Note: Descriptions are shown in the official language in which they were submitted.
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MODULATION OF PLATELET ACTIVATION
FIELD OF THE INVENTION
This invention relates to inhibition of platelet activation, and particularly
to such
inhibition mediated through thrombin receptors.
BACKGROUND OF THE INVENTION
Thrombin, a coagulation protease generated at sites of vascular injury,
activates
platelets, leukocytes, and mesenchymal cells (T.-K.H. Vu et al., Cell 64:1057-
1068 (1991)).
Activation of platelets by thrombin is thought to be critical for hemostasis
and thrombosis.
In animal models, thrombin inhibitors block platelet-dependent thrombosis,
which is the
cause of most heart attacks and strokes in humans. Available data in humans
suggests that
thrombosis in arteries can be blocked by inhibitors of platelet function and
by thrombin
inhibitors. Thus it is likely that thrombin's actions on platelets contribute
to the formation of
clots that cause heart attack and stroke. Thrombin's other actions on vascular
endothelial
cells and smooth muscle cells, leukocytes, and fibroblasts may mediate
inflammatory and
proliferative responses to injury, as occur in normal wound healing and a
variety of diseases
(atherosclerosis, restenosis, pulmonary inflammation CARDS),
glomerulosclerosis, etc.).
Thrombin signaling is mediated at least in part by a family of G protein-
coupled
protease-activated receptors (PARs) for which PART is the prototype. U. B.
Rasmussen et
al., FEBSLetts. 288:123-128 (1991). PAR1 is activated when thrombin binds to
and cleaves
its amino terminal exodomain to unmask a new receptor amino terminus. This new
amino
terminus then serves as a tethered peptide ligand, binding intramolecularly to
the body of the
receptor to effect transmembrane signaling. T.-K. Vu et al., Nature. 353:674-
677 (1991); J.
M. Chen et al., J. Biol. Chem., 269:16041-16045 (1994). The synthetic peptide
SFLLRN,
which mimics the first six amino acids of the new amino terminus unmasked by
receptor
cleavage, functions as a PART agonist and activates the receptor independent
of thrombin
and proteolysis. R. J. Vassallo et al., J. Biol. Chem., 267:6081-6085
(1994).R. M.
Scarborough et al., J. Biol Chem. 267:13146-9 (1992). Such peptides have been
used as
pharmacological probes of PAR function in various cell types.
Our understanding of the role of PARs in platelet activation is evolving
rapidly.
PART mRNA and protein were detected in human platelets. D. T. Hung et al., J.
Clinical
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Investigation. 89:1350-3 (1992); L. F. Brass et al., J. Biol. Chem. 267:13795-
13798 (1992);
M. Molino et al., J. Biol Chem. 272:6011-7 (1997). SFLLRN activated human
platelets 3, 7,
8, and PAR1-blocking antibodies inhibited human platelet activation by low,
but not high
concentrations of thrombin These data suggest a role for PART in activation of
human
platelets by thrombin but hold open the possibility that other receptors
contribute. PAR4
was recently identified. M. L. Kahn, Nature. 394:690-694 (1998); W. F. Xu et
al., PNAS
95:6642-6 (1998), and appears to function in both mouse arid human platelets.
There is a need for a better understanding of thrombin-mediated platelet
activation.
There is also a need for the identification and characterization of factors
that may either
contribute to or inhibit platelet-mediated pathologies such as platelet-
dependent arterial
thrombosis for use in treating associated pathologies.
SUMMARY OF THE INVENTION
Methods of modulating thrombin-mediated platelet activation by inhibition or
enhancement of protease-activated receptor 4 (PAR4) and protease-activated
receptor 1
(PART) activity are disclosed. This method provides a way of substantially
blocking all
thrombin-mediated activation of platelets by 1) inhibiting signaling through
PAR1 and 2)
inhibiting signaling through PAR4. Signaling through each PAR receptor may be
inhibited
at various molecular levels, including: the level of ligand binding (e.g. by
administration of
an antagonist), the level of the receptor activity (e.g. blocking expression
of the receptor in
the relevant cells) and/or intracellularly, (e.g. blocking expression or
activity of a molecule
required for activity of the receptor). Alternatively, this method provides a
method for
enhancing thrombin-mediated platelet activation by specifically activating
PAR1 and PAR4.
These methods may take place in vivo, through administration of the
appropriate compounds,
or in vitro, e.g. the ex vivo treatment of a sample.
The present invention provides compositions that are effective for prevention
of
thrombin-associated platelet activation. These compositions are comprised of
agents that
inhibit the activity of PAR1 and PAR4, e.g. antagonists of PART and PAR4, and
are
effective in the treatment of disorders. Examples of useful antagonists are
small molecules,
modeled proteins, and antibodies. In addition, compositions may comprise
dominant
negative PAR receptors, which may be employed to substantially decrease or
eliminate the
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expression of either PAR1 or PAR4. These compositions may also contain nucleic
acid
sequences that encode for antagonists of PAR1 and PAR4, which may be
administered for
expression in vivo.
The present invention also provides compositions that are effective for
enhancing
thrombin-associated platelet activation. either in vitro and/or in vivo. These
compositions
are comprised of agents that inhibit the activity of both PART and PAR4, e.g.
antagonists of
PAR1 and PAR4, and are effective in the treatment of disorders. Examples of
useful
antagonists are small molecules, modeled proteins, and antibodies. These
compositions may
also contain nucleic acid sequences that encode for antagonists of PART and
PAR4, which
may be administered for expression in vivo. These compositions are comprised
of agents
that enhance the activity of both PAR1 and PAR4, e.g. antagonists of PART and
PAR4, and
are effective in the treatment of disorders wherein there is insufficient
activation of platelets,
e.g. hemophilia. Examples of useful agonists are small molecules, modeled
proteins, and
antibodies. These compositions may also contain nucleic acid sequences that
encode for
antagonists of PAR1 and PAR4, which may be administered for expression in
vivo.
In one embodiment, the invention provides therapeutic uses of the inhibiting
compositions of the invention in the treatment of disorders such as such as
myocardial
infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral
arterial occlusion,
and other blood system thromboses. One method of treatment comprises the
administration
of protonated/acidified nucleic acids to the animal in an amount sufficient to
inhibit or
prevent tissue occlusion. Alternatively, a sample may be taken from an animal
(e.g. a blood
sample), treated ex vivo with the inhibiting composition of the invention, and
returned to the
animal.
In another embodiment, the invention provides therapeutic uses of the
activating
compositions of the invention in disorders involving insufficient clotting.
The dual
activation of PAR1 and PAR4 may increase the activation of platelets, since
thrombin has
the ability to activate both receptors.
The invention also provides specific PAR4 antibodies for use in the methods of
the
invention. Such antibodies effectively block signaling through PAR4 and thus
effectively
block PAR4's contribution to thrombin-mediated platelet activation.
It is an object of the invention to inhibit activity of PART and PAR4, thus
substantially eliminating thrombin mediated platelet activation.
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It is an advantage of the invention that the method can be practiced both in
vivo and
ex vivo.
It is a feature of the invention that the pharmaceutical compositions of the
invention
are effective at treating a variety of ailments.
These and other objects, advantages, and features of the invention will become
apparent to those skilled in the art upon reading the details of the nucleic
acids and uses
thereof as more fully described below.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is the summary of studies of a competitive RT-PCR assay of RNA
prepared
from platelets, neutrophils and Dami cells.
Figure 2 is a graph illustrating antibody binding to the surface of receptor-
expressing
COS cells.
Figures 3A - 3D is a series of graphs illustrating the results of flow
cytometric
analysis of human platelets.
Figures 4A - 4D is a series of graphs illustrating the results of flow
cytometric
analysis of Dami cells.
Figure 5 illustrates the ability of the PAR1 and PAR4 tethered ligand peptides
to
activate PAR1 and PAR4 heterologously expressed inXenopus oocytes.
Figures 6A - 6C illustrate the ability of the PART and PAR4 tethered ligand
peptides
to activate PAR1 and PAR4 human platelets.
Figure 7 is a graph illustrating the ability of the PART and PAR4 antibodies
to block
thrombin cleavage of PAR1 and PAR4in rat I fibroblasts expressing FLAG epitope-
tagged
PAR1 and PAR4.
Figures 8A and 8B illustrate the increases in cytoplasmic calcium in response
to
thrombin in cells expressing PAR 1 (Figure 8A) or PAR 4 (Figure 8B).
Figures 9A - 9E illustrate the contribution of PAR1 and PAR4 signaling to
thrombin
activation of human platelets with and without treatment with PAR activation
peptides or
antibodies.
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DESCRIPTION OF THE PREFERRED EMBODIMENTS
Before the present protease-activated receptor assays and methods of using
such are
described, it is to be understood that this invention is not limited to the
particular DNA
sequences, materials, methods, or processes described as such may, of course,
vary. It is also
to be understood that the terminology used herein is for the purpose of
describing particular
embodiments only, and is not intended to be limiting since the scope of the
present invention
will be limited only by the appended claims.
It must be noted that as used in this specification and the appended claims,
the
singular forms "a", "and," and "the" include plural referents unless the
contexts clearly
dictates otherwise. Thus, for example, reference to "a DNA sequence" includes
mixtures and
large numbers of such sequences, reference to "an assay" includes assays of
the same general
type, and reference to "the method" includes one or more methods or steps of
the type
described herein.
The publications discussed herein are provided solely for their disclosure
prior to the
filing date of the present application. Nothing herein is to be construed as
an admission that
the present invention is not entitled to antedate such publication by virtue
of prior invention.
Unless defined otherwise, all technical and scientific terms herein have the
same
meaning as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Although any methods and materials, similar or equivalent to those
described
herein, can be used in the practice or testing of the present invention, the
preferred methods
and materials are described herein. All publications cited herein are
incorporated herein by
reference for the purpose of disclosing and describing specific aspects of the
invention for
which the publication is cited in connection with.
DEFINITIONS
By "protease-activated receptor 1 ", "PART ", "PART receptor" and the like, is
meant
all or part of a vertebrate cell surface protein which is specifically
activated by thrombin or a
thrombin agonist thereby activating PART-mediated signaling events. The
polypeptide is
characterized as having the properties (including the agonist activating and
antagonist
inhibiting properties) described herein and in Vu et al. (1991) Cell, 64:1057-
1068, which is
incorporated herein by reference. PAR1 may refer to a naturally occurring form
of the
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receptor and/or a recombinantly produced form of the receptor. In addition,
the term may
include variants of the PAR1 protein that retain the same activity and
properties.
By "protease-activated receptor 4", "PAR4", "PAR4 receptor" and the like, is
meant
all or part of a vertebrate cell surface protein which is specifically
activated by thrombin or a
thrombin agonist thereby activating PAR4-mediated signaling events. The
polypeptide is
characterized as having the properties (including the agonist activating and
antagonist
inhibiting properties) described herein and in U.S. Application No.
09/032,397, which is
incorporated herein by reference. PAR4 may refer to a naturally occurring form
of the
receptor and/or a recombinantly produced form of the receptor. In addition,
the term may
include variants of the PAR4 protein that retain the same activity and
properties.
By a "polypeptide" is meant any chain of amino acids, regardless of length or
post-
translational modification (e.g., glycosylation).
By "substantially pure" is meant that the protease-activated receptor 4
polypeptide
provided by the invention is at least 60%, by weight, free from the proteins
and naturally-
occurnng organic molecules with which it is naturally associated. Preferably,
the preparation
is at least 75%, more preferably at least 90%, and most preferably at least
99%, by weight,
PAR polypeptide. A substantially pure PAR polypeptide may be obtained, for
example, by
extraction from a natural source, by expression of a recombinant nucleic acid
encoding a
PAR polypeptide, or by chemically synthesizing the protein. Purity can be
measured by any
appropriate method, e.g., column chromatography, polyacrylamide gel
electrophoresis, or
HPLC analysis. The protein is substantially pure if it can be isolated to a
band in a gel.
By a "substantially identical" amino acid sequence is meant an amino acid
sequence
which differs only by conservative amino acid substitutions, for example,
substitution of one
amino acid for another of the same class (e.g., valine for leucine, arginine
for lysine, etc.) or
by one or more non-conservative amino acid substitutions, deletions, or
insertions located at
positions of the amino acid sequence which do not destroy the biological
activity of the
receptor. Such equivalent receptors can be isolated by extraction from the
tissues or cells of
any animal which naturally produces such a receptor or which can be induced to
do so, using
the methods described below, or their equivalent; or can be isolated by
chemical synthesis; or
can be isolated by standard techniques of recombinant DNA technology, e.g., by
isolation of
cDNA or genomic DNA encoding such a receptor. Substantially identical
receptors have the
same biological function, e.g. are activated by the same compound.
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By "derived from" is meant encoded by the genome of that organism and present
on
the surface of a subset of that organism's cells.
By "isolated DNA" is meant DNA that is not in its native environment in terms
of not
being immediately contiguous with (i. e., covalently linked to) the complete
coding sequences
with which it is immediately contiguous (i. e., one at the 5' end and one at
the 3' end) in the
naturally-occurring genome of the organism from which the DNA of the invention
is derived.
The term therefore includes, for example, recombinant DNA which is
incorporated into a
vector; into an autonomously replicating plasmid or virus; or into the genomic
DNA of a
prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA
or a genomic
or cDNA fragment produced by PCR or restriction endonuclease digestion)
independent of
other sequences. It also includes any recombinant DNA which is part of a
hybrid gene
encoding additional polypeptide sequence.
By "transformed cell" "transfected cell", "genetically engineered cell", and
the like, is
meant a cell into which (or into an ancestor of which) has been introduced, by
means of
genetic engineering, a DNA molecule encoding PAR1, PAR4 or a biologically
active
fragment of either. Such a DNA molecule is "positioned for expression" meaning
that the
DNA molecule is positioned adjacent to a DNA sequence which directs
transcription and
translation of the sequence (i. e., facilitates the production of a PAR1 or
PAR4 protein, or
fragment or analog thereof).
By "antibody" is meant an immunoglobulin protein which is capable of binding
an
antigen. Antibody as used herein is meant to include the entire antibody as
well as any
antibody fragments (e.g. F(ab'~, Fab', Fab, Fv) capable of binding the
epitope, antigen or
antigenic fragment of interest.
Antibodies of the invention are immunoreactive or immunospecific for and
therefore
specifically and selectively bind to either PAR1 or PAR4 protein. Antibodies
for PAR1 or
PAR4 are preferably immunospecific -- i. e., not substantially cross-reactive
with related
materials, e.g., with each other. Although the term "antibody" encompasses all
types of
antibodies (e.g., monoclonal) the antibodies of the invention are preferably
produced using
the phage display methodology described herein. The preferred antibody of the
invention is a
purified antibody. By purified antibody is meant one which is sufficiently
free of other
proteins, carbohydrates, and lipids with which it is naturally associated.
Such an antibody
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"preferentially binds" to a specific PAR protein (or an antigenic fragment
thereof), i. e., does
not substantially recognize and bind to other antigenically-unrelated
molecules.
By "specifically activates", as used herein, is meant an agent, such as
thrombin, a
thrombin analog, a PAR agonist or other chemical agent including polypeptides
such as an
antibody, which activates protease-activated receptor, receptor polypeptide or
a fragment or
analog thereof to initiate PAR-mediated biological events as described herein,
but which
does not substantially bind other molecules in a sample, e.g., a biological
sample, which
naturally includes a protease-activated receptor polypeptide.
By "specifically inhibits", as used herein, is meant an agent, such as a
thrombin
analog, a PAR4 antagonist or other chemical agent including polypeptides such
as an
antibody, which inhibits activation of protease-activated receptor 4, receptor
polypeptide or a
fragment or analog thereof, such as by inhibiting thrombin or by blocking
activation of
PAR4 by thrombin or other PAR4 activator. Preferably, the agent activates or
inhibits the
biological activity in vivo or in vitro of the protein to which it binds.
By "biological activity" is meant the ability of the PAR to bind thrombin or
an
agonist and signal the appropriate cascade of biological events (e.g.,
phosphoinositide
hydrolysis, Ca2+ efflux, and platelet aggregation, and the like).
By "substantial increase" is meant an increase in activity or other measurable
phenotypic characteristic that is at least approximately a 2-fold increase
over control level
(where control assays are performed in the absence of activator), preferably
at least
approximately a 5-fold increase, more preferably at least approximately a 10-
fold increase in
activity over a control assay.
By "substantial decrease" or "substantial reduction" is meant a decrease or
reduction
in activity or other measurable phenotypic characteristic that is
approximately 80% or the
control level, preferably reduced to approximately 50% of the control level,
or more
preferably reduced to approximately 10% or less of the control level.
The terms "screening method" and "assay method" are used to describe a method
of
screening a candidate compound for its ability to act as an agonist or
antagonist of a PAR4
ligand. The method involves: a) contacting a candidate agonist compound with a
recombinant protease-activated receptor 4 (or PAR4 agonist-binding fragment or
analog);
b) measuring activation of the receptor, the receptor polypeptide or the
receptor fragment or
analog; and c) identifying agonist compounds as those which interact with the
recombinant
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receptor and trigger or block PAR4 activation. Interaction may be cleavage of
the receptor to
unmask an intramolecular receptor activating peptide or by mimicking the
intramolecular
receptor-activating peptide. A tethered ligand may be more difficult to block
than a free
agonist. Thus, blocking thrombin is the acid test for an antagonist which will
block
responses by other thrombin substrates. These terms include assays that
examine effects on
unoccupied receptors as well as assays that utilize displacement of a ligand
from an occupied
receptor.
By an "agonist" is meant a molecule which mimics a particular activity, in
this case,
interacting with a PAR or PAR ligand in a manner which activates thereby
triggering the
biological events which normally result from the interaction (e.g.,
phosphoinositide
hydrolysis, Ca2+ efflux, and platelet aggregation). Preferably, an agonist
initiates a
substantial increase in receptor activity relative to control assays in the
absence of activator
or candidate agonist. An agonist may possess the same, less, or greater
activity than a
naturally-occurring PAR ligand.
By an "antagonist" is meant a molecule which blocks activation of a PAR
receptor.
This can be done by inhibiting a particular activity such as the ability of
thrombin, for
example, to interact with a PAR thereby triggering the biological events
resulting from such
an interaction (e.g., phosphoinositide hydrolysis, Ca2+ efflux, and platelet
secretion, or
platelet aggregation). An antagonist may bind to and thereby block activation
of a PAR
receptor, either PAR1, PAR4 or both.
The terms "antagonist assay", "antagonist screening" and the like, refer to a
method
of screening a candidate compound for its ability to antagonize interaction
between a
naturally-occurring activating ligand or an agonist and the PAR, either PAR1,
PAR 4 or
both. The method involves: a) contacting a candidate antagonist compound with
a first
compound which includes a recombinant PAR (or agonist-binding fragment or
analog) on
the one hand and with a second compound which includes thrombin or a PAR
agonist on the
other hand; b) determining whether the first and second compounds interact or
are prevented
from interaction by the candidate compound; and c) identifying antagonistic
compounds as
those which interfere with the interaction of the first compound (PAR
receptor) to the second
compound (PAR agonist) and which thereby substantially reduce thrombin or PAR
agonist-
activated biological events (e.g., phosphoinositide hydrolysis, Ca2+ efflux,
and platelet
aggregation).
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The terms "treatment", "treating", "treat" and the like are used herein to
generally
mean obtaining a desired pharmacologic and/or physiologic effect. The effect
may be
prophylactic in terms of completely or partially preventing a disease or
symptom thereof
and/or may be therapeutic in terms of a partial or complete cure for a disease
and/or adverse
effect attributable to the disease. "Treatment" as used herein covers any
treatment of a
disease in a mammal, particular a human, and includes:
(a) preventing the disease or symptom from occurring in a subject which may be
predisposed to the disease or symptom but has not yet been diagnosed as having
it;
(b) inhibiting the disease symptom, i.e., arresting its development; or
(c) relieving the disease symptom, i.e., causing regression of the disease.
The term "extracorporeal blood" includes blood removed in line from a patient,
subjected to extracorporeal treatment, and returned to the patient in
processes such as
dialysis procedures or blood filtration or blood bypass during surgery. The
term also includes
blood products which are stored extracorporeally for eventual administration
to a patient.
Such products include whole blood, platelet concentrates and any other blood
fraction in
which inhibition or enhancement of platelet aggregation and platelet release
is desired.
GENERAL ASPECTS OF THE INVENTION
The present invention is based upon the discovery that inhibition of PART and
PAR4
virtually abolishes the ability of platelets to respond to thrombin.
Functional studies with
PART and PAR4 activating peptides confirmed that activation of either receptor
was
sufficient to trigger platelet secretion and aggregation. Given thrombin's
remarkable potency
as a platelet activator, blocking thrombin signaling in platelets is useful in
any number of
biological phenomena, such as prevention of thrombosis.
The invention thus provides a method of treatment for reducing the level of
thrombin response in a mammalian host by administering a composition which
inhibits
both PART and PAR4 activity. In general, such compounds will reduce the
activity of the
PAR1 and PAR4 receptors. Individuals treated may not presently exhibit
symptoms, but
prophylactic use is contemplated for individuals at risk for disorders such as
thromboembolism, such as the elderly and/or individuals with a history of
problems with
thrombi (i. e. blood clots).
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A number of mechanisms can be used to selectively block PAR1 signaling. For
example, peptides that selectively bind to and inactivate PAR1 may be used to
block PART
signaling. The availability of a potent and selective PAR1 peptide agonist
allows for
homologous desensitization of platelets to PARI activation. In another
example, antibodies
that selectively bind PART, but not the other PARs, can be used, e.g.
antibodies to PAR1's
hirudin-like domain, which inhibit PAR1 cleavage and activation by thrombin.
In a third
example, PART-specific antagonists permit direct blockade of PAR1 signaling,
while
leaving the signaling through the other PAR molecules generally unaffected.
Each of these
examples, as well as others known by those skilled in the art, may be used to
inhibit PART
signaling without interfering with other PAR function. Inhibition of PART
function by
antibody, antagonist, or desensitization markedly inhibits platelet responses
at low ( 1 nM)
concentrations of thrombin.
PAR4 may be inhibited using similar mechanisms, including but not limited to
peptide agonists, antibodies, and PAR4-specific antagonists. PAR4 inhibition
is preferably
accomplished using an antibody to PAR4's thrombin cleavage site. This antibody
preparation blocks PAR4 activation by thrombin without interfering with other
PAR activity.
Moreover, an antibody to this region of PAR4 does not inhibit activation of
either PAR4 or
platelets by the PAR4-activating peptide GYPGKF. In contrast to PART
inhibition,
inhibition of PAR4 alone had no significant effect on platelet aggregation.
The treatment of the present invention may take place in vivo, e. g. through
the
administration of PART and PAR4 antagonists or introduction and expression of
PART and
PAR4 antagonists. Alternatively, the treatment may take place ex vivo, e.g. a
patient's
extracorporeal blood may be treated with compositions of the invention and the
blood
replaced.
COMPOUNDS OF THE INVENTION
Compounds of the invention encompass numerous chemical classes, including but
not limited to the compounds described herein with known function.
Novel methods are provided which employ compounds that are effective in
decreasing the level of PAR1 and PAR4 in mammalian cells.
Candidate compounds can be obtained from a wide variety of sources including
libraries of synthetic or natural compounds. For example, numerous means are
available
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for random and directed synthesis of a wide variety of organic compounds and
biomolecules, including expression of randomized oligonucleotides and
oligopeptides.
Alternatively, libraries of natural compounds in the form of bacterial,
fungal, plant and
animal extracts are available or readily produced. Additionally, natural or
synthetically
produced libraries and compounds are readily modified through conventional
chemical,
physical and biochemical means, and may be used to produce combinatorial
libraries.
Known pharmacological compounds may be subjected to directed or random
chemical
modifications, such as acylation, alkylation, esterification, amidification,
etc. to produce
structural analogs.
The agonists or antagonists of the present invention may relate to synthetic
polypeptides that bind to of PAR1 and/or PAR4. Such synthetic antiplatelet
polypeptides
may be prepared by conventional chemical synthesis techniques, for example,
synthesis on a
solid support.
The present invention also relates to recombinant and synthetic DNA molecules
which encode molecules that either inhibit or enhance PAR activity. The
synthesis of these
DNA molecules may be achieved by methods well known in the art. For example,
the
recombinant DNA molecules may be isolated from a human hematopoetic cDNA
library.
The synthesis of cDNA libraries and the choice of vector into which the cDNA
molecules
may be cloned are conventional techniques, see e.g. T. Maniatis et al.,
"Molecular Cloning -
A Laboratory Manual", Cold Spring Harbor (1982). A wide variety of methods may
be used
in locating and identifying cDNA sequences corresponding to a the compositions
of the
present invention. The two most preferred techniques are the use of
oligonucleotide probe
based on the amino acid sequence of the of PAR1 or PAR4 ligands, and
immunoscreening,
which utilizes antibodies against the extracellular domains of PAR1 and PAR4
to detect
clones which express cDNA sequences corresponding to potential agonist or
antagonist
activity. It will be obvious to those of skill in the art that the choice of
oligonucleotides
probes will be based upon those strengths of amino acids which are encoded by
the least
redundant DNA sequences.
The immunoscreening technique requires that the cDNA library be contained in a
vector capable of expression. Such vectors include lambda gtl 1, lambda gtl0
and other
expression vectors known in the art. Antibodies employed in the
immunoscreening technique
include antibodies against intact PAR1 and PAR4, antibodies against denatured
PAR1 and
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PAR4 and antibodies against peptide portions of PAR1 and PAR4. Once a
potentially
interesting cDNA has been identified and isolated, it may be transcribed and
translated to
determine whether it encodes a peptide with PAR antagonist or agonist
activity. Partial
cDNAs may themselves be used to reprobe the cDNA library and to locate full-
length
cDNAs.
Alternatively, the DNA molecules of this invention may be synthesized from
nucleotides by chemical means using an synthesizer. Such nucleic acids may be
designed
based on identified amino acid sequence of the PAR agonists or antagonists.
Standard
methods may be applied to synthesize a gene encoding such a peptide. For
example, the
complete amino acid sequence may be used to construct a back-translated gene.
A DNA
oligomer containing a nucleotide sequence capable of coding for the desired
polypeptide may
be synthesized in a single step.
Alternatively, several smaller oligonucleotides coding for portions of the
PART and/or PAR4
agonist or antagonist may be synthesized and subsequently ligated together.
Preferably, the
antiplatelet polypeptide gene is synthesized as 10-20 separate
oligonucleotides which are
subsequently linked together. The individual oligonucleotides contain 5' or 3'
overhangs fox
complementary assembly.
Following synthesis of the nucleic acid and cleavage of the desired vector,
assembly
of the antiplatelet polypeptide gene may be achieved in one or more steps by
techniques well
known in the art. Once assembled, the gene will be characterized by sequences
which are
recognized by restriction endonucleases, including unique restriction sites
for direct
assembly into a cloning or an expression vector; preferential codons based
upon the host
expression system to be used: and a sequence which, when transcribed, produces
an mRNA
with minimal secondary structure. Proper assembly may be confirmed by
nucleotide
sequencing, restriction mapping, and expression of a biologically active
antagonist or agonist
in a platelet aggregation assay.
According to one embodiment, the present invention relates to compositions for
decreasing or preventing platelet aggregation and release and methods which
employ them.
These compositions may also contain a variety of other conventional
antiplatelet or
anti-thrombin compounds in addition to a naturally purified, recombinant or
synthetic
polypeptide inhibitor of platelet activation of this invention. The most
widely used
antiplatelet agent is aspirin, a cyclooxygenase inhibitor. Although aspirin
blocks ADP-
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and collagen-induced platelet aggregation, it fails to prevent cyclooxygenase-
independent
platelet aggregation initiated by agonists, such as thrombin. Alternative anti-
thrombin
compounds are hirudin derivatives.
The composition of the invention containing additional anti-platelet
activation
compounds may be a single dosage form, wherein a polypeptide inhibitor of
platelet
activation of this invention may be chemically conjugated to a conventional
polypeptide
platelet inhibitor or to a conventional anti-thrombin polypeptide.
Alternatively, a single
dosage form which contains the polypeptide inhibitor of platelet activation
and the other
polypeptide in the same composition, but as separate compounds. The
composition may also
contain multiple dosage forms, wherein the PART and PAR4 inhibitors and the
other
polypeptide that inhibits of platelet activation are administered separately,
but concurrently,
or wherein the two forms are administered sequentially.
A polypeptide inhibitor of PAR1 and/or PAR4 may also be cross-linked to a
conventional carrier polypeptide, which may be carned out by chemical cross-
linking
methods well known in the art. Most preferably, such combinations are formed
by
cross-linking a natural or recombinant polypeptide PAR antagonists to carriers
that have
been synthesized with a cross-linking moiety, such as dinitrofluorobenzene, at
its NHZ
terminus. Alternatively, the carrier peptide may be conjugated to a natural or
recombinant
PAR 1 and/or PAR4 antagonist by the use of agents such as glutaraldehyde,
dimethyladipimidate, or any other bifunctional cross-linkers known in the art.
The
conjugated antagonist preferably involves a 1:1 stoichiometry with the carrier
peptide.
The agonists or antagonists of the present invention may be present invention
relates
to synthetic polypeptides of platelet activation. Such synthetic antiplatelet
polypeptides may
be prepared by conventional chemical synthesis techniques, for example,
synthesis on a solid
support.
Methods of generating antibodies that may be used in the methods of the
present
invention are generally known to those skilled in the art. For example,
antibodies detecting
the extracellular domain of either PAR1 or PAR4 may be generated by immunizing
rabbits
or mice with a portion of the extracellular domain of each molecule, or a
peptide fragment
derived therefrom. Only antibodies with affinity at least 4 fold higher for
PAR1 or PAR4,
respectively as compared to their affinity for any other PAR molecules should
be selected.
The method of antibody generation, purification, labeling and detection may
vary.
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The IgG or Fab's may be purified from different sources by affinity HPLC using
protein A column and Size exclusion HPLC. The purified antibodies may be
labeled with
Europium and detected by time resolved fluorescence. The antibody binding to
different
PAR proteins may be measured by time-resolved, dissociation-enhanced
fluorescence.
However, the system of detection of PAR-bound IG on solid support in situ or
in solution
may vary. Further, it is possible to use direct or indirect immunological
methods including
direct radiolabels, fluorescence, luminescence, avidin-biotin amplification,
or enzyme-linked
assays with color or luminescent substrates. The preferred PAR1 antibody which
can be
used in the invention is disclosed in Hung et al. (1992) J. Clin. Invest.,
89:1350-1353.
For purposes of the invention an indication that no binding occurs means that
the
equilibrium or affinity constant Ka is 1061/mole or less. Further, binding
will be recognized
as existing when the Ka is at 10' 1/mole or greater, preferably 1 Og 1/mole or
greater. The
binding affinity of 10' 1/mole or more may be due to ( 1 ) a single monoclonal
antibody (i. e.,
large numbers of one kind of antibodies) or (2) a plurality of different
monoclonal antibodies
(e.g., large numbers of each of five different monoclonal antibodies) or (3)
large numbers of
polyclonal antibodies. It is also possible to use combinations of (1) - (3).
Selected preferred
antibodies will bind at least 4-fold more avidly to one PAR (e.g. PAR1) than
to a different
PAR (e.g. PAR4). The four fold differential in binding affinity may be
accomplished by
using several different.antibodies as per (1) - (3) above and as such some of
the antibodies in
a mixture could have less than a four fold difference.
The methods of the present invention may be practiced using one or more
different
antibodies to PAR1 and/or PAR4. Those skill in the art will recognize that
antibodies may
be labeled with known labels and used with currently available robotics,
sandwich assays,
electronic detectors, flow cytometry, and the like.
ADMINISTRATION OF THE COMPOUNDS
In the subject methods, the compound may be administered to the host using any
convenient means capable of resulting in the desired target protein activity
modulation.
Thus, the compound can be incorporated into a variety of formulations for
therapeutic
administration. More particularly, the compounds of the present invention can
be
formulated into pharmaceutical compositions by combination with appropriate,
pharmaceutically acceptable carriers or diluents, and may be formulated into
preparations
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in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules,
powders, granules,
ointments, solutions, transdermal patches, suppositories, injections,
inhalants and aerosols.
As such, administration of the compounds can be achieved in various ways,
including oral, buccal, rectal, parenteral, intraperitoneal, intradermal,
transdermal,
intratracheal, etc. , administration.
In pharmaceutical dosage forms, the compounds may be administered in the form
of their pharmaceutically acceptable salts, or they may also be used alone or
in appropriate
association, as well as in combination, with other pharmaceutically active
compounds.
The following methods and excipients are merely exemplary and are in no way
limiting.
For oral preparations, the compounds can be used alone or in combination with
appropriate additives to make tablets, powders, granules or capsules. Examples
of
additives are conventional additives, such as lactose, mannitol, corn starch
or potato
starch; binders, such as crystalline cellulose, cellulose derivatives, acacia,
corn starch or
gelatins; disintegrators, such as corn starch, potato starch or sodium
carboxymethylcellulose; lubricants, such as talc or magnesium stearate; and if
desired,
diluents, buffering agents, moistening agents, preservatives and flavoring
agents.
The compounds of the invention can be formulated into preparations for
injection
by dissolving, suspending or emulsifying them in an aqueous or nonaqueous
solvent, such
as vegetable or other similar oils, synthetic aliphatic acid glycerides,
esters of higher
aliphatic acids or propylene glycol. If desired, conventional additives such
as solubilizers,
isotonic agents, suspending agents, emulsifying agents, stabilizers and
preservatives may
also be added. The concentration of therapeutically active compound in the
formulation
may vary from about 0.5-100 wt. % .
The compounds can be utilized in aerosol formulation to be administered via
inhalation. The compounds of the present invention can be formulated into
pressurized
acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and
the like.
Furthermore, the compounds can be made into suppositories by mixing with a
variety of bases such as emulsifying bases or water-soluble bases. The
compounds of the
present invention can be administered rectally via a suppository. The
suppository can
include vehicles such as cocoa butter, carbowaxes and polyethylene glycols,
which melt at
body temperature, yet are solidified at room temperature.
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Unit dosage forms for oral or rectal administration such as syrups, elixirs,
and
suspensions may be provided wherein each dosage unit (e. g. , a teaspoonful,
tablespoonful,
tablet or suppository) contains a predetermined amount of the composition
containing one
or more inhibitors. Similarly, unit dosage forms for injection or intravenous
administration may comprise the inhibitors) in a composition as a solution in
sterile water,
normal saline or another pharmaceutically acceptable carrier.
The pharmaceutically acceptable excipients, such as vehicles, adjuvants,
carriers or
diluents, are readily available to the public. Moreover, pharmaceutically
acceptable
auxiliary substances, such as pH adjusting and buffering agents, tonicity
adjusting agents,
stabilizers, wetting agents and the like, are readily available to the public.
Compounds for use in the method of the invention may also be small organic
compounds having a molecular weight of more than 50 and less than about 2,500
daltons.
Candidate compounds comprise functional groups necessary for structural
interaction with
proteins, particularly hydrogen bonding, and typically include at least an
amine, carbonyl,
hydroxyl or carboxyl group, preferably at least two of the functional chemical
groups.
The candidate compounds often comprise cyclical carbon or heterocyclic
structures and/or
aromatic or polyaromatic structures substituted with one or more of the above
functional
groups. Candidate compounds are also found among biomolecules including, but
not
limited to: peptides, saccharides, fatty acids, steroids, purines,
pyrimidines, derivatives,
structural analogs or combinations thereof.
The compounds are added to a host in a physiologically acceptable carrier, at
a
dosage from 5 mg to 1400 mg, more usually from 100 mg to 1000 mg, preferably
500 to
700 for a dose of 0.5 to 20 mg/kg weight. The dosage for compounds suppressing
thrombin response is elected so that the PAR1 and PAR4 activity is reduced by
10 to 80 % ,
more preferably 20 to 70 % and even more preferably 25-50 % . The dosage for
compounds
inhibiting the activity of PART and PAR4 is elected so that the ability of
platelets to
respond to thrombin is reduced by about 20 to 80 % , preferably 40 to 50 % .
Platelet activation may be induced by a number of biological phenomenon,
including
injury, response to certain compounds, etc. The subject compositions will
generally be
administered daily, in an amount to provide at least about a 50 to 100% , more
preferably
75-95 % , even more preferably 80-90 % decrease in platelet activation.
Generally, the total
daily dosage will be at least about 10 mg, usually at least about 400 mg to
500 mg,
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preferably about 700 mg, and not more than about 1500 mg, usually not more
than about
1000 mg. The amount may vary with the general health of the patient, the
response of the
patient to the drug, whether the composition is used by itself or in
combination with other
drugs, and the like. Daily administrations may be one or more times, usually
not more
than about four times, particularly depending upon the level of drug which is
administered.
Administration of the compounds of the invention is particularly useful in the
treatment of diseases such as myocardial infarction, stroke, pulmonary
embolism, deep vein
thrombosis, peripheral arterial occlusion, and other blood thromboses.
Inhibition of platelet
activation in such disorders may allow localized treatment at the site of the
clotting, thus
eliminating some of the more unpleasant side effects of systemic treatment,
e.g. hemorrhage.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill
in the art
with a complete disclosure and description of how to make receptor proteins
and sequences
encoding such proteins and carry out the methodology for finding such DNA
sequences and
proteins, and are not intended to limit the scope of what is regarded as the
invention. Efforts
have been made to insure accuracy with respect to numbers used (e.g., amounts,
temperatures, etc.) but some experimental errors and deviations should be
accounted for.
Unless indicated otherwise, parts are parts by weight, molecular weight is
weight average
molecular weight; temperature is in degrees centigrade; and pressure is at or
near
atmospheric.
EXAMPLE 1: GENERATION AND CHARACTERIZATION OF PAR POLYCLONAL
ANTIBODIES
The synthetic peptides GGDDSTPSILPAPRGYPGQVC (PAR4 amino acids 34-55,
SEQ ID NO:1), AKPTLPIKTFRGAPPNSFEEFPFSALEGC (PAR3 amino acids 31-58 plus
carboxyl glycine-cysteine, SEQ ID N0:2) and NATLDPRSFLLRNPNDKYEPFWEDEEGC
(PART amino acids 35-61 plus carboxyl glycine-cysteine, SEQ ID N0:3) were used
to
generate polyclonal antisera in rabbits. Ig was purified by Protein-A affinity
chromatography
to generate the PAR4 IgG, PAR3 IgG and PAR1 IgG preparations used in this
study.
Binding of PAR1 PAR3, PAR4 immune IgGs and PAR4 pre-immune IgG to each
receptor
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was tested on COS cells transiently expressing FLAG epitope- tagged receptors
using an
enzyme-linked immunosorbent assay (ELISA) as previously described (H. Ishihara
et al.,
Blood, 91(11):4152-4157 (1998); K. Ishii et al., J. Biol. Chem., 268:9780-6
(1993). cDNA
for an epitope-tagged human PAR4 analogous to FLAG epitope-tagged PAR1 and was
constructed as previously described such that the FLAG epitope was fused to
amino acid 22
in PAR4 to yield the following sequence:
.DYKDDDDVE/TPSVYE...
where / indicates the junction with the PAR4 sequence.
EXAMPLE 2: EXPRESSION OF PAR mRNAS IN PLATELETS AND OTHER BLOOD CELLS
A competitive RT-PCR assay was developed to assess expression of mRNAs
encoding the known PARs. First, a competitor RNA (cRNA) was generated for each
PAR
mRNA. Each cRNA was identical to the sequence of native mRNA to be reverse
transcribed
and amplified by PCR except for mutation of a single restriction endonuclease
site; digestion
with the cognate restriction endonuclease thus allowed differentiation of PCR
products
generated from cRNA vs. mRNA. Varying amounts of competitor cRNAs were added
to
200ng of total cellular RNA and the mixtures were reverse transcribed and
amplified by PCR
using PAR-specific primers. In the absence of reverse transcription, no
products were
amplified. The products of reactions that included only native mRNA were
completely
cleaved by the appropriate restriction endonuclease while the products of
reactions that
included only cRNA remained undigested. The addition of increasing amounts of
competitor
cRNA to total cellular RNA prior to RT-PCR and comparison of the intensity of
the bands
obtained after restriction endonuclease digestion of the resulting PCR
products allowed
estimation of the amount of PAR mRNA in each sample.
Dami cells were grown in suspension in RPMI with 10% fetal bovine serum.
Platelets were separated from human blood as previously described. Platelet
preparations
contained less than 0.1 % leukocytes as assessed by light microscopic
analysis. A
discontinuous Percoll gradient was used to separate monocytes plus lymphocytes
from
neutrophils according to the manufacturer's instructions (Pharmacia). The
monocyte/
lymphocyte preparations contained less than 0.1 % neutrophils and the
neutrophil
preparations contained less than 0.1% monocytes or lymphocytes. Total RNA was
prepared
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from all cells using Trizol (GIBCO), treated with DNAse I (Boehringer-
Mannheim), and
quantified by OD26o.
Each receptor cDNA was mutated so as to ablate an endogenous restriction
endonuclease site. Sites were mutated by digesting cDNA encoding the receptor
with the
selected endonuclease, exposing the digested plasmid to T4 DNA polymerase then
religating.
Each of these mutant cDNAs was subcloned into Bluescript (KS- or SK-,
Stratagene) such
that sense cRNA could be transcribed in vitro using T7 RNA polymerase.
Competitor
cRNAs so generated were added to total cell RNA prior to reverse transcription
(RT)
reactions. RT reactions were performed at 42°C using 200 ng of total
RNA with varying
amounts of competitor cRNA in a 10 ,u1 reaction volume using a commercial kit
(GIBCO)
and receptor specific primers (see below). 2 ~cl of the RT product was then
subjected to PCR
amplification in a 50 ,u1 volume containing a final concentration of 2 ~cM
primers and SU of
Taq polymerase (GIBCO). Reaction conditions were as follows: 94°C for 4
min, 72°C for 1
min with addition of Taq; then 94°C for 45 sec, 55 °C for 1 min,
72°C for 1 min for 30-36
cycles (see below); then 72°C for 8 min. For each sample, a preliminary
experiment was
performed in which the number of amplification cycles was varied to determine
the cycle
number over which specific product was amplified and a cycle number in the
middle of the
dynamic range was chosen. For each analysis, the number of cycles chosen for
measurement
of PAR1, PAR2, PAR3, and PAR4 mRNA levels, respectively, in RNA from the
various
sources was:
PAR1 PAR2 PAR3 PAR4
Platelets: 31 36 36 36
Neutro hils: 36 27 31 36
Monocytes/ 31 31 33 36
L m hoc tes
Dami cells 30 32 32 33
Preliminary experiments also established the range of concentrations of
competitor RNA to
be added to each sample.
The primers used for RT and PCR of each receptor and the restriction
endonuclease
used to digest each PCR product were as follows. The convention for nucleotide
numbering
is that 1 = the A of the start methionine ATG.
PAR1, GenBank accession # M62424:
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Primer for RT: TAG ACG TAC CTC TGG CAC TC (1148-1129; SEQ ID N0:4).
Sense strand primer for PCR: CAG TTT GGG TCT GAA TTG TGT CG (SEQ ID NO:S).
Anti-sense primer for PCR: TGC ACG AGC TTA TGC TGC TGA C (SEQ ID N0:6).
Resulting PCR product: 505-1096.
Mutated site: AgeI at position 596.
PAR2, GenBank accession # U34038:
Primer for RT: CTG CTC AGG CAA AAC ATC (699-682; SEQ ID N0:7).
Sense strand primer for PCR: TGG ATG AGT TTT CTG CAT CTG TCC.(SEQ ID N0:8)
Anti-sense primer for PCR: CGT GAT GTT CAG GGC AGG AAT G (SEQ ID N0:9).
Resulting PCR product: 182-672.
Mutated site: SfiI at position 342.
PAR3 GenBank accession #U92972:
Primer for RT: TGA TGT CTG GCT GAA CAA G (727-709; SEQ ID NO:10).
Sense strand primer for PCR: TCC CCT TTT CTG CCT TGG AAG.(SEQ ID NO:11)
Anti-sense primer for PCR: AAA CTG TTG CCC ACA CCA GTC CAC (SEQ ID N0:12).
Resulting PCR product: 152-664.
Mutated site: NcoI at position 251.
PAR4, GenBank accession #AF080214:
Primer for RT: TGA GTA GCT GGG ATT ACA G (1519-1501; SEQ ID N0:13).
Sense strand primer for PCR: AAC CTC TAT GGT GCC TAC GTG C (SEQ ID N0:14).
Anti-sense primer for PCR: CCA AGC CCA GCT AAT TTT TG (SEQ ID NO:15).
Resulting PCR product: 949-1490.
Mutated site: BamHI at position 1005.
Following PCR amplification, 10 ,u1 of reaction product was digested overnight
with
the appropriate restriction endonuclease (AgeI, 2U at 25 °C; SfiI, 20U
at 50°C; NcoI, 10U at
37°C; or BamHI, 20U at 37°C). The products were then separated
by 1.5%, agarose gel
electrophoresis (Separide gel matrix, GIBCO) and visualized by ethidium
bromide staining.
The cRNA concentration at which the intensity of the cRNA-derived product
(uncleaved
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band) matched that of the endogenous mRNA-derived product (cleaved band) was
used to
estimate the quantity of each PAR mRNA in the original sample.
The summary of results of several studies of a competitive RT-PCR assay of RNA
prepared from platelets, neutrophils and Dami cells is presented in Fig. 1. To
validate the
assay, Dami cells, a human cell line that express some megakaryocyte markers
were analyzed
first. Competitive RT PCR of Dami cell RNA yielded results that were
concordant with
Northern and protein analysis. For Northern analysis, 2 ,ug of poly(A)+ RNA
isolated from
Dami cells was electrophoresed on a denaturing formaldehyde agarose gel and
transferred
onto a supported nitrocellulose membrane (Schleicher & Schuell). PAR1 mRNA was
detected with a 400 by PstI/PvuII cDNA probe; PAR2 mRNA was detected with a
260 by
SfiI/BstEII cDNA probe; PAR3 mRNA was detected with a 610 by KpnI/NsiI cDNA
probe;
PAR4 mRNA was detected using a 450 by SacI/PstI cDNA probe using high
stringency
conditions. In all such assays, PAR1, PAR3, and PAR4 were detected in Dami
cells while
PAR2 was not (Fig. 1 ).
Competitive RT-PCR of platelet RNA revealed PART mRNA to be present at
approximately one attomole/200 ng total RNA. Assuming mRNA is 1 % of total
platelet
RNA and an average mRNA size of 2kb, PAR1 mRNA represents one in three
thousand
platelet mRNAs. PAR4 mRNA was also readily detected in platelet RNA at 10-30%
of
PART mRNA levels. By contrast, PAR3 mRNA was undetectable in human platelet
RNA.
PAR3 competitor cRNA added to platelet RNA was detectable at 0.001
attomole/200ng total
RNA, suggesting that PAR3 mRNA was at least 1000 fold less abundant than PART
mRNA
in these samples. PAR2 mRNA was not detected in platelet RNA from one
individual and
only 0.001 attomole/200ng was detected in the other. The latter may be due to
trace
contamination of the platelet preparation by neutrophils, which do express
PAR2. Inability to
detect significant PAR2 mRNA in platelets is consistent with the observation
that the
specific PAR2 agonist peptide SLIGKV is unable to activate human platelets.
The pattern of PAR mRNA expression in neutrophils and mononuclear cells was
distinct from that seen in platelets, suggesting that contamination of
platelet preparations by
leukocytes did not significantly influence the PAR expression pattern detected
in platelets.
In particular, while virtually absent from platelets, substantial PAR2 mRNA
was detected in
RNA from both neutrophils and mononuclear cells. The relatively high PAR2 mRNA
level
in neutrophils is consistent with previous studies demonstrating neutrophil
responses to
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PAR2 activating peptide. In contrast to platelets, PAR3 mRNA was consistently
detected at
low levels in mononuclear cells. PAR4 mRNA was also found in mononuclear cell
preparations but was not detected in neutrophils. These results demonstrate
the presence of
mRNA encoding PAR1 and PAR4, but not PAR2 or PAR3, in human platelets.
EXAMPLE 3: Expression of PAR proteins on the surface of human platelets
IgG was purified from rabbit anti-peptide antisera directed against the amino
terminal
exodomains of PAR1, PAR3, or PAR4. To characterize the ability of each IgG
preparation
to recognize native PARs and to assess cross-reactivity, antibody binding to
the surface of
receptor-expressing COS cells was measured (Fig. 2). Each IgG preparation
bound to the
surface of cells expressing the appropriate receptor without significant cross-
reactivity.
The IgG preparations were then used for flow cytometric analysis of human
platelets
(Figs. 3A - 3D). Washed platelets were fixed in paraformaldehyde for 20
minutes at 40°C,
washed three times with platelet buffer (20mM Tris-HCl pH 7.4, 140mM NaCI,
2.SmM KC,
1mM MgCl2, lmg/ml glucose, 0.5% BSA), then incubated with primary IgG in
platelet
buffer at 40 ° C for 1 hour. PART and PAR3 IgG were used at 10 ,ug/ml
and PAR4 IgG at
100 ,ug/ml. Platelets were then washed and incubated with fluorescein
isothiocyanate
(FITC)-conjugated goat anti-rabbit IgG (Molecular Probes) at 4 ,ug/ml for 0.5
hour. Platelets
were then washed three times and analyzed by flow cytometry. Some fixed
platelet samples
were exposed to 30 nM thrombin at 37°C prior to incubation with primary
antibody. Dami
cells were treated like platelets for flow cytometry.
Significant surface binding was detected with PAR1 IgG compared with preimmune
IgG (Fig. 3A). A similar increase in platelet surface binding was detected
with PAR4 IgG
vs. preincubation the peptide antigen to preimmune IgG (Fig. 3C).
Preincubation of PAR4
with the peptide antigen to which it was raised abolished this increase (Figs.
3C and D).
Moreover, the epitope to which the PAR4 antiserum was raised spans PAR4's
thrombin
cleavage site, and treatment of platelets with thrombin indeed abolished PAR4
IgG binding
(Figs. 3C and D). These data strongly suggest that PAR1 and PAR4 are expressed
on the
surface of human platelets.
PAR3 immune IgG showed no specific binding to human platelets. To determine
whether this antibody could detect PAR3 expressed at "natural" levels, this
experiment was
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repeated with Dami cells (Figs. 4A - 4D), which had been shown by Northern
blot to express
PAR3 mRNA. As was observed for platelets, a significant increase in
fluorescence was seen
using PAR1 and PAR4 antibodies (Figs. 4A, 4C and 4D). In contrast to
platelets, a
significant increase in fluorescence was also observed with PAR3 antibody.
This result is
consistent with the presence of PAR3 mRNA in Dami cells by RT-PCR and Northern
blot
analysis. It further suggests that the absence of detectable PAR3 protein on
the surface of
human platelets is not due to insensitivity of the assay. These results are
consistent with the
analysis of platelet RNA and confirm the presence of PART and PAR4 but not
PAR3 on the
surface of human platelets.
EXAMPLE 4: ACTIVATION OF HUMAN PLATELETS BY PAR1 AND PAR4 TETHERED
LIGAND PEPTIDES
Synthetic peptides that mimic the tethered ligands of PART and PAR2 function
as
agonists for their respective receptors and have been used as pharmacological
tools to probe
the function of these receptors in various cell types. Unfortunately, the
cognate peptide for
PAR3 appears to be insufficiently avid to function as a free ligand. Peptides
mimicking the
tethered ligand for PAR4 can function as an agonist for that receptor, albeit
at a
concentration higher than that seen for the PAR1 and PAR2 peptides and their
cognate
receptors. To assist in the interpretation of experiments using the PART and
PAR4 tethered
ligand peptides, the ability to activate PART and PAR4 heterologously
expressed inXenopus
oocytes (a very sensitive assay system) was determined (Fig. 5). Agonist-
triggered calcium
mobilization was used an index of receptor activation. No responses were
detected in
oocytes expressing neither receptor. Both the human PAR4 peptide GYPGQV and
the
mouse PAR4 peptide GYPGKF activated oocytes expressing human PAR4, but with an
ECSo
roughly two orders of magnitude higher than that of SFLLRN for PART activation
(Fig. 5).
SFLLRN showed no activity at PAR4. At SOO~cM, the PAR4 peptide GYPGKF did show
minimal activity at PART. However, PAR1 is overexpressed in the oocytes and
the
sensitivity for detection of PAR1 activation in the oocyte assay is 10-100
fold greater than in
platelets; it is likely that PAR1 activation at SOO,uM GYPGKF is unimportant
in the platelet
studies described below.
The PAR1 peptide SFLLRN and the PAR4 peptides GYPGKF and GYPGQV all
activated human platelets (Fig. 6A). Both PAR4 peptides were considerably less
potent than
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the PART peptide for activating human platelets. GYPGKF was slightly more
potent than
GYPGQV (Fig. 6A and data not shown).
These data are consistent with the relative potencies with the activation of
the
receptors by their respective peptides in the oocyte system (Fig. S). In
short, FLAG epitope-
S tagged PAR4 cDNA was subcloned into pFROG3 and signaling studies performed
in
Xenopus oocytes as described. J. A. Williams et al., PNAS USA 85:4939-43
(1998). 2.0 ng
of PAR4 cRNA and 2S ng of PART cRNA was injected per oocyte.
Incubation of PGE,-treated platelets with SFLLRN rendered them refractory to
subsequent stimulation by SFLLRN but did not effect responsiveness to GYPGKF
(Fig. 6B).
Conversely, incubation with GYPGKF rendered platelets refractory to subsequent
stimulation by GYPGKF but did not effect responsiveness to SFLLRN (Fig. 6C).
These
results suggest that activation of either PAR1 or PAR4 with their cognate
peptide agonists is
sufficient to activate human platelets.
I S EXAMPLE S: PART AND PAR4 ANTIBODIES INHIBIT THROMBIN CLEAVAGE OF THEIR
RESPECTIVE RECEPTORS
Toward defining the necessary roles of PAR1 and PAR4 in platelet activation by
thrombin, blocking antibodies were developed. The previously described PART
antibody
raised against PARI's hirudin-like domain) is predicted to inhibit thrombin
cleavage of
PARl's amino terminal exodomain by disrupting binding to thrombin's anion-
binding
exosite. No analogous hirudin-like domain was apparent in the sequence of
PAR4's amino
terminal exodomain. To obtain an antibody that would inhibit thrombin cleavage
of PAR4,
antiserum was raised to a peptide that represented sequence spanning PAR4's
thrombin
2S cleavage site. This antiserum specifically recognized PAR4. To test the
ability of the PAR1
and PAR4 antibodies to block thrombin cleavage of PARI and PAR4, Rat I
fibroblasts
expressing FLAG epitope-tagged PART and PAR4 were preincubated with antibody
then
exposed to thrombin. Receptor cleavage by thrombin was measured as loss of
FLAG epitope
from the cell surface. PAR1 cleavage was markedly inhibited by PARI antibody
but not by
PAR4 antibody. Conversely, PAR4 cleavage was markedly inhibited by PAR4
antibody but
not by PARI antibody (Fig. 7). These data suggested that the PAR1 and PAR4
antibody
should selectively attenuate thrombin signaling via PAR1 and PAR4,
respectively.
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EXAMPLE 6: INHIBITION OF THROMBIN SIGNALING BY PAR1 AND PAR4 ANTIBODIES
AND BY A PART ANTAGONIST.
A Par 1-' mouse lung fibroblast cell line that showed no thrombin signaling
was
used to generate stable cell lines expressing FLAG epitope-tagged PART and
PAR4.
Increases in cytoplasmic calcium in response to thrombin were measured using
the calcium-
sensitive dye Fura-2 as previously described. A. J. Connolly, et al., Nature,
381:516-519
(1996). Because no thrombin responses were detectable in untransfected Parl-~
fibroblasts,
signaling in the transfected cells could be attributed to the transfected
receptor. In the PAR1-
expressing cell line, increases in cytoplasmic calcium were reliably elicited
by thrombin at
concentrations as low as 10 pM (Fig. 8A). PAR4 IgG had no inhibitory effect
even on these
threshold responses (Figs. 8A and 8B). PAR1 IgG markedly attenuated such
signaling and
non-immune antibody was without effect. Strikingly, the PAR1 antagonist
BMS20026121
attenuated PAR1 signaling even at high thrombin concentrations (Figs. 8A and
8B). The
1 S peptide-based PAR1 antagonist BNIS200261 was synthesized as previously
described in N.
I. Bernatowicz, et al., J. Med. Chem. 39:4879-87 (1996). Responsiveness to
lysophosphaticlic acid was unaffected by the antagonist (Figs. 8A and 8B),
suggesting that its
inhibitory effect was specific.
In the PAR4-expressing cell line, increases in cytoplasmic calcium were
reliably
triggered at 0.1 nM thrombin (Fig. 8B). PAR4 IgG blocked such responses but
had no effect
on responses to GYPGKF, consistent with the antibody's acting by preventing
receptor
cleavage by thrombin. PAR4 preimmune IgG, PAR1 IgG, and PAR1 antagonist (100
,uM)
failed to inhibit PAR4 signaling even at low thrombin concentrations (Fig.
8B).
Taken together, these results demonstrate that PAR4 IgG blocked PAR4 but not
PART signaling in response to thrombin, and PAR1 IgG attenuated PAR1 but not
PAR4
signaling in response to thrombin. Moreover, the PAR1 antagonist BMS 200261
was
remarkably effective in blocking PART signaling but was without effect on
PAR4. Lastly,
receptor desensitization studies with SFLLRN in PAR1 and PAR4 transfected
fibroblasts
(not shown) and in human platelets (Figs. 6A - 6C) demonstrated that prolonged
incubation
with SFLLRN was an additional means of attenuating PART signaling.
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EXAMPLE 7: INHIBITION OF PLATELET AGGREGATION BY INHIBITING PAR1 AND
PAR4 SIGNALING.
The contribution of PAR1 and PAR4 signaling to thrombin activation of human
platelets was tested to determine each molecule's relative contribution to the
process.
Platelets were prepared using whole blood from physiologically normal
volunteers and
aggregation and secretion were measured. For desensitization studies, SFLLRN
(100 ,uM) or
GYPGKF (500 ,uM) were added to platelets resuspended from the first platelet
pellet and
platelets were incubated at room temperature for 30 minutes without stirring
prior to repeat
centrifugation. For functional studies with PAR1 or PAR4 antibody, platelets
were
incubated with antibody or preimmune IgG for 60 minutes prior to measurement
of secretion
and aggregation. PART antagonist was added to stirring platelets 1-2 minutes
prior to the
addition of thrombin or other agonists.
By itself, PAR4 IgG had no effect on platelet aggregation even at low (1nM)
thrombin responses (Figs. 9A - 9E). By contrast, PAR1 IgG or PAR1 antagonist
markedly
inhibited platelet aggregation in response to 1 nM thrombin, as did prior
desensitization of
platelets with the PART agonist SFLLRN. None of these maneuvers inhibited
platelet
aggregation in response to GYPGKF or submaximal concentrations of ADP. These
data
suggest that PAR1 is the major mediator of platelet activation at low
concentrations of
thrombin.
In contrast to the case at 1 nM thrombin, at 30 nM thrombin, inhibition of
PAR1
signaling by either PART IgG, antagonist, or SFLLRN desensitization only
slowed
aggregation slightly such that shape change became detectable (see 0-30 second
portions of
the aggregation curves in Figures 9B, 9C, and 9D). Otherwise, these maneuvers
were without
inhibitory effect. Inhibition of PAR4 signaling with PAR4 IgG was similarly
ineffective
(Fig. 9B).
Strikingly, when signaling via PAR1 and PAR4 were blocked simultaneously,
aggregation in response to even high concentrations of thrombin was virtually
abolished
(Figs. 9A - 9E). Such synergy was seen regardless of the means by which PAR1
was blocked
(desensitization, PAR1 IgG, or antagonist). PAR4 preimmune concentrations as
high as 30
nM. They also suggest that PART is necessary for rapid platelet activation by
thrombin even
at high thrombin concentrations.
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The instant invention is shown and described herein in what is considered to
be the
most practical, and preferred embodiments. It is recognized, however, that
departures may
be made therefrom, which are within the scope of the invention, and that
obvious
modifications will occur to one skilled in the art upon reading this
disclosure, and thus the
invention is limited only by the appended claims.
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