WO1998016548A1 - Thrombin receptor peptides and uses thereof - Google Patents

Abstract

The invention is based on the discovery that isolated, e.g., synthetic, peptides corresponding to all or a portion of the first 41 amino acids of the thrombin receptor are potent agonists for platelets. These new TR1-41 peptides can be used to activate platelets in vitro and in vivo, to prepare TR1-41 peptide antagonists and antibodies, and in various assays for determining a patient's level of platelet reactivity and thrombin inhibitor efficacy.

Description

THROMBIN RECEPTOR PEPTIDES AND USES THEREOF

Background of the Invention The invention relates to isolated or synthetic thrombin receptor peptides and methods of using these peptides.

Thrombin is one of the most physiologically important platelet agonists, and is an essential component in the hemostatic, proliferative, and inflammatory responses to injury. The seven transmembrane domain G protein-linked thrombin receptor (proteinase-activated receptor-1) has been identified, and studies of this receptor revealed a novel proteolytic mechanism of activation (Vu et al., Cell , 64:1057-1068, 1991) . The receptor is activated when thrombin binds to a hirudin-like domain of the receptor via an anion binding exosite, and then cleaves the receptor between amino acids Arg-41 and Ser-42. This cut releases a thrombin receptor cleavage fragment and forms a new amino-terminal that acts as a so-called "tethered ligand." The tethered ligand then binds to another, as yet unknown, binding site of the thrombin receptor to activate the receptor (Vu et al., supra; Coughlin et al., J. Clin . Invest . , 89:351-355, 1992; Vu et al. , Nature , 353:674-677, 1991).

The tethered ligand hypothesis has been confirmed by a number of studies, including experiments in which synthetic peptides that correspond to the amino acid sequence of the new amino terminus were tested as thrombin receptor agonists. Examples of these thrombin receptor agonist peptides (TRAPs) include R₄_ ₇ (thrombin receptor peptide having amino acids 42 to 47 of the full receptor), TR₄₂_₄₈, and R₄₂_₅₅. These synthetic peptides mimic the new amino terminus in sequence, and in many instances, in effect as well. For example, TRAPs have been shown to activate the thrombin receptor and platelets to varying extents (Vu et al., Cell , 64:1057- 1068, 1991; Lau et al., Biochem . J. ,303 :91-400 1994), and to cause vascular contractions (Tesfamariam, Circulat . Res . , 74:930-936, 1994).

On the other hand, a synthetic peptide mimicking a portion PESKATNATLDPRSFLL (TR₂₉_₄₅, SEQ ID NO: 11) of the thrombin receptor cleavage fragment and a portion the cleavage site, was found to be devoid of agonist activity for the wild-type thrombin receptor expressed in Xenopus oocytes (Vu et al., Cell , 64:1057-1068, 1991).

Summary of the Invention The invention is based on the discovery that isolated, e.g., synthetic, peptides corresponding to all or a portion of the first 41 amino acids of the thrombin receptor are potent agonists for platelets. These new peptides will be referred to collectively as TR_1-41 peptides, although some do not include all 41 of the amino acids, and some include substituted amino acids (as described below) . The isolated peptides can be used to activate platelets in vitro and in vivo, to prepare TR_1-41 antibodies and antagonists, and in various assays for determining a patient's level of platelet reactivity, and thrombin inhibitor efficacy. In general, the invention features isolated peptides including a sequence of at least 6, 10, 15, or 21 consecutive amino acids, and up to 41, selected from the sequence:

MGPRRLLLVAACFSLCGPLLSARTRARRPESKATNATLDPR (SEQ ID N0:1)

and including a core region of consecutive amino acids that are the same as, or substantially the same as, amino acids 17 to 22, 15 to 24, 12 to 26, or 10 to 30 of SEQ ID N0:1. The peptides must include amino acids Lys-19 and Lys-20 of SEQ ID N0:1 without substitution. Thus, useful ^TR _l-₄₁ peptides include TR_1-41, TR₁₇_₂₂, TR₁₅_₂₄, TR₁₂_₂₆,

^TR10-30' ^and TR₅_₃₅, which include peptides that have the identical sequence of the correspondingly numbered amino acids in

SEQ ID N0:1, or are substantially identical to those sequences.

The isolated peptides can be synthetic peptides. One preferred peptide is TR_1-41 having the amino acid sequence of SEQ ID NO: 1.

In another aspect, the invention features a method of activating platelets by providing a TR_1-41 peptide and exposing the platelets to an amount of the peptide effective to achieve platelet activation. The method can be carried out in vitro, ex vivo, and in vivo, e.g., to induce thrombosis. The method also can be carried out by additionally exposing the platelets to a TR₄₂_₅₅ peptide in an amount sufficient to achieve a synergistic platelet activation effect together with the TR_1-41 peptide, either simultaneously or in succession with the TR_1-41 peptide. An alternative in vivo method of activating platelets involves administering to a patient, e.g., a mammal such as a human, a nucleic acid (e.g., an expression vector) containing a sequence that encodes a TR₁_₄₁ peptide.

In another aspect, the invention features a method of assaying platelet reactivity by obtaining a biological sample containing platelets, e.g., blood, platelet-rich- plasma, or washed platelets; incubating the sample with a TR_1-41 peptide, e.g., in an amount effective to activate normal platelets; and detecting platelet activation as a measure of platelet reactivity. Platelet activation can be detected by labeling the sample with an activation- dependent label, e.g., fluorescein isothiocyanate- fibrinogen, or an antibody, e.g., PAC1, F26, S12 and others; and detecting any labeled platelets, wherein the presence of labeled platelets indicates the reactivity of the platelets in the sample.

The platelet assay can also be carried out to detect platelet activation by labeling the sample with an activation-dependent label, detecting any labeled platelets, and comparing the percentage of labeling with control values of known activated platelets as an indication of the degree of reactivity of the platelets in the sample. Platelet activation also can be detected by measuring platelet aggregation, e.g., optically (for platelet-rich plasma or washed platelet samples) or electrically (for whole blood and other platelet samples) , or by measuring a change in calcium mobilization in the platelets.

In this method, the TR_1-41 peptide can be added to separate samples in a range of different concentrations to determine the degree of reactivity of the platelets in the sample. Moreover, platelets can be considered "activated" when the percentage of labeling compared to a known non-activated platelet control value is greater than two standard deviations from the control value.

The platelet reactivity assay can be carried out by further incubating the sample with a TR₂_₅₅ peptide. In another aspect, the invention features antagonist compounds, e.g., peptides or antibodies, that antagonize the action of the TR_1-41 peptides, e.g., TR_{1- 1} peptide-induced platelet activation. These compounds are antithrombotics, and include TR-_|_₄₁ peptide mutant analogs that specifically bind to TR_1-41 target cells, e.g., platelets, yet do not activate these cells. These compounds also include antibodies that bind specifically to naturally occurring TR_1-41 peptides so as to diminish the levels of TR_1-41 peptides capable of binding to their target cells. These compounds thus diminish or prevent ^TR ₁-₄₁ peptide-mediated events such as platelet aggregation.

The invention also features a method of inhibiting ^TRι-₄i peptide-induced platelet activation in a patient by administering to the patient an amount of an antagonist of TR_1-41 peptide effective to prevent TR_1-41 peptide- induced platelet activation. This method can be used, for example, in patients that have undergone surgery or angioplasty, or who have had deep venous thrombosis, venous stasis, a pulmonary embolism, angina, coronary artery disease, unstable angina, myocardial infarction, congestive heart failure, cardiomyopathy, ventricular or atrial arrhythmias, or a stroke. An alternative method of inhibiting TR_1-41 induced platelet activation in a patient involves administering to the patient a nucleic acid (e.g., an expression vector) containing a sequence that encodes an antagonist.

The "thrombin receptor" is the seven transmembrane domain G protein-linked thrombin receptor (proteinase activated receptor-1) .

A "TR₁_₄₁ peptide" is any peptide that has an amino acid sequence that is the same as, or substantially identical to, at least amino acids 17 to 22 within the first 41 amino acids of the naturally occurring thrombin receptor protein (SEQ ID N0:1), and that activates platelets to the same extent as, or at least to 50%, as the full length TR_1-41 peptide (in the flow cytometry and aggregation assays described below) . The term includes "TR₁_₄₁ peptide analogs," i.e., mutant analogs of TR_1-41 peptides, as well as peptide fragments of the full length ^TR ₁-₄₁ peptide and mutant analogs, as long as these peptide fragments and analogs include at least a sequence that is the same as, or substantially the same as, amino acids 17 to 22 within SEQ ID N0:1, and activate platelets to at least 50% as well as the full length TR_1-41 peptide. A "mutant analog of a TR₁_₄₁ peptide" is a peptide that includes any change in the amino acid sequence when compared to the wild-type sequence of the first 41 amino acids of the thrombin receptor (SEQ ID N0:1). Mutations include, e.g., amino acid changes, deletions, insertions, inversions, translocations, or duplications. Preferred mutations are conservative amino acid substitutions in one or more locations in the peptide. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

By "substantially identical" or "substantially the same as" is meant that a peptide exhibits at least 80% (e.g., 90 or 95%) identity to a reference amino acid sequence. For TR_1-41 peptides, the comparison sequences are the corresponding portions of the first 41 amino acids of the thrombin receptor (SEQ ID N0:1). Identity is typically measured visually or by using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) . Such software matches similar sequences by assigning degrees of identity or homology to the compared sequences.

An "isolated" TR₁_₄₁ peptide is a TR_1-41 peptide that has been separated or purified from components which naturally accompany it, e.g., in blood, serum, or urine. Typically, the peptide is considered "isolated" when it is at least 70%, by dry weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, a preparation of the peptide is at least 80%, more preferably at least 90%, and most preferably at least 99%, by dry weight, TR_X_ ₄₁ peptide. Since a TR_1-41 peptide that is chemically synthesized is separated from components which naturally accompany the first 41 amino acids of the thrombin receptor or the thrombin receptor cleaved fragment, the synthetic peptide is "isolated."

An isolated TR_1-41 peptide can be obtained, for example, by extraction from a natural source (e.g., from human blood, plasma, serum, or urine) ; by expression of a recombinant nucleic acid encoding a TR_1-41 peptide; or by chemical synthesis. A peptide that is produced in a cellular system different from the source from which it naturally originates is "isolated," because it will be separated from components which naturally accompany it. The extent of isolation or purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

A "TR₁_ ₁ peptide antibody" is any polyclonal or monoclonal antibody that specifically binds to a TR₁_₄₁ peptide. An antibody that "specifically binds" to a TR_χ_ ₄₁ peptide is an antibody which recognizes and binds to a ^TR ₁-₄₁ peptide, but which does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample, which naturally includes TR_1-41 peptides. A "purified antibody" is an antibody which is at least 70%, by dry weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 80%, more preferably at least 90%, and most preferably at least 99%, by dry weight, antibody, e.g., a ^TRι-₄i peptide antibody.

The use of TR_1-41 peptides enables the preparation of polyclonal and monoclonal antibodies that specifically bind to new epitopes on the peptide, which can be used in new diagnostic and therapeutic methods. Unless otherwise defined, 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 methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Brief Description of the Drawings Figs. 1A to 1C are a series of line graphs showing

TR.^-, TR₄₂_₅₅-, and thrombin-induced platelet activation. The platelet surface binding of the monoclonal antibodies S12 (directed against P-selectin, Fig. 1A) , PACI (directed against the activated glycoprotein (GP) Ilb-IIIa complex, Fig. IB) , and F26 (directed against fibrinogen bound to the GPIIb-IIIa complex, Fig. 1C) were determined by whole blood flow cytometry.

Figs. 2A to 2C are a series of graphs showing ^Rι-₄i~' ^TR ₄₂-₅₅ ^~' ^and thrombin-induced platelet aggregation. Washed platelets were analyzed in a platelet aggregometer after the addition (arrowhead) of thrombin 1 U/ml (Fig. 2A) , TR₄₂_₅₅ 20 μM (Fig. 2B) , or TR_1-41 μM (Fig. 2C) . Fig. 3 is a line graph showing that TR_1-41 and ^TR ₄₂-₅₅ activate platelets synergistically.

Fig. 4 is a bar graph showing intracellular signal transduction pathways involved in TR₁_₄₁-induced platelet activation. The platelet surface binding of the P- selectin-specific monoclonal antibody S12 was determined by whole blood flow cytometry.

Fig. 5 is a bar graph showing that TR_1-41-mediated platelet activation requires extracellular calcium and does not require either the GPIb-IX complex (absent in Bernard-Soulier syndrome) or the GPIIb-IIIa complex (absent in Glanzmann's thro basthenia) .

Fig. 6 is a graph showing that normal plasma inhibits TR₁_₄₁-induced platelet degranulation. Figs. 7A to 7C are a series of graphs showing the decrease over time in GPIb, GPIX, and GPV on platelets activated by TR_1-41 and thrombin.

Detailed Description The new peptides are potent agonists for platelets, and thus can be used to activate platelets in vitro and in vivo, as well as in methods for determining a patient's level of platelet reactivity, and thrombin inhibitor efficacy. The new peptides also can be used to prepare new antibodies for use in new diagnostic and therapeutic methods. The effects of the new peptides were demonstrated in various experiments, including whole blood flow cytometric studies and experiments using various inhibitors.

Whole blood flow cytometric studies established that the TR_1-41 peptide induces platelet activation. The ^TR _l-_4l peptide was shown to be more potent than the TR₄₂_₅₅ peptide and almost as potent as thrombin, as determined by the increase in: (i) platelet surface expression of P-selectin (reflecting α granule secretion) ; (ii) exposure of the fibrinogen binding site on the GPIIb-IIIa complex; and (iii) fibrinogen binding to the activated GPIIb-IIIa complex.

Experiments with inhibitors (PGI₂, staurosporine, wortmannin, EDRF, and genistein) , and with platelets obtained from Bernard-Soulier syndrome and Glanzmann's thrombasthenia patients, demonstrated that TR_1-41 peptide- induced platelet activation is: (i) inhibited by cyclic AMP; (ii) mediated by protein kinase C, phosphatidyl inositol-3 (PI₃) kinase and myosin light chain kinase; (iii) dependent on extracellular calcium and intracellular protein tyrosine kinase; and (iv) independent of the GPIb-IX complex and the GPIIb-IIa complex. Other tests established that TR_1-41 peptides and the TR₄₂_₅₅ peptide activate platelets synergistically.

Peptides

The new peptides were synthesized using a Rainin Symphony (Rainin, Woburn, MA) 12-port automated peptide synthesizer set to perform FMOC-chemistry with HBTU (N,N,N' ,N' ,-tetramethyl-0-[lH-Benzotriazol-l-yl] uronium hexafluorophosphate) -mediated coupling. The peptides were then obtained by automated cleavage from a standard resin using trifluoroacetate and appropriate scavengers. The peptides were purified using high performance liquid chromatography with a 25 x 100 mm DeltaPak™ C18 column (Waters Millipore, Milford, MA) and a linear gradient in CH₃CN with ultraviolet spectrophotometric detection at 280 nm. Other standard synthetic methods can be used to make the TR_1-41 peptides. The following peptides were synthesized: ^TR ₁-₄₁ " MGPRRLLLVAACFSLCGP LSART- RARRPESKATNATLDPR (SEQ ID N0:1), ^TR ₁-₁₀ " MGPRRLLLVA (SEQ ID NO: 2), ^TR ₁₁-₂₁ " ACFSLCGPLL (SEQ ID NO: 3), ^TR ₂₁-₃₀ " SARTRARRPE (SEQ ID NO: 4), ^TR ₃₁-₄₁ ~ SKATNAT DPR (SEQ ID NO: 5), ^TR ₁-₂₁ " MGPRRLL VAACFSLCGPL (SEQ ID NO: 6), ^TR ₂₁-₄₁ ~ SARTRARRPESKATNATLDPR (SEQ ID NO: 7), ^TR ₄₂-₅₅ " SFLLRNPNDKYEPF (SEQ ID NO: 8),

^TR ₁-₄₁ (S14A) - MGPRRL LVAACFALCGPLLSART- RARRPESKATNATLDPR (SEQ ID NO: 9) (where S14A indicates that the Ser residue normally found in the wild-type sequence at location 14 was replaced with an Ala residue, shown by underlining in the sequence) , and l^TNASLLVPFLTARAKSSGTRF^ADPPRLMCLRPLARRCG (SEQ ID NO: 10) (a scrambled peptide including all amino acids of ^TRι-₄i peptide, but in a random order) .

Other peptides have been made, including ^TR ₂₉-₄₅ " PESKATNATLDPRSFLL (SEQ ID NO: 11),

^TR ₄₄-₅₅ " LRNPNDKYEPF (SEQ ID NO: 12), and ^TR ₄₂-₅₅ (S42F, F43S) - FSLLRNPNDKYEPF (SEQ ID NO: 13) .

TR₄₄_₅₅ and TR₄₂_₅₅ (S42F, F43S) were used as controls and did not activate platelets.

Other useful peptides include TR₁₇_₂₂ TR₁₅_₂₄, TR₁₂_₂₆, TR₁₀__3Q and TR₅_₃₅, which can be synthesized using the standard techniques described above.

Antagonist peptides can be synthesized by selectively altering one or more amino acids compared to the naturally occurring first 41 amino acids of the thrombin receptor (SEQ ID NO:l). Candidate TR.^ peptide antagonists can then be tested for antagonistic activity using a modification of either the whole blood flow cytometry platelet activation assay or the platelet aggregation platelet activation assay described herein. In antagonist assays, platelets are exposed to the full length TR_1-41 peptide in the presence and absence of the test antagonist. Inhibition of TR_1-41 peptide-mediated platelet activation by the candidate antagonist would be an indication that it is indeed a TR_1-41 peptide antagonist.

The TR_1-41 peptides also can be prepared using standard techniques by isolating the thrombin receptor cleavage fragment naturally cleaved from the receptor by thrombin. In addition, the TR_{1- 1} peptides and antagonist peptides can be produced by standard in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination, using the nucleotide sequences encoding the appropriate portions of the first 41 amino acids of the thrombin receptor (see, e.g., Fig. 5 in Vu et al., Cell , 64:1057-1068, 1991), or mutant analogs thereof. Methods well known to those skilled in the art can be used to construct expression vectors containing differentially expressed or pathway gene protein coding sequences and appropriate transcriptional/translational control signals. See, for example, the techniques described in Maniatis et al., Molecular Cloning A Laboratory Manual (Cold Spring Harbor Laboratory, N.Y., 1989), and Ausubel et al., Current Protocols in Molecular Biology. (Green Publishing Associates and Wiley Interscience, N.Y., 1989).

A variety of host-expression vector systems can be used to express the TR_1-41 peptides and antagonist peptides. Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the differentially expressed or pathway gene protein of the invention in situ . These include, but are not limited to, microorganisms such as bacteria, e.g., E. coli or, B . subtilis , transformed with recombinant bacteriophage DNA, plasmid or cosmid DNA expression vectors containing TR_1-41 peptide coding sequences; yeast, e.g., Saccharomyces or Pichia , transformed with recombinant yeast expression vectors containing the appropriate coding sequences; insect cell systems infected with recombinant virus expression vectors, e.g., baculovirus; plant cell systems infected with recombinant virus expression vectors, e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV) , or transformed with recombinant plasmid expression vectors, e.g., Ti plasmids, containing the appropriate coding sequences; or mammalian cell systems, e.g., COS, CHO, BHK, 293 or 3T3 , harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells, e.g., metallothionine promoter, or from mammalian viruses, e.g., the adenovirus late promoter or the vaccinia virus 7.5K promoter. Peptides of the invention also include those described above, but modified for in vivo use by the addition, at either or both the amino- and carboxy- terminal ends, of a blocking agent to facilitate survival of the relevant peptide in vivo. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular or mitochondrial uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxy terminal residues of the peptide to be administered. This can be done either chemically during the synthesis of the peptide or by recombinant DNA technology by methods familiar to artisans of average skill. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxy terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxy terminus can be replaced with a different moiety. Likewise, the peptides can be covalently or noncovalently coupled to pharmaceutically acceptable "carrier" proteins prior to administration.

Also of interest are peptidomimetic compounds that are designed based upon the amino acid sequences of the new TR_1-41 peptides. Peptidomimetic compounds are synthetic compounds having a three-dimensional conformation (i.e., a "peptide motif") that is substantially the same as the three-dimensional conformation of a selected peptide. The peptide motif provides the peptidomimetic compound with the ability to activate platelets or to inhibit activation of platelets that is the same as or greater than the activity of the peptide from which the peptidomimetic was derived. Peptidomimetic compounds can have additional characteristics that enhance their therapeutic application such as increased cell permeability, greater affinity and/or avidity, and prolonged biological half- life.

The peptidomimetics typically have a backbone that is partially or completely non-peptide, but with side groups that are identical to the side groups of the amino acid residues that occur in the peptide on which the peptidomimetic is based. Several types of chemical bonds, e.g. ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, are known in the art to be generally useful substitutes for peptide bonds in the construction of protease- resistant peptidomimetics.

Antibodies Antibodies that specifically bind to one or more of the new TR_1-41 peptides can be produced by a variety of methods. Such antibodies can include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs) , humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂ fragments, fragments produced by a FAb expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Such antibodies can be used, for example, to detect the naturally occurring thrombin receptor cleaved fragment in a patient's circulating blood, which provides a direct measure of the level of thrombin receptor activation. Such antibodies also can be used in treatment methods for inhibiting excess activation of thrombin receptors and platelets by the thrombin receptor cleaved fragment, and to inhibit binding of the thrombin receptor cleaved fragment to its target cells.

To produce antibodies to the new TR_1-41 peptides, a host animal is immunized with any one of the peptides. Such host animals can include, but are not limited to, rabbits, mice, and rats. Various adjuvants can be used to increase the immunological response, depending on the host species, including Freund's (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active substances such as oil emulsions, keyhole limpet hemocyanin (KLH) , dinitrophenol (DNP) , and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Monoclonal antibodies can be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the standard hybridoma technique of Kohler and Milstein, (Nature , 256:495-497, 1975; and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., Immunology Today, 4:72, 1983; Cole et al., Proc . Natl . Acad. Sci . USA, 80:2026-2030, 1983), and the BV-hybridoma technique (Cole et al., Monoclonal Antibodies And Cancer Therapy (Alan R. Liss, Inc. 1985), pp. 77-96. Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention can be cultivated in vitro or in vivo . Production of high titers of mAbs in vivo makes this the presently preferred method of antibody production.

In addition, "chimeric antibodies" can be made by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity (see. Morrison et al., Proc. Natl . Acad . Sci . , 81:6851-6855, 1984; Neuberger et al., Nature , 312:604- 608, 1984; Takeda et al., Nature , 314:452-454, 1985; and U.S. Patent No. 4,816,567). A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a constant region derived from human immunoglobulin.

Alternatively, standard techniques described for the production of single chain antibodies (e.g., U.S. Patent 4,946,778; Bird, Science , 242:423-426, 1988; Huston et al., Proc . Natl . Acad . Sci . USA, 85:5879-5883, 1988; and Ward et al., Nature, 334:544-546, 1989), and for making humanized monoclonal antibodies (U.S. Patent No. 5,225,539), can be used for therapeutic antibodies. Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')₂ fragments that can be produced by pepsin digestion of the antibody, and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab expression libraries can be constructed (Huse et al., Science , 246:1275-1281, 1989) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

Specifically, monoclonal antibodies to TR₁_₄₁ can be prepared as follows. Mice were immunized with up to 40 μg of TR_1-41 conjugated to hemocyanin. The antiserum titers were tested by standard ELISA. Briefly, TR_1-41 is coated onto microtiter plates at 50 ng/well. After blocking the plates, diluted antisera is added and monitored by biotinylated anti-mouse antibody to detect mouse IgG bound to the plates. Streptavidin-alkaline phosphatase is then added and monitored for color development. Mouse spleen cells are fused with the myeloma cells once antiserum titers reach at least 1/10,000. Clones reacting with different domains of the TR_1-41 peptide are selected by observing the binding of the antibody with synthetic peptides corresponding to the ^TRι-₂₀ ^anα- ^TR ₂i-₄i* ^τhe binding characteristics of the selected clones of antibodies with TR_1-41 are then determined using standard techniques. Other monoclonal antibodies used in the following experiments were obtained as follows. Monoclonal antibody S12 is directed against P-selectin, and was obtained from Centocor (Malvern, PA) . P-selectin, also referred to as CD62P, GMP-140, and PADGEM protein, is a component of the α granule membrane of resting platelets that is expressed only on the platelet surface after platelet degranulation and secretion. PAC1 (Cell Center, University of Pennsylvania, Philadelphia, PA) is directed against the fibrinogen binding site exposed by a conformational change in the GPIIb-IIIa complex of activated platelets. F26 (Gralnick et al., J. Lab . Clin . Med. , 118:604-613, 1991) is directed against a conformational change in fibrinogen bound to the GPIIb- IIIa complex. Y2/51 (DAKO, Carpenteria, CA) is directed against GPIIIa and was purchased conjugated to fluorescein isothiocyanate (FITC) .

Unlike Y2/51, antibodies PAC1 and F26 do not bind to resting platelets. Y2/51 does not interfere with PAC1 and F26 binding and therefore can be used in the same assays. PAC1 and F26 were biotinylated using standard techniques (see, e.g., Michelson et al., Blood, 77:770- 779, 1991). S12, F26 and Y2/51 are IgG, whereas PAC1 is IgM.

TR₁_₄₁ Peptides Activate Platelets

^TR ₁-₄₁ peptides can be tested for platelet activation using a whole blood flow cytometry assay or a platelet aggregation assay. Other standard methods of measuring platelet activation include actin polymerization studies and calcium mobilization studies, e.g., as described in Watson et al. (ed.), Platelets: A Practical Approach (Oxford University Press, Inc., New York, 1996) .

Whole Blood Flow Cytometry Assay The new TR_1-41 peptides were analyzed in a whole blood flow cytometry assay previously described in detail in Michelson et al., supra . There were no centrifugation, gel filtration, vortexing, or stirring steps that could artifactually activate platelets. In brief, peripheral blood was drawn from healthy volunteers who had not ingested aspirin or other anti-platelet drugs during the previous ten days. As indicated, some samples were drawn from a boy with Bernard-Soulier syndrome, and some from a boy with Glanzmann's thrombasthenia. The first 2 ml of drawn blood were discarded. Blood was then drawn into a sodium citrate filled VACUTAINER™ (Becton Dickinson, Rutherford, NJ) . Within 15 minutes of drawing, the blood was diluted 1:20 in modified HEPES- Tyrode's buffer (137 mM NaCl, 2.8 mM KC1, 1 mM MgCl₂, 12 mM NaHC0₃, 0.4 mM Na₂HP0₄, 0.35% bovine serum albumin, 10 mM HEPES, 5.5 mM glucose), pH 7.4.

In some experiments the samples were diluted with autologous plasma along with modified HEPES-Tyrode's buffer, pH 7.4. In these experiments, plasma content varied from 0 to 45% by volume. The peptide glycine-L- prolyl-L-arginyl-L-proline (GPRP, Calbiochem, San Diego, CA) at a concentration of 2.5 mM was added to the samples to prevent fibrin polymerization. The samples were incubated for varying times at 22°C with various concentrations of either (a) the peptides TR_{1- 1}, TR₁_₁₀,

^TRll-20 ' ^TR21-30 ' ^TR31-41 ' ^TRl-20 ' ^TR21-41 ' ^{or TR}42-55 (also known as thrombin receptor activating peptide (TRAP) , Calbiochem) , (b) purified human α-thrombin, or (c) control buffer.

In some experiments, hirudin 10 U/ml (Calbiochem) , a specific thrombin inhibitor, was added concomitantly with the addition of the synthetic peptides or thrombin. In other experiments, the chelating agents EDTA (2 mM) or EGTA (2 mM) were added 10 minutes prior to addition of the synthetic peptides or thrombin.

At various time points up to 300 seconds after the addition of the agonists, all samples were fixed at 22 °C for 20 minutes with formaldehyde 1% (final concentration) . After fixation, samples were diluted 10- fold in modified Tyrode's buffer, pH 7.4. The samples were then incubated at 22 °C for 20 minutes with a near saturating concentration of FITC-conjugated monoclonal antibody Y2/51, and a saturating concentration of biotinylated monoclonal antibody S12, PAC-1, or F26, followed by an incubation at 22° for 20 minutes with 30 μg/ml of phycoerythrin-streptavidin (Jackson ImmunoResarch, West Grove, PA) .

Samples were analyzed in an EPICS Profile flow cytometer (Coulter Cytometry, Hialeah, FL) . The flow cytometer was equipped with a 500 mW argon laser (Cyonics, San Jose, CA) operated at 15 mW with an emission wavelength of 488 nm. The fluorescence of FITC and phycoerythrin were detected using 525 nm and 575 nm band pass filters, respectively.

After identification of platelets by gating on both Y2/51-FITC positivity (i.e., GPIIIa-positivity) and their characteristic light scatter, binding of the biotinylated monoclonal antibody (S12, PACl, or F26) was determined by analyzing 5,000 individual platelets for phycoerythrin fluorescence. Background binding, obtained from parallel samples run with FITC-Y2/51 and purified biotinylated mouse IgG (IgM for PACl assays) (Boehringer Mannheim, Indianapolis, IN) , was subtracted from each test sample.

Other antibodies that can be used to measure platelet activation include CD63 (Nieuwenhuis, Blood, 70:838, 1987)-, ligand-induced binding site- (LIBS) (Frelinger, J. Biol . Chem . , 263:12397, 1988, and Frelinger, J. Biol . Chem . , 265:6346, 1990) and receptor- induced binding site (RIBS) (Zamarron, Thromb . Haemost . , 64:41, 1990, Abrams, Blood, 75:128, 1990, and Gralnick, Br. J. Haematol . , 80:347, 1992)-, Factor Va (Sims, J. Biol . Chem . , 263:18205, 1988)-, Factor VIII (Gilbert, J^". Biol . Chem . , 266:17261, 1991)-, annexin V-, multimerin (Hayward, J. Biol . Chem . , 266:7114, 1991, and Hayward, J. Clin . Invest . , 91:2630, 1993)-, and thrombospondin (Boukerche, Eur. J. Biochem . , 171:383, 1988, and Aiken, Semin . Thromb . Hemost . , 13:307, 1987) -specific antibodies, as well as the 7E3 GPIIb-IIIa complex (Coller et al., Clin . Invest . , 72:325-338, 1983)-, 6D1 GPIb (Coller et al., Blood, 61:99-105, 1983)-, GRP GPIX (Michaelson et al., Blood, 87:1396-1408, 1996)-, and the SW16 GPV (Moderman et al., J. Biol . Chem . , 267:364, 1992) -specific antibodies. Platelet Aggregation Assay

A platelet aggregation assay also can be used to determine platelet activation. Washed platelets were analyzed in a platelet aggregometer after the addition (arrowhead) of thrombin 1 U/ml (Fig. 2A) , TR₄₂_₅₅ 20 μM (Fig. 2B) , or TR_1-41 μM (Fig. 2C) . Platelet aggregometry was performed in a Payton Series 1000B aggregometer (Payton, Buffalo, NY) . Aggregation was recorded as an increase in light transmission. Washed platelets were prepared as previously described in Michelson et al., Blood, 76:2005-2010, 1990. Blood was drawn by venipuncture into a VACUTAINER™ , as described above. The citrated blood was centrifuged (150 x g, 15 minutes, 22 °C) and the supernatant (platelet-rich plasma, "PRP") was separated. After addition to the PRP of citrate albumin wash buffer (128 mM NaCl, 4.3 mM Na₂H₂P0₄.H₂0, 7.5 mM Na₂HP0₄, 4.8 mM sodium citrate, 2.4 mM citric acid, 11 mM glucose, 0.35% bovine serum albumin), pH 6.5 with PGE_i 50 ng/ml, washed platelets were prepared by centrifugation.

The concentration of washed platelets was adjusted to 600,000/μL in modified HEPES-Tyrode's buffer, pH 7.4. The washed platelets were diluted in an equal volume of HEPES-Tyrode's buffer, pH 7.4, or autologous plasma. TR._]_ ₄₁ 20 μM, TR₄₂_₅₅ 20 μM, or thrombin 1 U/ml were added to a final volume of 500 μL of washed platelets.

TR₂_₄₁ Peptide Fragments and Mutant Analogs

To assess whether the entire 41 amino acid sequence of the thrombin receptor is required for platelet activation, various TR₁_₄₁ peptides were synthesized as described above that correspond to the first, second, third, and fourth sets of ten sequential amino acids, and the first and second sets of twenty sequential amino acids of the first 41 amino acids of the thrombin receptor. None of these peptides (TR^^, TR-^.

20' ^TR2i-30' ^TR3i-4i' ^TRι-20' ^TR2i-4θ) resulted in platelet activation as detected by whole blood flow cytometry (data not shown) . In addition, a scrambled peptide (SEQ ID NO: 10) was shown in the whole blood flow cytometry and platelet aggregation assays not to activate platelets (data not shown) . Other peptides (TR₄₄_₅₅ and TR₄₂_₅₅ (S42F, F43S) ) were also inactive. However, a mutant analog, TR_1-41 (S14A) (SEQ ID NO:9), did activate platelets (data not shown) . This finding shows that analogs of TR_1-41 containing a mutation, albeit a relatively conservative mutation (S14A) , retain agonist activity.

Modulation of the Intracellular Signaling Pathways in TR_X_ ₄₁ Peptide-Induced Platelet Activation

To explore the signal transduction mechanisms involved in TR_1-41 peptide-induced platelet activation, platelet activation by TR_1-41 peptide (and by thrombin for comparison) was assayed in the presence of agents that modify the intracellular environment, and potentially modulate the TR_1-41 peptide-induced platelet activation. In variations of the whole blood flow cytometry assay, either 10 μM prostaglandin (PGI₂) (Sigma, St. Louis, MO) or 10 μM S-nitroso-N-acetylcysteine (SNAC) (an endothelium-derived relaxing factor (EDRF) congener) , known inhibitors of platelet activation, were added concomitantly with the addition of the synthetic peptides or thrombin. The EDRF congener (SNAC) was prepared at 22°C by reacting equimolar concentrations of fresh N- acetylcysteine with NaN0₂ at acidic pH. SNAC was prepared within 10 minutes of use, kept at 4°C, and diluted as necessary into aqueous buffer immediately before addition to assay systems. In other experiments, the following agents were incubated at 22°C for 30 minutes prior to the addition of the synthetic peptides or thrombin: (a) staurosporine 10 μM (Sigma) , an inhibitor of protein kinase C; (b) wortmannin (Sigma) at 100 nM is an inhibitor of phosphatidyl inositol-3 (PI₃) -kinase and at 1 μM is an inhibitor of myosin light chain kinase (MLCK) and PI₃- kinase; or (c) genistein 100 μM (Sigma) , an inhibitor of protein tyrosine kinases. In addition to using normal blood samples, some samples were drawn from a boy with Bernard-Soulier syndrome, or a boy with Glanzmann's thrombasthenia.

Synergy of TR-^^ Peptides and TR^ -₅₅ Peptide

In other experiments, various concentrations of TRτ._4! peptide and TR₄₂_₅₅ peptide were added together for 10 minutes at 22°C to determine whether these two different peptides might interact in a greater than additive, i.e., synergistic, manner. Diluted whole blood was incubated (22°C, 10 minutes) with the TR_1-41 peptide at concentrations of 0, 5, 10, and 20 μM, and then incubated (22 °C, 10 minutes) with TR₄₂_₅₅ at concentrations of 0, 5, 10, and 20 μM. The platelet surface binding of the P-selectin-specific monoclonal antibody S12 was determined by flow cytometry. Binding was expressed as a percent of the binding with maximal thrombin (2 U/ml) . The results indicate that at least at the lower concentrations there is a synergistic effect (Fig. 3).

Effect of Plasma on TR-^^-Induced Platelet Activation The addition of increasing volumes of platelet- poor plasma to diluted whole blood resulted in increasing inhibition of the platelet activating effects of TR_1-41, as determined by the platelet surface binding of S12 (Fig. 6) and PACl (data not shown) . After dilution of whole blood with the indicated concentration of autologous platelet-poor plasma, samples were incubated (22°C, 10 minutes) with TR_χ_₄₁ 25 μM. The platelet surface binding of the P-selectin-specific monoclonal antibody S12 was determined by flow cytometry.

This plasma inhibitor of TR_1-41 may be a physiological mechanism for the prevention of undesirable TR₁_ ₁-induced platelet activation at points distal to local thrombus formation.

Uses of the TR.,,^ Peptides. Antagonist Peptides. and Antibodies

Coronary artery disease is increasingly being understood as a thrombotic disorder. Markers of platelet activation are present in patients with acute coronary syndromes and myocardial infarction, and platelets from patients with these conditions are more aggregable than platelets from normal subjects. However, it is unlikely that activated platelets circulate in the blood for a significant amount of time because a) activated platelets adhere to endothelial surfaces, b) aggregate to one another, c) adhere to monocytes and neutrophils, and/or d) are cleared via the reticulo-endothelial system. Thus, it is difficult to detect circulating activated platelets in patients at risk for the development of cardiovascular thrombotic disorders.

Not only is there evidence of platelet activation in patients with acute coronary artery syndromes, there is direct evidence of increased thrombin production in these syndromes. Thrombin is the most physiologically active and potent platelet activator. By cleaving its receptor between amino acids 41 and 42, thrombin activates platelets via two synergistic pathways: 1) via the tethered ligand which is mimicked using the thrombin receptor agonist peptides (TRAPs) such as the TR₄₂_₅₅ peptide; and 2) via the newly discovered TR₁_₄₁ peptide pathway. The enhanced thrombin generation in coronary artery disease results in increased platelet reactivity by generating subthreshold amounts of circulating TR_1-41 peptide that interact with the circulating platelets and lower their threshold for activation.

These "primed" platelets should be poised to become fully activated under conditions in which normal platelets would not be activated. Thus patients with

"primed" platelets may be at increased risk of thrombotic events such as unstable angina, myocardial infarction, and stroke. Identification of these patients allows individual tailoring of anti-platelet therapy (use of more potent agents for example) , or more careful clinical follow-up. Furthermore, identification of patients without "primed" platelets would identify a group at low risk for acute complications from interventional procedures such as percutaneous transluminal coronary angioplasty (PTCA) and intracoronary stent insertion.

Primed platelets can be detected using either flow cytometry or platelet aggregation as described above, and can be detected in whole blood, platelet rich plasma, or with washed platelets. Platelet activation is measured either as the binding of specific activation dependent monoclonal antibodies to the platelet surface (flow cytometry) , or by platelet aggregation (impedance method or optical method) . Platelet samples from the subject are activated at increasing concentrations of agonists, including thrombin, TRAP, TR₁_₄₁, epinephrine, ADP, the thromboxane A2 analog U46619, and others, and are compared to established normal controls.

For example, samples can be incubated for varying times at 22°C with various concentrations of a) TR_1-41 peptide (2.5, 5.0, 10, 15, 20, and 25 μM) ; b) TRAP (2.5, 5.0, 10, 15, 20, and 25 μM) ; c) epinephrine (1, 2, 5, and 10 μM) ; d) ADP (1, 2, 5, and 10 μM) ; e) U46619 (thromboxane A2 analog) (0.5, 1.0, 1.5, 2.0, and 5.0 μM) ; or f) thrombin (0.1 0.5, 1.0, and 2.0 U/ml). At 300 seconds after the addition of the agonists, all samples are fixed at 22 °C for 20 minutes with formaldehyde 1% (final concentration) . After fixation, samples are diluted 10-fold in modified Tyrode's buffer, pH 7.4. The samples are then incubated at 22°C for 20 minutes with a near saturating concentration of FITC-conjugated monoclonal antibody Y2/51 and a saturating concentration of biotinylated monoclonal antibody S12, PAC-l or F26, followed by an incubation at 22 °C for 20 minutes with 30 μg/ml of phycoerythrin-streptavidin (Jackson ImmunoResearch, West Grove, PA) . As described above, the samples are analyzed in an EPICS Profile flow cytometer (Coulter Cytometry, Hialeah, FL.).

Activation is expressed as either the percentage of fluorescent intensity compared to a maximally stimulated platelet, or as a percentage of platelets labelled with an activation-dependent antibody. A normal platelet control population is studied to obtain baseline normal values. Activated platelets are those whose percent maximal fluorescent antibody binding or percent positive cells is greater than two standard deviations from the values of normal, non-stimulated, non-activated platelets.

Thrombin inhibitors such as heparin, low molecular weight heparin, and hirudin are frequently used in the treatment of thrombotic disorders including unstable angina and myocardial infarction. They are also routinely used during angioplasty and cardiac catheterization. While these agents are effective anticoagulants, they have significant bleeding risks limiting their use. Development of specific anti- thrombin agents, such as those that inhibit either TRAP or TR_{1- 1} platelet activation pathways directly or competitively, may produce enough of an anti-platelet effect without significantly increasing the bleeding risk. For example, the TR_1-41 peptide mutant analog, TR_χ_ ₄₁ (S14A) , an inactive form of the TR_1-41 peptide, will competitively inhibit TR_1-41 peptide-induced platelet activation and thus be an effective anti-thrombin, or more specifically, an anti-TR₁_₄₁ peptide agent. Furthermore, development of any anti-thrombin compounds, either directed against the enzyme or its receptor, must be tested for their efficacy in blocking individually both pathways of thrombin-induced platelet activation, that is the pathway mediated via the tethered ligand (TRAP) and the pathway mediated by the TR_1-41 peptide. Full inhibition of thrombin interacting with its receptor will prevent both pathways from being activated. However, a significant anticoagulant effect will result, potentially limiting its usage. Pharmacologic compounds which inhibit either the tethered ligand pathway (TRAP) or TR_{1- 1} pathway should provide an anti-thrombin induced platelet effect without a significant anticoagulant defect. Any such compound must be tested for its ability to inhibit either TRAP, TR_{1- 1}, or thrombin-induced platelet activation.

For example, any specific TRAP inhibitor must a) inhibit TRAP-induced platelet activation, and b) allow ^TRi-_4l peptide-induced platelet activation to continue. Thus, TR_1-41 peptide can be used in the platelet activation assays described herein to test the TRAP specificity of the new compound. Similarly, a TR_j.^ inhibitor must inhibit TR_1-41-induced platelet activation. This too can be tested in the presence of TR₁_₄₁ peptide using the assays described herein. Thus, TR_1-41 peptides can be used to induce thrombosis in vivo as a hemostatic device. Artificially induced thrombus formation is beneficial in situations such as in surgery where wounds are continuously bleeding, and arterial punctures during interventional cardiovascular or radiological procedures. TR_1-41 peptides may prove more beneficial than either thrombin or TRAP compounds because the platelet activating effect is not as intense. TR_{1- 1} peptides can be given topically in either a gel, lotion, or solution to instigate platelet activation. Other modes of administration are described below.

In another aspect, the local concentration of endogenous TR_{1- 1} peptide at sites of thrombus formation, such as vessel injury, is likely to be sufficient to cause TR₁_₄₁-induced platelet activation. Thus, TR_1-41 peptides can be administered therapeutically to augment platelet activation in a growing thrombus. Such a role for the TR₁_₄₁ peptide is consistent with the slower kinetics of TR₁_₄₁-induced platelet activation compared to thrombin- or TR₂_₅₅-induced platelet activation. The inhibition of TR_1-41 peptide-induced platelet activation by a plasma component suggests a physiological mechanism for the prevention of undesirable TR₁_₄₁-induced platelet activation at points distal to a local thrombus.

Furthermore, certain medical situations such as cardiopulmonary bypass are associated with a defect in platelet function. TR₁_ ₁ peptides can be administered either intravenously or mixed into the bypass machine to stimulate platelets to a moderate degree, thus overriding the platelet defect.

On the other hand, the TR_1-41 peptide antagonists are useful therapeutically in clinical conditions in which thrombin-induced cellular activation is part of the pathophysiological process, for example, angina, coronary artery disease, unstable angina, myocardial infarction, congestive heart failure, cardiomyopathy, ventricular and atrial arrhythmias, and stroke. In addition, they can be administered to patients receiving thrombolytic therapy, and patients receiving angioplasty. Moreover, the antagonists can be beneficial for patients needing prophylaxis for venous thrombosis such as those having deep venous thrombosis, recent surgery (any type) , continuous bed rest, venous stasis, or pulmonary embolism. In addition, the antagonists can be used to reduce the restenosis rate in patients undergoing angioplasty.

The TR_{1- 1} peptides and peptide antagonists, as well as TR_1-41 peptide antibodies, are administered in conventional formulations for systemic administration as is known in the art. Typical formulations may be found, for example, in Remington's Pharmaceutical Sciences _r Mack Publishing Co., Easton PA, latest edition.

Preferred forms of systemic administration of peptides include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can also be used. More recently, alternative means for systemic administration of peptides have been devised which include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if properly formulated in enteric or encapsulated formulations, oral administration is also possible. The dosage range required depends on the choice of peptide, peptide antagonist or antibody, the route of administration, the nature of the formulation, the nature of the patient's illness, and the judgment of the attending physician. Suitable dosage ranges, however, are in the range of 0.1-100.0 μg/kg of the patient. Wide variations in the needed dosage, however, are to be expected in view of the variety of peptides and peptide antagonists available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. In addition to the above described methods of peptide administration, TR_1-41 peptides and peptide antagonists can be delivered to a patient by administration of a nucleic acid encoding the peptides, an expression vector encoding the peptides, or cells, e.g., a patient's own cells, transduced with such a vector.

Such expression vectors contain a nucleic acid in which a polynucleotide sequence encoding a TR_1-41 peptide or peptide antagonist is operatively linked to a promoter or enhancer-promoter combination. A promoter is a trancriptional regulatory element composed of a region of a DNA molecule, typically within 100 nucleotide pairs upstream of the point at which transcription starts.

Another transcriptional regulatory element is an enhancer, which provides expression specificity in terms of time, location, and level. Unlike a promoter, an enhancer can function when located at variable distances from the transcription site, provided a promoter is present. An enhancer can also be located downstream of the transcription initiation site. The coding sequence of the expression vector is operatively linked to a transcription terminating region. To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the peptide or polypeptide between one and about fifty nucleotides downstream (3') of the promoter. Suitable vectors include plas ids, and viral vectors such as herpes viruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses, canary pox viruses, adenoviruses and adeno-associated viruses, among others.

The application of agonist or antagonist encoding genes to the modulation of platelet activity in humans can utilize either in vivo or ex vivo based approaches. The ex vivo method includes the steps of harvesting cells (e.g., fibroblasts or hematopoietic stem cells) from a subject, culturing the cells, transducing them with an expression vector, and maintaining the cells under conditions suitable for expression of the therapeutic peptide. These methods are known in the art of molecular biology. The transduction step is accomplished by any standard means used for ex vivo gene therapy, including calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer. Cells that have been successfully transduced are then selected, for example, for expression of a drug resistance gene. The cells may then be lethally irradiated (if desired) and injected or implanted into the patient.

The in vivo approach requires delivery of a genetic construct encoding a therapeutic TR_1-41 peptide or antagonist into a patient, preferably targeted to the cells or tissue of interest (e.g., coronary arteries in the case of coronary artery disease) . This can be achieved by administering it directly to the relevant tissue (e.g., by injection into cardiac tissue during cardiac surgery) .

Tissue specific targeting can also be achieved by the use of a molecular conjugate composed of a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine binds to a ligand that can bind to a receptor on target cells (Cristiano et al. J. Mol . Med. 73:479, 1995). Similarly, cell-specific antibodies can be bound to vectors and thereby target them to the relevant cells. A promoter inducing tissue or cell-specific expression can be used to achieve a further level of targeting. Appropriate tissue-specific promoters include, for example, the cardiac myosin light chain promoter (Lee et al., J. Biol . Chem . 267:15875, 1992) or the α-cardiac myosin heavy chain promoter (Gulick et al., J. Biol . Chem . 266:9180, 1991). These promoters would allow production of the antagonists of the invention, for example, in cardiac tissue and could thus be useful in therapy of coronary heart disease or myocardial infarction, for example. Vectors can also be incorporated into liposomes or an appropriate biodegradable polymeric microparticle (also referred to as a "microsphere, " "nanosphere, " "nanoparticle, " or "microcapsule") and delivered in this form. The vectors can be incorporated alone into these delivery vehicles or co-incorporated with tissue-specific antibodies.

DNA or transfected cells can be administered in a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to a human, e.g., physiological saline. A therapeutically effective amount is an amount of the DNA of the invention which is capable of producing a medically desirable result in a treated animal. As is well known in the medical arts, the dosage for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages will vary, but a preferred dosage for administration of DNA is from approximately 10⁶ to 10¹² copies of the DNA molecule. This dose can be repeatedly administered, as needed. Routes of administration will be regional, as exemplified above, systemic (e.g., intravenous, subcutaneous, intramuscular or intraperitoneal) or transmucosal (e.g., intrarectal, intravaginal or intranasal) .

Current methods (including platelet aggrego etry and flow cytometry) of measuring platelet function use panels of platelet agonists, most of which are weaker agonists than TR_1-41 peptides. TR_1-41 peptide is one of the strongest platelet agonists and therefore, presumably one of the most physiologically important. Thus, TR_1-41 should be added to the panels of platelet agonists used in all platelet function tests. More generally, TR_1-41 peptides can be used to test ex vivo or in vitro platelet function and reactivity in clinical settings where altered platelet function may indicate a high risk of future thrombotic or clinical events. These situations include assaying platelet function in patients with angina, coronary artery disease, unstable angina, myocardial infarction, congestive heart failure, cardiomyopathy, ventricular and atrial arrhythmias, sudden death, stroke, patients receiving thrombolytic therapy, and patients receiving angioplasty. These tests are based on the whole blood flow cytometry and aggregation assays described herein. In addition, TR_1-41 peptides can be used to test ex vivo or in vitro platelet function and reactivity in research settings to better elucidate the signaling pathways of the thrombin receptor. Using standard cell biology, protein biochemistry, and molecular biology techniques, and activating platelets with either TRAP (representing the tethered ligand) or TR_1-41 peptides, the individual signal transduction mechanisms can be studied. ^TR ₁-₄₁ peptide antibodies, either polyclonal or monoclonal, can be used to inhibit the function of the naturally occurring thrombin receptor cleaved fragment on its target cells. TR₁_₄₁ peptide is thought to be involved in physiological and pathological processes. Like thrombin itself, TR_1-41 can regulate cell proliferation, growth factors, cytokines, wound healing, inflammation, etc. The monoclonal anti-TR₁_₄₁ antibodies should be capable of blocking the interactions of TR_{1- 1} with its target cells. The inhibition of TR₁_₄₁-mediated processes in vivo thus may exhibit therapeutic effects.

EXAMPLES 1. TR₁_₄₁ Peptides Activate Platelets

Exposure of diluted whole blood to TR_1-41 peptide resulted in a concentration-dependent increase in the surface expression of various activation-dependent antigens, as determined by the whole blood flow cytometry assay described above (Figs. 1A to 1C, binding is expressed as a percent of the binding with maximal thrombin (2 U/ml), and data are mean + S.E.M., n = 6). These results indicate that the platelets in the whole blood were activated.

^TRi-_4l peptide resulted in increased platelet surface expression of P-selectin (reflecting α-granule secretion; S12 monoclonal antibody binding) (Fig. 1A) . TR₁_ ₁ peptide also resulted in the increased expression of the activated conformation of the GPIIb-IIIa complex, as reported by PACl binding (Fig. IB) . The TR_1-41 peptide-induced increase in the platelet surface binding of monoclonal antibody F26 demonstrated that TR_1-41 resulted in fibrinogen binding to this activated GPIIb- IIIa complex (Fig. 1C) .

The maximal TR_1-41 peptide-induced binding of S12, PACl, and F26 (o at 25 μM) was significantly greater than with TR₄₂_₅₅ (D at 25 μM) and almost as great was with thrombin (■ at 0.5 to 1.0 U/ml) (Figs. 1A to 1C) . To assess whether thrombin is required for TR_1-41 peptide- induced platelet activation, platelets were activated with TR_1-41 peptide in the presence of saturating amounts of hirudin (10 U/ml) , a specific thrombin inhibitor. Hirudin did not inhibit the ability of TR_1-41 or TR₄₂_₅₅ to activate platelets but, as expected, did inhibit thrombin-induced platelet activation (data not shown) . The increase in platelet surface expression of P- selectin detected by flow cytometry was more rapid in response to thrombin and TR₂_₅₅ than to TR_1-41 (data not shown) . After one minute of agonist-induced platelet activation, thrombin 2 U/ml resulted in greater than 80% of maximal degranulation, TR₄₂_₅₅ 25 μM resulted in greater than 60% of maximal platelet degranulation, whereas TR_1-41 peptide (25 μM) resulted in only minimal platelet degranulation. Thrombin and TR₄₂_₅₅ resulted in maximal platelet degranulation within 1.5 minutes, whereas TR_1-41 peptide resulted in maximal platelet degranulation after 5 minutes.

2. TR-^-_t Peptides Cause Platelet Aggregation

Figs. 2A to 2C are a series of graphs showing the results of this TR₁_ ₁-, TR₄₂_₅₅-, and thrombin-induced platelet aggregation. Washed platelets were analyzed in a platelet aggregometer after the addition (arrowhead) of thrombin 1 U/ml (Fig. 2A) , TR₄₂_₅₅ 20 μM (Fig. 2B) , or ^TRi-_4l ²⁰ ^ (^Fi-9* ^2C) • ^As shown in Figs. 2A to 2C, the maximum amplitude of the aggregation curve is similar among the different agonists. However, the lag phase and slope of the TR₁_₄₁ aggregation curve were less than that of thrombin and TR₄₂_₅₅, demonstrating the slower kinetics of TR₁_₄₁-induced platelet aggregation. Similar results are also depicted in Tables 1 and 2 below, in which thrombin at a concentration of 2 U/ml was used as a 100% level of aggregation. Table 1 shows the aggregation results for 6 samples of washed platelets, whereas Table 2 shows the aggregation results for 6 samples of platelet rich plasma. The data in Table 1 shows that TR_1-41 peptide induces slower platelet aggregation and onset of activation than thrombin or TR₄_ ₅₅ peptide. The scrambled TR_1-41(s), TR₂₉_₄₅, TR_j.^, TR₄₄_ ₅₅, and TR₄₂_₅₅ are inactive.

In Table 2, the data show the plasma-inhibiting effect on the activity of the TR-ι_₄₁ peptide, which is described further in Example 6. Here, plasma inhibits the ability of TR_1-41 peptide to aggregate platelets.

3. TR₂_.₄₁ and R₄₂-₅₅ Peptides Interact Svnergistically

As shown in the graph of Fig. 3, TR_1-41 peptide augmented TR₄₂_₅₅-induced platelet activation, and TR₄₂_₅₅ augmented TR_1-41-induced platelet activation in a concentration-dependent manner, thereby demonstrating that these two peptides interact svnergistically, i.e., greater than additively. The graphs show the interaction of -_j.₄ peptide concentrations of 0 μM (O) , 5 μM (•) , 10 μM (v) , and 20 μM (T) , with TR_1-41 concentrations of 0, 5, 10, 15, and 20 μM. Data in Fig. 3 are mean ± S.E.M. (n = 3).

4. Intracellular Signalling Pathways Involved in TR₂.₄₁ Peptide-induced Platelet Activation

Platelet activation by TR_1-41 peptide (and by TR₄₂_ ₅₅, a combination of TR_1-41 and TR₄₂_₅₅ or thrombin for comparison) was assayed in the presence of agents that modify the intracellular environment. As shown in Fig. 4, these data demonstrate that TR₁_₄₁ peptide-induced platelet activation is modulated, at least in part, via cAMP, protein kinase C, phosphatidyl inositol-3-kinase, intracellular protein tyrosine kinases and myosin light chain kinase.

Figs. 4 and 5 are bar graphs showing intracellular signal transduction pathways involved in TR_1-41 peptide- induced platelet activation. After either a) preincubation (22 °C, 30 minutes) with staurosporine 10 μM, wortmannin 1 μM or 100 nM, or genistein 100 μM, or b) co-incubation with buffer only (control) , prostaglandin I₂ (PGI₂) 10 μM, or the EDRF congener SNAC 10 μM, diluted whole blood samples were incubated (22 °C, 10 minutes) with either TR_1-41 peptide 15 μM (dotted bars) , TR₄₂_₅₅ peptide 15 μM (diagonally hatched bars) , a mixture of TR_X_ ₄₁ 15 μM peptide and TR₄₂_₅₅ peptide 15 μM (horizontally hatched bars) or thrombin 0.1 U/ml (solid bars), and then fixed (Fig. 4) .

In studies of platelets of patients with Bernard- Soulier syndrome and Glanzmann's thrombasthenia, respectively, the blood samples were not preincubated with any inhibitors, but were incubated with TR_1-41 20 μM or thrombin 5 U/ml (Fig. 5) . The platelet surface binding of the P-selectin-specific monoclonal antibody S12 was determined by whole blood flow cytometry. Binding is expressed as a percent of the binding with maximal thrombin (2 U/ml) in normal volunteers. Data are mean ± S.E.M., n=6, except for data for Bernard-Soulier syndrome and Glanzmann's thrombasthenia which are means of two experiments.

Elevation of intraplatelet cyclic AMP (cAMP) by PGI₂ almost completely inhibited TR_1-41-, TR₄₂_₅₅~, concomitant

^TR ₁-₄₁ ^{and TR} ₄₂-₅₅ ^~' ^anc thrombin (0.1 U/ml) induced platelet degranulation (as detected by S12 binding) (Fig. 4) , and expression of activated GPIIb-IIIa (as detected by PACl and F26 binding, data not shown) . The EDRF congener SNAC partially inhibited thrombin- and TR₄₂_₅₅- induced platelet activation, but had only a minimal effect on TR_1-41- or concomitant TR_{1- 1} and TR₂_₅₅-induced platelet activation (Fig. 4) . The protein kinase C inhibitor staurosporine almost completely inhibited TR-_j_ ₄₁-, TR₄₂_₅₅-, concomitant

^TR _l-₄i ^{and TR} ₄₂-₅₅~' ^and thrombin-induced S12 binding (Fig. 4) and PAC-1 binding (data not shown) .

At nanomolar concentrations, wortmannin selectively inhibits PI₃ kinase but, at higher concentrations, it also inhibits myosin light chain kinase (Kovacsovics et al., J. Biol . Chem . 270:11358, 1995; Tho ason et al., J. Biol . Chem . 269:16525, 1994). At a concentration of 100 nM or 1 μM, wortmannin reduced platelet surface P-selectin expression in response to TR₂_ ₄₁ or TR₄₂_₅₅ more than platelet surface P-selectin expression in response to concomitant TR_1-41 and TR₄₂_₅₅ or thrombin (Fig. 4) .

Inhibition of intracellular tyrosine kinase activity by the addition of genistein resulted in a decrease in

^TRι-₄i-' ^TR ₄₂-₅₅~' concomitant TR_1-41 and TR₂_₅₅-, and thrombin-induced platelet activation (Fig. 4) .

The absence of the GPIb-IX complex (Bernard- Soulier syndrome) or the GPIIb-IIIa complex (Glanzmann's thrombasthenia) had no effect on TR_{1- 1}-induced platelet activation (Fig. 5) . In normal platelets, inhibition of the binding of fibrinogen to GPIIb-IIIa by the addition of RGD-containing peptides also did not interfere with TR₁_₄₁-induced platelet activation (data not shown) .

5. TR_j,^ Requires Extra-Cellular Calcium to Activate Platelets

In the presence of either the divalent cation chelator EDTA (2.8 mM) or the calcium chelator EGTA (2.8 mM) , the TR_1-41 peptide (25 μM) did not cause any platelet degranulation, as indicated by the absence of platelet surface P-selectin (Fig. 5) . Neither EDTA nor EGTA inhibited thrombin (2 U/ml) or TR₄₂_₅₅ (25 μM) induced platelet degranulation (Fig. 5) . Because the platelet surface expression of P-selectin is not calcium dependent, these experiments demonstrate that the TR_1-41 peptide requires extra-cellular calcium to activate platelets.

6. Plasma Inhibits TR-^^-Induced Platelet Activation

Addition of increasing volumes of platelet-poor plasma to diluted whole blood resulted in increasing inhibition of the platelet activating effects of the TR-ι_ ₁ peptide, as determined by the platelet surface binding of S12 (Fig. 6) and PACl (data not shown) . The added volumes of plasma also inhibited TR₄₂_₅₅- and thrombin- induced platelet degranulation and expression of the activated GPIIb-IIIa receptor, but not to the same extent as for TR_1-41 (data not shown) . Plasma inhibitors of thrombin include anti-thrombin III. Binding in Fig. 6 is expressed as a percent of the binding with maximal thrombin (2 U/ml). Data are mean ± S.E.M., n = 6.

7. TR_X_₄₁ and Thrombin Induced Decreases in Platelet Surface Expression of GPIb. GPIX and GPV

Exposure of platelets to either R^^ 20 μM (closed circles) or thrombin 1 U/ml (open circles) resulted in a decrease in platelet GPIb (Fig. 7A) , GPIX (Fig. 7B) , and GPV (Fig. 7C) expression, as indicated by surface binding of antibody 6D1, antibody GRP and antibody SW16, respectively. The samples were incubated with either TR_1-41 or thrombin for the indicated periods of time (up to 4 minutes) , fixed, stained with the FITC conjugated antibodies and analyzed by flow cytometry. Binding is expressed as a percent of maximal binding without thrombin or TR_1-41. Thus a decrease in surface expression of the GPIb-IX-V complex is an additional marker of platelet activation.

Other Embodiments

^TR ₁-₄₁ peptides are also agonists for cells other than platelets. For example, TR_χ_₄₁ peptides are chemotactic for monocytes and neutrophils. Other data indicate that GPIb and GPIIb-GPIIIa are not the binding site for the TR_1-41 peptides.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, that the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

What is claimed is:

1. An isolated TR_1-41 peptide consisting of a sequence of at least 6 and up to 41 amino acids, wherein said sequence is substantially identical to at least 6 consecutive amino acids within the sequence:

MGPRRLLLVAACFSLCGPLLSARTRARRPESKATNATLDPR (SEQ ID N0:1),

and including at least a core region of amino acids substantially identical to amino acids 17 to 22 of SEQ ID NO:l.

2. A peptide of claim 1, wherein said peptide consists of a sequence of at least 10 amino acids, said sequence being substantially identical to at least 10 consecutive amino acids within the sequence with SEQ ID NO:l and including at least a core region of amino acids substantially identical to amino acids 15 to 24 of SEQ ID NO:l.

3. A peptide of claim 1, wherein said peptide consists of a sequence of at least 15 amino acids, said sequence being substantially identical to at least 15 consecutive amino acids within the sequence with SEQ ID NO:l and including at least a core region of amino acids substantially identical to amino acids 12 to 26 of SEQ ID NO:l.

4. A peptide of claim 1, wherein said peptide consists of a sequence of at least 21 amino acids, said sequence being substantially identical to at least 21 consecutive amino acids within the sequence with SEQ ID NO:l and including at least a core region of amino acids substantially identical to amino acids 10 to 30 of SEQ ID NO:l.

5. A peptide of claim 1, wherein said peptide has a length of 41 amino acids, and has a sequence substantially identical to the sequence of SEQ ID NO:l.

6. A peptide of claim 1, wherein said peptide has the sequence of SEQ ID N0:1.

7. A peptide of claim 1, wherein said peptide has a length of 31 amino acids, and has a sequence substantially identical to the sequence of amino acids 5 to 35 of SEQ ID N0:l.

8. A peptide of claim 1, wherein said peptide has a length of 31 amino acids, and the sequence of amino acids 5 to 35 of SEQ ID NO:l.

9. A peptide of claim 1, wherein said peptide has a length of 21 amino acids, and has a sequence substantially identical to the sequence of amino acids 10 to 30 of SEQ ID N0:1.

10. A peptide of claim 1, wherein said peptide has a length of 21 amino acids, and the sequence of amino acids 10 to 30 of SEQ ID NO:l.

11. A method of activating platelets, the method comprising providing a TR_1-41 peptide of claim 1, and exposing the platelets to an amount of the peptide effective to achieve platelet activation.

12. A method of claim 11, wherein the method is carried out in vitro.

13. A method of claim 11, wherein the method is carried out ex vivo .

14. A method of claim 11, wherein the method is carried out in vivo and induces thrombosis.

15. A method of claim 11, further comprising the step of exposing the platelets to peptide TR₂_₅₅ in an amount sufficient to achieve a synergistic platelet activation effect together with the TR_1-41 peptide.

16. A method of assaying platelet reactivity, the method comprising obtaining a biological sample containing platelets; incubating the sample with a TR_{1- 1} peptide of claim 1; and detecting platelet activation as a measure of platelet reactivity.

17. A method of claim 16, wherein platelet activation is detected by labeling the sample with an activation-dependent label, and detecting any labeled platelets, wherein the presence of labeled platelets indicates the reactivity of the platelets in the sample.

18. A method of claim 17, wherein the activation- dependent label is fluorescein isothiocyanate, fibrinogen, or an antibody selected from the group consisting of PACl, F26, S12, 7E3, 6D1, SW16, ligand induced binding site-specific antibody, and receptor induced binding site-specific antibody.

19. A method of claim 16, wherein platelet activation is detected by labeling the sample with an activation-dependent label, detecting any labeled platelets, and comparing the percentage of labeling with control values of known activated platelets as an indication of the degree of reactivity of the platelets in the sample.

20. A method of claim 16, wherein platelet activation is detected by measuring platelet aggregation.

21. A method of claim 16, wherein platelet activation is detected by measuring a change in calcium mobilization in the platelets.

22. A method of claim 16, wherein the TR_1-41 peptide is added in an amount effective to activate normal platelets.

23. A method of claim 16, wherein said TR_1-41 peptide is added to separate samples in a range of different concentrations.

24. A method of claim 16, wherein the platelets are considered activated when the percentage of labeling compared to a non-activated platelet control value is greater than two standard deviations from the control value.

25. A method of claim 16, wherein the sample is from a patient having angina, coronary artery disease, unstable angina, myocardial infarction, congestive heart failure, cardiomyopathy, ventricular and atrial arrhythmias, or a stroke.

26. A method of claim 16, further comprising incubating the sample with a TR₄₂_₅₅ peptide.

27. An isolated antagonist of a TR_1-41 peptide, the antagonist comprising a compound that inhibits binding of a TR_1-41 peptide to platelets.

28. An antagonist of claim 27, wherein the antagonist is an antibody that binds specifically to a ^TRι-₄i peptide.

29. An antagonist of claim 27 for use in inhibiting TR_1-41 peptide-induced platelet activation in a patient, wherein the platelets are exposed to an amount of an antagonist effective to prevent TR_1-41 peptide- induced platelet activation.

30. A method of inhibiting TRι_₄ι peptide-induced platelet activation in a patient, the method comprising exposing the platelets of the patient to an amount of an antagonist of claim 27 effective to prevent TR_1-41 peptide-induced platelet activation in the patient.

31. A method of claim 30, wherein the patient has undergone surgery or angioplasty, or has had deep venous thrombosis, venous stasis, a pulmonary embolism, angina, coronary artery disease, unstable angina, myocardial infarction, congestive heart failure, cardiomyopathy, ventricular or atrial arrhythmias, or a stroke.

32. A method of claim 30, wherein the platelets are exposed to the antagonist by administration of the antagonist to the patient.

33. A method of claim 30, wherein the platelets are exposed to the antagonist by administration to the patient of a nucleic acid encoding the antagonist.

34. A nucleic acid comprising a nucleotide sequence that encodes the TR-^-L peptide for use in activating platelets in a patient, wherein the platelets are exposed to an amount of the peptide effective to achieve platelet activation.