JP2003526625A - Regulation of platelet activation - Google Patents

Abstract

  (57) [Summary] A method of modulating thrombin-mediated platelet activation by inhibiting or enhancing the activity of protease activated receptor 4 (PAR4) and protease activated receptor 1 (PAR1). The method provides a method to substantially block all thrombin-mediated platelet activation by 1) inhibiting PAR1 signaling and 2) inhibiting PAR4 signaling. . Signaling through each PAR receptor can be inhibited at various molecular levels, including: This method provides a method for enhancing thrombin-mediated platelet activation by specifically activating PAR1 and PAR4. These methods can be performed in vivo, through administration of the appropriate compound, or in vitro, for example, through ex vivo treatment of the sample.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to the inhibition of platelet activation and, in particular, such inhibition mediated through the thrombin receptor.

BACKGROUND OF THE INVENTION Thrombin, a coagulation protease produced at sites of vascular injury, activates platelets, leukocytes, and mesenchymal cells (T-KH Vu et al., Cell 64:
1057-1068 (1991)). Activation of platelets by thrombin is believed to be important for hemostasis and thrombosis. In animal models, thrombin inhibitors block platelet-dependent thrombosis. Platelet-dependent thrombosis is responsible for most heart attacks and strokes in humans. Available data in humans suggest that thrombosis in arteries may be blocked by inhibition of platelet function and by thrombin inhibitors. Thus, presumably, the action of thrombin on platelets is involved in the formation of blood clots resulting in heart attack and stroke. Other effects of thrombin on vascular endothelial and smooth muscle cells, leukocytes, and fibroblasts are associated with normal wound healing and various diseases (atherosclerosis, restenosis, pulmonary inflammation (ARDS), thread). When it occurs in sclerosis, etc., it may mediate inflammation and may mediate a proliferative response to injury.

Thrombin signaling is mediated, at least in part, by the family of G protein-coupled protease activated receptors (PAR), PAR1 being the prototype for that receptor. U. B. Rasmussen et al., FEBS
Letts. 288: 123-128 (1991). PAR1 is activated when thrombin binds to its amino-terminal exodomain and cleaves unmasked the amino terminus of the new receptor. This new amino terminus is then linked to the tethered peptide ligand
It acts as a peptide ligand and binds intramolecularly to the body of the receptor so as to effect transmembrane signaling. T. -K. Vu et al., Na
true. 353: 674-677 (1991); M. Chen et al. B
iol. Chem. , 269: 16041-16045 (1994). A synthetic peptide, SFLLRN, which mimics a new amino-terminal first six amino acids that is not masked by cleavage of the receptor, functions as a PAR1 agonist and acts on the receptor independently of thrombin and proteolysis. Activate. R. J. Vassallo et al. Biol. Chem. , 26
7: 6081-6085 (1994). R. M. Scarborough, et al., J
． Biol. Chem. 267: 13146-9 (1992). Such peptides are used as pharmacological probes of PAR function in various cell types.

Our understanding of the role of PAR in platelet activation is rapidly evolving. PAR1 mRNA and protein were detected in human platelets.
D. T. Hung et al. Clinical Investigation. 8
9: 1350-3 (1992); F. Brass et al. Biol. Che
m. 267: 13795-13798 (1992); Molino et al.
Biol. Chem. 272: 6011-7 (1997). SFLLRN activated human platelets 3, 7, 8 and the RAR1 blocking antibody inhibited human platelet activation at low concentrations not higher than that of thrombin. These data suggest a role for PAR1 in the activation of human platelets by thrombin, but the potential involvement of other receptors remains unsolved. PAR
4 was recently identified (ML Kahn, Nature. 394: 690-6.
94 (1998); F. Xu et al., PNAS 95: 6642-6 (1998).
)), And appears to function in both mouse and human platelets.

A better understanding of thrombin-mediated platelet activation is needed. Identification of any factor that may be involved in or inhibit platelet-mediated pathology (eg, platelet-dependent arterial thrombosis) for use in treating associated pathologies. And characterization is also needed.

SUMMARY OF THE INVENTION Modulation of thrombin-mediated platelet activation by inhibiting or enhancing the activity of protease activated receptor 4 (PAR4) and protease activated receptor 1 (PAR1) A method of doing is disclosed. This method provides a method of substantially blocking all thrombin-mediated platelet activation by: 1) inhibiting PAR1-mediated signaling and 2) inhibiting PAR4-mediated signaling. To do. Signaling through each PAR receptor can be inhibited at a variety of molecular levels, including: level of ligand binding (eg, by administration of an antagonist), level of activity of the receptor (eg, of receptor in relevant cells). Blocking expression) and / or intracellularly (eg, blocking the expression or activity of a molecule required for the 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 be carried out in vivo, via administration of the appropriate compound, or in vitro, for example via ex vivo treatment of the sample.

The present invention provides compositions that are effective in preventing thrombin-related platelet activation. These compositions are composed of reagents that inhibit the activity of PAR1 and PAR4 (eg, antagonists of PAR1 and PAR4) and are effective in treating disorders. Examples of useful antagonists are small molecules, modeled proteins, and antibodies. In addition, the composition may include a dominant negative PAR receptor, which may be used to substantially reduce or eliminate expression of either PAR1 or PAR4. These compositions may also include nucleic acid sequences encoding antagonists of PAR1 and PAR4. It can be administered for expression in vivo.

The present invention also provides compositions that are effective for enhancing thrombin-related platelet activation, either in vitro and / or in vivo. These compositions include reagents that inhibit the activity of both PAR1 and PAR4 (eg, PA
R1 and PAR4 antagonists) and are effective in treating disorders. Examples of useful antagonists are small molecules, modeled proteins, and antibodies. These compositions may also include nucleic acid sequences encoding antagonists of PAR1 and PAR4. It can be administered for expression in vivo. These compositions are composed of reagents that enhance the activity of both PAR1 and PAR4 (eg, antagonists of PAR1 and PAR4) and treat disorders in which platelet activation is insufficient (eg, hemophilia). Is effective in. Examples of useful agonists are small molecules, modeled proteins,
And antibodies. These compositions may also include nucleic acid sequences encoding antagonists of PAR1 and PAR4. It can be administered for expression in vivo.

In one embodiment, the present invention is in the treatment of disorders such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, and other blood system thrombosis. There is provided a therapeutic use of the inhibitor composition of. One method of treatment involves administration of the protonated / acidified nucleic acid to the animal in an amount sufficient to inhibit or prevent tissue obstruction. Alternatively, a sample (eg, a blood sample) can be taken from an animal and treated ex vivo with an inhibitory composition of the invention,
And can be returned to the animal again.

In another embodiment, the present invention provides therapeutic use of the activating composition of the present invention in disorders associated with insufficient coagulation. Dual activation of PAR1 and PAR4 can increase platelet activation. This is because thrombin has the ability to activate both receptors.

The present invention also provides specific PAR4 antibodies for use in the methods of the invention. Such antibodies effectively block signaling through PAR4, and thus PA against thrombin-mediated platelet activation.
Effectively blocks R4 involvement.

[0012] One object of the present invention is to inhibit the activity of PAR1 and PAR4, thus substantially eliminating thrombin-mediated platelet activation.

One advantage of the present invention is that the method of the present invention can be performed both in vivo and ex vivo.

One feature of the present invention is that the pharmaceutical composition of the present invention is effective in treating various diseases.

These and other objects, advantages, and features of the invention will be apparent to those of skill in the art upon reading the details of the nucleic acid and its use, as more fully described below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Before the receptor assay activated by the proteases of the present invention and methods of using the same are described, the present invention is directed to specific DNA sequences, materials, It should be understood that the method or process is not limited and can of course be varied. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. The scope of the invention is limited only by the appended claims.

As used in this specification and the appended claims, the singular forms “a,” “and,” and “the” are used unless the context clearly dictates otherwise. , It should be noted that it contains multiple indicators. So, for example,
References to "DNA sequences" include mixtures of such sequences and numerous such sequences, references to "assays" include assays of the same general type, and references to "methods" , One of the types described herein
Including one or more methods or steps.

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 prior invention is not entitled to antedate such publication by virtue of prior invention.

Unless defined otherwise, all technical and scientific terms used 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 certain aspects of the invention in which the publications are cited in combination. Has been done.

(Definition) “Receptor 1 activated by protease”, “PAR1”, “PA
By "R1 receptor" and the like is meant all or part of a vertebrate cell surface protein that is specifically activated by thrombin or a thrombin agonist, thereby activating a signaling event mediated by PAR1. Polypeptides are described herein and in Vu et al. (1991) Cell, 64: 1.
057-1068, which is incorporated herein by reference, including the properties of activating agonists and inhibiting antagonists. PAR1 is a naturally occurring form of the receptor,
And / or reference may be made to recombinantly produced forms of the receptor. Furthermore, the term may include variants of the PAR1 protein that retain the same activity and properties.

“Receptor 4 activated by protease”, “PAR4”, “PA
By "R4 receptor" and the like is meant all or part of a vertebrate cell surface protein that is specifically activated by thrombin or a thrombin agonist, thereby activating signaling events mediated by PAR4. Polypeptides are described herein and in US patent application Ser. No. 09 / 032,39.
No. 7 (which is incorporated herein by reference) (
(Including activation of agonists and inhibitory properties of antagonists). PAR4 may refer to the naturally occurring form of the receptor and / or the recombinantly produced form of the receptor. In addition, the term may include variants of PAR4 protein that retain the same activity and properties.

By “polypeptide” is meant an amino acid in any chain, regardless of length or post-translational modification (eg, glycosylation).

By “substantially pure” is meant that at least 60% by weight of the receptor 4 polypeptide activated by the protease provided by this invention is the protein with which it is naturally associated and naturally occurring. It is meant to contain no organic molecules present. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% PAR polypeptide by weight. A substantially pure PAR polypeptide can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding the PAR polypeptide, or by chemical synthesis of the protein. Purity is determined by any suitable method (eg, column chromatography, polyacrylamide gel electrophoresis,
Or HPLC analysis). A protein is substantially pure if it can be isolated as a band in the gel.

By “substantially identical” amino acid sequences, conservative amino acid substitutions (eg,
Placed by replacing one amino acid of the same class with another (eg, valine for leucine, arginine for lysine, etc.) or at amino acid positions that do not disrupt the biological activity of the receptor Amino acid sequences that differ only by one or more non-conservative amino acid substitutions, deletions, or insertions are meant. Such an equivalent receptor may be derived from any animal tissue or cell or its equivalent which naturally produces such a receptor or which can be introduced to produce using the methods described below. Or isolated by chemical synthesis; or by standard techniques of recombinant DNA technology (eg, a cDNA encoding such a receptor).
Or by isolation of genomic DNA). Substantially identical receptors have the same biological function and are, for example, activated by the same compound.

By “derived from” is meant encoded by the genome of the organism and present on the surface of a subset of cells of the organism.

By “isolated DNA” is it directly contiguous in the naturally occurring genome of the organism from which the DNA of the present invention is derived (ie, one of the 5 ′ ends and the 3 ′ end). One) and the complete coding sequence are not directly contiguous (covalently bound thereto) to DNA that is not in its natural environment. Thus, the term is, for example, incorporated into a vector, autonomously replicating plasmid or virus; or is incorporated into prokaryotic or eukaryotic genomic DNA; or other sequences. Independent of another molecule (
CDNA produced by, for example, PCR or restriction endonuclease digestion
Or a recombinant DNA present as a genomic or cDNA fragment). The term also includes any recombinant DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

By “transformed cells”, “transfected cells”, “genetically engineered cells” and the like, into which (or into its ancestors) PAR1, PAR4, or any by means of genetic engineering A cell into which a DNA molecule encoding a biologically active fragment thereof has been introduced is meant. Such a DNA molecule is "arranged for expression," which directs the transcription and translation of the sequence D.
It is meant to be placed adjacent to the NA sequence (ie, which facilitates the production of the PAR1 or PAR4 protein, or fragments or analogs thereof).

By “antibody” is meant an immunoglobulin protein capable of binding an antigen. Antibodies as used herein include whole antibodies, as well as epitopes of interest,
It is meant to include the antigen, or any antibody fragment capable of binding the antigenic fragment of interest (eg, F (Ab ′) ₂ , Fab ′, Fab, Fv).

Antibodies of the invention are immunoreactive or immunospecific for either PAR1 or PAR4 proteins, and thus PAR1 or PAR
It is specific for any of the four proteins and binds selectively. PAR
Antibodies to 1 or PAR4 are preferably immunospecific—ie, they are not substantially cross-reactive with the materials involved (eg, with each other). The term "antibody" includes all types of antibodies (eg, monoclonal), but the antibodies of the present invention are preferably produced using the phage display methodology described herein. The preferred antibody of the present invention is a purified antibody. By purified antibody is meant an antibody that is substantially free of other proteins, carbohydrates, and lipids with which it is naturally associated. Such antibodies "preferentially bind" to the specific PAR protein (or antigenic fragment thereof). That is,
It does not substantially recognize and bind to other antigenically unrelated molecules.

By “specifically activating”, as used herein, is mediated by a protease activated receptor, receptor polypeptide, or PAR described herein. A sample that activates a fragment or analog thereof that initiates a biological event, but which naturally contains a receptor polypeptide that is activated by a protease (eg, a biological sample)
Reagents comprising polypeptides such as antibodies that activate fragments or analogs thereof that do not substantially bind other molecules therein (eg, thrombin, thrombin analogs, PAR agonists or other chemical reagents) are meant It

By “specifically inhibit”, as used herein, other chemical reagents, including polypeptides such as thrombin analogs, PAR4 antagonists, or antibodies, which are proteases Receptor 4 activated by
Activation of a receptor polypeptide, or a fragment or analog thereof, eg, inhibiting thrombin, or thrombin or other RAP4
By blocking the activation of PAR4 by activators). Preferably, the reagent activates or inhibits the in vivo or in vitro biological activity of the protein to which it binds.

“Biological activity” binds thrombin or an agonist and signals the appropriate cascade of biological events such as phosphoinositide hydrolysis, Ca ²⁺ efflux, and platelet aggregation. The ability of PAR to communicate is implied.

By “substantial increase” is an at least about 2-fold increase over the level of control (when the control assay is performed in the absence of activator), preferably at least about about the control assay. An increase in activity or other measurable phenotypic characteristics is meant, which is a 5-fold increase, more preferably at least a 10-fold increase.

“Substantial decrease” or “substantial decrease” is about 80% of control level
And preferably reduced to about 50% of the control level, or more preferably to about 10% or less of the control level, a reduction or decrease in activity or other measurable phenotypic characteristics. Is meant.

The terms “screening method” and “assay method” are used to describe methods of screening candidate compounds for their ability to act as agonists or antagonists of PAR4 ligands. The method comprises the steps of: a) contacting a candidate agonist compound with recombinant protease-activated receptor 4 (or PAR4 agonist binding fragment or analog); b) receptor, receptor polypeptide , Or measuring the activation of a receptor fragment or analog; and c
3.) Identifying agonist compounds as those that interact with the recombinant receptor and induce or block activation of PAR4. The interaction may be the cleavage of the receptor in order not to mask the intramolecular receptor-activating peptide,
Alternatively, it can be obtained by mimicking an intramolecular receptor activating peptide. Tethered ligands can be more difficult to block than free agonists. Therefore, blocking thrombin is an acid test for antagonists that block the response by other thrombin substrates (
acid test). These terms include assays that test for effects on unoccupied receptors, as well as assays that utilize displacement of ligand from occupied receptors.

By “agonist” is meant a molecule that mimics a particular activation, where the molecule interacts with PAR or a PAR ligand in the following manner. This mode is one in which this interaction activates the induction of biological events that normally result from the interaction, such as phosphoinositide hydrolysis, Ca ²⁺ efflux, and platelet aggregation. Preferably, the agonist initiates a substantial increase in receptor activity relative to the control assay in the absence of activator or candidate agonist. An agonist may possess the same activity, less activity, or greater activity as a naturally occurring PAR ligand.

By “antagonist” is meant a molecule that blocks activation of the PAR receptor. This, for example, triggers biological events (eg, phosphoinositide hydrolysis, Ca ²⁺ efflux, and platelet secretion or platelet aggregation) that interact with PAR, thereby resulting in such interactions. Can be done by inhibiting a specific activity, such as thrombin's ability. Antagonists may bind to the PAR receptor, PAR1, PAR4, or both, and thereby block their activation.

The terms “antagonist assay”, “antagonist screening” and the like refer to naturally occurring activating ligands or agonists and PAR (PAR1
, PAR4, or both), to screen candidate compounds for their ability to antagonize their interaction with S., PAR4, or both. The method comprises the following steps: a) a candidate antagonist compound, on the one hand a recombinant PA
Contacting a first compound comprising R (or an agonist binding fragment or analog) and, on the other hand, a second compound comprising thrombin or a PAR agonist; b) interaction between the first compound and the second compound Whether to do
Or determining whether it is prevented from interacting with the candidate compound;
And c) the first compound (PAR) against the second compound (PAR agonist)
Receptor), and thereby thrombin or PAR
Identifying antagonistic compounds as those compounds that substantially reduce agonist-activated biological events such as phosphoinositide hydrolysis, Ca ²⁺ efflux, and platelet aggregation.

The terms “treatment”, “treating”, “treat”
) ”And the like are used herein to generally mean obtaining the desired pharmacological and / or physiological effect. This effect may be prophylactic with regard to the complete or partial obstruction of the disease or its symptoms, and / or with respect to the partial or complete cure of the disease, and / or the deleterious effects contributing to the disease. Can be therapeutic. "Treatment", as used herein, includes any treatment of mammalian (particularly human) disease, and this includes: (a) predisposing to the disease or condition. But preventing a disease or condition that is present in a subject who has not yet been diagnosed with it; (b) inhibiting the symptoms of the disease, ie preventing its development. Or (c) alleviating the symptoms of the disease, that is, causing regression of the disease.

The term “extracorporeal blood” refers to blood that is taken from a patient, subjected to extracorporeal treatment, and returned back to the patient in a process such as a dialysis procedure or blood filtration or blood bypass during surgery. Including. The term also includes blood products stored in vitro for the last administration to a patient. Such products include whole blood, platelet concentrates, and any other fraction of blood in which inhibition or enhancement of platelet aggregation and platelet release is desired.

General Aspects of the Invention The present invention is based on the discovery that inhibition of PAR1 and PAR4 substantially abrogates the ability of platelets to respond to thrombin. Functional studies with PAR1 and PAR4 activating peptides confirmed that activation of either receptor was sufficient to induce platelet secretion and aggregation. Given the prominent ability of thrombin as a platelet activating factor, blocking thrombin signaling in platelets is useful in a number of biological phenomena such as prevention of thrombosis.

Accordingly, the present invention provides a method of treatment for reducing the level of thrombin response in a mammalian host by administering a composition that inhibits the activity of both PAR1 and PAR4. In general, such compounds reduce the activity of PAR1 and PAR4 receptors. The treated individual may be symptomatic at present, but prophylactic use may be such as in the elderly and / or individuals who have a history of disorders associated with thrombus (ie blood clots). Intended for individuals at risk for disorders such as thromboembolism.

A number of mechanisms can be used to selectively block PAR1 signaling. For example, a peptide that selectively binds PAR1 and inactivates it
It can be used to block PAR1 signaling. The potency of potent and selective PAR1 peptide agonists allows homogeneous desensitization of platelets to PAR1 activation. In another example, an antibody that selectively binds PAR1 but not other PARs can be used. For example, PA
Antibodies against the hirudin-like domain of R1 can be used. It inhibits the cleavage and activation of PAR1 by thrombin. In the third example, PAR1
Specific antagonists allow direct blockade of PAR1 signaling,
Signaling through other PAR molecules generally remains unaffected.
Each of these examples, as well as others known to those of skill in the art, can be used to inhibit PAR1 signaling without interfering with other PAR functions. Inhibition of PAR1 function by antibodies, antagonists, or desensitization clearly inhibits the platelet response at low concentrations (1 nM) of thrombin.

PAR4 can be inhibited using similar mechanisms including, but not limited to, peptide agonists, antibodies, and PAR4-specific antagonists. PAR
Inhibition of 4 is preferably achieved using an antibody to the thrombin cleavage site of PAR4. This antibody preparation blocks the activation of PAR4 by thrombin without interfering with other PAR activities. Furthermore, antibodies to this region of PAR4 do not inhibit activation of either PAR4 or platelets by the PAR4 activating peptide GYPGKF. In contrast to PAR1 inhibition, PAR4 alone inhibition had no significant effect on platelet aggregation.

The treatment of the present invention may be performed in vivo, for example, through administration of PAR1 and PAR4 antagonists, or introduction and expression of PAR1 and PAR4 antagonists. Alternatively, the treatment can be performed ex vivo. For example, a patient's extracorporeal blood can be treated with the composition of the present invention and the blood returned again.

Compounds of the Invention The compounds of the invention include numerous chemical classes, including, but not limited to, the compounds described herein as having known functions.

Provided are novel methods of using compounds that are effective in reducing the levels 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. Numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including, for example, 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. In addition, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means, and used to produce combinatorial libraries. obtain. Known pharmacological compounds undergo directed or random chemical modifications, such as acylation, alkylation, esterification, amidation, etc., to produce structural analogs. Can be served.

The agonists or antagonists of the invention are PAR1 and / or PAR
It may involve a synthetic polypeptide that binds to 4. Such synthetic antiplatelet polypeptides can be prepared by conventional chemical synthesis techniques, such as synthesis on a solid support.

The present invention also relates to recombinant and synthetic DNA molecules that encode molecules that either inhibit or enhance PAR activity. Synthesis of these recombinant DNA molecules can be accomplished by methods well known in the art. For example,
Recombinant DNA molecules can be isolated from human hematopoietic cDNA libraries.
The synthesis of cDNA libraries and the selection of vectors into which cDNA molecules can be cloned is conventional. For example, T. Maniatis et al., “Molecu
la Cloning--A Laboratory Manual ", Co
See ld Spring Harbor (1982). A wide variety of methods can be used in locating and identifying the cDNA sequences corresponding to the compositions of the present invention. The two most preferred techniques are PAR1 or PAR
Use of oligonucleotide probes based on the amino acid sequences of 4 ligands, and immunoscreening. It utilizes antibodies to the extracellular domain of PAR1 and PAR4 to detect clones expressing cDNA sequences corresponding to potential agonist or antagonist activity. It will be apparent to those skilled in the art that the choice of oligonucleotide probe will be based on the strength of that probe with the amino acids encoded by the least overlapping DNA sequences.

Immunoscreening techniques require that a cDNA library be included in an expressible vector. Examples of such vectors include λgt11 and λgt10
, And other expression vectors known in the art. Antibodies used in immunoscreening techniques include antibodies to intact PAR1 and PAR4, denatured antibodies to PAR1 and PAR4, and antibodies to the peptide portion of PAR1 and PAR4. Once a potentially interesting cDNA is identified and isolated, it can be transcribed and translated to determine if it encodes a peptide with PAR antagonist or agonist activity. The partial cDNA itself can be used to reprobe a cDNA library and determine the location of the full-length cDNA.

Alternatively, the DNA molecule of the present invention can be synthesized from nucleotides by chemical means using a synthesizer. Such nucleic acids can be designed based on the identified amino acid sequences of PAR agonists or antagonists. Standard methods can be applied to synthesize the genes encoding such peptides. For example, the complete amino acid sequence can be used to construct the reverse translated gene. D containing a nucleotide sequence capable of encoding a desired polypeptide
NA oligomers can be synthesized in a single step.

Alternatively, several small oligonucleotides encoding part of the PAR1 and / or PAR4 agonists or antagonists can be synthesized and subsequently linked together. Preferably, the antiplatelet polypeptide gene is synthesized as 10-20 separate oligonucleotides that are then linked together. Individual oligonucleotides contain 5'or 3'overhangs for complete assembly.

After synthesis of the nucleic acid and cleavage of the desired vector, assembly of the antiplatelet polypeptide gene can be accomplished in one or more steps by techniques well known in the art. Once assembled, the gene is characterized by sequences that are recognized by restriction endonucleases, including unique restriction sites for direct assembly into cloning or expression vectors; preferred codons are the host used. Based on expression system: and the sequence, when transcribed, results in an mRNA with minimal secondary structure. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of biologically active antagonists or agonists in platelet aggregation assays.

According to one embodiment, the invention relates to compositions for reducing or preventing platelet aggregation and release, and methods of using them. These compositions also provide, in addition to the naturally-purified, recombinant, or synthetic polypeptide inhibitors of platelet activation of the present invention, various other conventional anti-platelet compounds or anti-platelet compounds. A thrombin compound may be included. The most widely used antiplatelet agent is aspirin, which is a cyclooxygenase inhibitor. Aspirin blocks platelet aggregation induced by ADP and collagen, which is an agonist (eg,
It cannot prevent cyclooxygenase-dependent platelet aggregation initiated by thrombin). An alternative antithrombin compound is a hirudin derivative.

The composition of the invention containing the additional antiplatelet activating compound may be in a single dosage form. Here, the polypeptide inhibitors of platelet activation of the present invention may be chemically conjugated to platelet inhibitors of conventional polypeptides or conventional antithrombin polypeptides. Alternatively, a single dosage form comprising a polypeptide inhibitor of platelet activation and another polypeptide in the same composition but as separate compounds. The composition may also include multiple dosage forms. Here, the PAR1 and PAR4 inhibitors, as well as other polypeptides that inhibit platelet activation, are administered separately but simultaneously, or here the two forms are administered sequentially.

Polypeptide inhibitors of PAR1 and / or PAR4 can also be cross-linked to conventional carrier polypeptides. This can be done by chemical crosslinking methods well known in the art. Most preferably, such a combination is formed by crosslinking a natural or recombinant polypeptide PAR antagonist to a carrier synthesized with a bridging moiety (eg, dinitrofluorobenzene) at its NH ₂ terminus. To be done. Alternatively, the carrier peptide is rendered a natural or recombinant PAR1 and / or PAR4 antagonist by the use of reagents such as glutaraldehyde, dimethyladipic acid, or any other bifunctional cross-linking agent known in the art. Can be conjugated to. The conjugated antagonist preferably comprises a 1: 1 stoichiometry with the carrier peptide.

The agonist or antagonist of the present invention may be an invention relating to a synthetic polypeptide for platelet activation. Such synthetic anti-platelet polypeptides can be prepared by conventional chemical synthesis techniques, such as synthesis on a solid support.

Methods of making antibodies that can be used in the methods of the invention are generally known to those of ordinary skill in the art. For example, antibodies that detect the extracellular domain of either PAR1 or PAR4 can be made by immunizing rabbits or mice with a portion of the extracellular domain of the respective molecule or the peptide fragment from which they are derived. Only antibodies with at least 4-fold higher affinity for PAR1 or PAR4, respectively, should be selected when compared to their affinity for any other PAR molecule. Methods of making, purifying, labeling, and detecting antibodies can be varied.

IgG or Fab ′ can be purified from different sources by affinity HPLC using a Protein A column and size exclusion HPLC. Purified antibody can be labeled with europium and detected by time-reduced fluorescence. Antibody binding to different PAR proteins can be measured by time-reduced, dissociation-enhanced fluorescence. However, the system of detection of IG bound to PAR on a solid support in situ or in solution can be modified. Furthermore, it is possible to use direct or indirect immunological methods including direct radioactive labeling, fluorescence, luminescence, avidin-biotin amplification, or enzyme-linked assays with dyes or luminescent substrates. A preferred PAR1 antibody that can be used in the present invention is described by Hung et al. Clin. I
nvest. , 89: 1350-1353.

For the purposes of the present invention, indicating that no binding occurs means that the equilibrium or affinity constant K _a is 10 ⁶ l / mol or less. further,
The binding is preferably 1 if the K _a is 10 ⁷ l / mol or greater.
0 ⁸ l / mol or is recognized to be present in greater than. 10 ⁷ l /
A binding affinity of mol or greater may be due to: (1) a single monoclonal antibody (ie, multiple antibodies of one type) or (2) multiple different monoclonal antibodies (eg, multiple antibodies). 5 different monoclonal antibodies), or (3) a large number of polyclonal antibodies. It is also possible to use the combination of (1) to (3). Preferred antibodies selected are for one PAR (eg PAR1) rather than for different PAR (eg PAR4),
It binds at least 4-fold more avidly. 4 in binding affinity
A fold difference can be achieved by using several different antibodies, such as (1)-(3) above, and some such antibodies in the mixture have less than a 4-fold difference. Can have

The method of the present invention may be carried out using one or more different antibodies against PAR1 and / or PAR4. Those skilled in the art will recognize that antibodies can be labeled with known labels and used with currently available robotics, sandwich assays, electronic detection equipment, flow cytometry, and the like.

Administration of Compounds In the methods of the invention, the compounds can be administered to the host using any convenient means that can result in the modulation of the desired target protein activity. Accordingly, the compounds may be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention are suitable for administration in a suitable pharmaceutically acceptable carrier or diluent.
uent) in combination with a pharmaceutical composition, and may be a solid, semi-solid, such as tablets, capsules, powders, granules, ointments, solutions, transdermal patches, suppositories, injections, inhalants and aerosols. It can be formulated into solid, liquid, or gaseous form preparations.

Thus, the administration of compounds can be oral, buccal, rectal, parenteral,
It can be achieved by various methods including intraperitoneal administration, intradermal administration, transdermal administration, intratracheal administration and the like.

In the pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts, or the compounds may also be alone or associated with other pharmaceutically active compounds. And in combination. The following methods and excipients are merely exemplary and are in no way limiting.

For oral preparations, the compounds may be used alone or in tablets, powders, granules,
Or it may be used in combination with suitable additives for making capsules. Examples of additives are conventional additives (eg lactose, mannitol, corn starch or potato starch); binders (eg crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatin); disintegrants (eg ,
Corn starch, potato starch, or sodium carboxymethyl cellulose); lubricants (eg, talc or magnesium stearate); and, if desired, diluents, buffers, wetting agents, preservatives, and flavors (
a flavoring agent).

The compounds of the invention may be dissolved, suspended or emulsified in an aqueous or non-aqueous solvent such as vegetable oils or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol. By allowing it to be formulated into a preparation for injection. If desired, conventional additives such as solubilizers, tonicity 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% by weight.

The compounds may be utilized in aerosol formulations which are administered via inhalation. The compounds of the present invention may be formulated in pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

In addition, 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 may be administered rectally via a suppository. Suppositories may include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature but solidify at room temperature.

Unit dosage forms for oral or rectal administration can be provided such as syrups, elixirs and suspensions, where an individual dosage unit (eg 1 teaspoon, 1 teaspoon, tablet) is provided. , Or a suppository) comprises a predetermined amount of the composition containing one or more inhibitors. Similarly, a unit dosage form for injection or intravenous administration may be the inhibitor (s) in the composition as a solution in sterile water, normal saline, or another pharmaceutically acceptable carrier. Can be included.

Pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents are readily available to the public. In addition, pharmaceutically acceptable auxiliary substances (eg pH adjusting agents and buffers, tonicity adjusting agents, stabilizers, wetting agents etc.)
However, it is publicly and easily available.

The compounds for use in the method of the present invention also have greater than 50 daltons and 2,5
It can also be a small organic compound having a molecular weight of less than 00 Daltons. Candidate compounds contain functional groups necessary for structural interaction with proteins, particularly hydrogen bonds, and typically contain at least one amine, carbonyl, hydroxyl, or carboxyl group. Preferably, it contains at least two of these chemical functional groups. Candidate compounds often include 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, their derivatives, structural analogs, or combinations.

The compound is administered in a physiologically acceptable carrier at a dose of 5 mg to 1400 mg, more usually 100 m, for a dose of 0.5-20 mg / kg body weight.
g to 1000 mg, preferably 500-700 mg, added to the host. The dose of the compound that suppresses the thrombin response is selected such that the activity of PAR1 and PAR4 is reduced by 10-80%, more preferably 20-70%, and even more preferably 25-50%. . PAR1 and PAR4
The dose for a compound that inhibits the activity of is selected such that the ability of platelets to respond to thrombin is reduced by about 20-80%, preferably 40-50%.

Platelet activation can be induced by a number of biological phenomena, including damage in response to specific compounds. The compositions of the invention are generally administered daily in an amount that provides a reduction in platelet activation of at least about 50-100%, more preferably 75-95%, and even more preferably 80-90%. To be done. Generally, the total daily dose will be at least about 10 mg, usually at least about 400 mg to 500 mg, preferably about 700 mg, and not more than about 1500 mg, usually not more than about 1000 mg. The amount can vary depending on the general health of the patient, the patient's response to the drug, whether the composition is used alone or in combination with other drugs, and the like. Daily administration may be more than once, usually up to about four times and may depend, among other things, on the level of drug administered.

Administration of the compounds of the invention is particularly useful in treating diseases such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, and other blood thrombosis. Inhibition of platelet activation in such disorders may allow localized treatment at the site of the clot, thereby eliminating some of the more unpleasant side effects of systemic treatment (eg, bleeding). obtain.

EXAMPLES The following examples provide a complete disclosure of methods for making receptor proteins and sequences encoding such proteins, and implementing methodologies for finding such DNA sequences and proteins. It is not intended to limit the scope of what is published and provided for providing the description to those skilled in the art and is recognized as the present invention. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, 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 Celsius; and pressure is at or near atmospheric.

Example 1: Generation and characterization of PAR polyclonal antibody Synthetic peptide GGDDSTPSILPAPRGYPGQVC (PAR4 amino acids 34-55, SEQ ID NO: 1), AKPTLPIKTFRGAPPNSFEEFP
FSALEGC (PAR3 amino acids 81-58 and carboxyglycine-cysteine, SEQ ID NO: 2), and NATLDPRSFLLRNPNDKKYEPF
WEDEEGC (PAR1 amino acids 35-61 and carboxyglycine-cysteine, SEQ ID NO: 3) was used to generate polyclonal antisera in rabbits. Ig was purified by protein A affinity chromatography to prepare the PAR4 IgG preparation, PAR3 IgG preparation used in this study,
And a PAR1 IgG preparation was made. PAR1 immune IgG, PAR3 immune IgG, PAR4 immune IgG and PAR4 preimmune Ig
The binding of G to its respective receptor has been previously described (H. Ishi.
hara et al., Blood, 91 (11): 4152-4157 (1998); K.
． Ishii et al. Biol. Chem. , 268: 9780-6 (1993).
)) Was tested on COS cells transiently expressing the FLAG epitope-tagged receptor using an enzyme-linked immunosorbent assay (ELISA). FLAG
A cDNA for epitope-tagged human PAR4 similar to epitope-tagged PAR1 was constructed as previously described, whereby the FLAG epitope was fused to amino acid 22 in PAR4 to yield the following sequence: Was :. ． ． DYKDDDDDVE / TPSVYE. ． ． Here, / indicates a connecting portion with the PAR4 sequence.

Example 2 PAR mRNA Expression in Platelets and Other Blood Cells A competitive RT-PCR assay was developed to evaluate the expression of known PAR-encoding mRNAs. First, the competitor RNA (cR
NA) was made for each PAR mRNA. Each cRNA
Was identical to the sequence of the native mRNA that was reverse transcribed and amplified by PCR, except for a single restriction endonuclease site mutation; therefore, the PCR product generated from the mRNA by digestion with a cognate restriction endonuclease. It was possible to distinguish the PCR products resulting from the cRNA against. Different amounts of competitive cRNA
Was added to 200 ng of total cellular RNA and the mixture was reverse transcribed and amplified by PCR using PCR specific primers. No product was amplified in the absence of reverse transcription. The reaction product containing only native mRNA was completely cleaved by the appropriate restriction endonuclease, while the reaction product containing only cRNA remained undigested. The amount of PAR mRNA in each sample was determined by addition of increasing amounts of competing cRNA to total cellular RNA prior to RT-PCR and comparison of the intensities of the bands obtained after restriction endonuclease digestion of the resulting PCR products. It is possible to estimate.

Dami cells were grown in suspension in RPMI containing 10% fetal bovine serum. Platelets were isolated from human blood as previously described. The platelet preparation contained less than 0.1% leukocytes as assessed by light microscopy analysis. The discontinuous Percoll gradient was tested with the manufacturer's instructions (Pharmacia).
Were used to separate monocytes and lymphocytes from neutrophils according to Monocyte / lymphocyte preparations contain less than 0.1% neutrophils and neutrophil preparations 0.1%.
Less than monocytes or lymphocytes. Total RNA is Trizol (GIBCO)
Were prepared from whole cells using DNAseI (Boehringer-Mann
heim) and quantified by OD ₂₆₀ .

Each receptor cDNA was mutated to remove an endogenous restriction endonuclease site. These sites are digested with a cDNA encoding the receptor using a selected endonuclease, and the digested plasmid is digested with T4 DNA.
It was mutated by exposure to a polymerase and then religation. Each of these mutant cDNAs was subcloned into Bluescript (KS- or SK-, Stratagene) so that the sense cRNA could be transcribed in vitro using T7 RNA polymerase. The competing cRNA thus produced was added to total cellular RNA prior to the reverse transcription (RT) reaction. The RT reaction was performed using a commercial kit (GIBCO) and receptor-specific primers (see below) in a reaction volume of 10 μl with 200 ng total RNA together with various amounts of competing cRNA. Performed at ° C. Then RT product 2μ
l of the final concentration of 2 μM primer and 5 U of Taq polymerase (GIBC
Subjected to PCR amplification in a volume of 50 μl containing O). The reaction conditions were as follows: 94 ° C. for 4 minutes, 72 ° C. for 1 minute with the addition of Taq; then 94 ° C. for 45 seconds, 55 ° C. for 1 minute, 72 ° C. for 1 minute for 30-36. Cycle (see below); then 72 ° C. for 8 minutes. A preliminary experiment was conducted for each sample. Here, the number of amplification cycles was varied to determine the number of cycles in which a specific product was amplified, and the number of cycles in the middle of the dynamic range was selected. For each analysis, PAR1 mR in RNA from different sources
The number of cycles selected for measuring the levels of NA, PAR2 mRNA, PAR3 mRNA, and PAR4 mRNA were as follows:

[0081]

[Table 1] Preliminary experiments also established a range of concentrations of competing RNA to add to each sample.

The primers used for RT and PCR of each receptor, and each P
The restriction endonucleases used to digest the CR products were as follows. The nucleotide numbering convention is that 1 is A for the starting methionine ATG.

[0083]   PAR1, GenBank registration number M62424:   Primers for RT: TAG ACG TAC CTC TGG CAC   TC (1148-1129; SEQ ID NO: 4).

Sense strand primer for PCR: CAG TTT GGG TCT GA
A TTG TGT CG (SEQ ID NO: 5).

Antisense primer for PCR: TGC ACG AGC TTA
TGC TGC TGA C (SEQ ID NO: 6).

[0086]   PCR products obtained: 505-1096.

[0087]   Mutated site: AgeI at position 596.

PAR2, GenBank Accession No. U34038: Primers for RT: CTG CTC AGG CAA AAC ATC
(699-682; SEQ ID NO: 7).

Sense strand primer for PCR: TGG ATG AGT TTT CT
G CAT CTG TCC (SEQ ID NO: 8).

Antisense primer for PCR: CGT GAT GTT CAG
GGC AGG AAT G (SEQ ID NO: 9).

[0091]   PCR products obtained: 182-672.

[0092]   Mutated site: SfiI at position 342.

[0093]   PAR3, GenBank registration number U92972:   Primers for RT: TGA TGT CTG GCT GAA CAA   G (727-709; SEQ ID NO: 10).

Sense strand primer for PCR: TCC CCT TTT CTG CC
T TGG AAG (SEQ ID NO: 11).

Antisense primer for PCR: AAA CTG TTG CCC
ACA CCA GTC CAC (SEQ ID NO: 12).

[0096]   PCR products obtained: 152-664.

[0097]   Mutated site: NcoI at position 251.

[0098]   PAR4, GenBank registration number AF080214:   Primers for RT: TGA GTA GCT GGG ATT ACA   G (1519-1501; SEQ ID NO: 13).

Sense strand primer for PCR: AAC CTC TAT GGT GC
C TAC GTG C (SEQ ID NO: 14).

Antisense primer for PCR: CCA AGC CCA GCT
AAT TTT TG (SEQ ID NO: 15).

[0101]   PCR products obtained: 949 to 1490.

[0102]   Mutated site: BamHI at position 1005.

After PCR amplification, 10 μl of the reaction product was digested with the appropriate restriction endonucleases overnight (AgeI, 2U, 25 °; SfiI, 20U, 50 ° C; NcoI, 10).
U, 37 ° C; or BamHI, 20U, 37 ° C). The product is then added to 1.5
% Agarose gel electrophoresis (Separide gel matrix; GIB)
CO) and visualized by ethidium bromide staining. The intensity of the cRNA-derived product (uncleaved band) is determined by the intrinsic m
The cRNA concentration that matched the intensity of the RNA-derived product (truncated band) was used to estimate the amount of each PAR mRNA in the initial sample.

Competitive RT-PCR of RNA prepared from platelets, neutrophils, and Dami cells
A summary of the results of several studies in the assay is shown in Figure 1. To confirm the assay, Dami cells, a human cell line expressing several megakaryocyte markers, were first analyzed. Competitive RT PCR of RNA from Dami cells yielded results consistent with Northern and protein analyses. D for Northern analysis
2 μg of poly (A) + RNA isolated from ami cells was electrophoresed on a denaturing formaldehyde agarose gel and supported on a nitrocellulose membrane (
Schleicher & Schuell). PAR1 mRNA
Was detected with a 400 bp PstI / PvuII cDNA probe;
PAR2 mRNA was detected with a 260 bp SfiI / BstEII cDNA probe: PAR3 mRNA was detected with a 610 bp KpnI / NsiI cDNA probe; PAR4 mRNA was SacI / PstI using high stringency conditions. It was detected using a cDNA probe. In all such assays, PAR1, PCR3, and PA
R4 was detected in Dami cells, but not PAR2 (Fig. 1).

Competitive RT-PCR of platelet RNA revealed that PAR1 mRNA was present at approximately 1 attomole / 200 ng total RNA. Assuming that the mRNA is 1% of the total platelet RNA and the average mRNA size is 2 kb, PAR1 mRNA shows 3000 platelet mRN.
One of A is shown. PAR4 mRNA was also readily detected in platelet RNA at 10-30% of PAR1 mRNA levels. In contrast, PCR3m
RNA was undetectable in human platelet RNA. PAR3 competing cRNA added to platelet RNA was detectable at 0.001 attomole / 200 ng total RNA, suggesting that PAR3 mRNA was at least 1000 times less than PAR1 mRNA in these samples. did. PAR2 m
RNA was not detected in platelet RNA from one individual, and only 0.001 attomole / 200 ng was detected in the other. The latter is P
It may be due to trace contamination of the platelet preparation by neutrophils expressing AR2. The 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 different from that seen in platelets. This suggests that contamination of the platelet preparation with leukocytes does not significantly affect the expression pattern of PAR detected in platelets. In particular, significant PAR2 mRNA was detected in RNA derived from both neutrophils and mononuclear cells, although it was substantially absent from platelets. Relatively high PAR2 mRNA levels in neutrophils are
Consistent with previous studies showing a neutrophil response to AR2 activating peptides. 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 it was not detected in neutrophils. These results show that PA in human platelets
Demonstrating the presence of mRNA encoding R1 and PAR4, PCR2 or PC
Demonstrate the absence of R3.

Example 3 Expression of PAR Proteins on the Surface of Human Platelets Rabbit anti-peptide antibodies directed against IgG against the amino-terminal exodomain of PAR1, PAR3, or PAR4. Purified from serum. To characterize the ability of each IgG preparation to recognize native PAR and to assess cross-reactivity, antibody binding to the surface of COS cells expressing the receptor was measured (FIG. 2). Each IgG preparation bound to the surface of cells expressing the appropriate receptor without significant cross-reactivity.

The IgG preparation was then used for flow cytometric analysis of human platelets (FIGS. 3A-3D). The washed platelets were fixed in paraformaldehyde at 40 ° C. for 20 minutes, and the platelet buffer solution (20 mM Tris-HCl, pH 7.4, 1) was added.
40 mM NaCl, 2.5 mM KCl, 1 mM MgCl ₂ , 1 mg / ml
Of glucose, 0.5% BSA), and then incubated with primary IgG in platelet buffer for 1 hour at 40 ° C. PAR1 IgG and P
AR3 IgG at 10 μg / ml and PAR4 IgG at 100 μg / m
Used in 1. Platelets were then washed and 4 μg / ml fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit IgG (Molecul).
ar Probes) and incubated for 0.5 hours. Platelets were then washed 3 times and analyzed by flow cytometry. Some fixed platelet samples were incubated at 37 ° C. at 30 ° C. before incubation with primary antibody.
Exposed to nM thrombin. Dami cells were treated similarly to platelets for flow cytometry.

Significant surface binding compared to pre-immune IgG was detected with PAR1 IgG (FIG. 3A). A similar increase in surface binding of platelets to preincubation of peptide antigens against preimmune IgG was detected with PAR4 IgG (Fig. 3C). Preincubation of PAR4 with the peptide antigen that raised it circumvented this increase (FIGS. 3C and D). Furthermore, PAR4
Antisera-evoked epitopes spread to the thrombin cleavage site of PAR4, and treatment of platelets with thrombin actually abrogated PCR4 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. This experiment was repeated with Dami cells to determine if this antibody could detect PCR3 expressed at "native" levels (Figures 4A-4D). D
Ami cells were shown to express PAR3 mRNA by Northern blot. 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 the PAR3 antibody. This result is consistent with the presence of PCR3 mRNA in Dami cells by RT-PCR and Northern blot analysis. This further
It is suggested that the absence of detectable PAR3 protein on the surface of human platelets is not due to the insensitivity of the assay. These results are consistent with the analysis of platelet RNA, and PAR on the surface of human platelets.
Make sure that 1 and PAR4 are present, but PAR3 is not.

Example 4 Activation of Human Platelets by PAR1- and PAR4-Linked Ligand Peptides Synthetic peptides that mimic PAR1- and PAR2-linked ligands function as agonists for their respective receptors. And has been used as a pharmacological tool to explore the function of these receptors in various cell types. Unfortunately, PAR3 cognate peptides appear to be insufficiently strong to function as free ligands. Peptides that mimic the ligands linked to PAR4 can function as agonists of the PAR1 and PAR2 peptides, even at concentrations higher than those found for their cognate receptors. To aid in the interpretation of experiments using PAR1 and PAR4 linked ligand peptides, the ability to activate heterologously expressed PAR1 and PAR4 in Xenopus oocytes, a highly sensitive assay system, was determined. (Fig. 5). Agonist-induced calcium metabolism was used as an indicator of receptor activation. No response was detected in oocytes that did not express either receptor. Both the human PAR4 peptide GYPGQV and the mouse PAR4 peptide GYPGKF activated oocytes expressing human PAR4, but E
The C ₅₀ was almost two orders of magnitude greater than SFLLRN for PAR1 activation (FIG. 5). SFLLRN showed no activity at PAR4. At 500 μM, P
The AR4 peptide GYPGKF showed minimal activity at PAR1. But PA
R1 is overexpressed in oocytes and PAR in the oocyte assay
The sensitivity for the detection of 1 activation is 10-100 fold greater than in platelets; presumably the activation of PAR1 at 500 μM GYPGKF is not important 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 significantly less potent than the PAR1 peptide in activating human platelets. GYPGKF was slightly more potent than GYPGQV (FIG. 6A, data not shown).

These data are consistent with the relative potency of receptor activation by their respective peptides in the oocyte line (FIG. 5). Briefly, the FLAG epitope-tagged PAR4 cDNA was subcloned into pFROG ³ and signaling studies were performed in Xenopus oocytes as described. J.
A. Williams et al., PNAS USA 85: 4939-43 (1998).
). 2.0 ng PAR4 cRNA and 25 ng P per oocyte
AR1 cRNA was injected.

Incubation of PGE ₁ -treated platelets with SFLLRN rendered them refractory to subsequent stimulation by SFLLRN, but GYPG
There was no effect on responsiveness to KF (Fig. 6B). Conversely, incubation with GYPGKF rendered platelets refractory to subsequent stimulation by GYPGKF, but did not affect 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.

Example 5 PAR1 and PAR4 Antibodies Inhibit Thrombin Cleavage of Their Receptors To define the essential role of PAR1 and PAR4 in thrombin-induced platelet activation, a blocking antibody (blocking antibody) was used. )
Was developed. The previously described PAR1 antibody (raised against the hirudin-like domain of PAR1) inhibits thrombin cleavage of the amino-terminal ectodomain of PAR1 by disrupting the binding of the anion-binding exosite of thrombin. It is supposed to be. A similar hirudin-like domain is PAR
It was not apparent in the amino terminal ectodomain sequence of 4. To obtain antibodies that inhibit PAR4 thrombin cleavage, antisera were raised against peptides displaying sequences spanning the thrombin cleavage site of PAR4. This antiserum is PAR4
Was specifically recognized. To test the ability of PAR1 and PAR4 antibodies to block thrombin cleavage of PAR1 and PAR4, RatI fibroblasts expressing FLAG epitope-tagged PAR1 and FLAG epitope-tagged PAR4 were preincubated with the antibody and then thrombin. Exposed to. Receptor cleavage by thrombin was measured as a deletion of the FLAG epitope from the cell surface. Cleavage of PAR1 was markedly inhibited by PAR1 antibody but not PAR4 antibody. Conversely, PAR4 cleavage was markedly inhibited by PAR4 antibody but not PAR1 antibody (FIG. 7). These data suggested that PAR1 and PAR4 antibodies should selectively attenuate thrombin signaling through PAR1 and PAR4, respectively.

Example 6 Inhibition of Thrombin Signaling by PAR1 and PAR4 Antibodies and PAR1 Antagonist Par1 ^{− / −} mouse lung fibroblast cell line, which showed no thrombin signaling. It was used to generate stable cell lines expressing FLAG epitope-tagged PAR1 and FLAG epitope-tagged PAR4. The increase in cytosolic calcium in response to thrombin was measured using the calcium-sensitive dye Fura-2 as previously described. A. J. C
onnolly et al., Nature, 381: 516-519 (1996). Signaling in the transfected cells may be due to the transfected receptor, as the thrombin response was not detectable in untransfected Par1 ^{− / −} fibroblasts. In cell lines expressing PAR1,
The increase in cytosolic calcium was indeed induced 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 significantly attenuated such signaling, and non-immune antibodies did not have this effect. Surprisingly, the PAR1 agonist BMS20026121
High thrombin concentrations still attenuated PAR1 signaling (FIGS. 8A and 8B).
), A peptide-based PAR1 antagonist, BNIS200261,
N. I. Bernatowicz et al. Med. Chem. 39: 4879-
87 (1996) as previously described. The responsiveness to lysophosphatidic acid is
It was unaffected by the antagonist (Figures 8A and 8B). This suggested that the inhibitory effect was specific.

In cell lines expressing PAR4, the increase in cytosolic calcium was
It was indeed induced by 0.1 nM thrombin (Fig. 8B). PAR4 IgG blocked such a response, but had no effect on the response to GYPGKF. This is consistent with the action of the antibody by blocking the cleavage of the receptor by thrombin. PAR4 preimmune IgG, PAR1 IgG, and PAR1 antagonist (100 μM) were still unable to inhibit PAR4 signaling at low thrombin concentrations (FIG. 8B).

Taken together, these results indicate that PAR4 IgG blocked PAR4 but not PAR1 signaling in response to thrombin, and PAR1 IgG attenuated PAR1 but not PAR4 signal in response to thrombin. Demonstrate that it did not weaken transmission. In addition, the PAR1 antagonist BMS200261 was significantly effective in blocking PAR1 signaling, but not PAR4. Finally, in fibroblasts (not shown) transfected with PAR1 and PAR4,
And a study of receptor desensitization with SFLLRN in human platelets (FIGS. 6A-6C) demonstrated that long incubation with SFLLRN was an additional means of attenuating PAR1 signaling.

Example 7 Inhibition of Platelet Aggregation by Inhibiting PAR1 and PAR4 Signaling PAR1 Signaling and PAR on Thrombin Activation of Human Platelets
The contribution of 4 signaling was examined to determine the relative contribution of each molecule to this process. Platelets were prepared using whole blood from physiologically normal volunteers and aggregation and secretion measured. For desensitization studies, SFLLRN (100 μM) or GYPGKF (500 μM) was added to the resuspended platelets from the initial platelet pellet, and the platelets were incubated at room temperature for 30 minutes without agitation, then The centrifugation was repeated. For functional studies with PAR1 or PAR4 antibodies, platelets were incubated with antibody or preimmune IgG for 60 minutes before measuring secretion and aggregation. 1 to 2 of PAR1 antagonist with addition of thrombin or other agonist
Minutes ago, added to stirring platelets.

Alone, PAR4 IgG had no effect on platelet aggregation, even at low (1 nM) thrombin response (FIGS. 9A-9E). In contrast, PAR
1 IgG or PAR1 antagonist is a PAR agonist SFLL
It significantly inhibited platelet aggregation in response to 1 nM thrombin, as did previous desensitization of platelets with RN. All of these operations are GYPGKF or A
It did not inhibit platelet aggregation in response to submaximal concentrations of DP. These data suggest that PAR1 is a major regulator of platelet activation at low concentrations of thrombin.

In contrast to 1 nM thrombin, at 30 nM thrombin, PA
P by either R1 IgG, antagonist, or SFLLRN desensitization
Inhibition of AR1 signaling showed only slight aggregation, such that shape changes were detectable (see the 0-30 second part of the aggregation curve in Figures 9B, 9C, and 9D). On the other hand, these manipulations did not have an inhibitory effect. PAR4 IgG
Inhibition of PAR4 signal transduction using E. coli was similarly ineffective (Fig. 9B).

Remarkably, when PAR1 and PAR4 signaling were simultaneously blocked, aggregates responsive to even high concentrations of thrombin were virtually eliminated (FIGS. 9A-9E). Such synergistic effects were observed regardless of the means by which PAR1 was blocked (desensitization, PAR1 IgG, or antagonist). The PAR4 pre-immune concentration was as high as 30 nM. These also suggest that PAR1 is essential for rapid platelet activation by thrombin even at high thrombin concentrations.

The invention is shown and described herein in what is considered to be the most practical and preferred embodiments. However, departures therefrom may be made, and it is within the scope of the present invention, and that modifications apparent to those of ordinary skill in the art upon reading the disclosure herein will occur, and thus the present invention will be covered by the appended patents. It will be appreciated that it is limited only by the scope of the claims.

[Brief description of drawings]

FIG. 1. Competitive R of RNA prepared from platelets, neutrophils, and Dami cells.
It is an outline of the experiment of a T-PCR assay.

FIG. 2 is a graph showing antibodies that bind to the surface of COS cells that express the receptor.

3A-3D are a series of graphs showing the results of flow cytometric analysis of human platelets.

4A-4D are a series of graphs showing the results of flow cytometric analysis of Dami cells.

FIG. 5: PAR1 and PAR4 heterologously expressed in Xenopus oocytes.
3 shows the ability of PAR1 and PAR4 linked ligand peptides to activate A.

6A-6C show the ability of PAR1 and PAR4 linked ligand peptides to activate PAR1 and PAR4 human platelets.

FIG. 7 is a graph showing the ability of PAR1 and PAR4 antibodies to block thrombin cleavage of PAR1 and PAR4 in rat I fibroblasts expressing FLAG epitope-tagged PAR1 and PAR4.

8A and 8B show an increase in cytoplasmic calcium in response to thrombin in cells expressing PAR1 (FIG. 8A) or PAR4 (FIG. 8B).

9A-9E show PAR1 signaling and P1 on thrombin activation of human platelets with and without treatment with PAR activating peptides or antibodies.
The contribution of AR4 signaling is shown.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 7/04 A61P 9/08 9/08 9/10 9/10 11/00 11/00 29/00 29 / 00 C07K 16/28 ZNA C07K 16/28 ZNA C12N 15/00 A C12N 15/09 A61K 37/02 (72) Inventor Khan, Mark USA California 94010, Burlingame, Carlos Avenue 1337 F Term (reference) 4B024 AA01 BA61 CA04 DA02 EA04 GA11 HA01 4C084 AA02 AA14 AA19 AA20 BA01 BA08 BA23 CA18 CA25 CA53 CA59 MA02 NA05 NA14 ZA392 ZA452 ZA512 ZA532 ZA542 ZA592 4C085 AA12 AA14 A40 AA12 A14 A40 A20 AA20 A10 A40 A20 A4 AA20 A4

Claims (12)

[Claims] 1. A method for influencing the activation of platelets, comprising: administering an effective amount of a first compound characterized by its ability to specifically modulate the activity of PAR1; and PAR4. Administering an effective amount of a second compound characterized by its ability to specifically modulate the activity of. 2. The method of claim 1, wherein the first compound specifically inhibits the activity of PAR1 and the second compound specifically inhibits the activity of PAR4. 3. The method of claim 1, wherein the first compound specifically activates the activity of PAR1 and the second compound specifically activates the activity of PAR4. 4. The method of claim 1, 2 or 3 wherein said first compound and second compound are administered to a subject simultaneously. 5. A pharmaceutical composition comprising: a therapeutically effective amount of a first compound characterized by its ability to specifically modulate the activity of PAR1; and that which specifically modulates the activity of PAR4. A pharmaceutical composition comprising a therapeutically effective amount of a second compound characterized by potency. 6. The pharmaceutical composition according to claim 5, wherein the first compound specifically inhibits the activity of PAR1 and the second compound specifically inhibits the activity of PAR4. 7. The first compound is a PAR1 antagonist BMS2002.
61. The pharmaceutical composition of claim 6, which is 61 and the second compound is a PAR4 antibody directed against the thrombin binding site of PAR4. 8. The pharmaceutical composition of claim 6, wherein the first compound is the desensitizing peptide SFLLRN and the second compound is the desensitizing peptide GYPGKF. 9. The pharmaceutical composition of claim 5, wherein the first compound specifically activates the activity of PAR1 and the second compound specifically activates the activity of PAR4. 10. The pharmaceutical composition of claim 9, wherein the first compound is a PAR1 agonist and the second compound is a PAR4 agonist. 11. A PAR4 antibody directed against all or part of the thrombin binding site of PAR4. 12. An antibody according to claim 11 directed against the sequence GGDDSTPSILPAPRGYPGQVC.