for collagen is composed from two chains of GPVI. Furthermore, they suggest that the binding sites for Cvx are different from the collagen-binding sites and do not need to be formed by two GPVI molecules. Because dimeric GPVI is the only form that shows high affinity to fibrous collagen, our results indicate that GPVI would be present as a dimeric form on the platelet. Moreover, surface plasmon resonance indicated that there is no detectable interaction between soluble collagen and GPVI, supporting our previous observation that GPVI only reacts with fibrous collagen.
- Published
- 2002
- Full Text
- View/download PDF
19. Association of Fyn and Lyn with the proline-rich domain of glycoprotein VI regulates intracellular signaling.
- Author
-
Suzuki-Inoue, Katsue, Tulasne, David, Shen, Yang, Bori-Sanz, Teresa, Inoue, Osamu, Jung, Stephanie M, Moroi, Masaaki, Andrews, Robert K, Berndt, Michael C, and Watson, Steve P
- Abstract
The glycoprotein VI (GPVI)-Fc receptor (FcR) gamma-chain complex, a key activatory receptor for collagen on platelet surface membranes, is constitutively associated with the Src family kinases Fyn and Lyn. Molecular cloning of GPVI has revealed the presence of a proline-rich domain in the sequence of GPVI cytoplasmic tail which has the consensus for interaction with the Src homology 3 (SH3) domains of Fyn and Lyn. A series of in vitro experiments demonstrated the ability of the SH3 domains of both Src kinases to bind the proline-rich domain of GPVI. Furthermore, depletion of the proline-rich domain in GPVI (Pro(-)-GPVI) prevented binding of Fyn and Lyn and markedly reduced phosphorylation of FcR gamma-chain in transiently transfected COS-7 cells, but did not affect the association of the gamma-chain with GPVI. Jurkat cells stably transfected with wild type GPVI show robust increases in tyrosine phosphorylation and intracellular Ca2+ in response to the snake venom convulxin that targets GPVI. Importantly, convulxin is not able to activate cells transfected with Pro(-)-GPVI, even though the association with the immunoreceptor tyrosine-based activation motif-containing chains is maintained. These findings demonstrate that the proline-rich domain of GPVI mediates the association with Fyn/Lyn via their SH3 domain and that this interaction initiates activation signals through GPVI.
- Published
- 2002
- Full Text
- View/download PDF
20. Signal-transducing Mechanisms Involved in Activation of the Platelet Collagen Receptor Integrin α2β1*
- Author
-
Jung, Stephanie M. and Moroi, Masaaki
- Abstract
Evidence was obtained about the mechanism responsible for platelet integrin α2β activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin α2β1activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin α2β1, but those operating via glycoprotein Ib cannot. Activation of α2β1induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin α2β1activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin α2β1activation, while at the high agonist concentrations, there would be several pathways through which integrin α2β1activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (Bmax) as thrombin-induced platelets, but their affinity (Kd) for soluble collagen was 3.7–12.7-fold lower, suggesting that activated integrin α2β1induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin α2β1; these activated forms of integrin α2β1would have different conformations that determine their ligand affinity.
- Published
- 2000
- Full Text
- View/download PDF
21. Involvement of Activated Integrin α2β1 in the Firm Adhesion of Platelets onto a Surface of Immobilized Collagen under Flow Conditions
- Author
-
Moroi, Masaaki, Onitsuka, Ichiro, Imaizumi, Tsutomu, and Jung, Stephanie M.
- Published
- 2000
- Full Text
- View/download PDF
22. Analysis of the Involvement of the von Willebrand Factor–Glycoprotein Ib Interaction in Platelet Adhesion to a Collagen-Coated Surface Under Flow Conditions
- Author
-
Moroi, Masaaki, Jung, Stephanie M., Nomura, Shosaku, Sekiguchi, Sadayoshi, Ordinas, Antonio, and Diaz-Ricart, Maribel
- Abstract
The requisite initial reaction for in vivo thrombus formation in flowing blood is platelet adhesion to the exposed surface of the extracellular matrix. The contribution of von Willebrand factor (vWF ) in plasma and glycoprotein (GP) Ib on the platelet membrane to platelet adhesion has been well-documented. We have recently developed a procedure (the “flow adhesion assay”) for measuring platelet adhesion under flow conditions that allowed us to characterize platelet adhesion to a collagen-coated surface. Here, we apply our method to analyze platelet adhesion to a vWF-coated surface to determine how this might differ from adhesion to a collagen-coated surface. Platelet adhesion to the vWF-coated surface was monitored as the linear increase in the area occupied by adherent platelets. The fluorescence image showed that platelets adhering to the vWF surface were mainly single platelets, and if any were present, the platelet aggregates were small, this being the primary difference from the adhesion to a collagen surface, where adherent platelets were mostly in aggregates. The flow adhesion assay detected the movement of platelets on the vWF surface, suggesting the reversible binding of vWF with platelets. The velocity of the platelets increased at higher shear rates or at lower vWF densities on the surface. Treatment of the vWF-coated surface with the aggregating agent botrocetin before initiation of blood flow increased platelet adhesion while dramatically decreasing the velocity of platelet movement. The present observations on the adhesion of platelets to the vWF-pretreated collagen surface and measurements of the velocity of platelets moving on the collagen surface suggest that the first interaction on the collagen-coated surface is the binding of vWF molecules to the collagen surface. This small number of vWF molecules would serve to attract and slow platelets flowing near the surface. This would facilitate the actual adhesion to the collagen surface that is mainly generated by the interaction between platelet collagen receptors, including GP Ia/IIa and GP VI, with collagen.
- Published
- 1997
- Full Text
- View/download PDF
23. Genetic Polymorphism of Platelet Glycoprotein Ib
- Author
-
Moroi, Masaaki, Jung, Stephanie M., and Yoshida, Nobuhiko
- Abstract
Platelet glycoprotein (GP) lb from 131 healthy Japanese was analyzed using SDS-polyacrylamide gel electrophoresis and specific staining with peroxidase-coupled wheat germ agglutinin after it was transferred to nitrocellulose membranes. Four slightly different species of GPIb were observed and designated as A, B, C, and D for glycoproteins with molecular weights of 168,000, 162,000, 159,000, and 153,000 daltons, respectively. The respective gene frequencies were calculated to be .073, .011, .561, and .355 for A-, B-, C-, and D-type GPIb. Portions from each type of GPIb molecule (α-chain and glycocalicin) showed heterogeneity with the same molecular weight difference, indicating that the variance would be derived from the polypeptide portion that is exposed to the outer medium. The different types of GPIb were the same with respect to their accessibility to lactoperoxidase, reactivity to lectins, and affinity to TLCK-thrombin. Although Bolin et al reported patients with a bleeding tendency whose platelets have double GPIb bands, here we found that platelets with different GPIb phenotypes showed no significant differences in aggregating activity and platelet retention. Analysis of GPIb phenotype should be important for structural and physiologic studies on GPIb and glycocalicin.
- Published
- 1984
- Full Text
- View/download PDF
24. Molecular Basis for Glanzmann’s Thrombasthenia (GT) in a Compound Heterozygote With Glycoprotein lib Gene: A Proposal for the Classification of GT Based on the Biosynthetic Pathway of Glycoprotein Ilb-IIIa Complex
- Author
-
Kato, Atushi, Yamamoto, Koh, Miyazaki, Sumio, Jung, Stephanie M., Moroi, Masaaki, and Aoki, Nobuo
- Abstract
The genetic basis for Glanzmann's thrombasthenia (GT) was elucidated on a compound heterozygote with glycoprotein (GP)IIb gene: an opal mutation at the end of exon 17 (CGA→ TGA) results in only a trace amount of GPIIb mRNA, and a splicing mutation at the acceptor site of exon 26 (CAG → GAG) causes an in-frame, exon skipping process from exon 25 to 27. This aberrant transcript encodes a single-chain polypeptide characterized by a 42-amino acid deletion, which includes the proteolytic cleavage site(s) and a unique, proline-rich region at the location corresponding to the carboxyl-terminal of the normal GPIIb α-chain. These characteristics are shared by a previously reported defective GPIIb molecule, which is neither assembled with GPIIIa nor transported to the cellular surface. Despite its normal transcription level, expression of the present defective GPIIb molecule was significantly decreased (~6% of the control level). Because the precursor GPIIb molecule is assembled with GPIIIa in the endoplasmic reticulum (ER) and its processing, as well as stability, is dependent on the GPIIIa subunit, the defective GPIIb molecule may be rapidly degraded by the intrinsic quality control system of the ER due to its inability to form a stable heterodimer complex as a consequence of its mis-folded structure. Although we did not confirm that the GPIIIa genes of this individual were normal, GPIIIa may be secondarily decreased (-11% of control), because a large part of it could not be complexed, making it vulnerable to proteolysis. To elucidate the molecular basis for GT, we propose here a classification of GT based on the biosynthetic pathway of the GPIIb-IIIa complex.
- Published
- 1992
- Full Text
- View/download PDF
25. Platelets Interact with Soluble and Insoluble Collagens through Characteristically Different Reactions*
- Author
-
Jung, Stephanie M. and Moroi, Masaaki
- Abstract
Platelet interaction with soluble and insoluble collagens was characterized through binding studies. In contrast to resting platelets, cells reacted with activators, TS2/16 (integrin α2β1-activating antibody), thrombin, collagen-related peptide, or ADP, exhibited specific soluble collagen binding that is Mg2+-dependent, but inhibited by prostaglandin I2, Ca2+, and Gi9 (anti-integrin α2β1antibody). Each platelet has 1500–3500 soluble collagen binding sites, with a dissociation constant of 3.5–9 × 10−8m. This is the first study to show the specific binding of soluble collagen to platelets; our data strongly suggest that the receptor is integrin α2β1after it becomes activated upon platelet activation. These results suggest that activation of platelets transforms integrin α2β1to a state with higher affinity binding sites for soluble collagen. The soluble collagen-platelet interaction was compared with the platelet interaction with fibrillar collagen, which has until now not been demonstrated to bind specifically to platelets. Here, we demonstrated specific, biphasic fibrillar collagen binding. One phase is rapid and metal ion-independent, and accounts for most of the binding. The other phase is slow and Mg2+-dependent. The characteristic differences in the specific bindings of soluble and fibrous collagens demonstrate the different contributions of two different collagen receptors.
- Published
- 1998
- Full Text
- View/download PDF
26. The Electrophoretic Mobility Heterogeneity of Human Platelet Subpopulations of Different Buoyant Densities
- Author
-
Jung, Stephanie M, Tanoue, Kenjiro, and Yamazaki, Hiroh
- Published
- 1985
- Full Text
- View/download PDF
27. Role of Surface Negative Charge in Platelet Function Related to the Hyperreactive State in Estrogen-Treated Prostatic Carcinoma
- Author
-
Jung, Stephanie M, Kinoshita, Kenji, Tanoue, Kenjiro, Isohisa, Ichiro, and Yamazaki, Hiroh
- Published
- 1982
- Full Text
- View/download PDF
28. Analysis of Platelet Adhesion to a Collagen-Coated Surface Under Flow Conditions: The Involvement of Glycoprotein VI in the Platelet Adhesion
- Author
-
Moroi, Masaaki, Jung, Stephanie M., Shinmyozu, Koichi, Tomiyama, Yoshiaki, Ordinas, Antonio, and Diaz-Ricart, Maribel
- Abstract
Platelet adhesion to the exposed surface of the extracellular matrix in flowing blood is the first and critical reaction for in vivo thrombus formation. However, the mechanism of this in vivo platelet adhesion has yet to be studied extensively. One of the reasons for this is the lack of a practical assay method for assessing platelet adhesion under flow conditions. We have devised an assay method (the fluorescent adhesion assay) that is based on the technique originally reported by Hubbell and McIntire (Biomaterials7:354, 1986) with some modifications to make it more amenable for assaying small samples and have developed an analysis method to quantify the extent of platelet adhesion and aggregation from fluorescence images by using a computer-assisted image analysis system. In our assay, platelet adhesion, expressed as the percentage of the area covered by adhered platelets, was found to increase biphasically as a function of time. In the first phase, platelets interacted with the coated collagen, transiently stopping on the surface; we called this reaction the temporary arrest. In the second phase, platelets adhered much more rapidly and permanently on the surface, and this adhesion was dependent on the shear rate; platelets formed aggregates in this phase. We used our assay to analyze the effects of platelet aggregation inhibitors on platelet adhesion. All three examined inhibitors, EDTA (10 mmol/L), antiglycoprotein (GP) llb/llla, and GRGDS peptide (1 mmol/L), inhibited the second phase adhesion in flowing blood. Furthermore, GPVI-deficient platelets also showed defective second-phase adhesion under the same conditions. These results suggested that GPIIb/llla activation and GPVI contribute to the reaction inducing the second phase. The second-phase adhesion has been extensively investigated, and the consensus is that this reaction is mainly attributable to the platelet-platelet interaction. In this report, we were able to detect an earlier reaction, the temporary arrest. This temporary arrest would reflect the fast and weak interaction between platelet GPIb/IX and collagen-von Willebrand factor complexes on the collagen-coated surface.
- Published
- 1996
- Full Text
- View/download PDF
29. Thrombasthenia With an Abnormal Platelet Membrane Glycoprotein lib of Different Molecular Weight
- Author
-
Jung, Stephanie M., Yoshida, Nobuhiko, Aoki, Nobuo, Tanoue, Kenjiro, Yamazaki, Hiroh, and Moroi, Masaaki
- Abstract
We describe an individual with abnormal platelet glycoprotein (GP) lIb of different molecular weight (mol wt), a defect that distinguishes this patient from previously reported thrombasthenics. The patient, a 21-year-old female, has a mild bleeding tendency; her platelets lack adenosine diphosphate (ADP) aggregation and have severely suppressed collagen aggregation but a normal response to ristocetin. Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis of her platelets indicates that they contain two types of GPIIb molecules: one with an abnormal mol wt (122 kd, unreduced; 128 kd, reduced) and one with a normal mol wt (128 kd, unreduced; 118 kd, reduced). Relative to the amount of GPIIb in normal platelets, her platelets contain approximately 35% abnormal GPIIb and 20% normal GPIIb. Fibrinogen binding assays on the patient's platelets indicated that they contained 25% of the normal amount of fibrinogen receptors. Crossed immunoelectrophoresis of the patient's platelets demonstrated the formation of a GPIIb/IlIa complex that was mainly composed of normal mol wt GPIIb and GPIIIa. The patient's father has decreased ADP aggregability, and his platelets also contained both abnormal and normal GPIIb (about 50% of the normal level and about 50% of the normal number of fibrinogen receptors); her mother has only normal GPIIb. These results indicate that the patient has heterozygous GPIIb molecules with an abnormality of GPIIb at the molecular level. Studies on this abnormal GPIIb should provide information about the function of GPIIb and the mechanism of its biosynthesis.1988 by Grune& Stratton, Inc. 0006-4971/88/7104-0039$3.00/0
- Published
- 1988
- Full Text
- View/download PDF
30. The Increase of the Electrophoretic Mobility of Platelets after Laparotomy
- Author
-
Yamazaki, Hiroh, Tsukui, Ryoko, Motomiya, Takeshi, Jung, Stephanie May, Sonoda, Minoru, Watanabe, Chifuyu, Ogino, Masahiro, and Miyagawa, Noboru
- Published
- 1980
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.