U.S. patent number 7,524,826 [Application Number 09/834,760] was granted by the patent office on 2009-04-28 for method of inhibiting the generation of active thrombin on the surface of a cell within an atherosclerotic plaque.
This patent grant is currently assigned to McMaster University and Hamilton Health Sciences Corporation. Invention is credited to Richard C. Austin, Leslie Roy Berry, Anthony K. C. Chan.
United States Patent |
7,524,826 |
Austin , et al. |
April 28, 2009 |
Method of inhibiting the generation of active thrombin on the
surface of a cell within an atherosclerotic plaque
Abstract
The present invention provides methods for preventing the
generation of active thrombin on the surface of cells. The present
methods are based upon the surprising discovery that expression or
activation of an ER resident chaperone protein in a cell inhibits
the generation of thrombin on the surface of the cell. Accordingly,
expressing or activating an ER resident chaperone protein can be
used to treat or prevent any of a number of thrombotic
diseases.
Inventors: |
Austin; Richard C. (Ancaster,
CA), Chan; Anthony K. C. (Ancaster, CA),
Berry; Leslie Roy (Burlington, CA) |
Assignee: |
McMaster University and Hamilton
Health Sciences Corporation (Hamilton, CA)
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Family
ID: |
26892605 |
Appl.
No.: |
09/834,760 |
Filed: |
April 12, 2001 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20020039791 A1 |
Apr 4, 2002 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60197146 |
Apr 14, 2000 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61K
38/52 (20130101); A61K 38/19 (20130101); A61K
38/1709 (20130101); Y02A 50/395 (20180101); A61K
48/00 (20130101); Y02A 50/30 (20180101); G01N
2500/10 (20130101) |
Current International
Class: |
A61K
31/70 (20060101) |
Field of
Search: |
;435/69.1 ;514/44 |
References Cited
[Referenced By]
U.S. Patent Documents
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Primary Examiner: Angell; J. E
Attorney, Agent or Firm: Townsend and Townsend and Crew
LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. Provisional Application No.
60/197,146, filed Apr. 14, 2000, which application is incorporated
herein by reference for all purposes.
Claims
What is claimed is:
1. A method of inhibiting the generation of active thrombin on the
surface of a cell within an atherosclerotic plaque within a mammal,
the method comprising increasing the expression or activity of
GRP78/BiP, an ER resident calcium-binding protein in said cell by
directly administering to said cell a polynucleotide operably
linked to a promoter, wherein said polynucleotide encodes
GRP78/BiP, whereby said GRP78/BiP is produced in said cell and the
generation of active thrombin on the surface of said cell is
inhibited.
2. The method of claim 1, wherein said cell is an endothelial
cell.
3. The method of claim 1, wherein said cell is a smooth muscle
cell.
4. The method of claim 1, wherein said cell is a macrophage.
5. The method of claim 1, wherein said cell is a monocyte.
6. The method of claim 1, wherein the increase in the expression or
activity of said GRP78/BiP within said cell results in a decrease
in the level of tissue factor procoagulant activity on the surface
of said cell.
7. The method of claim 1, wherein said polynucleotide is introduced
into said cell using a viral vector.
8. The method of claim 7, wherein said viral vector is an
adenoviral vector.
9. The method of claim 1, wherein said polynucleotide is introduced
into said cell using a nonviral vector.
10. The method of claim 9, wherein said nonviral vector is
introduced into said cell as naked DNA or using liposome-mediated
transfection.
Description
BACKGROUND OF THE INVENTION
Regulation of coagulation is essential for animal survival:
deficiencies in coagulation, such as those associated with
hemophilia, can produce life-threatening hemorrhage events, whereas
excessive coagulation can lead to the formation of dangerous blood
clots or can contribute to deleterious processes such as
atherosclerosis. Clearly, the ability to modulate the extent of
coagulation in cells, e.g., in endothelial cells, within a patient
would provide a powerful tool for the treatment or prevention of a
large number of common human diseases and conditions.
One key, rate limiting factor in the coagulation pathway is the
generation of active thrombin. Indeed, tissue factor (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. Accordingly, regulation
of thrombin is critical for the prevention of thromboembolic
disease and is dependent on an intricate balance between
coagulation factors and inhibitors present in plasma and on the
surface of cells. Thrombin generation in vivo occurs predominantly
on cell surfaces and involves the interaction of tissue factor
(TF), an integral membrane glycoprotein, with factor VII/VIIa to
initiate blood coagulation. TF is present on the cell surface of
lipid-laden macrophages (foam cells) and smooth muscle cells within
human atherosclerotic plaques, and contributes to the
thrombogenicity of the ruptured plaque. The generation of thrombin
is subsequently regulated at the cell surface by thrombomodulin
(TM), a cell surface receptor which promotes thrombin-dependent
activation of the anticoagulant protein C pathway. Alterations in
cell surface levels and/or activity of TF or TM can enhance
thrombin generation, thereby increasing the risk of thromboembolic
disease. Accordingly, a decrease in TF levels or activity, or an
increase in TM levels or activity, would decrease the potential for
thrombosis.
The biosynthesis and maturation of TF and TM, as well as other
coagulation factors and inhibitors, occurs within the cellular
secretory pathway which consists of the ER, Golgi complex and a
variety of intermediate transport vesicles. The ER is the cellular
organelle where these factors and inhibitors undergo a series of
modifications, including folding, proteolytic processing,
glycosylation, disulfide-bond formation and oligomeric assembly.
Failure to fold properly or to assemble into correct oligomeric
complexes can result in the generation of dysfunctional molecules
and/or the retention of these molecules in the ER, followed by
degradation.
To assist in the proper folding and assembly of newly synthesized
proteins, the ER lumen contains a number of molecular chaperones,
including GRP78/BiP, the 94 kDa glucose-regulated protein (GRP94),
Calnexin, Calreticulin and ERp72. These molecular chaperones are
important components of the quality control system of the ER that
allows export of correctly folded and assembled proteins.
Overexpression of GRP78/BiP in cultured mammalian cells can affect
the processing and secretion of several coagulation and
fibrinolytic factors, including von Willebrand factor (vWf), factor
VIII and tissue plasminogen activator (tPA). Recent studies using
cultured human vascular endothelial cells have also shown that
homocysteine, a thiol-containing amino acid implicated in the
progression of atherothrombosis, induces the expression of
GRP78/BiP while decreasing the processing and secretion of vWf.
SUMMARY OF THE INVENTION
The present invention provides novel methods for inhibiting
thrombin on the surface of cells. This invention is based on the
surprising discovery that the expression or activation of an ER
resident chaperone in a cell prevents the generation of thrombin on
the cell surface. As thrombin is the rate-limiting factor in
coagulation, this invention thus provides methods for inhibiting
coagulation, thereby providing a treatment for any of a number of
thrombotic diseases and conditions.
Thus, in one aspect, the present invention provides a method of
inhibiting the generation of active thrombin on the surface of a
cell of a mammal, the method comprising producing an ER resident
chaperone protein in the cell.
In one embodiment, the cell is an endothelial cell. In another
embodiment, the cell is a smooth muscle cell. In another
embodiment, the cell is a macrophage. In another embodiment, the
cell is a monocyte. In another embodiment, the ER resident
chaperone protein is GRP78/BiP. In another embodiment, the ER
resident chaperone protein is selected from the group consisting of
GRP94, GRP72, Calreticulin, Calnexin, Protein disulfide isomerase,
cis/trans-Prolyl isomerase, and HSP47. In another embodiment, the
production of the ER resident chaperone protein within the cell
results in a decrease in the level of tissue factor activity on the
surface of the cell.
In another embodiment, the cell is present within the mammal. In
another embodiment, the cell is present within an atherosclerotic
plaque in the mammal. In another embodiment, a polynucleotide
encoding the ER resident chaperone, operably linked to a promoter,
is introduced into the cell, whereby the ER resident chaperone
protein is produced. In another embodiment, the polynucleotide is
introduced into the cell using a viral vector. In another
embodiment, the viral vector is an adenoviral vector. In another
embodiment, the polynucleotide is introduced into the cell using a
nonviral vector. In another embodiment, the nonviral vector is
introduced into the cell as naked DNA or using liposome-mediated
transfection. In another embodiment, the ER resident chaperone
protein is produced by administering to the cell a compound that
induces the expression or activation of an endogenous ER resident
chaperone protein. In another embodiment, the compound is a
cytokine.
In another aspect, the present invention provides a method of
preventing or treating a thrombotic disease or condition in a
mammal, the method comprising producing an ER resident chaperone
protein within a population of cells of the mammal, whereby the
generation of thrombin on the surface of the cells is
inhibited.
In one embodiment, the population of cells comprises endothelial
cells. In another embodiment, the population of cells comprises
smooth muscle cells. In another embodiment, the population of cells
comprises macrophages. In another embodiment, the cells comprise
monocytes. In another embodiment, the ER resident chaperone protein
is GRP78/BiP. In another embodiment, the ER resident chaperone
protein is selected from the group consisting of GRP94, GRP72,
Calreticulin, Calnexin, Protein disulfide isomerase,
cis/trans-Prolyl isomerase, and HSP47. In another embodiment, the
production of the ER resident chaperone protein within the
population of cells results in a decrease in the level of tissue
factor activity on the surface of the population of cells. In
another embodiment, the population of cells is present within an
atherosclerotic plaque within the mammal.
In another embodiment, the mammal has had a myocardial infarction
and is undergoing angioplasty or stenting, and the inhibition of
the generation of thrombin on the surface of the population of
cells reduces the risk of thrombosis of the mammal during the
angioplasty or stenting. In another embodiment, the mammal is
undergoing stenting and the population of cells is present on the
surface of a stent. In another embodiment, the mammal is undergoing
cranial radiation, and the inhibition of the generation of thrombin
on the surface of the population of cells reduces the risk of
radiation-related thrombotic events during the cranial radiation.
In another embodiment, the mammal is undergoing vascular surgery,
and the inhibition of the generation of thrombin on the surface of
the population of cells reduces the risk of surgery-related
thrombotic events during the vascular surgery.
In another embodiment, a polynucleotide encoding the ER resident
chaperone protein, operably linked to a promoter, is introduced
into the population of cells, whereby the ER resident chaperone
protein is produced. In another embodiment, the polynucleotide is
introduced into the population of cells using a viral vector. In
another embodiment, the viral vector is an adenoviral vector. In
another embodiment, the polynucleotide is introduced into the
population of cells using a nonviral vector. In another embodiment,
the nonviral vector is introduced into the cell as naked DNA or
using liposome-mediated transfection. In another embodiment, the ER
resident chaperone protein is produced by administering to the
population of cells a compound that induces the expression or
activation of an endogenous ER resident chaperone protein. In
another embodiment, the compound is a cytokine.
In another aspect, the present invention provides a method of
identifying a compound that is useful in the treatment or
prevention of a thrombotic disease or condition, the method
comprising (1) contacting a cell that expresses an ER resident
chaperone protein, or that is capable of expressing an ER resident
chaperone protein; and (2) detecting the functional effect of the
compound on the ER resident chaperone protein; wherein an increase
in the expression or activity of the ER resident chaperone protein
in the cell indicates that the compound would be useful in the
treatment or prevention of the thrombotic disease or condition.
In one embodiment, the ER resident protein is GRP78/BiP. In another
embodiment, the ER resident protein is selected from the group
consisting of GRP94, GRP72, Calreticulin, Calnexin, Protein
disulfide isomerase, cis/trans-Prolyl isomerase, and HSP47. In
another embodiment, the cell is an endothelial cell. In another
embodiment, the cell is a smooth muscle cell. In another
embodiment, the cell is a macrophage. In another embodiment, the
cell is a monocyte. In another embodiment, the compound induces the
expression or activation of the ER resident chaperone protein in
the cell without inducing ER stress in the cell.
In another aspect, the present invention provides a method of
treating or preventing a thrombotic disease in a mammal, the method
comprising administering to the mammal a therapeutically or
prophylactically effective amount of a compound identified using a
method comprising (1) contacting a cell that expresses an ER
resident chaperone protein, or that is capable of expressing an ER
resident chaperone protein; and (2) detecting the functional effect
of the compound on the ER resident chaperone protein; wherein an
increase in the expression or activity of the ER resident chaperone
protein in the cell indicates that the compound would be useful in
the treatment or prevention of the thrombotic disease or
condition.
BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
FIG. 1. Stable overexpression of human GRP78/BiP in T24/83 cells.
Total protein lysates (30 .mu.g/lane) from wild-type T24/83 cells
(T24/83), or cells stably transfected with either the vector
pcDNA3.1(+) (T24/83-pcDNA) or pcDNA3.1(+) containing the
full-length human GRP78/BiP cDNA (T24/83-GRP78c1 or c2) 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 mAb which recognizes
GRP78/BiP and GRP94 (lower panel). The migration positions of
GRP78/BiP and GRP94 are shown by the arrowheads.
FIG. 2. Immunolocalization of GRP78/BiP in T24/83 cells. Wild-type
(top panel) or GRP78/BiP overexpressing T24/83 cells (lower panel)
plated onto gelatin-coated glass coverslips were fixed,
permeabilized and incubated with an anti-KDEL mAb. Antibody
localization was detected with a FITC-conjugated goat anti-mouse
IgG. Magnification .times.1000.
FIG. 3. Overexpression of GRP78/BiP suppresses total (A) and free
(B) thrombin generation on T24/83 cell surfaces. Normal pooled
human plasma was used to measure total and free thrombin generated
on the surface of T24/83 cells. Wild type (.tangle-solidup.),
vector transfected (.box-solid.), or GRP78/BiP overexpressing cells
(.circle-solid.). Data represent mean .A-inverted. SEM (standard
error of the mean, i.e., the standard deviation divided by the
square root of sample size) of triplicate measurements from four
separate experiments. GRP78/BiP overexpressing cells generated
significantly less thrombin compared with wild-type or
vector-transfected cells (p<0.001).
FIG. 4. Overexpression of GRP78/BiP decreases prothrombin
consumption on T24/83 cell surfaces. Normal pooled human plasma was
used to measure prothrombin consumption on the surface of T24/83
cells. Wild type (.tangle-solidup.), vector transfected
(.box-solid.), or GRP78/BiP overexpressing cells (.circle-solid.).
Data represent mean .A-inverted. SEM of triplicate measurements
from four separate experiments. GRP78/BiP overexpressing cells
consumed significantly less prothrombin after 4 min, compared with
wild-type or vector-transfected cells (p<0.001).
FIG. 5. Effect of GRP78/BiP overexpression on free thrombin
generation in normal (A) or factor VII-deficient (B) plasma
containing APTT reagent. Normal or factor VII-deficient human
plasma, in the presence of APTT reagent, was used to measure free
thrombin generation on the surface of T24/83 cells. Wild type
(.tangle-solidup.), vector transfected (.box-solid.), or GRP78/BiP
overexpressing cells (.circle-solid.). Data representing mean
.A-inverted. SEM of triplicate measurements from four separate
experiments. In the presence of normal (A), but not factor
VII-deficient plasma (B), peak free thrombin generation was
significantly decreased in the GRP78/BiP overexpressing cells,
compared to wild-type or vector-transfected cells (p<0.001).
FIG. 6. Overexpression of GRP78/BiP increases TF expression and
synthesis. (A) Northern blot hybridization of total RNA (10
.mu.g/lane) from wild type (T24/83), vector transfected
(T24/83-pcDNA) or GRP78/BiP overexpressing T24/83 cells
(T24/83-GRP78) using a radiolabeled human TF cDNA probe. The
migration position of TF mRNA is shown by the arrowhead. To control
for RNA loading, blots were hybridized with a radiolabeled GAPDII
cDNA probe (lower panel). (B) Immunoblot analysis of total protein
lysates (30 .mu.g/lane) from wild-type (T24/83), vector-transfected
(T24/83-pcDNA) or GRP78/BiP overexpressing T24/83 cells
(T24/83-GRP78). Lysates were separated by SDS-polyacrylamide gel
electrophoresis under reducing conditions and gels were either
stained with Coomassie Blue (upper panel) or immunostained with an
anti-human TF mAb (lower panel). The migration position of TF is
shown by the arrowhead. The asterisk indicates the migration
position of GRP78/BiP, which is increased in lane 3.
FIG. 7. Increased cell surface levels of TF in GRP78/BiP
overexpressing cells. Wild type (A), vector-transfected (B) or
GRP78/BiP overexpressing T24/83 cells (C) were grown on coverslips
and incubated for 1 hr at 4.degree. C. with an anti-human TF mAb.
Following incubation with a goat Alexa-conjugated anti-mouse IgG,
cells were fixed in 1% paraformaldehyde. Magnification
.times.1000.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
I. Introduction
The present invention provides novel methods for preventing the
tissue-factor dependent generation of thrombin on the surface of
cells. These methods are based upon the surprising discovery that
the expression or activation of an ER resident chaperone protein in
a cell inhibits the generation of thrombin on the surface of the
cell, and that this inhibition occurs in conjunction with a
decrease in the level of prothrombin consumption, tissue factor
procoagulant activity, and thrombin-inhibitor complexes.
Accordingly, expressing or activating an ER resident chaperone
protein in a cell can be used to inhibit coagulation in the
vicinity of the cell, thereby providing a method for the treatment
or prevention of any of a number of thromboembolic conditions and
diseases. In addition to inhibiting thrombin generation, the
overexpression of ER resident chaperone proteins can also be used
to decrease tissue factor (TF) procoagulant activity (PCA),
cell-surface-mediated prothrombin consumption, or the formation of
thrombin-inhibitor complexes. In addition to these effects, ER
resident chaperones such as GRP78/BiP also protect the cells from
oxidative and ER stress. Thus, cells expressing the chaperones will
also be resistant to physiological perturbants known to cause cell
injury or dysfunction.
In numerous embodiments of this invention, the expression of an ER
resident chaperone protein, such as GRP78/BiP, is induced in a
population of cells of a mammal. The expression or activity of such
proteins can be induced in any of a number of ways, including by
introducing a polynucleotide encoding the chaperone protein into
the cells or by introducing into the cells a polynucleotide that
encodes a protein that induces the chaperone protein.
Alternatively, the cells can be treated with compounds, such as
proteins or small molecules, that induce the activity or expression
of these proteins.
Without being bound by the presently offered theories, it is
hypothesized that, at least in part, the effect of ER resident
chaperones such as GRP78/BiP on the generation of active thrombin
on the surface of cells is mediated by TF, i.e. the expression of
the chaperones inhibits the generation of active TF on the surface
of the cells, thereby preventing the conversion of prothrombin to
thrombin. Accordingly, the present methods are particularly useful
in the prevention of the generation or accumulation of active
thrombin on the surface of TF-expressing cells. It is further
speculated that the effect of the chaperones such as GRP78/BiP on
TF and thrombin may be mediated by calcium, i.e. overexpression of
the calcium-binding GRP78/BiP causes a reduction in the level of
available calcium within the ER, thereby preventing the production
of active TF on the cell surface. As a result, the generation of
active thrombin on the cell surface is inhibited. Accordingly,
thrombin can be inhibited in the surface of cells by the
application of any treatment or compound that causes a decrease in
the level of free calcium in the ER, such as the expression of ER
resident calcium binding proteins such as Calreticulin or
Calretuxin.
Other possible mechanisms for the observed effect include a
conformational change in TF quaternary structure, thereby
decreasing its procoagulant activity (PCA), or a modulation of the
accessibility of anionic phospholipid essential for TF PCA. Another
possible mechanism follows from recent studies using smooth muscle
cells that have shown that LDL increases steady-state levels of TF
mRNA and cell surface protein, but does not affect TF PCA (Penn et
al., (1999) Circulation 99:1753-1759), and that hydrogen peroxide
stimulates TF activity on the cell surface but does not increase TF
mRNA or cell surface protein. These findings support a 2-step
activation pathway for increased TF activity in which lipoproteins
(or other cellular factors) regulate synthesis of latent TF while
oxidants mediate post-translational modifications that enhance cell
surface TF PCA. Given that GRP78/BiP prevents oxidant-induced cell
dysfunction/injury (see, e.g., Liu et al., (1997) J. Biol. Chem.
272:21751-21759; Liu et al., (1998) J. Biol. Chem. 273:12858-12862;
Morris et al., (1997) J. Biol. Chem. 272:4327-4334), it is possible
that overexpression of GRP78/BiP inhibits the oxidant-dependent
activation of latent TF at the cell surface.
It is also speculated that the effect of ER chaperones on thrombin
generation is mediated by thrombomodulin (TM), which promotes
thrombin-dependent activation of the anticoagulant protein C (APC)
pathway, i.e., an increase in the level of an ER chaperone causes
an increase in the level of TM on the cell surface, thereby causing
a decrease in thrombin activity on the cell surface. Accordingly,
the present methods are particularly useful in the prevention of
the accumulation of active thrombin on the surface of TM-expressing
cells.
The present invention also provides methods for identifying
compounds useful in the treatment or prevention of thromboembolic
diseases and conditions. In numerous embodiments, these methods
involve contacting a cell with a test agent, wherein the cell
either expresses, or is capable of expressing, an ER resident
chaperone protein, and determining the effect of the test agent on
the level of expression or activity of the ER resident chaperone in
the cell. In other embodiments, the methods involve contacting an
ER resident chaperone protein with a test agent, and determining
the effect of the test agent on the activity of the ER resident
chaperone protein. In each of these methods, a compound that is
found to increase the level or activity of the ER resident
chaperone represents a candidate for a compound useful in the
treatment or prevention of thromboembolic diseases and
conditions.
It will be appreciated that the herein-described relationship
between ER resident chaperones is valid in either direction, i.e.
an increase in the level or activity of an ER resident chaperone
can be used to decrease the level of active thrombin or TF on the
surface of cells, and a decrease in the ER resident chaperone can
be used to increase the level of active thrombin or TF on the cell
surface.
The inhibition of thrombin generation on the surface of cells can
be used to prevent or treat any of a number of thrombotic or other
cardiovascular diseases or conditions. For example, as
intravascular coagulation is associated with atherosclerosis,
myocardial infarction, and acute arterial thrombosis following
plaque disruption, the present methods can be used to prevent or
treat any of these conditions or diseases. In addition, as
TF-induced thrombin generation plays a critical role in hemostasis
following tissue injury and also in the pathogenesis of multiple
thrombotic disorders associated with a wide range of diseases
including, but not limited to, cardiovascular disease, sepsis, and
cancer, the present methods can also be used to treat or prevent
these diseases and conditions. The level of an ER resident
chaperone can thus be used to prevent thrombin accumulation on the
surface of cells in the endothelium of patients who have had a
myocardial infarction and who are undergoing angioplasty or
stenting, thereby reducing the risk of thrombosis. In addition, the
level can be increased in the endothelium of patients undergoing
cranial radiation to prevent radiation-related thrombotic events.
In addition, the level of ER resident chaperone protein or protein
activity can be increased in the endothelium of patients undergoing
vascular surgery to prevent surgery related thrombosis.
In addition, the present invention provides methods for using
cells, such as the endothelial cell lines overexpressing GRP78/BiP,
as model systems to better understand the factors which mediate
cell surface thrombin generation in endothelial cells.
The therapeutic and prophylactic methods of this invention can be
used in any mammal, including, but not limited to, humans and other
primates, canines, felines, murines, bovines, equines, ovines,
porcines, and lagomorphs.
Kits are also provided for carrying out the herein-disclosed
diagnostic and therapeutic methods.
II. Definitions
An "ER resident chaperone protein" refers to any protein, present
in, or associated with, the ER, that acts to facilitate the
folding, assembly, or translocation of proteins (see, e.g., Ellis
et al., (1989) Trends Biochem Sci 14(8):339-42; Ruddon et al.,
(1997) J. Biol. Chem. 272:3125-3128). As used herein, "ER resident
chaperone proteins" can refer to any protein that facilitates
protein folding, assembly, or translocation, and which is naturally
present in the ER or which is modified to be present in the ER, for
example by the recombinant addition of a signal sequence and/or
other ER localization domains. Examples of ER resident chaperone
proteins include, but are not limited to, BiP/GRP78 (see, e.g.,
GenBank accession No. AJ271729; see, also, SEQ ID NOs: 1 and 2),
GRP94, GRP72, Calreticulin, Calnexin (p88, IP90), TRAP or p28,
cis/trans-Prolyl isomerase, Protein disulfide isomerase, and others
(see, e.g., Ruddon et al., supra). The term "ER resident chaperone
protein" also refers to any variant or derivative of any of the
above proteins.
The "ER," or "endoplasmic reticulum," refers to a highly convoluted
membrane, present in nearly every eukaryotic cell, that is
organized into a netlike meshwork that extends throughout the
cytoplasm. Transmembrane and secreted proteins are synthesized in
association with the ER membrane, and proteins destined to remain
in the ER lumen, the Golgi apparatus, or lysosomes are at least
initially delivered to the ER lumen, the internal space enclosed by
the ER membrane. The ER is also involved in the synthesis of
lipids, including those used to form membranes such as the plasma,
mitochondrial, and peroxisomal membranes. The ER is also the site
of numerous modifications to secreted or membrane associated
proteins, including secreted coagulation factors such as TF and TM;
such modifications include folding, proteolytic processing,
glycosylation, disulphide-bond formation, and oligomeric assembly.
The ER comprises two types of endoplasmic reticulum, called "rough
ER" and "smooth ER." The rough ER corresponds to regions of the ER
in which proteins are synthesized and immediately translocated
across the membrane. The ER membrane in such regions are dotted
with ribosomes, giving it a "rough" appearance. The smooth ER
corresponds to regions of the ER that lack bound ribosomes. As used
herein, "ER" refers to rough ER, smooth ER, as well as transitional
elements that include partly rough and partly smooth regions. A
factor that is said to be "in" or "associated with" the ER refers
to any factor that is permanently, transiently, and/or inducibly
located in the ER lumen, in the ER membrane, or associated with the
ER membrane, e.g., a cytosolic protein in close proximity to the ER
membrane.
"Control cells," in the context of an assay, refers to a population
of cells grown under standard conditions, i.e., in the absence of a
test agent. The "level" of a protein or mRNA of interest can be
measured in such cells expressly for use in the methods described
in this invention, or can refer to an established level of, e.g.,
ER resident chaperone mRNA, protein, or protein activity, which has
been previously established based on measurements from untreated
cells grown under similar conditions. If a detection method is used
that only detects a level of polypeptide or polynucleotide when a
level higher than that typical of control cells is present, i.e.,
an immunohistochemical assay giving a simple positive or negative
result, this is considered to be assessing the level of the
polypeptide or polynucleotide in comparison to the control cells,
as the level typical of the control cells is inherent in the
assay.
An "increased" or "elevated" level of a polypeptide or
polynucleotide, e.g., an ER resident chaperone or thrombin, or of a
detectable activity of the polypeptide, refers to a level of the
polynucleotide or polypeptide, that, in comparison with a control
level, is detectably higher. Similarly, a "decreased" level refers
to a level that, in comparison with a control level, is detectably
lower. The method of comparison can be statistical, using
quantified values, or can be compared using nonstatistical means,
such as by a visual, subjective assessment by a human.
The phrase "functional effects" in the context of assays for
testing compounds that modulate ER resident chaperone levels or
activity includes the determination of any parameter that is
indirectly or directly under the influence of the chaperone, e.g.,
functional, physical and chemical effects. It includes binding to
other proteins or other compounds, changes in TF, TM, or thrombin
activity or levels, chaperone phosphorylation or dephosphorylation,
the ability to counteract ER stress, or inducers of ER stress such
as unfolded proteins, Ca.sup.2+ levels, and also includes other
physiologic effects such as coagulation and associated conditions
including atherosclerosis.
An "inducer" or "activator" of an ER resident chaperone refers to
any compound, e.g., protein, polynucleotide, small organic or
inorganic molecule, that causes a net increase in the level of DNA,
RNA, protein, or protein activity of an ER resident chaperone. Such
inducers can act, e.g., by increasing the transcription,
translation, stability, or protein activity of the chaperone,
directly or indirectly, by any mechanism, and in vivo, ex vivo, or
in vitro. Inducers include genetically modified versions of ER
resident chaperones, e.g., with altered activity, as well as
naturally occurring and synthetic small chemical molecules and the
like. Such assays for inducers include, e.g., applying putative
modulator compounds to a cell that expresses an ER resident
chaperone, or that is capable of expressing an ER resident
chaperone, and determining the functional effects on the expression
or activity of the ER resident chaperone. Samples or assays that
are treated with a potential inducer are compared to control
samples without the inducer to examine the extent of induction.
Control samples (untreated with inhibitors) are assigned a relative
value of 100%. Induction of an ER resident chaperone is achieved
when the value relative to the control is about 110%, optionally
150%, optionally 200-500%, or 1000-3000% higher.
"Biologically active" ER resident chaperone refers to a chaperone
having activity as described herein, in particular regarding the
capacity to modulate the accumulation of active thrombin on the
surface of cells.
The terms "isolated" "purified" or "biologically pure" refer to
material that is substantially or essentially free from components
which normally accompany it as found in its native state. Purity
and homogeneity are typically determined using analytical chemistry
techniques such as polyacrylamide gel electrophoresis or high
performance liquid chromatography. A protein that is the
predominant species present in a preparation is substantially
purified. In particular, an isolated nucleic acid is separated from
open reading frames that flank the gene and which encode
heterologous proteins. The term "purified" denotes that a nucleic
acid or protein gives rise to essentially one band in an
electrophoretic gel. Particularly, it means that the nucleic acid
or protein is at least 85% pure, optionally at least 95% pure, and
optionally at least 99% pure.
"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides
and polymers thereof in either single- or double-stranded form. The
term encompasses nucleic acids containing known nucleotide analogs
or modified backbone residues or linkages, which are synthetic,
naturally occurring, and non-naturally occurring, which have
similar binding properties as the reference nucleic acid, and which
are metabolized in a manner similar to the reference nucleotides.
Examples of such analogs include, without limitation,
phosphorothioates, phosphoramidates, methyl phosphonates,
chiral-methyl phosphonates, 2-O-methyl ribonucleotides,
peptide-nucleic acids (PNAs).
Unless otherwise indicated, a particular nucleic acid sequence also
implicitly encompasses conservatively modified variants thereof
(e.g., degenerate codon substitutions) and complementary sequences,
as well as the sequence explicitly indicated. Specifically,
degenerate codon substitutions may be achieved by generating
sequences in which the third position of one or more selected (or
all) codons is substituted with mixed-base and/or deoxyinosine
residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka
et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol.
Cell. Probes 8:91-98 (1994)). The term nucleic acid is used
interchangeably with gene, cDNA, mRNA, oligonucleotide, and
polynucleotide.
The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
The term "amino acid" refers to naturally occurring and synthetic
amino acids, as well as amino acid analogs and amino acid mimetics
that function in a manner similar to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified,
e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
Amino acids may be referred to herein by either their commonly
known three letter symbols or by the one-letter symbols recommended
by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, may be referred to by their commonly accepted
single-letter codes.
"Conservatively modified variants" applies to both amino acid and
nucleic acid sequences. With respect to particular nucleic acid
sequences, conservatively modified variants refers to those nucleic
acids which encode identical or essentially identical amino acid
sequences, or where the nucleic acid does not encode an amino acid
sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence.
As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
The following eight groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
Macromolecular structures such as polypeptide structures can be
described in terms of various levels of organization. For a general
discussion of this organization, see, e.g., Alberts et al.,
Molecular Biology of the Cell (3.sup.rd ed., 1994) and Cantor and
Schimmel, Biophysical Chemistry Part I: The Conformation of
Biological Macromolecules (1980). "Primary structure" refers to the
amino acid sequence of a particular peptide. "Secondary structure"
refers to locally ordered, three dimensional structures within a
polypeptide. These structures are commonly known as domains.
Domains are portions of a polypeptide that form a compact unit of
the polypeptide and are typically 50 to 350 amino acids long.
Typical domains are made up of sections of lesser organization such
as stretches of .beta.-sheet and .alpha.-helices. "Tertiary
structure" refers to the complete three dimensional structure of a
polypeptide monomer. "Quaternary structure" refers to the three
dimensional structure formed by the noncovalent association of
independent tertiary units. Anisotropic terms are also known as
energy terms.
A "label" or a "detectable moiety" is a composition detectable by
spectroscopic, photochemical, biochemical, immunochemical, or
chemical means. For example, useful labels include .sup.32P,
fluorescent dyes, electron-dense reagents, enzymes (e.g., as
commonly used in an ELISA), biotin, digoxigenin, or haptens and
proteins which can be made detectable, e.g., by incorporating a
radiolabel into the peptide or used to detect antibodies
specifically reactive with the peptide.
I. Expressing or Activating ER Resident Chaperone Proteins
In numerous embodiments of the present invention, an ER resident
chaperone, e.g., GRP78/BiP, is expressed or activated within one or
more mammalian cells. Preferably, the cells are present within a
mammal, i.e., the methods are in vivo methods, and the cells are
associated with an existing, or potential, thromboembolic condition
or disease, for example endothelial or macrophage cells (including
foam cells) at an atherosclerotic lesion.
Any ER resident chaperone protein can be used in the present
methods. In a particularly preferred embodiment, the expression or
activity of GRP78/BiP is increased (see, e.g., Kozutsumi et al.
(1989) J Cell Sci Suppl 11:115-37; Ting et al. (1988) DNA
7(4):275-86; GenBank Accession No. M19645). In addition to
GRP78/BiP, any other ER resident chaperone protein, such as GRP94 (
see, e.g., Sorger et al. (1987) J Mol Biol 194(2):341-4; see, e.g.,
GenBank Accession No. M26596), Calnexin (see, e.g., Wada et al.
(1991) J. Biol. Chem. 266:19599-19610; GenBank Accession No.
M94859), and Calreticulin (see, e.g., Michalak et al. (1992)
Biochem J 285 (Pt 3):681-92; Fliegel et al. (1989) J Biol Chem
264(36):21522-8; GenBank Accession No. NM.sub.--004343), can be
used. In addition, reticulocalbin, an ER-resident calcium-binding
protein can be used (see, e.g., Ozawa et al. (1993) J. Biol. Chem.
268:699-705). It will be appreciated that any variant, derivative,
fragment, or allele of any of these genes or gene products can be
used.
As discussed supra, without being bound by the following theory, it
is speculated that the herein described effects of ER resident
chaperones on the generation of active thrombin on the surface of
cells is mediated by calcium, i.e., the chaperones sequester free
calcium within the secretory pathway, thereby preventing the
generation of active TF on the surface of the cells and, in turn,
preventing the generation of active thrombin. Accordingly, any
treatment, compound, protein, or polynucleotide can be used that
decreases the level of free calcium in the secretory pathway, e.g.,
GRP78/BiP, reticulocalbin, Calreticulin, or Calnexin.
Further, any treatment, factor, or condition that increases the
level or activity of an ER resident chaperone protein can be used,
including those that adversely affect protein processing and
folding in the ER (e.g., homocysteine and other sulfhydryl-reducing
agents, unassembled protein subunits within the ER, overexpression
and aggregation of proteins within the ER), treatments known to
affect cellular glycosylation (e.g., glucose starvation,
tunicamycin, 2-deoxyglucose, glucosamine), treatments known to
affect intracellular Ca.sup.2+ levels (e.g., calcium ionophores
A23187 and ionomycin, calcium chelating agents, EGTA, calcium
ATPase inhibitors, thapsigargin), or other treatments and/or
conditions including ethanol, hypoxia, insulin, tissue injury, and
low extracellular pH.
In certain embodiments, a growth factor will be administered to the
cell that induces the expression of ER chaperone proteins. For
example, IL-3 and other cytokines have been shown to induce the
expression of ER chaperones such as GRP78/BiP and GRP94. See, e.g.,
Brewer et al., (1997) EMBO J. 16:7207-7216. Significantly, this
effect of IL-3 occurs without inducing the unfolded protein
response (UPR) pathway, as evidenced by the absence of expression
of the transcription factor CHOP.
The expression of any of the herein-described genes can be induced
using any of a number of methods, including, but not limited to,
introducing nucleic acids encoding the gene product into cells,
introducing nucleic acids encoding a protein that induces the
expression or activation of the protein of interest, or by
administering to a mammal a compound that induces the expression of
the gene.
1. Introducing Nucleic Acids Into Cells
In numerous embodiments, one or more nucleic acids, e.g., a
GRP78/BiP-encoding polynucleotide, is introduced into cells in
vitro or in vivo. Accordingly, the present invention provides
methods, reagents, vectors, and cells useful for the expression of
GRP78/BiP and other ER resident chaperone proteins and nucleic
acids using in vitro (cell-free), ex vivo or in vivo (cell or
organism-based) recombinant expression systems. Such methods are
useful for a number of applications, for example to treat or
prevent a thromboembolic condition or disease in a patient, to
modulate the level of TF activity and/or thrombin activity on the
surface of a cell, e.g., to investigate the role of TF or thrombin
in coagulation, or to create cells useful in the screening of test
agents for the ability to modulate ER resident chaperone expression
or activity.
For use in the present invention, any standard procedure for
introducing foreign nucleotide sequences into host cells may be
used including, but not limited to, calcium phosphate transfection,
spheroplasts, electroporation, liposomes, microinjection, plasma
vectors, viral vectors, and any other method for introducing cloned
genomic DNA, cDNA, synthetic DNA or other foreign genetic material
into a host cell (see, e.g., Berger and Kimmel, Guide to Molecular
Cloning Techniques, Methods in Enzymology volume 152 Academic
Press, Inc., San Diego, Calif. (Berger), F. M. Ausubel et al.,
eds., Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (supplemented
through 1999), and Sambrook et al., Molecular Cloning--A Laboratory
Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., 1989.
Preparation of various polynucleotides and vectors useful in the
present invention are well known. General texts which describe
methods of making recombinant nucleic acids include Sambrook et
al., supra; Ausubel et al., supra; and Berger and Kimmel, Guide to
Molecular Cloning Techniques, Methods in Enzymology, volume 152
Academic Press, Inc., San Diego, Calif. (Berger). In numerous
embodiments of this invention, nucleic acids will be inserted into
vectors using standard molecular biological techniques. Vectors may
be used at multiple stages of the practice of the invention,
including for subcloning nucleic acids encoding, e.g., components
of proteins or additional elements controlling protein expression,
vector selectability, etc. Vectors may also be used to maintain or
amplify the nucleic acids, for example by inserting the vector into
prokaryotic or eukaryotic cells and growing the cells in
culture.
Product information from manufacturers of biological reagents and
experimental equipment also provide information useful in known
biological methods such as cloning. Such manufacturers include the
SIGMA chemical company (Saint Louis, Mo.), R&D systems
(Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway,
N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes
Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research,
Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.), Fluka
Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),
Invitrogen, San Diego, Calif., Applied Biosystems (Foster City,
Calif.), Digene Diagnostics, Inc. (Beltsville, Md.) as well as many
other commercial sources known to one of skill. These commercial
suppliers produce extensive catalogues of compounds, products,
kits, techniques and the like for performing a variety of standard
methods.
A convenient method of introducing the polynucleotides into cells
in vivo and in vitro involves the use of viral vectors, e.g.,
adenoviral vector mediated gene delivery (see, e.g., Chen et al.
(1994) Proc. Nat'l. Acad. Sci. USA 91:3054-3057; Tong et al. (1996)
Gynecol. Oncol. 61:175-179; Clayman et al. (1995) Cancer Res.
5:1-6; O'Malley et al. (1995) Cancer Res. 55:1080-1085; Hwang et
al. (1995) Am. J. Respir. Cell Mol. Biol. 13:7-16; Haddada et al.
(1995) Curr. Top. Microbiol. Immunol. 199 (Pt. 3):297-306; Addison
et al. (1995) Proc. Nat'l. Acad. Sci. USA 92:8522-8526; Colak et
al. (1995) Brain Res. 691:76-82; Crystal (1995) Science
270:404-410; Elshami et al. (1996) Human Gene Ther. 7:141-148;
Vincent et al. (1996) J. Neurosurg. 85:648-654); and retroviral
vectors (see, e.g., Marx et al. Hum Gene Ther 1999 May
1;10(7):1163-73; Mason et al. (1998) Gene Ther. 5(8):1098-104). In
addition, replication-defective retroviral vectors harboring a
therapeutic polynucleotide sequence as part of the retroviral
genome have also been used, particularly with regard to simple MuLV
vectors. See, e.g., Miller et al. (1990) Mol. Cell. Biol. 10:4239;
Kolberg (1992) J. NIH Res. 4:43, and Cornetta et al. Hum. (1991)
Gene Ther. 2:215. Other suitable retroviral vectors include
lentiviruses (Klimatcheva et al., (1999) Front Biosci 4:D481-96).
Other viral vectors that can be used in the present invention
include vectors derived from adeno-associated viruses (Bueler
(1999) Biol Chem 380(6):613-22; Robbins and Ghivizzani (1998)
Pharmacol. Ther. 80(1):35-47), herpes simplex viruses (Krisky et
al. (1998) Gene Ther. 5(11):1517-30), and others.
Plasmid vectors can also be delivered as "naked" DNA or combined
with various transfection-facilitating agents. Numerous studies
have demonstrated the direct administration of naked DNA, e.g.,
plasmid DNA, to cells in vivo (see, e.g., Wolff (1997) Neuromuscul.
Disord. 7(5):314-8, Nomura et al. (1999) Gene Ther. 6(1):121-9).
For certain applications it is possible to coat the DNA onto small
particles and project genes into cells using a device known as a
gene gun.
Plasmid DNA can also be combined with any of a number of
transfection-facilitating agents. The most commonly used
transfection facilitating agents for plasmid DNA in vivo have been
charged and/or neutral lipids (Debs and Zhu (1993) WO 93/24640 and
U.S. Pat. No. 5,641,662; Debs U.S. Pat. No. 5,756,353; Debs and Zhu
Published EP Appl. No. 93903386; Mannino and Gould-Fogerite (1988)
BioTechniques 6(7):682-691; Rose U.S. Pat. No. 5,279,833; Brigham
(1991) WO 91/06309 and U.S. Pat. No. 5,676,954; and Felgner et al.
(1987) Proc. Natl. Acad. Sci. USA 84:7413-7414). Additional useful
liposome-mediated DNA transfer methods, other than the references
noted above, are described in U.S. Pat. Nos. 5,049,386; 4,946,787;
and 4,897,355; PCT publications WO 91/17424, WO 91/16024; Wang and
Huang (1987) Biochem. Biophys. Res. Commun. 147:980; Wang and Huang
(1989) Biochemistry 28:9508; Litzinger and Huang (1992) Biochem.
Biophys. Acta 1113:201; Gao and Huang (1991) Biochem. Biophys. Res.
Commun. 179:280. Immunoliposomes have been described as carriers of
exogenous polynucleotides (Wang and Huang (1987) Proc. Natl. Acad.
Sci. U.S.A. 84:7851; Trubetskoy et al. (1992) Biochem. Biophys.
Acta 1131:311) and may have improved cell type specificity as
compared to liposomes by virtue of the inclusion of specific
antibodies which presumably bind to surface antigens on specific
cell types. Behr et al. (1989) Proc. Natl. Acad. Sci. U.S.A.
86:6982 report using lipopolyamine as a reagent to mediate
transfection itself, without the necessity of any additional
phospholipid to form liposomes.
Lipid carriers usually contain a cationic lipid and a neutral
lipid. Most in vivo transfection protocols involve forming
liposomes made up of a mixture of cationic and neutral lipid and
complexing the mixture with a nucleic acid. The neutral lipid is
often helpful in maintaining a stable lipid bilayer in liposomes
used to make the nucleic acid:lipid complexes, and can
significantly affect transfection efficiency. Liposomes may have a
single lipid bilayer (unilamellar) or more than one bilayer
(multilamellar). They are generally categorized according to size,
where those having diameters up to about 50 to 80 nm are termed
"small" and those greater than about 80 to 1000 nm, or larger, are
termed "large." Thus, liposomes are typically referred to as large
unilamellar vesicles (LUVs), multilamellar vesicles (MLVs) or small
unilamellar vesicles (SUVs).
Cationic liposomes are typically mixed with polyanionic compounds
(including nucleic acids) for delivery to cells. Complexes form by
charge interactions between the cationic lipid components and the
negative charges of the polyanionic compounds.
A wide variety of liposomal formulations are known and commercially
available and can be tested in the assays of the present invention
for precipitation, DNA protection, pH effects and the like. Because
liposomal formulations are widely available, no attempt will be
made here to describe the synthesis of liposomes in general. Two
references which describe a number of therapeutic formulations and
methods are WO 96/40962 and WO 96/40963.
Cationic lipid-nucleic acid transfection complexes can be prepared
in various formulations depending on the target cells to be
transfected. While a range of lipid-nucleic acid complex
formulations will be effective in cell transfection, optimal
conditions are determined empirically in the desired system. Lipid
carrier compositions are evaluated, e.g., by their ability to
deliver a reporter gene (e.g., CAT, which encodes chloramphenicol
acetyltransferase, luciferase, .beta.-galactosidase, or GFP) in
vitro, or in vivo to a given tissue type in an animal, or in assays
which test stability, protection of nucleic acids, and the
like.
The lipid mixtures are complexed with nucleic acids in different
ratios depending on the target cell type, generally ranging from
about 6:1 to 1:20 .mu.g nucleic acid:nmole cationic lipid.
For mammalian host cells, viral-based and nonviral, e.g.,
plasmid-based, expression systems are provided. Nonviral vectors
and systems include plasmids and episomal vectors, typically with
an expression cassette for expressing a protein or RNA, and human
artificial chromosomes (see, e.g., Harrington et al. (1997) Nat.
Genet. 15:345). For example, plasmids useful for expression of
polynucleotides and polypeptides in mammalian (e.g., human) cells
include pcDNA3.1/His, pEBVHis A, B & C, (Invitrogen, San Diego
Calif.), MPSV vectors, others described in the Invitrogen 1997
Catalog (Invitrogen Inc, San Diego Calif.), which is incorporated
in its entirety herein, and numerous others known in the art for
other proteins.
Useful viral vectors include vectors based on retroviruses,
adenoviruses, adeno-associated viruses, herpes viruses, vectors
based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia
virus vectors and Semliki Forest virus (SFV). SFV and vaccinia
vectors are discussed generally in Ausubel et al., supra, Ch. 16.
These vectors are often made up of two components, a modified viral
genome and a coat structure surrounding it (see generally, Smith
(1995) Ann. Rev. Microbiol. 49:807), although sometimes viral
vectors are introduced in naked form or coated with proteins other
than viral proteins. However, the viral nucleic acid in a vector
may be changed in many ways, for example, when designed for gene
therapy. The goals of these changes are to disable growth of the
virus in target cells while maintaining its ability to grow in
vector form in available packaging or helper cells, to provide
space within the viral genome for insertion of exogenous DNA
sequences, and to incorporate new sequences that encode and enable
appropriate expression of the gene of interest.
Thus, viral vector nucleic acids generally comprise two components:
essential cis-acting viral sequences for replication and packaging
in a helper line and the transcription unit for the exogenous gene.
Other viral functions are expressed in trans in a specific
packaging or helper cell line. Adenoviral vectors (e.g., for use in
human gene therapy) are described in, e.g., Rosenfeld et al. (1992)
Cell 68:143; PCT publications WO 94/12650; 94/12649; and 94/12629.
In cases where an adenovirus is used as an expression vector, a
sequence may be ligated into an adenovirus
transcription/translation complex consisting of the late promoter
and tripartite leader sequence. Insertion in a nonessential E1 or
E3 region of the viral genome will result in a viable virus capable
of expressing in infected host cells (Logan and Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655). Replication-defective retroviral
vectors harboring a therapeutic polynucleotide sequence as part of
the retroviral genome are described in, e.g., Miller et al. (1990)
Mol. Cell. Biol. 10:4239; Kolberg (1992) J. NIH Res. 4:43; and
Cornetta et al. (1991) Hum. Gene Ther. 2:215. In certain
embodiments, the surface of the virus can be coated, e.g., by
covalent attachment, with polyethylene glycol (PEG; see, e.g.,
O'Riordan et al. (1999) Hum. Gene Ther. 10(8):1349-58.). Such
"PEGylation" of viruses can impart various benefits, including
increasing the infectivity of the virus, and lowering the host
immune response to the virus.
A variety of commercially or commonly available vectors and vector
nucleic acids can be converted into a vector of the invention by
cloning a polynucleotide of this invention into the commercially or
commonly available vector. A variety of common vectors suitable for
this purpose are well known in the art. For cloning in bacteria,
common vectors include pBR322 derived vectors such as
pBLUESCRIPT.TM., and bacteriophage derived vectors. In yeast,
vectors include Yeast Integrating plasmids (e.g., YIp5) and Yeast
Replicating plasmids (the YRp series plasmids) and pGPD-2.
Expression in mammalian cells can be achieved using a variety of
commonly available plasmids, including pSV2, pBC12BI, and p91023,
as well as lytic virus vectors (e.g., vaccinia virus, adeno virus,
and baculovirus), episomal virus vectors (e.g., bovine
papillomavirus), and retroviral vectors (e.g., murine
retroviruses).
Typically, a nucleic acid subsequence encoding a polypeptide, e.g.,
an ER resident chaperone protein, is placed under the control of a
promoter. A nucleic acid is "operably linked" to a promoter when it
is placed into a functional relationship with the promoter. For
instance, a promoter or enhancer is operably linked to a coding
sequence if it increases or otherwise regulates the transcription
of the coding sequence. Similarly, a "recombinant expression
cassette" or simply an "expression cassette" is a nucleic acid
construct, generated recombinantly or synthetically, with nucleic
acid elements that are capable of effecting expression of a
structural gene in hosts compatible with such sequences. Expression
cassettes include promoters and, optionally, introns,
polyadenylation signals, and transcription termination signals.
Additional factors necessary or helpful in effecting expression may
also be used as described herein. For example, an expression
cassette can also include nucleotide sequences that encode a signal
sequence that directs secretion of an expressed protein from the
host cell. Transcription termination signals, enhancers, and other
nucleic acid sequences that influence gene expression, can also be
included in an expression cassette.
An extremely wide variety of promoters are well known, and can be
used in the vectors of the invention, depending on the particular
application. Ordinarily, the promoter selected depends upon the
cell in which the promoter is to be active. In mammalian cell
systems, promoters from mammalian genes or from mammalian viruses
are often appropriate. Suitable promoters may be constitutive, cell
type-specific, stage-specific, and/or inducible or repressible
(e.g., by hormones such as glucocorticoids). Useful promoters
include, but are not limited to, the metallothionein promoter, the
constitutive adenovirus major late promoter, the
dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP
polIII promoter, the constitutive MPSV promoter, the
tetracycline-inducible CMV promoter (such as the human
immediate-early CMV promoter), the constitutive CMV promoter, and
promoter-enhancer combinations known in the art. In one embodiment,
the promoter normally associated with the polynucleotide is used,
e.g., to express GRP78/BiP, a GRP78/BiP promoter is used (see,
e.g., GenBank Accession No. X59969).
Other expression control sequences such as ribosome binding sites,
transcription termination sites and the like are also optionally
included. For E. coli, example control sequences include the T7,
trp, or lambda promoters, a ribosome binding site and preferably a
transcription termination signal. For eukaryotic cells, the control
sequences typically include a promoter which optionally includes an
enhancer derived from immunoglobulin genes, SV40, cytomegalovirus,
a retrovirus (e.g., an LTR based promoter) etc., and a
polyadenylation sequence, and may include splice donor and acceptor
sequences.
2. Inducing ER Resident Chaperones Using Heterologous Compounds
In numerous embodiments of this invention, the level of active
thrombin generation on the surface of a cell will be induced by
administering to the cell, in vivo or in vitro, any of a large
number of potential ER resident chaperone-inducing molecules, e.g.,
polypeptides, antibodies, amino acids, nucleotides, lipids,
carbohydrates, or any organic or inorganic molecule. Such
modulators are particularly useful in the prevention or treatment
of any of a large number of thromboembolic diseases or
conditions.
In one embodiment, a growth factor, e.g., a cytokine such as IL-3
is used to induce the expression of a chaperone such as GRP78/BiP
(see, e.g., Brewer et al., supra).
To identify molecules capable of modulating ER resident chaperones,
assays will be performed to detect the effect of various compounds
on the level or activity of the chaperone. Such assays can involve
the identification of compounds that interact with the chaperone
proteins, either physically or genetically, and can thus rely on
any of a number of standard methods to detect physical or genetic
interactions between compounds. Such assays can also involve the
identification of compounds that affect ER resident chaperone
expression, activity or other properties, such as its
phosphorylation state or ability to bind other proteins. Such
assays can also involve the detection of ER resident chaperone
activity in a cell, either in vitro or in vivo. Such cell-based
assays can be performed in any type of cell, e.g., a cell that
naturally expresses an ER resident chaperone, or a cultured cell
that produces the chaperone due to recombinant expression.
In a preferred embodiment, the cells used in such assays are
bladder carcinoma cell lines such as T24/83 cells (see, Examples
section).
In numerous embodiments, a compound will be identified that induces
the expression of an ER resident chaperone protein such as
GRP78/BiP, e.g., by increasing its transcription, translation, mRNA
stability, etc. For example, to identify agents that induce the
expression of, e.g., GRP78/BiP, a preferred "screening" method
involves (i) contacting a cell capable of expressing GRP78/BiP with
a test agent; and (ii) detecting the level of GRP78/BiP expression
in the presence and absence of the test agent, where an increased
level of expression in the presence of the test agent in comparison
to the level of expression in the absence of the test agent
indicates that the test agent increases or induces the expression
of the protein.
Such modulators of expression or activity of an ER resident
chaperone proteins can also involve detecting the ability of a test
agent to bind to or otherwise interact with the protein, or with a
nucleic acid sequence, e.g., a promoter, encoding or regulating the
expression of the protein.
In any of the herein described embodiments, a compound that causes
an increase in the level or activity of the ER resident chaperone
protein can be used to decrease the accumulation of active thrombin
or TF PCA on the surface of cells. In contrast, compounds that
cause a decrease in the level or activity of the ER resident
chaperone protein can be used to increase the accumulation of
active thrombin or TF PCA on the surface of cells.
In preferred embodiments, compounds found to increase the
expression or activity of an ER resident chaperone will not cause
an associated increase in ER stress (i.e., the induction of the
chaperone is preferably direct and not occurring in response to an
induction of ER stress). The effect of a candidate ER resident
chaperone inducer on ER stress can be detected, using any of a
number of methods, including by detecting the expression or
activation of any ER stress-responsive gene or gene product,
including, but not limited to, GRP78/BiP, NF.kappa.B, GADD153,
GADD45, Id-1, ATF-4, YY1, cyclin D1, FRA-2, glutathione peroxidase,
NKEF-B PAG, superoxide dismutase, and clusterin (Outinen et al.
(1999) Blood 94:959-967; Outinen et al. (1998) Biochem. J.
332:213-221). In addition, ER stress-inducing ability can be
detected using a "cell-killing" type assay, where the ability of an
agent to kill a cell by ER stress can be determined by comparing
the ability of the agent to kill cells in normal cells or in cells
expressing an ER protecting factor, such as GRP78/BiP. Agents that
kill cells only in the absence of such protective factors are
identified as ER stress-inducing factors. See, e.g., Morris et al.
(1997) J. Biol. Chem. 272:4327-34). Agents that affect the level of
misfolded proteins can also be used, e.g., to detect modulation of
ER stress, by, e.g., detecting misfolded proteins by virtue of
their ability to bind to GRP78/BiP.
a) Detection of Polynucleotides
In numerous embodiments, the ability of a test agent to induce an
ER resident chaperone protein is monitored by detecting the level
of mRNA encoding the ER resident chaperone itself, or of mRNA
encoding a protein whose expression is regulated by the ER resident
chaperone (e.g., TF, TM, or thrombin). Methods of detecting and/or
quantifying the level of a gene transcript using nucleic acid
hybridization techniques are known to those of skill in the art
(see, Sambrook et al (1989) Molecular Cloning: A Laboratory Manual,
2d Ed., vols 1-3, Cold Spring Harbor Press, New York).
For example, one method for evaluating the presence, absence, or
quantity of an ER resident chaperone associated cDNA involves a
Southern Blot. Briefly, the mRNA is isolated using standard methods
and reverse transcribed to produce cDNA. The cDNA is then
optionally digested, run on a gel, and transferred to a membrane.
Hybridization is then carried out using nucleic acid probes
specific for the cDNA and detected using standard techniques (see,
e.g., Sambrook et al., supra).
Similarly, a Northern blot may be used to detect an mRNA directly.
In brief, in a typical embodiment, mRNA is isolated from a
population of cells, electrophoresed to separate the mRNA species,
and transferred from the gel to a nitrocellulose membrane. As with
the Southern blots, labeled probes are then hybridized to the
membrane to identify and/or quantify the mRNA.
In another preferred embodiment, a transcript (e.g., mRNA) is
detected using amplification-based methods (e.g., RT-PCR). RT-PCR
methods are well known to those of skill (see, e.g., Ausubel et al,
supra). Preferably, quantitative RT-PCR is used, thereby allowing
the comparison of the level of mRNA in a sample with a control
sample or value.
b) Detection of Polypeptides
In other embodiments, the functional effect of a test agent is
monitored by detecting the level of an ER resident chaperone
protein or of a protein that is regulated by an ER resident
chaperone protein, e.g., thrombin, TF, or TM. The level of any
polypeptide, e.g., an ER resident chaperone protein, thrombin, TF,
or TM, can be detected and quantified by any of a number of means
well known to those of skill in the art. These include analytic
biochemical methods such as electrophoresis, capillary
electrophoresis, high performance liquid chromatography (HPLC),
thin layer chromatography (TLC), hyperdiffusion chromatography, and
the like, or various immunological methods such as fluid or gel
precipitin reactions, immunodiffusion (single or double),
immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked
immunosorbent assays (ELISAs), immunofluorescent assays, western
blotting, and the like.
In a preferred embodiment, one of the above-recited polypeptides is
detected using an immunoassay such as an ELISA assay (see, e.g.,
Crowther, John R. ELISA Theory and Practice. Humana Press: New
Jersey, 1995). As used herein, an "immunoassay" is an assay that
utilizes an antibody to specifically bind to the analyte (i.e., the
polypeptide). The immunoassay is thus characterized by detection of
specific binding of a polypeptide to an antibody specific to the
polypeptide.
In an immunoassay, a polypeptide can be detected and/or quantified
using any of a number of well recognized immunological binding
assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288;
and 4,837,168). For a review of the general immunoassays, see also
Asai (1993) Methods in Cell Biology Volume 37: Antibodies in Cell
Biology, Academic Press, Inc. New York; Stites & Terr (1991)
Basic and Clinical Immunology 7th Edition, Enzyme Immunoassay
(Maggio, ed., 1980); and Harlow & Lane, supra.
Immunoassays typically rely on direct or indirect labeling methods
to detect antibody-analyte binding. For example, an anti-GRP78/BiP
antibody can be directly labeled, thereby allowing detection.
Alternatively, the anti-GRP78/BiP antibody may itself be unlabeled,
but may, in turn, be bound by a labeled third antibody specific to
antibodies of the species from which the second antibody is
derived. The second or third antibodies can also be modified with a
detectable moiety, e.g., as biotin, to which a third labeled
molecule can specifically bind, such as enzyme-labeled
streptavidin. Also, other antibody-binding molecules can be used,
e.g., labeled protein A or G (see, generally Kronval, et al. (1973)
J. Immunol. 111:1401-1406, and Akerstrom (1985) J. Immunol.
135:2589-2542).
Throughout the assays, incubation and/or washing steps may be
required after each combination of reagents. Incubation steps can
vary from about 5 seconds to several hours, preferably from about 5
minutes to about 24 hours. However, the incubation time will depend
upon the assay format, antigen, volume of solution, concentrations,
and the like. Usually, the assays will be carried out at ambient
temperature, although they can be conducted over a range of
temperatures, such as 10.degree. C. to 40.degree. C.
Immunoassays for detecting a polypeptide can be competitive or
noncompetitive. Noncompetitive immunoassays are assays in which the
amount of captured analyte is directly measured. In a preferred
embodiment, "sandwich" assays will be used, for example, wherein
antibodies specific for the analyte are bound directly to a solid
substrate where they are immobilized. These immobilized antibodies
then capture the protein of interest present in a test sample. The
protein thus immobilized is then bound by a labeling agent, such as
a second specific antibody bearing a label.
In competitive assays, the amount of protein present in a sample is
measured indirectly, e.g., by measuring the amount of added
(exogenous) protein displaced (or competed away) from a specific
antibody by protein present in a sample. For example, a known
amount of labeled GRP78/BiP polypeptide is added to a sample and
the sample is then contacted with an anti-GRP78/BiP antibody. The
amount of labeled GRP78/BiP polypeptide bound to the antibody is
inversely proportional to the concentration of GRP78/BiP
polypeptide present in the sample.
Any of a number of labels can be used in any of the immunoassays of
this invention, including fluorescent labels, radioisotope labels,
or enzyme-based labels, wherein a detectable product of enzyme
activity is detected (e.g., peroxidase, alkaline phosphatase,
.beta.-galactosidase, etc.).
One of skill in the art will appreciate that it is often desirable
to minimize nonspecific binding in immunoassays. Particularly,
where the assay involves an antigen or antibody immobilized on a
solid substrate it is desirable to minimize the amount of
nonspecific binding to the substrate. Means of reducing such
nonspecific binding are well known to those of skill in the art.
Typically, this technique involves coating the substrate with a
proteinaceous composition. In particular, protein compositions such
as bovine serum albumin (BSA), nonfat powdered milk, and gelatin
are widely used.
Methods of producing polyclonal and monoclonal antibodies that
react specifically with a protein are known to those of skill in
the art (see, e.g., Coligan (1991) Current Protocols in Immunology;
Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principles
and Practice (2d ed. 1986); and Kohler & Milstein (1975) Nature
256:495-497. Such techniques include antibody preparation by
selection of antibodies from libraries of recombinant antibodies in
phage or similar vectors, as well as preparation of polyclonal and
monoclonal antibodies by immunizing rabbits or mice (see, e.g.,
Huse et al. (1989) Science 246:1275-1281; Ward et al. (1989) Nature
341:544-546).
A number of peptides or a full length protein may be used to
produce antibodies specifically reactive with a protein of
interest. For example, recombinant protein can be expressed in
eukaryotic or prokaryotic cells and purified using standard
methods. Recombinant protein is the preferred immunogen for the
production of monoclonal or polyclonal antibodies. Alternatively, a
synthetic peptide derived from any amino acid sequence can be
conjugated to a carrier protein and used as an immunogen. Naturally
occurring protein may also be used either in pure or impure form.
The product is then injected into an animal capable of producing
antibodies. Either monoclonal or polyclonal antibodies may be
generated, for subsequent use in immunoassays to measure the
protein.
Methods of production of polyclonal antibodies are known to those
of skill in the art. An inbred strain of mice (e.g., BALB/C mice)
or rabbits is immunized with the protein using a standard adjuvant,
such as Freund's adjuvant, and a standard immunization protocol.
The animal's immune response to the immunogen preparation is
monitored by taking test bleeds and determining the titer of
reactivity to the protein. When appropriately high titers of
antibody to the immunogen are obtained, blood is collected from the
animal and antisera are prepared. Further fractionation of the
antisera to enrich for antibodies reactive to the protein can be
done if desired (see, Harlow & Lane, supra).
Monoclonal antibodies may be obtained by various techniques
familiar to those skilled in the art. Briefly, spleen cells from an
animal immunized with a desired antigen are immortalized, commonly
by fusion with a myeloma cell (see, Kohler & Milstein (1976)
Eur. J. Immunol. 6:511-519). Alternative methods of immortalization
include transformation with Epstein Barr Virus, oncogenes, or
retroviruses, or other methods well known in the art. Colonies
arising from single immortalized cells are screened for production
of antibodies of the desired specificity and affinity for the
antigen, and yield of the monoclonal antibodies produced by such
cells may be enhanced by various techniques, including injection
into the peritoneal cavity of a vertebrate host. Alternatively, one
may isolate DNA sequences which encode a monoclonal antibody or a
binding fragment thereof by screening a DNA library from human B
cells according to the general protocol outlined by Huse et al.
(1989) Science 246:1275-1281.
Monoclonal antibodies and polyclonal sera are collected and titered
against the immunogen protein in an immunoassay, for example, a
solid phase immunoassay with the immunogen immobilized on a solid
support. Typically, polyclonal antisera with a titer of 10.sup.4 or
greater are selected and tested for their cross reactivity against
non-specific proteins or even other related proteins from other
organisms, using a competitive binding immunoassay. Specific
polyclonal antisera and monoclonal antibodies will usually bind
with a K.sub.d of at least about 0.1 mM, more usually at least
about 1 .mu.M, preferably at least about 0.1 .mu.M or better, and
most preferably, 0.01 .mu.M or better.
c) Detection of ER Resident Chaperone-Binding Compounds
In certain embodiments, assays will be performed to identify
molecules that physically or genetically interact with ER resident
chaperone proteins. Such molecules can be any type of molecule,
including polypeptides, polynucleotides, amino acids, nucleotides,
carbohydrates, lipids, or any other organic or inorganic molecule.
Such molecules may represent molecules that normally interact with
ER resident chaperones, or may be synthetic or other molecules that
are capable of interacting with ER resident chaperones and that can
potentially be used to modulate ER resident chaperone activity in
cells, or used as lead compounds to identify classes of molecules
that can interact with and/or modulate ER resident chaperones. Such
assays may represent physical binding assays, such as affinity
chromatography, immunoprecipitation, two-hybrid screens, or other
binding assays, or may represent genetic assays as described
infra.
In any of the binding or functional assays described herein, in
vivo or in vitro, any ER resident chaperone protein, or any
derivative, variant, homolog, or fragment of an ER resident
chaperone protein, can be used. In numerous embodiments, a fragment
of an ER resident chaperone protein is used.
Compounds that interact with ER resident chaperone proteins can be
isolated based on an ability to specifically bind to an ER resident
chaperone protein or fragment thereof. In numerous embodiments, the
ER resident chaperone protein or protein fragment is attached to a
solid support. In one embodiment, affinity columns are made using
the ER resident chaperone polypeptide, and physically-interacting
molecules are identified. It will be apparent to one of skill that
chromatographic techniques can be performed at any scale and using
equipment from many different manufacturers (e.g., Pharmacia
Biotech). In addition, molecules that interact with ER resident
chaperone proteins in vivo can be identified by
co-immunoprecipitation or other methods, i.e., immunoprecipitating
ER resident chaperone proteins using anti-ER resident chaperone
antibodies, e.g., anti GRP78/BiP antibodies, from a cell or cell
extract, and identifying compounds, e.g., proteins, that are
precipitated along with the ER resident chaperone protein. Such
methods are well known to those of skill in the art and are taught,
e.g., in Ausubel et al., Sambrook et al., Harlow & Lane, all
supra.
Two-hybrid screens can also be used to identify polypeptides that
interact in vivo with an ER resident chaperone polypeptide or a
fragment thereof (Fields et al. (1989) Nature 340:245-246). Such
screens comprise two discrete, modular domains of a transcription
factor protein, e.g., a DNA binding domain and a transcriptional
activation domain, which are produced in a cell as two separate
polypeptides, each of which also comprises one of two potentially
binding polypeptides. If the two potentially binding polypeptides
do in fact interact in vivo, then the DNA binding and the
transcriptional activating domain of the transcription factor are
united, thereby producing expression of a target gene in the cell.
The target gene typically encodes an easily detectable gene
product, e.g., .beta.-galactosidase, GFP, or luciferase, which can
be detected using standard methods. In the present invention, an ER
resident chaperone polypeptide, or fragment thereof, is fused to
one of the two domains of the transcription factor, and the
potential ER resident chaperone-binding polypeptides (e.g., encoded
by a cDNA library) are fused to the other domain. Such methods are
well known to those of skill in the art, and are taught, e.g., in
Ausubel et al., supra.
d) Assays for ER Resident Chaperone Protein Activity
The effects of a test compound upon the function of an ER resident
chaperone protein can also be measured by examining any suitable
physiological change that is affected by ER resident chaperone
activity. For example, the ability of ER resident chaperones to
protect cells from oxidative or ER stress can be detected. When the
functional consequences are determined using intact cells or
animals, one can also measure a variety of effects that are
indicative of decreased thrombin levels, e.g., inhibition of
atherosclerosis or other cardiovascular conditions, prevention of
thrombosis, etc.
3. Test Agents
Virtually any agent can be tested in the presently described
assays, including, but not limited to, natural or synthetic nucleic
acids, natural or synthetic polypeptides, natural or synthetic
lipids, natural or synthetic small organic molecules, and the like.
In one preferred format, test agents are provided as members of a
combinatorial library. In preferred embodiments, a collection of
small molecules are tested for the ability to modulate the
expression or activity of an ER resident chaperone gene or gene
product. A "small molecule" refers to any molecule, e.g., a
carbohydrate, nucleotide, amino acid, oligonucleotide,
oligopeptide, lipid, inorganic compound, etc. that can be tested in
such an assay. Such molecules can modulate the expression or
activity of any of the herein-recited genes or gene products by any
of a number of mechanisms, e.g., by binding to a promoter and
modulating the expression of the encoded protein, by binding to an
mRNA and affecting its stability or translation, or by binding to a
protein and competitively or non-competitively affecting its
interaction with, e.g., other proteins in the cell. Further, such
molecules can affect the protein directly or indirectly, i.e., by
affecting the expression or activity of a regulatory of the
protein. Preferably, such "small molecule inhibitors" are smaller
than about 10 kD, preferably 5, 2, or 1 kD or less.
As discussed above, test agents can be screened based on any of a
number of factors, including, but not limited to, a level of a
polynucleotide, e.g., mRNA, of interest, a level of a polypeptide,
the degree of binding of a compound to a polynucleotide or
polypeptide, the intracellular localization of a polynucleotide or
polypeptide, any biochemical properties of a polypeptide, e.g.,
phosphorylation or glycosylation, or any functional properties of a
protein, such as the ability of the protein to induce the
expression of other genes or to induce cholesterol biosynthesis.
Such direct and indirect measures of protein activity in vivo can
readily be used to detect and screen for molecules that modulate
the activity of the protein.
a) Combinatorial Libraries
In certain embodiments, combinatorial libraries of potential
modulators will be screened for an ability to bind to a polypeptide
or to modulate the activity of the polypeptide. Conventionally, new
chemical entities with useful properties are generated by
identifying a chemical compound (called a "lead compound") with
some desirable property or activity, e.g., GRP78/BiP activating
activity, creating variants of the lead compound, and evaluating
the property and activity of those variant compounds. However, the
current trend is to shorten the time scale for all aspects of drug
discovery. Because of the ability to test large numbers quickly and
efficiently, high throughput screening (HTS) methods are replacing
conventional lead compound identification methods.
In one preferred embodiment, high throughput screening methods
involve providing a library containing a large number of potential
therapeutic compounds (candidate compounds). Such "combinatorial
chemical libraries" are then screened in one or more assays to
identify those library members particular chemical species or
subclasses) that display a desired characteristic activity. The
compounds thus identified can serve as conventional "lead
compounds" or can themselves be used as potential or actual
therapeutics.
A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library, such as a polypeptide (e.g., mutein) library, is
formed by combining a set of chemical building blocks called amino
acids in every possible way for a given compound length (i.e., the
number of amino acids in a polypeptide compound). Millions of
chemical compounds can be synthesized through such combinatorial
mixing of chemical building blocks (Gallop et al. (1994) J. Med.
Chem. 37(9):1233-1251).
Preparation and screening of combinatorial chemical libraries is
well known to those of skill in the art. Such combinatorial
chemical libraries include, but are not limited to, peptide
libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka (1991) Int. J.
Pept. Prot. Res., 37:487-493, Houghton et al. (1991) Nature,
354:84-88), peptoids (PCT Publication No WO 91/19735, Dec. 26,
1991), encoded peptides (PCT Publication WO 93/20242, Oct. 14.
1993), random bio-oligomers (PCT Publication WO 92/00091, Jan. 9,
1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such
as hydantoins, benzodiazepines and dipeptides (Hobbs et al (1993)
Proc. Nat. Acad. Sci. USA 90:6909-6913), vinylogous polypeptides
(Hagihara et al (1992) J. Amer. Chem. Soc. 114:6568), nonpeptidal
peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et
al. (1992) J. Amer. Chem. Soc. 114:9217-9218), analogous organic
syntheses of small compound libraries (Chen et al. (1994) J. Amer.
Chem. Soc. 116:2661), oligocarbamates (Cho, et al. (1993) Science
261:1303), and/or peptidyl phosphonates (Campbell et al. (1994) J.
Org. Chem. 59:658). See, generally, Gordon et al. (1994) J. Med.
Chem. 37:1385, nucleic acid libraries (see, e.g., Strategene
Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat. No.
5,539,083), antibody libraries (see, e.g., Vaughn et al. (1996)
Nature Biotechnology, 14(3):309-314), and PCT/US96/10287),
carbohydrate libraries (see, e.g., Liang et al. (1996) Science
274:1520-1522, and U.S. Pat. No. 5,593,853), and small organic
molecule libraries (see, e.g., benzodiazepines, Baum (1993)
C&EN, January 18, page 33; isoprenoids, U.S. Pat. No.
5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No.
5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;
morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines,
U.S. Pat. No. 5,288,514; and the like).
Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.).
A number of well-known robotic systems have also been developed for
solution phase chemistries. These systems include automated
workstations like the automated synthesis apparatus developed by
Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic
systems utilizing robotic arms (Zymate II, Zymark Corporation,
Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.), which
mimic the manual synthetic operations performed by a chemist. Any
of the above devices are suitable for use with the present
invention. The nature and implementation of modifications to these
devices (if any) so that they can operate as discussed herein will
be apparent to persons skilled in the relevant art. In addition,
numerous combinatorial libraries are themselves commercially
available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow,
Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D
Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md.,
etc.).
b) High Throughput Screening
Any of the assays to identify compounds capable of modulating the
expression or activity of any of the genes or gene products
described herein are easily amenable to high throughput
screening.
High throughput assays for the presence, absence, quantification,
or other properties of test agents on cells are well known to those
of skill in the art. Similarly, binding assays and reporter gene
assays are similarly well known. Thus, for example, U.S. Pat. No.
5,559,410 discloses high throughput screening methods for proteins,
U.S. Pat. No. 5,585,639 discloses high throughput screening methods
for nucleic acid binding (i.e., in arrays), while U.S. Pat. Nos.
5,576,220 and 5,541,061 disclose high throughput methods of
screening for ligand/antibody binding.
In addition, high throughput screening systems are commercially
available (see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical
Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton,
Calif.; Precision Systems, Inc., Natick, Mass., etc.). These
systems typically automate entire procedures, including all sample
and reagent pipetting, liquid dispensing, timed incubations, and
final readings of the microplate in detector(s) appropriate for the
assay. These configurable systems provide high throughput and rapid
start up as well as a high degree of flexibility and customization.
The manufacturers of such systems provide detailed protocols for
various high throughput systems. Thus, for example, Zymark Corp.
provides technical bulletins describing screening systems for
detecting the modulation of gene transcription, ligand binding, and
the like.
III. Administration of ER Resident Chaperone-Modulating
Compounds
In numerous embodiments of the present invention, a compound useful
in the treatment or prevention of a thromboembolic disorder, i.e.,
a polynucleotide, polypeptide, test agent, or any compound that
increases levels of GRP78/BiP mRNA, polypeptide and/or protein
activity, is administered to a mammal. Such compounds can be
administered to the mammal to inhibit the generation of thrombin on
cells within the mammal, e.g., endothelial cells present at an
atherosclerotic plaque within the mammal.
The administration of such compounds can be used to prevent or
treat any of a number of thrombotic conditions or diseases. For
example, as intravascular coagulation is associated with
atherosclerosis, myocardial infarction, and acute arterial
thrombosis following plaque disruption, the compounds can be used
to prevent or treat any of these conditions or diseases. The
compounds can thus be used to prevent thrombin accumulation on the
surface of cells in the endothelium of patients who have had a
myocardial infarction and who are undergoing angioplasty or
stenting, thereby reducing the risk of thrombosis. In addition, the
compounds can be administered to patients undergoing cranial
radiation to prevent radiation-related thrombotic events. In
addition, the compounds can be administered to patients undergoing
vascular surgery to prevent surgery related thrombosis. In
addition, as TF-induced thrombin generation plays a critical role
in hemostasis following tissue injury and also in the pathogenesis
of multiple thrombotic disorders associated with a wide range of
diseases including, but not limited to, cardiovascular disease,
sepsis, and cancer, the present methods can also be used to treat
or prevent these diseases and conditions.
Such compounds can be administered by a variety of methods
including, but not limited to, parenteral, topical, oral, or local
administration, such as by aerosol or transdermally, for
prophylactic and/or therapeutic treatment. The pharmaceutical
compositions can be administered in a variety of unit dosage forms
depending upon the method of administration. For example, unit
dosage forms suitable for oral administration include, but are not
limited to, powder, tablets, pills, capsules and lozenges. It is
recognized that the modulators (e.g., antibodies, antisense
constructs, ribozymes, small organic molecules, etc.) when
administered orally, must be protected from digestion. This is
typically accomplished either by complexing the molecule(s) with a
composition to render it resistant to acidic and enzymatic
hydrolysis, or by packaging the molecule(s) in an appropriately
resistant carrier, such as a liposome. Means of protecting agents
from digestion are well known in the art.
The compositions for administration will commonly comprise an
inducer of an ER resident chaperone dissolved in a pharmaceutically
acceptable carrier, preferably an aqueous carrier. A variety of
aqueous carriers can be used, e.g., buffered saline and the like.
These solutions are sterile and generally free of undesirable
matter. These compositions may be sterilized by conventional, well
known sterilization techniques. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, toxicity adjusting agents and the like, for
example, sodium acetate, sodium chloride, potassium chloride,
calcium chloride, sodium lactate and the like. The concentration of
active agent in these formulations can vary widely, and will be
selected primarily based on fluid volumes, viscosities, body weight
and the like in accordance with the particular mode of
administration selected and the patient's needs.
Thus, a typical pharmaceutical composition for intravenous
administration would be about 0.1 to 10 mg per patient per day.
Dosages from 0.1 up to about 100 mg per patient per day may be
used, particularly when the drug is administered to a secluded site
and not into the blood stream, such as into a body cavity or into a
lumen of an organ. Substantially higher dosages are possible in
topical administration. Actual methods for preparing parenterally
administrable compositions will be known or apparent to those
skilled in the art and are described in more detail in such
publications as Remington's Pharmaceutical Science, 15th ed., Mack
Publishing Company, Easton, Pa. (1980).
The compositions containing inducers of ER resident chaperones can
be administered for therapeutic or prophylactic treatments. In
therapeutic applications, compositions are administered to a
patient suffering from a disease (e.g., atherosclerosis) in an
amount sufficient to cure or at least partially arrest the disease
and its complications. An amount adequate to accomplish this is
defined as a "therapeutically effective dose." Amounts effective
for this use will depend upon the severity of the disease and the
general state of the patient's health. Single or multiple
administrations of the compositions may be administered depending
on the dosage and frequency as required and tolerated by the
patient. In any event, the composition should provide a sufficient
quantity of the agents of this invention to effectively treat the
patient. An amount of an ER resident chaperone inducer that is
capable of preventing or slowing the development of the disease or
condition in a mammal is referred to as a "prophylactically
effective dose." The particular dose required for a prophylactic
treatment will depend upon the medical condition and history of the
mammal, the particular disease or condition being prevented, as
well as other factors such as age, weight, gender, etc. Such
prophylactic treatments may be used, e.g., in a mammal who has
previously had the disease or condition to prevent a recurrence of
the disease or condition, or in a mammal who is suspected of having
a significant likelihood of developing the disease or
condition.
It will be appreciated that any of the present ER resident
chaperone inducing compounds can be administered alone or in
combination with additional ER resident chaperone inducing
compounds or with any other therapeutic agent, e.g., other
anti-atherosclerotic or other cholesterol-reducing agents or
treatments.
IV. Kits
For use in the prophylactic and therapeutic applications suggested
above, kits are also provided by the invention. In the diagnostic
and research applications such kits may include any or all of the
following: assay reagents, buffers, ER resident chaperone nucleic
acids or antibodies, hybridization probes and/or primers, small
molecule inducers of ER resident chaperone proteins, etc. A
therapeutic product may include sterile saline or another
pharmaceutically acceptable emulsion and suspension base.
In addition, the kits may include instructional materials
containing directions (i.e., protocols) for the practice of the
methods of this invention. While the instructional materials
typically comprise written or printed materials they are not
limited to such. Any medium capable of storing such instructions
and communicating them to an end user is contemplated by this
invention. Such media include, but are not limited to electronic
storage media (e.g., magnetic discs, tapes, cartridges, chips),
optical media (e.g., CD ROM), and the like. Such media may include
addresses to Internet sites that provide such instructional
materials.
II. EXAMPLES
A. Stable Overexpression of Human GRP78/BiP in T24/83 Cells
In order to address the role of GRP78/BiP in cell surface thrombin
generation and/or inactivation, human GRP78/BiP was stably
overexpressed in cells of the human transitional bladder carcinoma
cell lines T24/83 (obtained from the American Type Culture
Collection; ATCC; Rockville, Md.). T24/83 cells constitutively
overexpress active TF on their cell surface and are considered to
be procoagulant. Because thrombin generation at the surface of
cells is dependent on active TF, these cells were selected as a
model system to study the effect of GRP78/BiP overexpression on
TF-dependent thrombin generation.
1. Preparation of GRP78/BiP Encoding Vector
To obtain cells stably expressing GRP78/BiP, T24/83 cells were
transfected with either the mammalian cell expression vector
pcDNA3.1(+) or pcDNA3.1(+) containing the open reading frame of
human GRP78/BiP. The latter vector was obtained by amplifying the
cDNA encoding the open-reading frame of human GRP78/BiP
(approximately 1.95 kb) by reverse transcriptase-PCR using total
RNA from primary HUVEC. GRP78/BiP cDNA was generated using
SuperScript RNase H reverse transcriptase (Gibco/BRL) and a primer
complimentary to a sequence in the 3'-untranslated region of the
human GRP78/BiP mRNA transcript (AB10230;
5'-TATTACAGCACTAGCAGATCAGTG-3') (SEQ ID NO:1). For PCR
amplification, the forward primer
AB10231(5'-CTTAAGCTTGCCACCATGAAGCTCTCCCTGGTGGCCGCG-3') (SEQ ID
NO:2) contained a Kozak consensus sequence (bold) prior to the
initiating ATG and a terminal HindIII restriction site
(underlined). The reverse primer AB10232
(5'-AGGCCTCGAGCTACAACTCATCTTTTTCTGCTGT-3') (SEQ ID NO:3) contained
a terminal XhoI restriction site (underlined) adjacent to the
authentic termination codon of the GRP78/BiP cDNA. PCR reactions
took place in a final volume of 50 .mu.l containing 2 .mu.l of the
RT reaction, 100 ng of primers, 2.5 U Taq polymerase (Perkin-Elmer,
Mississauga, ON) in a buffer consisting of 1.5 mM MgCl.sub.2, 50 mM
KCl, 10 mM Tris-HCl (pH 8.8) and 0.5 mM of each dNTP. All samples
were subjected to amplification in a DNA thermal cycler 480
(Perkin-Elmer) with a step programme of 30 cycles of 94.degree. C.
for 1 min 58.degree. C. for 1 min, and 72.degree. C. for 1 min. The
amplified GRP78/BiP cDNA was separated on a 0.8% agarose-TBE gel
containing ethidium bromide, purified from the agarose gel using
the QIAEX gel extraction kit (Qiagen, Mississauga, ON) and ligated
into T-ended pBluescript (KS) (Stratagene, La Jolla, Calif.). The
ligation mixture was then used to transform competent DH5.alpha.
cells (Gibco/BRL). Plasmid DNA was isolated from transformed cells
using the QIAEX miniprep kit (Qiagen), digested with HindIII and
XhoI, and the GRP78/BiP cDNA insert purified from agarose. The
GRP78/BiP cDNA insert was ligated into the HindIII/XhoI site of the
mammalian expression vector pcDNA3.1(+) (Invitrogen, Carlsbad,
Calif.) to produce the recombinant plasmid, pcDNA3.1(+)-GRP78/BiP.
Authenticity of the GRP78/BiP cDNA sequence was confirmed by
fluorescence-based double stranded DNA sequencing (MOBIX).
2. Isolation of Stably Transfected Cells
T24/83 cells grown to 30% confluency were transfected with 5 .mu.g
of the pcDNA3.1I (+)-GRP78/BiP expression plasmid using 30 .mu.l of
SuperFect Transfection reagent (Qiagen) as described by the
manufacturer. As a vector control, pcDNA3.1l (+) was used to
transfect T24/83 under the same conditions. Stable transfectants
were selected in complete medium containing 1.2 mg/ml G418
(Gibco/BRL) for two weeks. G418-resistant clones were subsequently
identified, isolated and cultured in complete medium containing
G418. Overexpression of GRP78/BiP was assessed using immunoblot
analysis using an anti-KDEL mAB (SPA-827; StressGen
Biotechnologies, Victoria, B. C.) which recognizes both human
GRP78/BiP and GRP94. Total protein lysates from T24/83 cells were
solubilized in SDS-PAGE sample buffer, heated to 95.degree. for 2
min, separated on SDS-polyacrylamide gels under reducing conditions
and transferred to nitrocellulose membranes (Bio-Rad). After
incubation with the primary (1 .mu.g/ml) and horseradish peroxidase
(HRP)-conjugated secondary antibodies (Gibco/BRL), the membranes
were developed using the Renaissance chemiluminescence reagent kit
(DuPont/NEN).
As shown in FIG. 1, 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
to wild-type or vector-transfected cells. In contrast, GRP94
protein levels were unchanged in all cell lines, suggesting that
alterations in GRP78/BiP protein levels do not affect endogenous
GRP94 protein levels. Unless otherwise stated, the two GRP78/BiP
overexpressing T24/83 cell lines C1 and C2, were used
interchangeably throughout these studies. Stable cell lines and
their vector-transfected counterpart were maintained in a
humidified incubator at 37.degree. C. with 5% CO.sub.2 in M199
medium (Gibco/BRL) containing 10% fetal bovine serum, 100 .mu.g/ml
penicillin and 100 .mu.g/ml streptomycin.
3. Detection of GRP78/BiP Protein Expression in Transfected
Cells
To compare the levels and distribution of GRP78/BiP protein, wild
type or GRP78/BiP overexpressing cells were cultured on coverslips,
fixed, permeabilized and examined in situ by indirect
immunofluorescence using anti-GRP78/BiP polyclonal antibodies
(Santa Cruz Biotechnology; Santa Cruz, Calif.) (Outinen et al.
(1998) Biochem. J., 332:213-221). For detection of cell surface
levels of TF, cells were washed with ice-cold 1.times.PBS for 2 min
and incubated with anti-human TF mAb (5 .mu.g/ml) for 1 hr at
4.degree. C. After 3 washes with 1.times.PBS containing 0.5% FBS,
cells were incubated with the appropriate Alexa-labeled secondary
antibody (BioLynx, Brockville, Ontario) for 1 hr at 4.degree. C.,
washed again and fixed with 1% paraformaldehyde. Images were
captured and analyzed using Northern Exposure image
analysis/archival software (Mississauga, ON)
In wild-type cells and GRP78/BiP overexpressing cells, GRP78/BiP
was concentrated in the perinuclear region, consistent with its
location in the endoplasmic reticulum (FIG. 2). However, the
intensity of the immunofluorescent signal was much greater in the
GRP78/BiP overexpressing cells, a result consistent with the
immunoblot analysis. Non-specific staining was not observed in
T24/83 cells immunostained with normal mouse IgG. Overexpression of
GRP78/BiP in T24/83 cells suppressed the induction of endogenous
GRP78/BiP mRNA levels and increased survival of cell exposed to the
ER stress-inducing agent, A23187.
B. Overexpression of GRP78/BiP Decreases Thrombin Generation,
Prothrombin Consumption and the Formation of Thrombin-Inhibitor
Complexes on T24/83 Cell Surfaces
Thrombin generation, prothrombin consumption, and formation of
thrombin-inhibitor complexes were determined in normal defibrinated
human plasma. Prior to use in the thrombin generation assay,
plasmas were defibrinated using arvin, as described previously
(Chan et al. (1998) Lung Cell. Mol. Physiol. 18:L914-L921).
Prothrombin concentrations were determined for each time point
during the thrombin generation experiments using a commercially
available ELISA kit (Affinity Biologicals). Control plasma with a
known concentration of prothrombin was used as a standard.
1. Detecting Thrombin Generation
Total amidolytic activity of thrombin generated on T24/83 cell
surfaces was measured as previously described (Ling et al. (1995)
Pediatr. Res. 37:373-378); Chan et al. (1998) Lung Cell. Mol.
Physiol. 18:L914-L921). T24183 cell monolayers in 24 well plates
were placed on a Thermolyne dri-bath set at 37.degree. C. until the
pH of the culture medium had increased to 7.2. After washing twice
with 1 ml of acetate-barbital-saline (ABS) buffer, pH 7.4 (0.036 M
Na acetate, 0.036 M sodium diethylbarbitarate and 0.145 M NaCl),
monolayers were incubated for 3 min with 100 .mu.l of ABS buffer
and 200 .mu.l of defibrinated plasma, in the absence or presence of
10% activated partial thromboplastin time (APTT) reagent (Organon
Teknika Corp., Durham, N.C.). At various times following the
addition of 100 .mu.l of 0.04 M CaCl.sub.2 in ABS buffer, 25 .mu.l
aliquots of the reaction mixture on the surface of the cells were
removed and mixed with 475 .mu.l of 0.005 M Na.sub.2 EDTA on ice.
Twenty-five .mu.l of each EDTA sample were then mixed with 775
.mu.l of 0.00016 M S-2238 (KabiVitum, Stockholm, Sweden) in buffer
and heated to 37.degree. C. for 10 min prior to termination of the
amidolytic reaction with 200 .mu.l 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, Hamilton,
ON). Because thrombin bound to .alpha.2macroglobulin (.alpha.2M)
retains amidolytic activity against S-2238, the contribution of
thrombin-.alpha.2M to total thrombin activity was measured.
Briefly, the amidolytic activity of total thrombin was measured as
described above, except that the 25 .mu.l reaction mixture taken at
each time point was incubated with 3.5 .mu.l of 0.15 M NaCl
containing 0.25 U standard heparin and 0.042 U antithrombin (to
inhibit any free thrombin) for 1 min on ice, followed by the
addition of 475 .mu.l Na.sub.2 EDTA. Thrombin activity due to
.alpha.2M-bound thrombin was then subtracted from the total
thrombin activity to give the amount of free thrombin generated by
the cell surface.
In control plasma, after the addition of calcium, the concentration
of total thrombin generated on the cell surface of T24/83 cells
overexpressing GRP78/BiP was significantly less after 2 min,
relative to wild-type or vector-transfected cells (p<0.001)
(FIG. 3A). The concentration of free thrombin generated on the
surface of T24/83 cells overexpressing GRP78/BiP was negligible for
all time points examined, up to 25 min (FIG. 3B). In contrast to
the GRP78/BiP overexpressing cells, free thrombin levels generated
on the surface of wild-type or vector-transfected T24/83 cells were
significantly higher between 2 and 22 min with concentrations
reaching 110.+-.16 and 131.+-.2 nM, respectively, by 4 min after
the addition of calcium (p<0.001).
Consistent with these findings, both prothrombin consumption (FIG.
4) and thrombin-inhibitor complexes (Table 1) were significantly
reduced in the GRP78/BiP overexpressing cells, compared to
wild-type or vector-transfected cells. The observation that the
initial rates of thrombin generation (FIG. 3A) and prothrombin
consumption (FIG. 4) are similar among all cell lines suggest that
cellular factors which either promote or inhibit TF activity,
subsequent to initiation, are altered in the GRP78/BiP
overexpressing cells.
In the presence of APTT reagent, which potentiates thrombin
generation through activation of the intrinsic coagulation pathway,
concentrations of free thrombin increased among all cell lines
examined, with peak levels achieved by 2 min (FIG. 5A). However,
peak levels were significantly decreased in T24/83 cells
overexpressing GRP78/BiP (480 nM), compared to wild-type (590 nM)
and vector-transfected cells (570 nM) (p<0.05; n=4).
TABLE-US-00001 TABLE 1 Thrombin-inhibitor complex formation on
T24/83 cell surfaces Total inhibitor Cell Type (nM) TAT (nM)
IIa-.alpha..sub.2M (nM) IIa-HCII (nM) Wild type 759 .+-. 18 631
.+-. 13 112 .+-. 5 16 .+-. 0.3 pcDNA 752 .+-. 57 610 .+-. 52 124
.+-. 7 18 .+-. 0.4 GRP78/BiP 266 .+-. 18.sup.a 161 .+-. 15 93 .+-.
3 12 .+-. 0.6 Concentrations of thrombin covalently bound to plasma
inhibitors were measured in the EDTA samples collected during the
thrombin generation end points. Thrombin-antithrombin (TAT) and
thrombin-heparin cofactor II (IIa-HCII) were measured by ELISA, and
thrombin-.alpha..sub.2 macroglobulin (IIa-.alpha..sub.2M) was
measured chromogenically after inhibiting free thrombin with
antithrombin and heparin. Data are presented as the mean .+-. SEM
of triplicate measurements from four separate experiments. .sup.ap
= 0.001, compared to wild-type or vector-transfected (pcDNA)
controls.
C. Overexpression of GRP78/BiP Inhibits the Procoagulant Activity
(PCA) of Tissue Factor
To determine if the effect of GRP78 overexpression on thrombin
generation was dependent on the procoagulant activity (PCA) of
tissue factor (TF), Factor VII-depleted plasma was used and the
results were compared with normal plasma in the absence or presence
of APTT reagent. In the absence of APTT reagent, both wild-type and
vector-transfected cells had negligible levels of free thrombin
generated over the 25 min time period. In the presence of APTT
reagent, peak concentrations of free thrombin generated in
FVII-depleted plasma was both delayed (4 vs. 2 min) and decreased
(FIG. 5B), compared to normal plasma (FIG. 5A). However, there was
no significant difference in peak thrombin levels generated in
either the wild-type T24/83 cells (460 nM), vector-transfected (410
nM) or GRP78/BiP overexpressing cells (430 nM) in the factor
VII-depleted plasma.
The TF-dependent binding and conversion of factor VII to VIIa is
the initial step in the extrinsic pathway of blood coagulation. To
directly determine whether overexpression of GRP78/BiP decreases TF
PCA, the conversion of factor VII to VIIa was measured by a
one-step assay using the chromogenic substrate S-2288. Monolayers
of wild-type, vector-transfected or GRP78/BiP-overexpressing T24/83
cells were washed in ABS buffer and incubated for time periods up
to 30 min in the presence of 108 nM recombinant factor VII and 5 mM
Ca.sup.2+. The amount of factor VIIa generated in the subsamples of
the cell supernatant was assessed by measuring the amidolysis of
S-2288 at 405 nm. As shown in Table 2, the conversion of factor VII
to VIIa at 30 min on the surface of GRP78/BiP overexpressing cells
was decreased approximately 48- and 23-fold compared to wild-type
and vector-transfected cells, respectively (p<0.03). The
observation that the amidolysis of S-2288 did not occur in the
absence of factor VII by 30 min (blank control) suggests 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). Taken together, these data suggest that the
reduction in the procoagulant response on the GRP78/BiP
overexpressing cells is mediated by a reduction in TF PCA.
TABLE-US-00002 TABLE 2 Tissue factor activity on T24/83 cell
surfaces Cell Type .DELTA.A.sub.405 (A.sub.405 sample - A.sub.405
blank [factor VIIa] generated (nM) Wild type 0.026 0.01025 pcDNA
0.024 0.00493 GRP78/BiP 0.017.sup.a 0.00021.sup.a Plasma
membrane-associated tissue factor (TF) activity was determined as
the ability to convert factor VII to factor VIIa. Monolayers were
incubated with factor VII in ABS buffer followed by measurement of
generated factor VIIa by reaction with a chromogenic substrate
(S-2288) and recording the absorbance at 405 nm. Similar reactions
were carried out in the absence of factor VII (blank). Results were
converted to the concentration of factor VIIa generated (nM) using
a standard curve from reactions of S-2288 with known amounts of
purified factor VIIa. Data are presented as the average of
triplicate measurements from two independent experiments. .sup.ap
< 0.03, compared to wild-type or vector-transfected (pcDNA)
controls.
D. GRP78/BiP Overexpression Leads to an Increase in TF Expression
and Synthesis
Northern blot hybridization and immunoblot analysis were used to
determine whether the decrease in TF PCA resulted from a decrease
in TF expression and/or synthesis. Steady-state TF mRNA levels from
wild-type, vector-transfected or GRP78/BiP overexpressing cells
were analyzed by Northern blot hybridization using a human TF cDNA
probe. Total RNA was isolated from cells using the RNeasy total RNA
kit (Qiagen) and separated (10 .mu.g/lane) on 2.2M
formaldehyde/1.2% agarose gels. Following transfer on to Zeta-Probe
GT nylon membranes (Bio-Rad), the RNA was cross-linked to the
membrane using a UV crosslinker (PDI Bioscience, Toronto). cDNA
fragments encoding either human GRP78/BiP or TF (1.05 kb
NarI/HindIII fragment of human TF cDNA) were labeled with
[.alpha.-.sup.32P]dCTP (NEN) using a random primed DNA labeling kit
(Boehringer Mannheim). After overnight hybridization at 43.degree.
C., the membranes were washed as described by the manufacturer,
exposed to X-ray film and subjected to autoradiography. Changes in
gene expression were quantified using a Storm 860 PhosphorImager
and ImageQuant image analysis software (Molecular Dynamics,
Sunnyvale, Calif.). As a control for RNA loading, integrated
optical densities were normalized to human glyceraldehyde
3-phosphate dehydrogenase (GAPDH).
In contrast to the decreased TF PCA observed in the GRP78/BiP
overexpressing cells, TF mRNA levels were increased approximately
4-fold in these cells, compared to wild-type or vector-transfected
cells (FIG. 6A). Consistent with the Northern blot results,
immunoblot analysis using anti-human TF mAbs demonstrated that
GRP78/BiP overexpressing cells had approximately 2.5-fold greater
TF antigen levels, compared to wild-type or vector-transfected
cells (FIG. 6B). This increase in total TF antigen levels was
confirmed by ELISA. Although total TF protein levels were increased
in the GRP78/BiP overexpressing cells, the electrophoretic mobility
of TF observed on immunoblots was similar to that of wild-type and
vector-transfected cells.
This observation that TF expression, synthesis, and cell surface
levels are elevated in GRP78/BiP overexpressing cells suggests that
the above-described decrease in thrombin generation does not result
from the retention of TF in the ER by GRP78/BiP and that elevated
levels of cell surface TF do not compensate for the
GRP78/BiP-dependent decrease in TF PCA.
E. Localization of TF on the Cell Surface of T24/83 Cells
To assess whether increased TF antigen levels in the GRP78/BiP
overexpressing cells correlated with increased cell surface protein
levels, immunofluorescence studies using anti-human TF mAbs were
performed on intact wild-type, vector-transfected, or GRP78/BiP
overexpressing cells (FIG. 7). All cell types exhibited clusters of
TF in defined patches distributed over the cell surface. However,
the intensity of TF staining was much greater in the GRP78/BiP
overexpressing cells (FIG. 7C) compared to wild type (FIG. 7A) or
vector-transfected cells (FIG. 7B). Computer-assisted analysis of
the fluorescence intensity/cell area revealed that GRP78/BiP
overexpressing cells had a 2.2-fold increase in cell surface TF,
compared to wild-type or vector-transfected cells; a result
consistent with the increased total TF antigen levels shown in FIG.
6B. Non-specific immunostaining was not observed in cells incubated
with normal mouse IgG.
While the foregoing invention has been described in some detail for
purposes of clarity and understanding, it will be clear to one
skilled in the art from a reading of this disclosure that various
changes in form and detail can be made without departing from the
true scope of the invention. For example, all the techniques and
apparatus described above may be used in various combinations. All
publications and patent documents cited in this application are
incorporated by reference in their entirety for all purposes to the
same extent as if each individual publication or patent document
were so individually denoted.
SEQUENCE LISTINGS
1
5 1 24 DNA Artificial Sequence Description of Artificial Sequence
Primer AB10230 1 tattacagca ctagcagatc agtg 24 2 39 DNA Artificial
Sequence Description of Artificial Sequence Primer AB10231 2
cttaagcttg ccaccatgaa gctctccctg gtggccgcg 39 3 34 DNA Artificial
Sequence Description of Artificial Sequence Primer AB10232 3
aggcctcgag ctacaactca tctttttctg ctgt 34 4 654 PRT Homo sapiens
GRP78/BiP amino acid sequence 4 Met Lys Leu Ser Leu Val Ala Ala Met
Leu Leu Leu Leu Ser Ala Ala 1 5 10 15 Arg Ala Glu Glu Glu Asp Lys
Lys Glu Asp Val Gly Thr Val Val Gly 20 25 30 Ile Asp Leu Gly Thr
Thr Tyr Ser Cys Val Gly Val Phe Lys Asn Gly 35 40 45 Arg Val Glu
Ile Ile Ala Asn Asp Gln Gly Asn Arg Ile Thr Pro Ser 50 55 60 Tyr
Val Ala Phe Thr Pro Glu Gly Glu Arg Leu Ile Gly Asp Ala Ala 65 70
75 80 Lys Asn Gln Leu Thr Ser Asn Pro Glu Asn Thr Val Phe Asp Ala
Lys 85 90 95 Arg Leu Ile Gly Arg Thr Trp Asn Asp Pro Ser Val Gln
Gln Asp Ile 100 105 110 Lys Phe Leu Pro Phe Lys Val Val Glu Lys Lys
Thr Lys Pro Tyr Ile 115 120 125 Gln Val Asp Ile Gly Gly Gly Gln Thr
Lys Thr Phe Ala Pro Glu Glu 130 135 140 Ile Ser Ala Met Val Leu Thr
Lys Met Lys Glu Thr Ala Glu Ala Tyr 145 150 155 160 Leu Gly Lys Lys
Val Thr His Ala Val Val Thr Val Pro Ala Tyr Phe 165 170 175 Asn Asp
Ala Gln Arg Gln Ala Thr Lys Asp Ala Gly Thr Ile Ala Gly 180 185 190
Leu Asn Val Met Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala Ile Ala 195
200 205 Tyr Gly Leu Asp Lys Arg Glu Gly Glu Lys Asn Ile Leu Val Phe
Asp 210 215 220 Leu Gly Gly Gly Thr Phe Asp Val Ser Leu Leu Thr Ile
Asp Asn Gly 225 230 235 240 Val Phe Glu Val Val Ala Thr Asn Gly Asp
Thr His Leu Gly Gly Glu 245 250 255 Asp Phe Asp Gln Arg Val Met Glu
His Phe Ile Lys Leu Tyr Lys Lys 260 265 270 Lys Thr Gly Lys Asp Val
Arg Lys Asp Asn Arg Ala Val Gln Lys Leu 275 280 285 Arg Arg Glu Val
Glu Lys Ala Lys Arg Ala Leu Ser Ser Gln His Gln 290 295 300 Ala Arg
Ile Glu Ile Glu Ser Phe Tyr Glu Gly Glu Asp Phe Ser Glu 305 310 315
320 Thr Leu Thr Arg Ala Lys Phe Glu Glu Leu Asn Met Asp Leu Phe Arg
325 330 335 Ser Thr Met Lys Pro Val Gln Lys Val Leu Glu Asp Ser Asp
Leu Lys 340 345 350 Lys Ser Asp Ile Asp Glu Ile Val Leu Val Gly Gly
Ser Thr Arg Ile 355 360 365 Pro Lys Ile Gln Gln Leu Val Lys Glu Phe
Phe Asn Gly Lys Glu Pro 370 375 380 Ser Arg Gly Ile Asn Pro Asp Glu
Ala Val Ala Tyr Gly Ala Ala Val 385 390 395 400 Gln Ala Gly Val Leu
Ser Gly Asp Gln Asp Thr Gly Asp Leu Val Leu 405 410 415 Leu Asp Val
Cys Pro Leu Thr Leu Gly Ile Glu Thr Val Gly Gly Val 420 425 430 Met
Thr Lys Leu Ile Pro Arg Asn Thr Val Val Pro Thr Lys Lys Ser 435 440
445 Gln Ile Phe Ser Thr Ala Ser Asp Asn Gln Pro Thr Val Thr Ile Lys
450 455 460 Val Tyr Glu Gly Glu Arg Pro Leu Thr Lys Asp Asn His Leu
Leu Gly 465 470 475 480 Thr Phe Asp Leu Thr Gly Ile Pro Pro Ala Pro
Arg Gly Val Pro Gln 485 490 495 Ile Glu Val Thr Phe Glu Ile Asp Val
Asn Gly Ile Leu Arg Val Thr 500 505 510 Ala Glu Asp Lys Gly Thr Gly
Asn Lys Asn Lys Ile Thr Ile Thr Asn 515 520 525 Asp Gln Asn Arg Leu
Thr Pro Glu Glu Ile Glu Arg Met Val Asn Asp 530 535 540 Ala Glu Lys
Phe Ala Glu Glu Asp Lys Lys Leu Lys Glu Arg Ile Asp 545 550 555 560
Thr Arg Asn Glu Leu Glu Ser Tyr Ala Tyr Ser Leu Lys Asn Gln Ile 565
570 575 Gly Asp Lys Glu Lys Leu Gly Gly Lys Leu Ser Ser Glu Asp Lys
Glu 580 585 590 Thr Met Glu Lys Ala Val Glu Glu Lys Ile Glu Trp Leu
Glu Ser His 595 600 605 Gln Asp Ala Asp Ile Glu Asp Phe Lys Ala Lys
Lys Lys Glu Leu Glu 610 615 620 Glu Ile Val Gln Pro Ile Ile Ser Lys
Leu Tyr Gly Ser Ala Gly Pro 625 630 635 640 Pro Pro Thr Gly Glu Glu
Asp Thr Ala Glu Lys Asp Glu Leu 645 650 5 2007 DNA Homo sapiens
Human GRP78/BiP mRNA sequence 5 actggctggc aagatgaagc tctccctggt
ggccgcgatg ctgctgctgc tcagcgcggc 60 gcgggccgag gaggaggaca
agaaggagga cgtgggcacg gtggtcggca tcgacctggg 120 gaccacctac
tcctgcgtcg gcgtgttcaa gaacggccgc gtggagatca tcgccaacga 180
tcagggcaac cgcatcacgc cgtcctatgt cgccttcact cctgaagggg aacgtctgat
240 tggcgatgcc gccaagaacc agctcacctc caaccccgag aacacggtct
ttgacgccaa 300 gcggctcatc ggccgcacgt ggaatgaccc gtctgtgcag
caggacatca agttcttgcc 360 gttcaaggtg gttgaaaaga aaactaaacc
atacattcaa gttgatattg gaggtgggca 420 aacaaagaca tttgctcctg
aagaaatttc tgccatggtt ctcactaaaa tgaaagaaac 480 cgctgaggct
tatttgggaa agaaggttac ccatgcagtt gttactgtac cagcctattt 540
taatgatgcc caacgccaag caaccaaaga cgctggaact attgctggcc taaatgttat
600 gaggatcatc aacgagccta cggcagctgc tattgcttat ggcctggata
agagggaggg 660 ggagaagaac atcctggtgt ttgacctggg tggcggaacc
ttcgatgtgt ctcttctcac 720 cattgacaat ggtgtcttcg aagttgtggc
cactaatgga gatactcatc tgggtggaga 780 agactttgac cagcgtgtca
tggaacactt catcaaactg tacaaaaaga agacgggcaa 840 agatgtcagg
aaagacaata gagctgtgca gaaactccgg cgcgaggtag aaaaggccaa 900
acgggccctg tcttctcagc atcaagcaag aattgaaatt gagtccttct atgaaggaga
960 agacttttct gagaccctga ctcgggccaa atttgaagag ctcaacatgg
atctgttccg 1020 gtctactatg aagcccgtcc agaaagtgtt ggaagattct
gatttgaaga agtctgatat 1080 tgatgaaatt gttcttgttg gtggctcgac
tcgaattcca aagattcagc aactggttaa 1140 agagttcttc aatggcaagg
aaccatcccg tggcataaac ccagatgaag ctgtagcgta 1200 tggtgctgct
gtccaggctg gtgtgctctc tggtgatcaa gatacaggtg acctggtact 1260
gcttgatgta tgtcccctta cacttggtat tgaaactgtg ggaggtgtca tgaccaaact
1320 gattccaagg aacacagtgg tgcctaccaa gaagtctcag atcttttcta
cagcttctga 1380 taatcaacca actgttacaa tcaaggtcta tgaaggtgaa
agacccctga caaaagacaa 1440 tcatcttctg ggtacatttg atctgactgg
aattcctcct gctcctcgtg gggtcccaca 1500 gattgaagtc acctttgaga
tagatgtgaa tggtattctt cgagtgacag ctgaagacaa 1560 gggtacaggg
aacaaaaata agatcacaat caccaatgac cagaatcgcc tgacacctga 1620
agaaatcgaa aggatggtta atgatgctga gaagtttgct gaggaagaca aaaagctcaa
1680 ggagcgcatt gatactagaa atgagttgga aagctatgcc tattctctaa
agaatcagat 1740 tggagataaa gaaaagctgg gaggtaaact ttcctctgaa
gataaggaga ccatggaaaa 1800 agctgtagaa gaaaagattg aatggctgga
aagccaccaa gatgctgaca ttgaagactt 1860 caaagctaag aagaaggaac
tggaagaaat tgttcaacca attatcagca aactctatgg 1920 aagtgcaggc
cctcccccaa ctggtgaaga ggatacagca gaaaaagatg agttgtagac 1980
actgatctgc tagtgctgta atattgt 2007
* * * * *