From the Departments of Pathology and Molecular
Medicine, ¶ Pediatrics, and
Medicine, McMaster University
and the Henderson Research Centre, Hamilton, Ontario L8V 1C3 and the
** Division of Experimental Therapeutics, Ontario Cancer
Institute, Toronto, Ontario M5G 2M9, Canada
Received for publication, January 29, 2003
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ABSTRACT |
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Previous studies have
demonstrated that overexpression of GRP78/BiP, an endoplasmic
reticulum (ER)-resident molecular chaperone, in mammalian cells
inhibits the secretion of specific coagulation factors. However, the
effects of GRP78/BiP on activation of the coagulation cascade
leading to thrombin generation are not known. In this study, we
examined whether GRP78/BiP overexpression mediates cell surface
thrombin generation in a human bladder cancer cell line T24/83 having
prothrombotic characteristics. We report here that cells overexpressing
GRP78/BiP exhibited significant decreases in cell surface-mediated
thrombin generation, prothrombin consumption and the formation of
thrombin-inhibitor complexes, compared with wild-type or
vector-transfected cells. This effect was attributed to the ability of
GRP78/BiP to inhibit cell surface tissue factor (TF) procoagulant
activity (PCA) because conversion of factor X to Xa and factor VII to
VIIa were significantly lower on the surface of
GRP78/BiP-overexpressing cells. The additional findings that (i) cell
surface factor Xa generation was inhibited in the absence of factor
VIIa and (ii) TF PCA was inhibited by a neutralizing antibody to human
TF suggests that thrombin generation is mediated exclusively by TF.
GRP78/BiP overexpression did not decrease cell surface levels of TF,
suggesting that the inhibition in TF PCA does not result from retention
of TF in the ER by GRP78/BiP. The additional observations that both
adenovirus-mediated and stable GRP78/BiP overexpression attenuated TF
PCA stimulated by ionomycin or hydrogen peroxide suggest that GRP78/BiP
indirectly alters TF PCA through a mechanism involving cellular
Ca2+ and/or oxidative stress. Similar results were
also observed in human aortic smooth muscle cells transfected with the
GRP78/BiP adenovirus. Taken together, these findings demonstrate
that overexpression of GRP78/BiP decreases thrombin generation by
inhibiting cell surface TF PCA, thereby suppressing the prothrombotic
potential of cells.
In eukaryotic cells, the endoplasmic reticulum
(ER)1 is the cellular
organelle where secretory proteins or proteins destined for the plasma
membrane undergo a variety of modifications, including disulfide bond
formation, glycosylation, folding, and oligomeric assembly. The
inability of nascent polypeptide chains to fold into their native
conformation generally leads to retention in the ER and degradation
(1). To assist in the correct folding of newly synthesized proteins and
to prevent aggregation of folding intermediates, the ER contains a wide
range of molecular chaperones such as the glucose-regulated proteins,
calnexin, calreticulin, protein-disulfide isomerase, and
Erp72 (2, 3). These chaperones are postulated to act as a quality
control system by ensuring that only correctly folded proteins are
processed prior to entering the Golgi apparatus for further processing
and secretion (2-4). In addition to their function as molecular
chaperones, recent studies have demonstrated that some of these ER
proteins protect cells against oxidative stress (5-7).
Prominent among the ER-resident chaperones is the 78-kDa
glucose-regulated protein/immunoglobulin-binding protein
(GRP78/BiP), a highly conserved member of the 70-kDa heat
shock protein family (8, 9) and a major ER luminal Ca2+
storage protein (10, 11). GRP78/BiP transiently associates with
correctly folded proteins but forms more stable complexes with
misfolded or incompletely assembled proteins (1, 4, 12). This
association involves the binding of GRP78/BiP to hydrophobic motifs
exposed on unfolded or unassembled polypeptides (13-15). Proteins
stably bound to GRP78/BiP are subsequently translocated from the ER into the cytosol for proteasome-dependent
degradation (16, 17). The observation that GRP78/BiP is induced by
conditions of stress that result in the accumulation of misfolded or
underglycosylated proteins in the ER (8, 9) provides further evidence
that GRP78/BiP plays a critical role in quality control processes
during protein synthesis and folding.
Recent studies have demonstrated that alterations in GRP78/BiP protein
levels mediate selective changes in the abundance of membrane or
secretory proteins that transit the ER. In mammalian cells, reduced
levels of GRP78/BiP increase the secretion of factor VIII and a mutant
form of tissue plasminogen activator lacking glycosylation sites
(18-20). In contrast, elevated levels of GRP78/BiP decrease the
secretion of von Willebrand factor (vWf) and factor VIII (18-20).
Although these studies provide convincing evidence that alterations in
GRP78/BiP levels lead to selective changes in the processing and
secretion of certain coagulation factors, the effect of GRP78/BiP on
the prothrombotic potential of cells was not investigated.
The major physiological initiator of the extrinsic coagulation cascade
is tissue factor (TF), a 47-kDa transmembrane glycoprotein (21-24). TF
initiates the coagulation cascade by complexing with factor VII/VIIa on
the surface of cells, leading to the activation of factors IX and X and
the subsequent generation of thrombin. TF expression is increased in
atherosclerotic plaques (25-31) and in vascular smooth muscle cells
following balloon injury (32). Infection with adenovirus, as well as
other respiratory viruses, increases TF expression and procoagulant
activity (PCA) (33). Growth factors (34) and cytokines (35, 36),
endotoxin (37, 38), hypoxia (39), cell injury (40), reactive oxygen
species (ROS) (41-43), oxidized LDL (44-46), and increases in
intracellular free Ca2+ (47-50) are also known to induce
TF PCA in a variety of cell types, including vascular endothelial
cells, smooth muscle cells, and circulating monocytes. Furthermore, the
majority of tumor cells express TF (51-54), and the prothrombotic
state observed in cancer patients has been largely attributed to this
expression (53).
Given that TF transits the ER prior to its localization in the plasma
membrane, we have postulated that GRP78/BiP overexpression could
potentially alter the prothrombotic potential of cells. In this study,
GRP78/BiP was stably overexpressed in a human bladder cancer cell line
T24/83 having prothrombotic characteristics. Our findings indicate that
overexpression of GRP78/BiP significantly decreases TF-mediated cell
surface thrombin generation. The finding that GRP78/BiP overexpression
fails to decrease cell surface levels of TF suggests that the
inhibition of TF PCA by GRP78/BiP does not result from the retention of
TF in the ER. The ability of GRP78/BiP overexpression to attenuate TF
PCA induced by ionomycin, hydrogen peroxide, or adenovirus supports a
mechanism involving cellular Ca2+ and/or oxidant stress.
These findings show that GRP78/BiP overexpression decreases TF PCA and
provides novel evidence that alterations in the levels of GRP78/BiP can
have profound effects on the prothrombotic potential of cells.
Cell Culture and Treatment Conditions--
The human
transitional bladder carcinoma cell line T24/83 was obtained from the
American Type Culture Collection and cultured in M199 medium containing
10% fetal bovine serum (FBS), 100 µg/ml penicillin, and 100 µg/ml
streptomycin in a humidified incubator at 37 °C with 5%
CO2. Although previous studies classified T24/83 cells as
an immortalized human umbilical vein endothelial cell line, designated
ECV304 (55), recent genetic analysis and functional responses have
demonstrated that ECV304 cells are indeed T24/83 (56). Human aortic
smooth muscle cells (HASMC) were purchased from Cascade Biologicals
(Portland, OR) and cultured in medium 231 containing 20% smooth muscle
growth supplement and PSA solution (100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin) (Cascade Biologicals).
Establishment of Stable T24/83 Cell Lines Overexpressing
GRP78/BiP--
Construction of the mammalian expression
vector, pcDNA3.1(+)- GRP78/BiP, encoding human GRP78/BiP and the
establishment of stable T24/83 cell lines overexpressing human
GRP78/BiP have been described previously (57). As a vector control,
T24/83 cells were transfected with pcDNA3.1(+) under the same
conditions. Overexpression of GRP78/BiP was assessed by indirect
immunofluorescence and immunoblot analysis, as described below.
Immunoblot Analysis--
Monoclonal and polyclonal antibodies to
human TF were purchased from American Diagnostica (Greenwich, CT).
The anti-KDEL monoclonal antibody (SPA-827), which recognizes both
GRP78/BiP and GRP94, was purchased from StressGen Biotechnologies
(Victoria, BC). Total protein lysates from T24/83 cells were
solubilized in Laemmli sample buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 1% Indirect Immunofluorescence and Image Analysis--
Polyclonal
antibodies to GRP78/BiP (sc-1050) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Immunofluorescence for GRP78/BiP was
performed as described previously (58, 59). For detection of cell
surface levels of TF, cells were washed with ice-cold 1× PBS for 2 min, blocked with 3% BSA in 1× PBS for 1 h at 4 °C and
incubated with an anti-human TF monoclonal antibody (5 µg/ml) for
1 h at 4 °C. After three washes with 1× PBS containing 0.5%
BSA, cells were incubated with the appropriate Alexa-labeled secondary
antibody (BioLynx, Brockville, ON) for 1 h at 4 °C, washed
again, and fixed with 1% paraformaldehyde. Images were subsequently
captured and analyzed using Northern Exposure image analysis/archival
software (Mississauga, ON).
Immunoprecipitation of Cell Surface TF--
Cell surface TF was
immunoprecipitated essentially as described previously (42). Briefly,
anti-human TF polyclonal antibodies at 0.5 µg/ml in FBS-free M199
medium were added to cell monolayers at 4 °C for 2 h. After
washing twice with ice-cold 1× PBS, cells were lysed in ice-cold RIPA
buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet
P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitors (Roche Diagnostics, Laval, QC), and cell surface TF protein
was immunoprecipitated from total cell protein by overnight incubation
with protein A-Sepharose-linked beads (Pierce). After washing with RIPA
buffer, the beads were boiled in Laemmli sample buffer, and the
immunoprecipitates were separated on 10% SDS-polyacrylamide gels under
reducing conditions. Gels were transferred to nitrocellulose membranes
and immunoblotted with anti-human TF polyclonal antibodies, as
described above.
Biotinylation of Cell Surface Proteins--
Cell surface
biotinylation was performed as described previously (60, 61). Briefly,
cell monolayers were washed once with ice-cold FBS-free M199 medium and
three times with ice-cold 1× PBS to remove residual FBS and other
proteins from the culture medium. PBS containing 1 mg/ml EZ Link
NHS-SS-Biotin (Pierce) was added to the monolayers, and the
biotinylation reaction was carried out at room temperature for 30 min
with gentle shaking. Following three washes with ice-cold 1× PBS,
cells were solubilized in Laemmli sample buffer. Proteins were
separated on 10% SDS-polyacrylamide gels and transferred to
nitrocellulose membranes. To detect biotinylated cell surface proteins,
immunoblot analysis was performed using HRP-conjugated ExtrAvidin (Sigma).
Thrombin Generation--
Thrombin generation studies were
conducted with either control pooled plasma from healthy adults or
factor VII-deficient plasma (Affinity Biologicals). Factor VII levels
in the deficient plasmas were Prothrombin Consumption--
EDTA samples were used to determine
prothrombin consumption during the experiments. Prothrombin
concentrations were determined for each time point during the thrombin
generation experiments using a commercially available ELISA kit.
Control plasma with a known concentration of prothrombin was used as a standard.
Factor VII Activation Assay--
Factor VIIa generation on the
surface of T24/83 cells was performed by a one-step assay, which
directly measures the conversion of factor VII to factor VIIa, using
the chromogenic substrate S-2288 (65). Cell monolayers were washed
twice in ABS buffer and incubated for time periods up to 30 min in the
absence (blank control) or presence of 108 nM recombinant
human factor VII (Enzyme Research Laboratories, South Bend, IN) and 5 mM Ca2+ in ABS buffer. The generation of factor
VIIa was assessed by subsampling cell surface supernatant into S-2288,
followed by incubation at 37 °C for 30 min. After termination with
50% acetic acid, absorbance at 405 nm was determined for each sample.
The rates of factor VIIa formation in the various samples were
calculated based on a standard curve using known amounts of human
factor VIIa (Enzyme Research Laboratories) and TF (Thromborel S, Dade Behring, Newark, DE).
Cell Surface Tissue Factor Activity Assay--
Cell surface TF
activity, measured as the amount of factor Xa generated, was performed
as described previously (66, 67). Briefly, cells were seeded onto
24-well tissue culture plates and upon reaching confluence, the culture
medium was removed, and the cells were washed once with 1× PBS and
incubated with FBS-free medium for 1 h at 37 °C. Cells in
FBS-free medium were untreated or treated with ionomycin (5-10
µM for 10 min) or hydrogen peroxide (10 mM
for 1 h). Cells were washed twice with 1× TBS (50 mM
Tris, 120 mM NaCl, 2.7 mM KCl, 3 mg/ml BSA, pH
7.4) and incubated with 300 µl of 1× TBS containing 0.5 nM human factor VIIa and 15 nM human factor X
(Enzyme Research Laboratories). Activation of factor X was initiated by
the addition of 5 mM CaCl2 for 30 min at
37 °C. 250-µl aliquots were removed and incubated with 25 µl of
chromogenic substrate S-2765 (0.2 mM) (DiaPharma, West
Chester, OH) for 3 min at 37 °C. The reaction was terminated using
20 µl of 50% acetic acid, and the absorbance at 405 nm was measured
using a microplate reader. Standards containing the reaction mixture
and various amounts of rabbit brain thromboplastin (Thromboplastin C
Plus, Dade Behring) were also prepared and incubated with S-2765 as
described above. The absorbance at 405 nm was determined and used to
generate a standard curve where 100 units of TF activity was defined as
the amount of activity in 1 µl of rabbit brain thromboplastin. Cells
in the 24-well plates were lysed in RIPA buffer, and the amount of
total protein was measured using the DC Protein Assay
(Bio-Rad) according to the manufacturer's instructions. Absorbances
were measured at 750 nm and the amount of total protein calculated by
comparing the OD750 with reference standards of Bio-Rad
Protein Assay Standard II. Cell surface TF PCA was calculated as the
amount of factor Xa generated per µg of total protein
(units/µg).
Adenoviral Infection--
The construction and large scale
preparation of recombinant adenoviral vectors expressing GRP78/BiP
(AdV-GRP78) or Statistical Analysis--
Data represent the mean ± S.E.
Significance of differences between control and
GRP78/BiP-overexpressing cells was determined by analysis of variance
(ANOVA). On finding significance with ANOVA, unpaired Student's
t test was performed. For all analyses, p < 0.05 was considered significant.
Previous studies have demonstrated that non-stimulated T24/83
cells constitutively express both TF mRNA and antigen, which correlate with an increase in cell surface TF levels and activity (69-71). TF PCA in unstimulated T24/83 cells is significantly greater than other cells lines, including human umbilical vein endothelial cells and unstimulated monocytes (69, 70). Furthermore, TF expression
in T24/83 cells was increased by phorbol myristate acetate, a known
inducer of TF mRNA and protein in a wide variety of cultured cells
(69). Based on these findings and given the important role of TF in
blood coagulation and thrombosis, T24/83 cells were selected as our
model system to study the effect of GRP78/BiP overexpression on
TF-dependent thrombin generation.
Stable Overexpression of GRP78/BiP in
T24/83 Cells--
T24/83 cells were transfected with either
the pcDNA3.1(+)-GRP78/BiP expression vector or pcDNA3.1(+)
alone and stable transfectants selected in complete medium containing
G418. Total cell lysates isolated from wild-type or stably transfected
T24/83 cells were analyzed for GRP78/BiP protein by immunoblot analysis
using an anti-KDEL monoclonal antibody, which recognizes both human
GRP78/BiP and GRP94. As shown in Fig.
1A, two independently isolated
G418-resistant cell lines, c1 and c2 (designated T24/83-GRP78c1 and c2,
respectively) exhibited a 3.3-fold increase in GRP78/BiP protein
levels, compared with wild-type or vector-transfected cells. In
contrast, GRP94 was unchanged in all cell lines, suggesting that
alterations in GRP78/BiP protein levels do not alter endogenous GRP94
protein levels. To compare the cellular levels and distribution of
GRP78/BiP protein, wild-type, vector-transfected, or
GRP78/BiP-overexpressing cells cultured on coverslips were fixed,
permeabilized, and examined by indirect immunofluorescence using
anti-GRP78/BiP polyclonal antibodies. In both wild-type and
GRP78/BiP-overexpressing cells, GRP78/BiP was concentrated in the
perinuclear region, consistent with its location in the ER (Fig.
1B). However, the fluorescence intensity was much greater in
the GRP78/BiP-overexpressing cells, compared with wild-type and
vector-transfected cells. Nonspecific immunostaining was not detected
in cells in which the primary antibody had been omitted or in which
preimmune mouse IgG was substituted for the primary antibody (data not
shown). In terms of cellular function, overexpression of GRP78/BiP
suppressed the induction of endogenous GRP78/BiP mRNA levels and
increased survival of T24/83 cells exposed to the ER stress-inducing
agent, A23187 (data not shown), findings consistent with those observed
in GRP78/BiP-overexpressing CHO cells (7).
GRP78/BiP protein levels present in the stable GRP78/BiP-overexpressing
cell lines were comparable to levels observed in wild-type cells
treated with known ER stress-inducing agents, including homocysteine,
dithiothreitol, and tunicamycin (Fig. 1C). This observation
suggests that the levels of GRP78/BiP observed in these stable cell
lines are within the range that can be attained under physiological
conditions of ER stress. However, unlike the GRP78/BiP-overexpressing
cell lines, cells treated with ER stress-inducing agents had an
expected increase in GRP94 protein levels.
Overexpression of GRP78/BiP Decreases Thrombin
Generation, Prothrombin Consumption, and the Formation of
Thrombin-Inhibitor Complexes on the Surface of T24/83
Cells--
Thrombin generation, prothrombin consumption, and
formation of thrombin-inhibitor complexes were determined using
normal defibrinated human plasma. In control plasma, after the addition
of Ca2+, the concentration of physiologically active free
thrombin generated on the surface of wild-type or vector-transfected
cells increased significantly between 2 and 22 min (p < 0.001), with peak concentrations reaching 110 ± 16 and
131 ± 2 nM, respectively, by 4 min (Fig. 2A). In contrast, the
concentration of free thrombin generated on the surface of the stable
GRP78/BiP-overexpressing cell lines was negligible for all time points
examined, up to 25 min. Consistent with these findings, both
prothrombin consumption (Fig. 2B) and formation of
thrombin-inhibitor complexes (Table I)
were significantly reduced in the stable GRP78/BiP-overexpressing
cells, compared with wild-type or vector-transfected cells
(p < 0.001).
Overexpression of GRP78/BiP Inhibits
TF-dependent Factor VII and Factor X
Activation--
Factor VII-depleted plasma was used to initially
determine whether GRP78/BiP overexpression may have selectively
impaired a component of the extrinsic pathway such as TF. Thus, in the presence of factor VII-depleted plasma, free thrombin generation and
prothrombin consumption were negligible for all cell lines over the
25-min time period (data not shown), a result consistent with the
requirement of factor VIIa for TF-dependent thrombin generation (62-65). To specifically examine TF PCA on cell surfaces, wild-type, vector-transfected, or GRP78/BiP-overexpressing cells were
grown to confluency and factor Xa generation was measured over 30 min
using the chromogenic substrate S-2765. As shown in Fig.
3A, the rate of factor Xa
generation was significantly decreased in the GRP78/BiP-overexpressing
cells, compared with wild-type and vector-transfected cells
(p < 0.005).
To determine whether GRP78/BiP overexpression directly impaired the
conversion of factor VII to VIIa at the cell surface by TF, factor VIIa
generation was measured over 30 min using the chromogenic substrate
S-2288. As shown in Fig. 3B, the rate of factor VIIa
generation on the surface of GRP78/BiP-overexpressing cells was
significantly decreased, compared with wild-type and vector-transfected
cells (p < 0.005). The observation that the amidolysis
of S-2288 was negligible in the absence of factor VII by 30 min
indicates that amidolysis of the substrate correlates with the
generation of factor VIIa and is not due to the presence of other
cellular factors known to hydrolyze the substrate (i.e. tissue plasminogen activator, kallikrein).
To ensure that the PCA measured was mediated by TF, control experiments
measuring the amount of factor Xa generated were performed on wild-type
T24/83 cells. Negligible factor Xa was generated on the cell surface
upon exclusion of factor VIIa from the reaction, indicating that the
generation of factor Xa (which corresponds to PCA) was factor VIIa-TF
dependent (Fig. 4A).
Furthermore, the addition of increasing concentrations of a
neutralizing anti-human TF antibody prior to measuring PCA decreased
the amount of factor Xa generated in a dose-dependent
manner, with complete inhibition occurring at 10 µg/ml (Fig.
4B). No inhibition was observed using rabbit IgG (data not
shown). These findings are consistent with previously published data
that PCA on the surface of T24/83 cells is TF-dependent
(69, 70) and provides strong evidence that the generation of factor Xa
was mediated exclusively by TF.
Localization of TF on the Cell Surface of T24/83
Cells--
To determine whether alterations in TF PCA were the result
of changes in the cellular levels of TF, both non-permeabilized (cell
surface) and permeabilized (total) T24/83 cells were examined by
indirect immunofluorescence using an anti-human TF monoclonal antibody
(Fig. 5). Non-permeabilized cells were
characterized by clusters of TF in defined patches distributed over the
cell surface (Fig. 5, A-C). The intensity of TF staining
was not decreased in the GRP78/BiP-overexpressing cells (Fig.
5C), compared with wild-type (Fig. 5A) or
vector-transfected (Fig. 5B) cells. Permeabilized cells
exhibited both cell surface and perinuclear staining of TF, a finding
consistent with previous studies (72). Again, the intensity of staining
was not decreased in the GRP78/BiP-overexpressing cells (Fig.
5F), compared with wild-type (Fig. 5D) or
vector-transfected (Fig. 5E) cells. Nonspecific
immunostaining was not detected in cells in which the primary antibody
had been omitted or in which preimmune mouse IgG was substituted for
the primary antibody (data not shown). Consistent with these findings,
immunoprecipitation experiments did not show a decrease in cell
surface TF on GRP78/BiP-overexpressing cells, compared with wild-type
or vector-transfected cells (Fig. 6A).
Effects of GRP78/BiP Overexpression on Cell Surface
Protein Levels--
To determine whether GRP78/BiP overexpression
mediates the levels of other cell surface proteins, intact wild-type,
vector-transfected, or GRP78/BiP-overexpressing cells were labeled
using NHS-SS-Biotin, a membrane impermeable form of biotin. Following
biotinylation, cells were washed in 1× PBS, and total cell lysates
were separated on 10% SDS-polyacrylamide gels, transferred to
nitrocellulose membranes, and biotinylated proteins detected using
HRP-conjugated ExtrAvidin. As shown in Fig. 6B, there was no
significant difference in the migration pattern or intensity of
staining of cell surface biotinylated proteins among the different cell
types (lanes 4-8). No staining was observed in the lysates
from cells that were not pretreated with NHS-SS-Biotin (lanes
1-4). Consistent with these findings, the levels of insulin
receptor, caveolin-1, and Fas were not altered among the different cell
lines (data not shown).
Overexpression of GRP78/BiP Attenuates Ionomycin- or
Hydrogen Peroxide-induced TF PCA--
Previous studies have
demonstrated that increases in cytosolic Ca2+ levels by
treatment with the Ca2+ ionophores, ionomycin or A23187,
increases TF PCA, at a rate independent of de novo protein
synthesis (47-50). Treatment of cells with hydrogen peroxide also
increases TF PCA, which is independent of an increase in TF protein
levels (42, 46). Given that GRP78/BiP is a Ca2+-binding
protein and protects cells from oxidative stress (5-7), the effects of
ionomycin and hydrogen peroxide on cell surface TF PCA were examined.
Factor Xa generation was significantly increased in all cell lines
exposed to ionomycin or hydrogen peroxide, compared with untreated
cells (Fig. 7). However, the absolute
increase in TF PCA upon ionomycin (Fig. 7A) or hydrogen
peroxide (Fig. 7B) treatment was significantly lower
(p < 0.05) in the GRP78/BiP-overexpressing cells,
compared with wild-type and vector-transfected control cells. These
findings provide evidence that GRP78/BiP overexpression inhibits the
stimulation of TF PCA by agents that are known to increase
intracellular Ca2+ or oxidative stress.
Adenovirus-mediated GRP78/BiP Overexpression Inhibits TF
PCA Induced by Viral Infection--
To confirm the effect of stable
overexpression of GRP78/BiP on TF PCA, an additional approach using
adenovirus to overexpress GRP78/BiP was performed. A recombinant
adenovirus containing the complete human GRP78/BiP open reading frame
under the control of the constitutively expressed CMV promoter
(AdV-GRP78) was constructed. As shown in Fig.
8A, GRP78/BiP protein levels
were increased ~3-fold in T24/83 cells and HASMC following infection
with AdV-GRP78, compared with infection with the control virus
expressing
Given that adenovirus infection has been shown to increase TF PCA (33),
the effect of GRP78/BiP overexpression on adenovirus-mediated TF PCA
was examined. Wild-type T24/83 cells or HASMC were infected with
AdV-GRP78 or AdV- Adenovirus-mediated GRP78/BiP Overexpression Inhibits TF
PCA Induced by Ionomycin or Hydrogen Peroxide--
To confirm the
observations from the stable cell lines, the effect of
adenovirus-mediated GRP78/BiP overexpression on the induction of TF PCA
by ionomycin or hydrogen peroxide was examined. Wild-type T24/83 cells
and HASMC were infected with AdV- TF-dependent thrombin generation plays a critical role
in hemostasis after tissue injury and also in the pathogenesis of
multiple thrombotic disorders associated with a wide range of diseases, including cardiovascular disease, sepsis and cancer (21-24,73). Despite its importance in coagulation and human disease, the
cellular factors that regulate TF expression and/or activity are
relatively unknown. In this report, we provide novel evidence that
overexpression of GRP78/BiP in the prothrombotic cell line T24/83
prevents cell surface thrombin generation by inhibiting TF PCA. The
observation that GRP78/BiP overexpression does not decrease cell
surface TF suggests that the inhibition of TF PCA is not due to an
impairment in the trafficking of TF through the ER. However, the
ability of GRP78/BiP overexpression to attenuate TF PCA induced by
ionomycin, hydrogen peroxide or viral infection supports a mechanism
involving cellular Ca2+ and/or oxidative stress. These
findings demonstrate that alterations in the expression of GRP78/BiP
can have profound effects on the prothrombotic characteristics of cells.
Stable overexpression of GRP78/BiP does not cause a decrease in cell
surface levels of TF in T24/83 cells. In fact, TF levels are somewhat
increased in GRP78/BiP-overexpressing cells, compared with wild-type or
vector-transfected cells. This finding is consistent with previous
studies demonstrating that GRP78/BiP overexpression increases
macrophage colony-stimulating factor (MCSF) expression and secretion in
CHO cells (7, 20). Since overexpression of GRP78/BiP can inhibit the
secretion of factor VIII and vWf (7, 20), structural differences among
proteins which affect their association with GRP78/BiP are likely to
occur. Indeed, both factor VIII and vWf, but not MCSF, have been shown
to stably associate with GRP78/BiP in the ER lumen (7, 20). Several lines of evidence suggest that GRP78/BiP does not stably associate with
TF or directly alter its activity. First, immunoprecipitation experiments have failed to identify stable complexes between TF and
GRP78/BiP in T24/83 cells.2
Second, the BiP Score program (13), which has been successfully used to
predict BiP binding sites within antibodies and HIV gp160 (13-15),
indicates a low probability of TF-GRP78/BiP association. Third, TF PCA
in rabbit brain thromboplastin is not inhibited by recombinant
GRP78/BiP.2 Taken together, these findings demonstrate that
GRP78/BiP chaperone function is not required for the folding/processing
of all proteins that transit the ER. However, given that GRP78/BiP has
been shown to bind a number of proteins, GRP78/BiP could potentially
regulate TF PCA through its interactions with other proteins/factors,
the identities of which are as yet unknown.
The observation that GRP78/BiP overexpression decreases TF PCA without
a corresponding decrease in cell surface TF levels may be partly
explained given that TF expression does not necessarily correlate
with activity. Tissue factor expression and activity are considered to
be independently regulated by a two-step activation pathway in which
certain cellular factors (such as lipoproteins) regulate synthesis of
latent TF while other factors (such as ROS) mediate post-translational
modifications of existing cell surface TF to an active form (42, 46).
Based on our findings, it is unlikely that a mechanism involving
decreased TF synthesis or impaired trafficking of TF through the ER is
responsible for a decrease in TF PCA. This would imply that GRP78/BiP
overexpression indirectly alters TF PCA through a mechanism independent
of its chaperone activity. In support of this concept, GRP78/BiP
overexpression attenuates TF PCA induced by ionomycin, suggesting that
alterations in ER Ca2+ stores play an important role in the
regulation of TF PCA by GRP78/BiP. Given that GRP78/BiP is a major
Ca2+-binding protein (10, 11) and that TF PCA is mediated
by changes in intracellular levels of free Ca2+ (47-50),
overexpression of GRP78/BiP could potentially inhibit TF PCA indirectly
by sequestering intracellular Ca2+. Although it is not
completely understood as to how alterations in intracellular
Ca2+ increase TF PCA, it may involve the production of ROS.
It is well established that efflux of Ca2+ from the ER
enhances the peroxidase activity of cyclooxygenases and lipoxygenases,
thereby leading to the production of ROS (74). Furthermore, elevated
levels of intracellular Ca2+ lead to mitochondrial
Ca2+ uptake, mitochondrial dysfunction and the generation
of ROS (5, 6, 74). Oxidative stress has been shown to increase TF PCA (42, 46), suggesting that changes in intracellular Ca2+
could indirectly alter TF PCA through the generation of ROS. This
mechanism is also supported by findings that in contrast to ionomycin,
thapsigargin, an inhibitor of the ER Ca2+-ATPase, does not
significantly increase TF PCA and induces a lower intracellular
Ca2+ response than
ionomycin.3 The observation
that GRP78/BiP overexpression attenuates TF PCA induced by both
ionomycin and hydrogen peroxide also suggests a link between
intracellular Ca2+ and oxidative stress. In addition,
GRP78/BiP overexpression could potentially limit the accessibility of
anionic phospholipids essential for TF PCA. Exposure of anionic
phospholipids on the outer plasma membrane increases TF PCA (47,
66, 75, 76) and is regulated by Ca2+ levels (47, 75, 77).
Therefore, modulation of any component/cofactors involved in this
pathway by GRP78/BiP could ultimately attenuate TF PCA.
Infection of cultured human vascular endothelial cells with adenovirus,
as well as other respiratory viruses, increases TF PCA and expression
(33). Consistent with these findings, we demonstrated that infection of
T24/83 cells and HASMC with a recombinant adenovirus expressing
On the basis of our findings, is there a plausible biological link
between GRP78/BiP overexpression and TF PCA? Numerous agents and/or
conditions, including viral infection, Ca2+ ionophores and
homocysteine, that activate TF expression and activity can also induce
ER stress (9). The ability of ER stress to increase TF expression and
activity could involve the activation of NF In summary, we have provided novel evidence that
overexpression of GRP78/BiP can inhibit TF-mediated thrombin generation
at the cell surface. Because TF is a primary determinant of the
thrombogenicity of human atherosclerotic plaques, the ability to
inhibit TF PCA by modulating GRP78/BiP protein levels could potentially
alleviate many TF-dependent pathological conditions,
including myocardial infarction and acute arterial injury.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 0.01% bromphenol
blue), separated on SDS-polyacrylamide gels under reducing conditions
and transferred to nitrocellulose membranes (Bio-Rad), as described
previously (58). After incubation with the appropriate primary and
horseradish peroxidase (HRP)-conjugated secondary antibodies (Affinity
Biologicals, Hamilton, ON), the membranes were developed using the
Renaissance chemiluminescence reagent kit (PerkinElmer Life Sciences).
1%, according to the manufacturer.
Prior to use in the thrombin generation assay, plasmas were
defibrinated using arvin and total amidolytic activity of thrombin
generated on cell surfaces was measured as previously described (62,
63). Cell monolayers in 24-well plates were placed on a Thermolyne
dri-bath set at 37 °C. After washing twice with 1 ml of
acetate/barbital/saline (ABS) buffer (0.036 M sodium
acetate, 0.036 M sodium diethylbarbitarate, 0.145 M NaCl, pH 7.4), monolayers were incubated for 3 min with 100 µl of ABS buffer and 200 µl of defibrinated plasma, in the absence or presence of 10% activated partial thromboplastin time (APTT) reagent (Organon Teknika Corp., Durham, NC). At time periods up
to 30 min following the addition of 100 µl of 40 mM
CaCl2 in ABS buffer, 25-µl aliquots of the reaction
mixture on the surface of the cells were removed and mixed with 475 µl of 5 mM EDTA on ice. 25 µl of each EDTA sample were
then mixed with 775 µl of 0.16 mM S-2238 (KabiVitum,
Stockholm, Sweden) in buffer and heated at 37 °C for 10 min prior to
termination of the amidolytic reaction with 200 µl of 50% acetic
acid. The absorbance at 405 nm was measured and the concentration of
total thrombin determined by comparing results to a standard curve
generated with purified thrombin in S-2238. EDTA samples were also used
to measure the concentrations of prothrombin, thrombin-antithrombin
(TAT) complexes and thrombin-heparin cofactor II complexes.
Prothrombin, TAT, and thrombin-heparin cofactor II complexes were
assayed using commercially available ELISA kits (Affinity Biologicals).
Because thrombin bound to
2 macroglobulin
(
2M) retains amidolytic activity against S-2238 (64),
the contribution of thrombin-
2M to total thrombin
activity was measured as previously described (63). Briefly, the
amidolytic activity of total thrombin was measured as described above,
except that the 25-µl reaction mixture taken at each time point was
incubated with 3.5 µl of 0.15 M NaCl containing 0.25 units of standard heparin and 0.042 units of antithrombin (to inhibit
any free thrombin) for 1 min on ice, followed by the addition of 475 µl of 5 mM EDTA.
2M-dependent
thrombin activity was then subtracted from the total thrombin activity
to give the amount of physiologically active free thrombin generated by
the cell surface.
-galactosidase (AdV-
-Gal)
were performed essentially as previously described (68). Wild-type
T24/83 cells or HASMC were grown in appropriate medium until reaching
70% confluence. Cells were washed once with 1× PBS and infected with
either AdV-GRP78 or AdV-
-Gal at multiplicities of infection (m.o.i.)
of 100, 300, or 500 pfu/cell, in FBS-free medium for 30 min at
37 °C. Cells were incubated in medium containing FBS for 72 h
at 37 °C. Media was removed, and cells were washed once in 1× PBS
prior to cell surface TF PCA assay or lysate preparation for
immunoblotting, as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Stable overexpression of human GRP78/BiP in
T24/83 cells. A, immunoblot analysis of GRP78/BiP.
Total protein lysates (30 µg/lane) from wild-type
(T24/83), vector-transfected
(T24/83-pcDNA) or GRP78/BiP-overexpressing
(T24/83-GRP78c1 or c2) cells
were separated by SDS-polyacrylamide gel electrophoresis under reducing
conditions. Gels were either stained with Coomassie Blue (upper
panel) or immunostained with an anti-KDEL monoclonal antibody,
which recognizes GRP78/BiP and GRP94 (lower panel). The
migration positions of GRP78/BiP and GRP94 are shown by the
arrowheads. B, indirect immunofluorescence
detection of GRP78/BiP. Wild-type (T24/83),
vector-transfected (pcDNA) or GRP78/BiP-overexpressing
(GRP78c1) cells grown on glass coverslips were fixed,
permeabilized, and incubated with an anti-human GRP78/BiP antibody.
Antibody localization was detected with an Alexa-conjugated rabbit
anti-goat IgG. Original magnification, ×630. C, comparison
of GRP78/BiP protein levels in cells treated with various ER
stress-inducing agents. Total protein lysates (30 µg/ml) from
wild-type (T24/83), vector-transfected
(T24/83-pcDNA), GRP78/BiP-overexpressing
cells (T24/83-GRP78c1) or wild-type cells
cultured in the presence of either homocysteine (5 mM),
dithiothreitol (2.5 mM), or tunicamycin (10 µg/ml) for
18 h were separated by SDS-polyacrylamide gel electrophoresis
under reducing conditions. Gels were either stained with Coomassie Blue
(upper panel) or immunostained with an anti-KDEL monoclonal
antibody, which recognizes GRP78/BiP and GRP94 (lower
panel). The migration positions of GRP78/BiP and GRP94 are shown
by arrowheads.
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Fig. 2.
Overexpression of GRP78/BiP suppresses free
thrombin generation and prothrombin consumption on the surface of
T24/83 cells. A, free thrombin generation. Normal
pooled human plasma was used to measure free thrombin generated on the
surface of wild-type ( ), vector-transfected (
), or the
GRP78/BiP-overexpressing cell lines, c1 (
) and c2 (
). Data
represent mean ± S.E. of triplicate measurements from four
separate experiments. *, p < 0.001 versus
wild-type or vector-transfected cells. B, prothrombin
consumption. Normal pooled human plasma was used to measure prothrombin
consumption on the surface of wild-type (
), vector-transfected
(
), or the GRP78/BiP-overexpressing cells, c1 and c2 (
). Data
represent mean ± S.E. of triplicate measurements from four
separate experiments. *, p < 0.001 versus
wild-type or vector-transfected cells.
Thrombin-inhibitor complex formation on T24/83 cell surface
2
macroglobulin (IIa-
2M) was measured chromogenically after
inhibiting free thrombin with antithrombin and heparin. Data are
presented as the mean ± S.E. of triplicate measurements from four
separate experiments. GRP78/BiP represents the combined measurements
from two GRP78/BiP-overexpressing clones, c1 and c2.
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Fig. 3.
Overexpression of GRP78/BiP attenuates TF PCA
on the surface of T24/83 cells. A, rate of Xa
generation. Factor Xa generation on the surface of wild-type
(T24/83), vector-transfected
(T24/83-pcDNA) or GRP78/BiP
(T24/83-GRP78) cells was performed by a one-step
assay, which directly measures the conversion of factor X to factor Xa,
using the chromogenic substrate S-2765. B, rate of VIIa
generation. Factor VIIa generation on cell surfaces was performed by a
one-step assay that directly measures the conversion of factor VII to
factor VIIa, using the chromogenic substrate S-2288. Data represent
mean ± S.E. of triplicate measurements from three separate
experiments. *, p < 0.05 versus wild-type
or vector-transfected cells.
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Fig. 4.
Procoagulant activity on T24/83 cells is
specific for TF. Wild-type T24/83 cells were incubated in the
absence of factor VIIa (panel A) or with increasing
concentrations of TF neutralizing antibody (panel B) prior
to the determination of amount of factor Xa generated, using the
chromogenic substrate S-2765. Cell surface TF PCA was measured as the
amount of factor Xa generated per microgram of total protein
(units/µg). Data represent mean ± S.E. of three separate
experiments. *, p < 0.05 versus wild-type
cells incubated in the presence of factor VIIa (panel A) or
the absence of TF neutralizing antibody (panel B) (0 µg/ml).
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Fig. 5.
Indirect immunofluorescence detection of TF
in T24/83 cells. Wild-type (A and D),
vector-transfected (B and E) or
GRP78/BiP-overexpressing T24/83 cells (C and F)
were grown on glass coverslips and immunostained with an anti-human TF
monoclonal antibody without (panels A-C) or with
(panels D-F) permeabilization using Triton X-100. Primary
antibody was detected with a goat Alexa-conjugated anti-mouse IgG.
Original magnification, ×1000.
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Fig. 6.
Effect of GRP78/BiP overexpression on cell
surface proteins from T24/83 cells. A, cell surface TF
was immunoprecipitated from wild-type (T24/83),
vector-transfected (T24/83-pcDNA), or
GRP78/BiP-overexpressing (T24/83-GRP78c1 or
c2) cell monolayers as described under "Experimental
Procedures." Supernatants were separated on 10% SDS-polyacrylamide
gels under reducing conditions, transferred to nitrocellulose membranes
and immunostained with an anti-human TF antibody. The migration
position of TF is shown by the arrowhead. B,
identification of cell surface-biotinylated proteins from T24/83 cells.
Wild-type (T24/83), vector-transfected
(T24/83-pcDNA), or GRP78/BiP-overexpressing
(T24/83-GRP78c1 or c2) cells were
treated in the absence ( ) or presence (+) of NHS-SS-biotin to
biotinylate cell surface proteins, as described under "Experimental
Procedures." Equivalent amounts of total cell lysates were separated
on 10% SDS-polyacrylamide gels under reducing conditions, transferred
to nitrocellulose membranes and immunostained with HRP-conjugated
ExtrAvidin to detect biotinylated cell surface proteins.
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Fig. 7.
Effect of GRP78/BiP overexpression on TF PCA
stimulated by ionomycin or hydrogen peroxide. Wild-type
(T24/83), vector-transfected
(pcDNA), or GRP78/BiP-overexpressing cells
(GRP78c1 and GRP78c2) were treated
with 10 µM ionomycin for 10 min (panel A) or
10 mM hydrogen peroxide for 1 h (panel B).
Untreated cells were used as a control. Cell surface TF PCA was
measured as the amount of factor Xa generated per microgram of total
protein (units/µg). Data represent the mean ± S.E. from three
separate experiments. p < 0.05 versus
wild-type untreated cells (*) or wild-type-treated cells (**).
-galactosidase (AdV-
-Gal) or uninfected (no virus)
cells. In addition, adenovirus-mediated overexpression of GRP78/BiP was
not associated with a discernable change in cell surface TF protein
levels, compared with uninfected cells or cells infected with
AdV-
-Gal (Fig. 8B). Adenovirus infection did not
significantly affect cell proliferation or cell death (data not
shown).
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Fig. 8.
Effect of AdV-GRP78 or
AdV- -Gal infection on GRP78/BiP and TF
expression. A, wild-type T24/83 cells or HASMC were
infected with adenovirus expressing GRP78/BiP (AdV-GRP78) or
-galactosidase (AdV-
-Gal) at 100, 300, or 500 pfu/cell
for 72 h. Uninfected wild-type cells (T24/83
or HASMC) were used as a control. Total protein lysates were
separated by SDS-PAGE under reducing conditions and immunoblotted with
an anti-KDEL monoclonal antibody (lower panel). To control
for protein loading, total protein lysates separated by SDS-PAGE were
stained with Coomassie Blue (upper panel). The migration
position of
-galactosidase in cells infected with AdV-
-Gal is
denoted by the asterisk (upper panel).
B, wild-type cells were infected with adenovirus expressing
GRP78/BiP (AdV-GRP78) or
-galactosidase
(AdV-
-Gal) at 300 pfu/cell for 72 h. Uninfected
wild-type cells (T24/83 or HASMC) were
used as a control. Cell surface TF was immunoprecipitated from
wild-type or adenovirus infected cells using a rabbit anti-human TF
polyclonal antibody, as described under "Experimental Procedures."
Immunoprecipitation of wild-type cells with preimmune rabbit IgG was
used to control for nonspecific binding (rabbit IgG). The
migration position of TF is shown by the arrowhead.
-Gal and TF PCA was determined. T24/83 cells (Fig.
9A) and HASMC (Fig.
9B) infected with AdV-
-Gal exhibited an increase in TF
PCA, compared with uninfected cells, a finding consistent with previous
studies showing that adenoviral infection increases TF PCA (33). In
contrast, TF PCA was not increased in cells infected with AdV-GRP78.
Therefore, in addition to attenuating TF PCA stimulated by ionomycin or
hydrogen peroxide, GRP78/BiP overexpression also decreases
adenovirus-mediated TF PCA.
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Fig. 9.
TF PCA is attenuated in T24/83 cells or HASMC
infected with AdV-GRP78. Wild-type T24/83 cells (panel
A) or HASMC (panel B) were infected with AdV-GRP78 or
AdV- -Gal at 300 pfu/cell for 72 h. Uninfected wild-type cells
were used as a control for basal TF PCA. Cell surface TF PCA was
measured as the amount of factor Xa generated per microgram of total
protein (units/µg). Data represent the mean ± S.E. from three
separate experiments. p < 0.05 compared with
uninfected wild-type cells (*) or AdV-
-Gal (**).
-Gal or AdV-GRP78 followed by
treatment with ionomycin or hydrogen peroxide and measurement of TF
PCA. In both T24/83 cells and HASMC, AdV-GRP78 inhibited the
stimulation of TF PCA by ionomycin (Fig. 10A) or hydrogen peroxide
(Fig. 10B). These findings confirm the results from the
stable cell lines and provide further evidence that intracellular
Ca2+ and/or oxidative stress play a role in the regulation
of TF PCA by GRP78/BiP. Furthermore, the similar effects of GRP78/BiP
overexpression on TF PCA in these different cell lines provides
evidence for a general cellular mechanism.
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Fig. 10.
AdV-GRP78 attenuates the induction of TF PCA
by ionomycin or hydrogen peroxide. T24/83 cells and HASMC were
infected with AdV-GRP78 or AdV- -Gal at 300 pfu/cell for 72 h.
Uninfected wild-type cells were used as a control. Cells were treated
with 10 µM (T24/83) or 5 µM (HASMC) ionomycin for 10 min (panel
A) or 10 mM hydrogen peroxide for 1 h
(panel B). Untreated cells were used as a control. Cell
surface TF PCA was measured as the amount of factor Xa generated per
µg of total protein (units/µg). Data represent the mean ± S.E. from three separate experiments. *, p < 0.05 versus wild-type-treated cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase increases TF PCA in a dose-dependent manner. However, TF PCA did not increase in T24/83 cells or HASMC infected with a recombinant adenovirus expressing GRP78/BiP. Given that
viral infection could potentially increase intracellular Ca2+ concentration (74), which could lead to the production
of ROS, the inhibitory effect of GRP78/BiP on TF PCA induced by viral infection further supports a mechanism involving Ca2+
and/or oxidative stress. This mechanism is also suggested by the
observation that adenovirus-mediated delivery of GRP78/BiP inhibits a
rise in intracellular Ca2+ caused by hydrogen peroxide,
thereby protecting neuronal cells from hydrogen peroxide-mediated cell
death (78).
B. It has been reported
that ER stress activates NF
B through a mechanism involving release
of Ca2+ from the ER and the subsequent generation of ROS
(74). This is consistent with previous studies demonstrating that TF
PCA is increased by elevations in intracellular Ca2+ and
oxidative stress (42, 46-50). However, the induction of GRP78/BiP, as
well as other ER-resident molecular chaperones, is considered to be a
protective mechanism elicited by cells to alleviate the adverse effects
of ER stress (58, 59). Based on our findings, GRP78/BiP overexpression
may thus impair TF PCA in an attempt to reduce the thrombotic potential
often associated with ER stress agents/conditions.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Jack Hirsh and Jeffrey Weitz for valuable suggestions throughout the course of this study and during preparation of the article. We also thank Duc Ngo for the production and purification of recombinant adenoviral stocks.
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FOOTNOTES |
---|
* This research was supported in part by Research Grants NA-4842 (to R. C. A.) and NA4020 (to A. K. C. C.) from the Heart and Stroke Foundation of Ontario.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ A recipient of a Premier's Research Excellence Award Studentship and an Ontario Graduate Scholarship.
A Career Investigator of the Heart and Stroke Foundation of
Ontario (HSFO). To whom correspondence should be addressed: Henderson Research Centre, 711 Concession St., Hamilton, Ontario L8V 1C3, Canada.
Tel.: 905-527-2299 (ext. 42628); Fax: 905-575-2646; E-mail: raustin@thrombosis.hhscr.org.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M301006200
2 L. M. Watson and R. C. Austin, unpublished results.
3 L. M. Watson, J. G. Dickhout, and R. C. Austin, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: ER, endoplasmic reticulum; GRP78/BiP, 78-kDa glucose-regulated protein/immunoglobulin-binding protein; vWf, von Willebrand factor; TF, tissue factor; PCA, procoagulant activity; APTT, activated partial thromboplastin time; TAT, thrombin-antithrombin; MCSF, macrophage colony-stimulating factor; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; RIPA, radioimmune precipitation assay buffer; pfu, plaque-forming unit; HRP, horseradish peroxidase; ROS, reactive oxygen species; CHO, Chinese hamster ovary; HASMC, human aortic smooth muscle cells.
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