WO1998016548A1 - Peptides recepteurs de thrombine et utilisations correspondantes - Google Patents

Peptides recepteurs de thrombine et utilisations correspondantes Download PDF

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Publication number
WO1998016548A1
WO1998016548A1 PCT/US1997/018742 US9718742W WO9816548A1 WO 1998016548 A1 WO1998016548 A1 WO 1998016548A1 US 9718742 W US9718742 W US 9718742W WO 9816548 A1 WO9816548 A1 WO 9816548A1
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peptide
platelets
amino acids
sequence
platelet
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PCT/US1997/018742
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English (en)
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Mark I. Furman
Longbin Liu
Stephen E. Benoit
Alan D. Michelson
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University Of Massachusetts
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Priority to AU49860/97A priority Critical patent/AU4986097A/en
Publication of WO1998016548A1 publication Critical patent/WO1998016548A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • thrombin receptor cleavage fragment 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).
  • thrombin receptor agonist peptides include R 4 _ 7 (thrombin receptor peptide having amino acids 42 to 47 of the full receptor), TR 42 _ 48 , and R 42 _ 55 . These synthetic peptides mimic the new amino terminus in sequence, and in many instances, in effect as well.
  • 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).
  • 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) .
  • TR 1-41 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.
  • 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:
  • TR l - 41 peptides include TR 1-41 , TR 17 _ 22 , TR 15 _ 24 , TR 12 _ 26 ,
  • TR 10-30' and TR 5 _ 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.
  • 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 TR 42 _ 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 TR 1 _ 41 peptide.
  • a patient e.g., a mammal such as a human
  • a nucleic acid e.g., an expression vector
  • 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.
  • a biological sample containing platelets e.g., blood, platelet-rich- plasma, or washed platelets
  • a TR 1-41 peptide e.g., in an amount effective to activate normal platelets
  • 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.
  • an activation- dependent label e.g., fluorescein isothiocyanate- fibrinogen
  • an antibody e.g., PAC1, F26, S12 and others
  • 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.
  • 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.
  • 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 TR 2 _ 55 peptide.
  • 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 TR ⁇ - 4 i 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) .
  • TR 1 _ 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) .
  • TR 1 _ 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 TR 1 _ 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.
  • a peptide exhibits at least 80% (e.g., 90 or 95%) identity to a reference amino acid sequence.
  • 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.
  • TR 1 _ 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 natural source e.g., from human blood, plasma, serum, or urine
  • 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.
  • TR 1 _ 1 peptide antibody is any polyclonal or monoclonal antibody that specifically binds to a TR 1 _ 41 peptide.
  • An antibody that "specifically binds" to a TR ⁇ _ 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.
  • the preparation is at least 80%, more preferably at least 90%, and most preferably at least 99%, by dry weight, antibody, e.g., a TR ⁇ - 4 i peptide antibody.
  • 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.
  • 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.
  • 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.
  • Figs. 1A to 1C are a series of line graphs showing
  • TR. ⁇ -, TR 42 _ 55 -, and thrombin-induced platelet activation were determined by whole blood flow cytometry.
  • Figs. 2A to 2C are a series of graphs showing R ⁇ - 4 i ⁇ ' 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) , TR 42 _ 55 20 ⁇ M (Fig. 2B) , or TR 1-41 ⁇ M (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 TR 1 _ 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 TR 1 _ 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.
  • 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.
  • TR 1-41 peptide induces platelet activation.
  • the TR l - 4l peptide was shown to be more potent than the TR 42 _ 55 peptide and almost as potent as thrombin, as determined by the increase in: (i) platelet surface expression of P-selectin (reflecting ⁇ granule secretion) ; (ii) exposure of the fibrinogen binding site on the GPIIb-IIIa complex; and (iii) fibrinogen binding to the activated GPIIb-IIIa complex.
  • TR 1-41 peptide-induced platelet activation is: (i) inhibited by cyclic AMP; (ii) mediated by protein kinase C, phosphatidyl inositol-3 (PI 3 ) 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 TR 42 _ 55 peptide activate platelets synergistically.
  • 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 DeltaPakTM C18 column (Waters Millipore, Milford, MA) and a linear gradient in CH 3 CN with ultraviolet spectrophotometric detection at 280 nm. Other standard synthetic methods can be used to make the TR 1-41 peptides.
  • 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)
  • 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
  • l ⁇ TNASLLVPFLTARAKSSGTRF ⁇ ADPPRLMCLRPLARRCG (SEQ ID NO: 10) (a scrambled peptide including all amino acids of TR ⁇ - 4 i peptide, but in a random order) .
  • 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) .
  • TR 44 _ 55 and TR 42 _ 55 were used as controls and did not activate platelets.
  • TR 17 _ 22 TR 15 _ 24 , TR 12 _ 26 , TR 10 _ 3Q and TR 5 _ 35 are useful peptides.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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., CHO
  • 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.
  • a blocking agent to facilitate survival of the relevant peptide in vivo.
  • 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.
  • 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.
  • the peptides can be covalently or noncovalently coupled to pharmaceutically acceptable "carrier" proteins prior to administration.
  • 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 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.
  • mAbs monoclonal antibodies
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • monoclonal antibodies to TR 1 _ 41 can be prepared as follows. Mice were immunized with up to 40 ⁇ g 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 TR ⁇ - 20 an ⁇ - TR 2 i- 4 i* ⁇ he 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
  • CD62P Cell Center, University of Pennsylvania, Philadelphia, PA
  • PAC1 Cell Center, University of Pennsylvania, Philadelphia, PA
  • F26 is directed against a conformational change in fibrinogen bound to the GPIIb- IIIa complex
  • Y2/51 DAKO, Carpenteria, CA
  • FITC fluorescein isothiocyanate
  • 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.
  • 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) .
  • VACUTAINERTM Becton Dickinson, Rutherford, NJ
  • the blood was diluted 1:20 in modified HEPES- Tyrode's buffer (137 mM NaCl, 2.8 mM KC1, 1 mM MgCl 2 , 12 mM NaHC0 3 , 0.4 mM Na 2 HP0 4 , 0.35% bovine serum albumin, 10 mM HEPES, 5.5 mM glucose), pH 7.4.
  • 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 , TR 1 _ 10 ,
  • TR ll-20 ' TR 21-30 ' TR 31-41 ' TR l-20 ' TR 21-41 ' or TR 42-55 also known as thrombin receptor activating peptide (TRAP) , Calbiochem
  • TR 42-55 also known as thrombin receptor activating peptide (TRAP) , Calbiochem
  • TRAP thrombin receptor activating peptide
  • Calbiochem Calbiochem
  • purified human ⁇ -thrombin or
  • hirudin 10 U/ml (Calbiochem) , a specific thrombin inhibitor, was added concomitantly with the addition of the synthetic peptides or thrombin.
  • the chelating agents EDTA (2 mM) or EGTA (2 mM) were added 10 minutes prior to addition of the synthetic peptides or thrombin.
  • binding of the biotinylated monoclonal antibody 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.
  • 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) , TR 42 _ 55 20 ⁇ M (Fig. 2B) , or TR 1-41 ⁇ M (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 VACUTAINERTM , as described above.
  • the citrated blood was centrifuged (150 x g, 15 minutes, 22 °C) and the supernatant (platelet-rich plasma, "PRP" was separated.
  • PRP platelet-rich plasma
  • citrate albumin wash buffer (128 mM NaCl, 4.3 mM Na 2 H 2 P0 4 .H 2 0, 7.5 mM Na 2 HP0 4 , 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.
  • washed platelets The concentration of washed platelets was adjusted to 600,000/ ⁇ L 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 ⁇ M, TR 42 _ 55 20 ⁇ M, or thrombin 1 U/ml were added to a final volume of 500 ⁇ L of washed platelets.
  • TR 1 _ 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- ⁇ .
  • TR 1-41 peptide-induced platelet activation was assayed in the presence of agents that modify the intracellular environment, and potentially modulate the TR 1-41 peptide-induced platelet activation.
  • PKI 2 prostaglandin
  • SNAC S-nitroso-N-acetylcysteine
  • EDRF endothelium-derived relaxing factor
  • This plasma inhibitor of TR 1-41 may be a physiological mechanism for the prevention of undesirable TR 1 _ 1 -induced platelet activation at points distal to local thrombus formation.
  • 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.
  • thrombin is the most physiologically active and potent platelet activator.
  • 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 TR 42 _ 55 peptide; and 2) via the newly discovered TR 1 _ 41 peptide pathway.
  • TREPs thrombin receptor agonist peptides
  • 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.
  • “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.
  • PTCA percutaneous transluminal coronary angioplasty
  • 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, TR 1 _ 41 , epinephrine, ADP, the thromboxane A2 analog U46619, and others, and are compared to established normal controls.
  • agonists including thrombin, TRAP, TR 1 _ 41 , epinephrine, ADP, the thromboxane A2 analog U46619, and others, and are compared to established normal controls.
  • 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 ⁇ M) ; b) TRAP (2.5, 5.0, 10, 15, 20, and 25 ⁇ M) ; c) epinephrine (1, 2, 5, and 10 ⁇ M) ; d) ADP (1, 2, 5, and 10 ⁇ M) ; e) U46619 (thromboxane A2 analog) (0.5, 1.0, 1.5, 2.0, and 5.0 ⁇ M) ; or f) thrombin (0.1 0.5, 1.0, and 2.0 U/ml).
  • 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.
  • the TR 1-41 peptide mutant analog, TR ⁇ _ 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-TR 1 _ 41 peptide agent.
  • 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.
  • TRAP tethered ligand pathway
  • TR 1- 1 tethered ligand pathway
  • any specific TRAP inhibitor must a) inhibit TRAP-induced platelet activation, and b) allow TR i- 4l peptide-induced platelet activation to continue.
  • TR 1-41 peptide can be used in the platelet activation assays described herein to test the TRAP specificity of the new compound.
  • a TR j . ⁇ inhibitor must inhibit TR 1-41 -induced platelet activation. This too can be tested in the presence of TR 1 _ 41 peptide using the assays described herein.
  • TR 1-41 peptides can be used to induce thrombosis in vivo as a hemostatic device.
  • 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.
  • 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 TR 1 _ 41 -induced platelet activation.
  • TR 1-41 peptides can be administered therapeutically to augment platelet activation in a growing thrombus.
  • Such a role for the TR 1 _ 41 peptide is consistent with the slower kinetics of TR 1 _ 41 -induced platelet activation compared to thrombin- or TR 2 _ 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 TR 1 _ 41 -induced platelet activation at points distal to a local thrombus.
  • TR 1 _ 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.
  • 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.
  • they can be administered to patients receiving thrombolytic therapy, and patients receiving angioplasty.
  • 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.
  • the antagonists can be used to reduce the restenosis rate in patients undergoing angioplasty.
  • TR 1- 1 peptides and peptide antagonists 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.
  • systemic administration of peptides include injection, typically by intravenous injection.
  • Other injection routes such as subcutaneous, intramuscular, or intraperitoneal, can also be used.
  • 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.
  • penetrants such as bile salts or fusidic acids or other detergents.
  • 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 are in the range of 0.1-100.0 ⁇ g/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.
  • 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.
  • 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 of interest e.g., coronary arteries in the case of coronary artery disease
  • 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).
  • 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 .
  • 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.
  • 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 10 6 to 10 12 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) .
  • systemic e.g., intravenous, subcutaneous, intramuscular or intraperitoneal
  • transmucosal e.g., intrarectal, intravaginal or intranasal
  • 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.
  • 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.
  • TR 1 - 41 peptide antibodies can be used to inhibit the function of the naturally occurring thrombin receptor cleaved fragment on its target cells.
  • TR 1 _ 41 peptide is thought to be involved in physiological and pathological processes.
  • TR 1-41 can regulate cell proliferation, growth factors, cytokines, wound healing, inflammation, etc.
  • the monoclonal anti-TR 1 _ 41 antibodies should be capable of blocking the interactions of TR 1- 1 with its target cells. The inhibition of TR 1 _ 41 -mediated processes in vivo thus may exhibit therapeutic effects.
  • TR i- 4l peptide resulted in increased platelet surface expression of P-selectin (reflecting ⁇ -granule secretion; S12 monoclonal antibody binding) (Fig. 1A) .
  • TR 1 _ 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) .
  • TR 1-41 peptide-induced binding of S12, PACl, and F26 was significantly greater than with TR 42 _ 55 (D at 25 ⁇ M) and almost as great was with thrombin ( ⁇ at 0.5 to 1.0 U/ml) (Figs. 1A to 1C) .
  • thrombin ⁇ at 0.5 to 1.0 U/ml
  • 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 TR 42 _ 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 TR 2 _ 55 than to TR 1-41 (data not shown) .
  • thrombin 2 U/ml resulted in greater than 80% of maximal degranulation
  • TR 42 _ 55 25 ⁇ M resulted in greater than 60% of maximal platelet degranulation
  • TR 1-41 peptide 25 ⁇ M
  • TR 1-41 peptide 25 ⁇ M
  • Figs. 2A to 2C are a series of graphs showing the results of this TR 1 _ 1 -, 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) , TR 42 _ 55 20 ⁇ M (Fig. 2B) , or TR i- 4l 20 ⁇ ( F i-9* 2C ) • As shown in Figs. 2A to 2C, the maximum amplitude of the aggregation curve is similar among the different agonists.
  • Table 2 the data show the plasma-inhibiting effect on the activity of the TR- ⁇ _ 41 peptide, which is described further in Example 6.
  • plasma inhibits the ability of TR 1-41 peptide to aggregate platelets.
  • TR 2 _. 41 and R 42 - 55 Peptides Interact Svnergistically
  • TR 1-41 peptide augmented TR 42 _ 55 -induced platelet activation As shown in the graph of Fig. 3, TR 1-41 peptide augmented TR 42 _ 55 -induced platelet activation, and TR 42 _ 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 ⁇ M (O) , 5 ⁇ M (•) , 10 ⁇ M (v) , and 20 ⁇ M (T) , with TR 1-41 concentrations of 0, 5, 10, 15, and 20 ⁇ M.
  • TR 1 _ 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 ⁇ M, wortmannin 1 ⁇ M or 100 nM, or genistein 100 ⁇ M, or b) co-incubation with buffer only (control) , prostaglandin I 2 (PGI 2 ) 10 ⁇ M, or the EDRF congener SNAC 10 ⁇ M, diluted whole blood samples were incubated (22 °C, 10 minutes) with either TR 1-41 peptide 15 ⁇ M (dotted bars) , TR 42 _ 55 peptide 15 ⁇ M (diagonally hatched bars) , a mixture of TR X _ 41 15 ⁇ M peptide and TR 42 _ 55 peptide 15 ⁇ M (horizontally hatched bars) or thrombin 0.1 U/ml (solid bars), and then fixed (
  • the EDRF congener SNAC partially inhibited thrombin- and TR 42 _ 55 - induced platelet activation, but had only a minimal effect on TR 1-41 - or concomitant TR 1- 1 and TR 2 _ 55 -induced platelet activation (Fig. 4) .
  • the protein kinase C inhibitor staurosporine almost completely inhibited TR- j _ 41 -, TR 42 _ 55 -, concomitant
  • wortmannin selectively inhibits PI 3 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).
  • wortmannin reduced platelet surface P-selectin expression in response to TR 2 _ 41 or TR 42 _ 55 more than platelet surface P-selectin expression in response to concomitant TR 1-41 and TR 42 _ 55 or thrombin (Fig. 4) .
  • TR ⁇ - 4 i-' TR 42 - 55 ⁇ ' concomitant TR 1-41 and TR 2 _ 55 -, and thrombin-induced platelet activation (Fig. 4) .
  • the TR 1-41 peptide 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 ⁇ M) 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 TR 42 _ 55 (25 ⁇ M) 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.
  • TR 1 - 41 peptides are also agonists for cells other than platelets.
  • TR ⁇ _ 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.

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Abstract

Cette invention se rapporte à de nouveaux peptides isolés, ou à des peptides de synthèse, correspondant à tout ou partie des 41 premiers acides aminés du récepteur de la thrombine, qui s'avèrent être de puissants agonistes plaquettaires. Ces nouveaux peptides TR1-41 s'avèrent utiles s'agissant d'activer les plaquettes in vitro et in vivo, de préparer des antagonistes et des anticorps dirigés contre les peptides TR1-41, et de procéder à des analyses variées destinées à la détermination du taux de réactivité plaquettaire d'un patient et à celle de l'efficacité d'inhibiteurs de la thrombine.
PCT/US1997/018742 1996-10-11 1997-10-10 Peptides recepteurs de thrombine et utilisations correspondantes WO1998016548A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006021942A2 (fr) * 2004-08-26 2006-03-02 Royal College Of Surgeons In Ireland Peptides bioactifs
WO2009098689A1 (fr) * 2008-02-07 2009-08-13 Hadasit Medical Research Services & Development Limited Immunodétection d'un état cancéreux chez un sujet
US20100197580A1 (en) * 2007-03-29 2010-08-05 Johns Hopkins University Parstatin peptides and uses thereof
US8227412B2 (en) * 2007-03-29 2012-07-24 Tsopanoglou Nikos E Bioactive parstatin peptides and methods of use
US9180163B2 (en) 2007-03-29 2015-11-10 Nikos Tsopanoglou Parstatin peptides

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5256766A (en) * 1991-02-19 1993-10-26 The Regents Of The University Of California Recombinant thrombin receptor and related pharmaceuticals

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5256766A (en) * 1991-02-19 1993-10-26 The Regents Of The University Of California Recombinant thrombin receptor and related pharmaceuticals

Non-Patent Citations (1)

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Title
CELL, 22 March 1991, Vol. 64, VU T.-K.H. et al., "Molecular Cloning of a Functional Thrombin Receptor Reveals a Novel Proteolytic Mechanism of Receptor Activation", pages 1057-1068. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006021942A2 (fr) * 2004-08-26 2006-03-02 Royal College Of Surgeons In Ireland Peptides bioactifs
WO2006021942A3 (fr) * 2004-08-26 2007-03-15 Royal College Of Surgeons Ie Peptides bioactifs
US20100197580A1 (en) * 2007-03-29 2010-08-05 Johns Hopkins University Parstatin peptides and uses thereof
US8227412B2 (en) * 2007-03-29 2012-07-24 Tsopanoglou Nikos E Bioactive parstatin peptides and methods of use
US8389476B2 (en) * 2007-03-29 2013-03-05 The Johns Hopkins University School Of Medicine Parstatin peptides and uses thereof
US9180163B2 (en) 2007-03-29 2015-11-10 Nikos Tsopanoglou Parstatin peptides
WO2009098689A1 (fr) * 2008-02-07 2009-08-13 Hadasit Medical Research Services & Development Limited Immunodétection d'un état cancéreux chez un sujet

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