Purification and functional characterization of homodimeric gamma B- gamma B fibrinogen from rat plasma

Lawrence, SO; Tw, Wright; Francis, C.W.; Fay, PJ; Haidaris, PJ

doi:10.1182/blood.v82.8.2406.bloodjournal8282406

Cited by 8 publications

(12 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…After immunopurification, samples were analyzed by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) on 8% gels according to Laemmli as described in detail (19). For 2D-gel electrophoresis, nonreduced protein was resolved in the first dimension on horizontal 2% agarose slab gels prepared in 0.1% SDS and 50 mM phosphate buffer, pH 7.0; the gel running buffer consisted of 100 mM phosphate buffer, pH 7.0 with 0.1% SDS.…”

Section: Electrophoretic Analysis Of Immunopurified Fbgmentioning

confidence: 99%

“…The agarose strip was transferred to the top of an 8% polyacrylamide gel and overlaid with 0.5% agarose with 0.1 mM dithiothreitol for the second dimension electrophoresis in the discontinuous buffer system of Laemmli. Continuous buffer Weber-Osborn SDS-PAGE was carried out using 7% gels as recently described in detail (19). Electrophoresis reagents were purchased from BioRad (Hercules, CA, USA).…”

Section: Electrophoretic Analysis Of Immunopurified Fbgmentioning

confidence: 99%

See 1 more Smart Citation

Regulated de novo biosynthesis of fibrinogen in extrahepatic epithelial cells in response to inflammation

Lawrence¹,

Simpson-Haidaris²

2004

Thromb Haemost

View full text Add to dashboard Cite

Hepatic fibrinogen (FBG) is upregulated during an acute phase response (APR) induced by glucocorticoids and interleukin (IL)-6. Furthermore, intestine and lung epithelium synthesize FBG after exposure to inflammatory mediators, and both plasma and lung cell-derived FBG, along with fibronectin, assemble in detergent-insoluble extracellular matrices (ECM) of pneumocytes and fibroblasts independent of thrombin or plasmin cleavage. An epitope cryptic in soluble FBG (beta(15-21)) but exposed in matrix-FBG and fibrin induces cell proliferation and actin cytoskeleton reorganization during wound repair and angiogenesis. Although fibrin(ogen) is involved in hemostasis and homeostasis, mechanisms regulating extrahepatic FBG expression remain unexplored. Herein we examined FBG production by lung compared to liver epithelial cell lines in response to dexamethasone (DEX)+IL-6. Regulated synthesis of HepG2-FBG follows the pathway shown for constitutive synthesis by liver epithelium. Constitutive A549-FBG expression was not detectable, however, intracellular FBG precursors in DEX+IL-6-treated A549 lung cells were similar to HepG2 cells with two notable exceptions. The relative rate of chain synthesis in HepG2 cells was unequal, whereas nascent synthesis of all three chains occurred at equivalent rates in stimulated A549 cells. Unlike HepG2 cells, which rapidly secreted intact FBG, nascent dimeric FBG accumulated in the A549 cell-associated fraction prior to release into medium. Furthermore, soluble A549-FBG was susceptible to thrombin and plasmin cleavage. Interestingly, many functionally diverse proteins possess FBG-related domains that direct cell-fate determination during development or wound repair, suggesting that extrahepatic FBG biosynthesis evoked only during inflammation plays such a role during localized injury and repair to restore tissue homeostasis.

show abstract

Section: Electrophoretic Analysis Of Immunopurified Fbgmentioning

confidence: 99%

Section: Electrophoretic Analysis Of Immunopurified Fbgmentioning

confidence: 99%

Regulated de novo biosynthesis of fibrinogen in extrahepatic epithelial cells in response to inflammation

Lawrence¹,

Simpson-Haidaris²

2004

Thromb Haemost

View full text Add to dashboard Cite

show abstract

“…Mouse, rat, guinea pig, and bovine FBG were purchased from Sigma (St. Louis, MO). Ferret FBG was purified as previously described (15,16). SDS-PAGE was performed according to Laemmli as more recently described in detail (15).…”

Section: Western Blot Analysismentioning

confidence: 99%

“…Ferret FBG was purified as previously described (15,16). SDS-PAGE was performed according to Laemmli as more recently described in detail (15). Total protein was visualized by staining gels with either silver or Coomassie Brilliant Blue.…”

Section: Western Blot Analysismentioning

confidence: 99%

Characterization of a Monoclonal Antibody, D73H, that Maps to a Highly Conserved Region on Fibrinogen Bβ Chain

Rybarczyk

Pereira²,

Simpson-Haidaris³

2000

Thromb Haemost

View full text Add to dashboard Cite

SummaryThe primary structure of fibrinogen is highly conserved across species, yet often times monoclonal antibodies produced against the fibrinogen of one species will not crossreact with the fibrinogen of another. Herein, we describe the production and characterization of murine MAb, D73H, raised against human fibrinogen. D73H crossreacts with a highly conserved epitope on the Bβ chain of fibrinogen from human, rat, bovine, guinea pig, and mouse. Western blotting revealed that D73H reacted with the Bβ chain of plasmin fragment D, localizing its epitope to Bβ134-461. A 7 kDa band was identified by D73H in Western blots of reduced fibrinogen CNBr-fragments. N-terminal sequencing mapped this fragment to Bβ243-253, further localizing the epitope to Bβ243-305. In silico analysis indicated that Bβ243-305 is predominantly hydrophilic, and surface probability prediction indicated three potential antigenic determinants corresponding to Bβ252-258, Bβ262-269, and Bβ279-286. Further in silico analysis of the crystal structure of fibrinogen fragment D-D indicated that Bβ262-269 (FGRKWDPY) is predominantly α-helical and located on the surface of the molecule adjacent to a bend imposed in the β chain at residue 260, which is near the junction between the rigid coiled-coil domain and the globular C-terminus. A synthetic peptide corresponding to Bβ261-272 competitively inhibited the binding of D73H to the Bβ chain of denatured intact fibrinogen and reduced and denatured Bβ chain in Western blots, experimentally proving the validity of these predictive algorithms. Together these data indicate that, although plasmin resistant, Bβ chain residues Bβ261-272 comprising the D73H epitope are highly conserved across species, surface exposed, and immunogenic.

show abstract

“…IEM. Polyclonal antiserum was raised in rabbits against purified mouse FBG, and the immunoglobulin G (IgG) fraction was affinity purified (28,46). P. cariniiinfected SCID mice were sacrificed, and the lungs were inflation fixed in situ with 4% paraformaldehyde-0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, as previously described (57).…”

Section: Sds-page and Western Blot Analysis Of Human And Ferret Plasmmentioning

confidence: 99%

Induction of Fibrinogen Expression in the Lung Epithelium during Pneumocystis carinii Pneumonia

et al. 1998

View full text Add to dashboard Cite

Pneumocystis carinii is an important pulmonary pathogen responsible for morbidity and mortality in patients with AIDS. The acute-phase response (APR), the primary mechanism used by the body to restore homeostasis following infection, is characterized by increased levels of circulating fibrinogen (FBG). Although the liver is the primary site of increased FBG synthesis during the APR, we unexpectedly discovered that FBG is synthesized and secreted by lung alveolar epithelial cells in vitro during an inflammatory stimulus. Therefore, we sought to determine whether lung epithelial cells produce FBG in vivo using animal models of P. carinii pneumonia (PCP). Inflammation was noted by an influx of macrophages to P. carinii-infected alveoli. Northern hybridization revealed that ␥-FBG mRNA increased two-to fivefold in P. carinii-infected lung tissue, while RNA in situ hybridization demonstrated increased levels of ␥-FBG mRNA in the lung epithelium. Immunoelectron microscopy detected lung epithelial cell-specific production of FBG, suggesting induction of a localized inflammatory response resembling the APR. A systemic APR was confirmed by a two-to fivefold upregulation of the levels of hepatic ␥-FBG mRNA in animals with PCP, resulting in a corresponding increase in levels of FBG in plasma. Furthermore, immunoelectron microscopy revealed the presence of FBG at the junction of cell membranes of trophic forms of P. carinii organisms aggregated along the alveolar epithelium. These results implicate FBG in the pathogenesis of PCP in a manner similar to that of the adhesive glycoproteins fibronectin and vitronectin, which are known to participate in intra-alveolar aggregation of organisms and adherence of P. carinii to the lung epithelium.

show abstract

Purification and functional characterization of homodimeric gamma B- gamma B fibrinogen from rat plasma

Cited by 8 publications

References 0 publications

Regulated de novo biosynthesis of fibrinogen in extrahepatic epithelial cells in response to inflammation

Regulated de novo biosynthesis of fibrinogen in extrahepatic epithelial cells in response to inflammation

Characterization of a Monoclonal Antibody, D73H, that Maps to a Highly Conserved Region on Fibrinogen Bβ Chain

Induction of Fibrinogen Expression in the Lung Epithelium during Pneumocystis carinii Pneumonia

Contact Info

Product

Resources

About