EP1254160A1 - Par4 peptides and polypeptides - Google Patents

Abstract

Members of the protease-activated receptor (PAR) family mediate cellular signaling in response to proteases. These receptors are characterized by a tethered peptide ligand at the extracellular amino terminus that is generated by minor proteolysis. Structure-activity analysis revealed that peptides and polypeptides can be designed to activate human PAR4.

Description

PAR4 Peptides and Polypeptides

TECHNICAL FIELD

The present invention relates generally to new peptides and polypeptides capable of stimulating protease-activated receptor 4.

BACKGROUND OF THE INVENTION

Researchers have identified a subfamily of G protein-coupled receptors that mediate cellular signaling in response to proteases (Nu et al, Cell 64:1051 (1991); Rasmussen et al, FEBS Lett. 288:123 (1991); Νystedt et al, Proc. Nat'l Acad. Sci. USA 91:9208 (1994); Ishihara et al, Nature 353:614 (1997)). Members of this unique G protein-coupled receptor family include protease-activated receptors PARl, PAR2, PAR3, and PAR4. These receptors are characterized by a tethered peptide ligand at the extracellular amino terminus that is generated by minor proteolysis.

The first identified member of this family was the thrombin receptor, presently designated protease-activated receptor 1 (PARl). Thrombin cleaves an amino-terminal extracellular extension of PARl to create a new amino terminus that functions as a tethered ligand and intramolecularly activates the receptor (Nu et al, Cell 64:1051 (1991)). PAR2 mediates signaling following minor proteolysis by trypsin or tryptase, but not thrombin (Νystedt et al, Proc. Nat'l Acad. Sci. USA 91:9208 (1994)). Knockout of the gene coding for PARl provided definitive evidence for a second thrombin receptor in mouse platelets and for tissue-specific roles for different thrombin receptors (Connolly et al, Nature 381:516 (1996)). PAR3 was identified as a second thrombin receptor, which mediates phophatidyl inositol 4,5 diphosphate hydrolysis, and the receptor was found to be expressed in a variety of tissues (Ishihara et al, Nature 353:614 (1997)). More recently, Xu et al, J. Biol. Chem. 95:6642 (1998), reported the isolation of protease-activated receptor-4 (PAR4) (also see, Kahn et al, Nature 394:690 (1998)). The protease cleavage site (Arg47/Gly48) was identified within the extracellular amino terminus.

Functional studies indicate that PARl is important for the activation of human platelets by thrombin (see, for example, Hung et al, J. Clin. Invest. 59:1350 (1992); Scarborough et al, J. Biol. Chem. 267:13146 (1992)). Human platelets also appear to use PAR4 for thrombin signaling, and studies suggest that PARl and PAR4 account for most thrombin signaling in human platelets (Kahn et al, Nature 394:690 (1998); Kahn et al, J. Clin. Invest. 103:819 (1999)).

The discovery of new PAR4-activating peptides and polypeptides fulfills a need in the art by providing new compositions useful in diagnosis and therapy. The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel peptides and polypeptides that can activate PAR4. The present invention also provides methods of producing these peptides and polypeptides.

DESCRIPTION OF THE INVENTION

1. Overview Upon activation by thrombin, PAR4 yields an anchored N-terminal sequence, which ends in the N-terminal hexapeptide sequence GYPGQV (SEQ ID NO:3). Preliminary studies indicated that GYPGQV is capable of activating PAR4, but only at extremely high concentrations. The corresponding murine PAR4, upon activation by thrombin, yields the anchored N-terminal sequence GYPGKF (SEQ ID NO: 13). This hexapeptide activates murine PAR4 at similarly high concentrations. Thus, a challenge was to design new peptides that could activate human PAR4 at relatively low μM concentrations.

As described herein, the present invention provides peptides and polypeptides, which mimic the N-terminus of the activated form of PAR4. Illustrative peptides include peptides that comprise an amino acid sequence selected from the group consisting of: (a) Gly-Tyr-Pro-Gly-Gln-Val-Cys-NH (SEQ ID NO:4), (b) Gly-Tyr-Pro- Gly-Gln-Val-Cys-Ala-NH₂ (SEQ ID NO:5), and (c) Gly-Xaa Pro-Gly-Lys-Xaa₂-Xaa₃- NH₂ (SEQ ID NO:56), wherein Xaa_t is selected from the group consisting of Tyr, Tyr(Me), Bip, and 2-Nal, Xaa₂ is selected from the group consisting of Phe, hPhe, Phe(4-F), Phe(4-Me), Thi, 1-Nal, 2-Nal, and Bip, and Xaa₃ is Cys or Pen. Examples of peptide (c) include: Gly-Tyr-Pro-Gly-Lys-hPhe-Cys-NH₂ (SEQ ID NO:31), Gly- Tyr(Me)-Pro-Gly-Lys-Phe-Cys-NH₂ (SEQ ID NO:37), Gly-Tvr-Pro-Gly-Lys-Phe(4-F)- Cys-NH₂ (SEQ ID NO:39), Gly-Tyr-Pro-Gly-Lys-Phe(4-Me)-Cys-NH₂ (SEQ ID NO:42), Gly-Bip-Pro-Gly-Lys-Phe-Cys-NH₂ (SEQ ID NO:44), Gly-2Nal-Pro-Gly-Lys- Phe-Cys-NH₂ (SEQ ID NO:50), Gly-Tvr-Pro-Gly-Lys-Thi-Cys-NH₂ (SEQ ID NO:53), Gly-Tyr-Pro-Gly-Lys-Phe-Pen-NH₂ (SEQ ID NO:54), Gly-2Nal-Pro-Gly-Lys-Phe-Pen- NH₂ (SEQ ID NO:55), and the like.

The present invention also presents compositions that comprise a PAR4 activating peptide, or polypeptide, and a carrier. The present invention further provides methods of stimulating platelet aggregation, comprising administering to platelets a composition comprising a carrier and a PAR4 activating peptide, or polypeptide. For example, the composition can be administered in vitro or to a mammalian subject.

The present invention also provides methods of inhibiting the proliferation of tumor cells, comprising administering to tumor cells a composition comprising a carrier and a PAR4 activating peptide, or polypeptide. Such a composition can be administered in vitro, or to a mammalian subject.

These and other aspects of the invention will become evident upon reference to the following detailed description. In addition, various references are identified below.

2. Definitions

In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the invention.

As used herein, "nucleic acid" or "nucleic acid molecule" refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally- occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., α-enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. The term "nucleic acid molecule" also includes so- called "peptide nucleic acids," which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.

The term "complement of a nucleic acid molecule" refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence. The term "structural gene" refers to a nucleic acid molecule that is transcribed into messenger RNA (mRNA), which is then translated into a sequence of amino acids characteristic of a specific polypeptide.

An "isolated nucleic acid molecule" is a nucleic acid molecule that is not integrated in the genomic DNA of an organism. For example, a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism. A nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species. A "nucleic acid molecule construct" is a nucleic acid molecule, either single- or double-stranded, that has been modified through human intervention to contain segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature.

"Linear DNA" denotes non-circular DNA molecules having free 5' and 3' ends. Linear DNA can be prepared from closed circular DNA molecules, such as plas ids, by enzymatic digestion or physical disruption.

"Complementary DNA (cDNA)" is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription. Those skilled in the art also use the term "cDNA" to refer to a double- stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand. The term "cDNA" also refers to a clone of a cDNA molecule synthesized from an RNA template.

A "promoter" is a nucleotide sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee et al, Mol. Endocrinol. 7:551 (1993)), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman, Seminars in Cancer Biol 1:41 (1990)), glucocorticoid response elements (GREs), and binding sites for other transcription factors, such as CRE/ATF (O'Reilly et al, J. Biol. Chem. 267:19938 (1992)), AP2 (Ye et al, J. Biol. Chem. 269:25128 (1994)), SP1, cAMP response element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamer factors (see, in general, Watson et al, eds., Molecular Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J. 303:1 (1994)). If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter. Repressible promoters are also known. A "core promoter" contains essential nucleotide sequences for promoter function, including the TATA box and start of transcription. By this definition, a core promoter may or may not have detectable activity in the absence of specific sequences that may enhance the activity or confer tissue specific activity.

A "regulatory element" is a nucleotide sequence that modulates the activity of a core promoter. For example, a regulatory element may contain a nucleotide sequence that binds with cellular factors enabling transcription exclusively or preferentially in particular cells, tissues, or organelles. These types of regulatory elements are normally associated with genes that are expressed in a "cell-specific," "tissue-specific," or "organelle-specific" manner. An "enhancer" is a type of regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.

"Heterologous DNA" refers to a DNA molecule, or a population of DNA molecules, that does not exist naturally within a given host cell. DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species (i.e., endogenous DNA) so long as that host DNA is combined with non-host DNA (I.e., exogenous DNA). For example, a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a transcription promoter is considered to be a heterologous DNA molecule. Conversely, a heterologous DNA molecule can comprise an endogenous gene operably linked with an exogenous promoter. As another illustration, a DNA molecule comprising a gene derived from a wild-type cell is considered to be heterologous DNA if that DNA molecule is introduced into a mutant cell that lacks the wild-type gene.

A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides."

A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a "heterologous" peptide or polypeptide. An "integrated genetic element" is a segment of DNA that has been incorporated into a chromosome of a host cell after that element is introduced into the cell through human manipulation. Within the present invention, integrated genetic elements are most commonly derived from linearized plasmids that are introduced into the cells by electroporation or other techniques. Integrated genetic elements are passed from the original host cell to its progeny.

A "cloning vector" is a nucleic acid molecule, such as a plasmid, cosmid, or bacteriophage, that has the capability of replicating autonomously in a host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites that allow insertion of a nucleic acid molecule in a deterrninable fashion without loss of an essential biological function of the vector, as well as nucleotide sequences encoding a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.

An "expression vector" is a nucleic acid molecule encoding a gene that is expressed in a host cell. Typically, an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be "operably linked to" the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity ofthe core promoter. A "recombinant host" is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or expression vector. In the present context, an example of a recombinant host is a cell that produces PAR4 from an expression vector. In contrast, PAR4 can be produced by a cell that is a "natural source" of PAR4, and that lacks an expression vector.

"Integrative transformants" are recombinant host cells, in which heterologous DNA has become integrated into the genomic DNA of the cells. A "fusion protein" is a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes. For example, a fusion protein can comprise at least part of a PAR4 polypeptide fused with a polypeptide that binds an affinity matrix. Such a fusion protein provides a means to isolate large quantities of PAR4 using affinity chromatography. The term "receptor" denotes a cell-associated protein that binds to a bioactive molecule termed a "ligand." This interaction mediates the effect of the ligand on the cell. Receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor). Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. In certain membrane-bound receptors, the extracellular ligand-binding domain and the intracellular effector domain are located in separate polypeptides that comprise the complete functional receptor.

In general, the binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell, which in turn leads to an alteration in the metabolism of the cell. Metabolic events that are often linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.

The term "secretory signal sequence" denotes a nucleotide sequence that encodes a peptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.

An "isolated polypeptide" is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature. Typically, a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. One way to show that a particular protein preparation contains an isolated polypeptide is by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining of the gel. However, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.

The terms "amino-terminal" and "carboxyl-terminal" are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.

The term "expression" refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.

The term "complement/anti-complement pair" denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair. Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. If subsequent dissociation of the complement/anti-complement pair is desirable, then the complement/anti-complement pair preferably is characterized by a binding affinity of less than 10⁹ M^"1. An "antibody fragment" is a portion of an antibody such as F(ab')₂, F(ab)₂,

Fab', Fab, and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody.

The term "antibody fragment" also includes a synthetic or a genetically engineered polypeptide that binds to a specific antigen, such as polypeptides consisting of the light chain variable region, "Fv" fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("scFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region. A "chimeric antibody" is a recombinant protein that contains the variable domains and complementary determining regions derived from a rodent antibody, while the remainder ofthe antibody molecule is derived from a human antibody. "Humanized antibodies" are recombinant proteins in which murine complementarity determining regions of a monoclonal antibody have been transferred from heavy and light variable chains ofthe murine immunoglobulin into a human variable domain. A "detectable label" is a molecule or atom which can be conjugated to an antibody moiety to produce a molecule useful for diagnosis. Examples of detectable labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, or other marker moieties.

The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly- histidine tract, protein A (Nilsson et al, EMBO J. 4:1015 (1985); Nilsson et al, Methods Enzymol 198:3 (1991)), glutathione S transferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al, Proc. Natl. Acad. Sci. USA 82:1952 (1985)), substance P, FLAG peptide (Hopp et al, Biotechnology 5:1204 (1988)), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al, Protein Expression and Purification 2:95 (1991). Nucleic acid molecules encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ).

A "naked antibody" is an entire antibody, as opposed to an antibody fragment, which is not conjugated with a therapeutic agent. Naked antibodies include both polyclonal and monoclonal antibodies, as well as certain recombina it antibodies, such as chimeric and humanized antibodies.

As used herein, the term "antibody component" includes both an entire antibody and an antibody fragment.

An "immunoconjugate" is a conjugate of an antibody component with a therapeutic agent or a detectable label. Due to the imprecision of standard analytical methods, molecular weights and lengths of polymers are understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to ±10%. 3. Synthetic PAR-4 Peptides and Polypeptides

An examination of the hexapeptide, Gly-Tvr-Pro-Gly-Gln-Val-NH₂, showed that it only had two amino acids with functional side chains: Tyr and Gin. The remaining amino acids (Gly, Pro, and Val) are structural amino acids. It was hypothesized that additional side chains might be required for optimal activation of PAR4. Following this approach, the first step was to lengthen the peptide by adding amino acids found in the tethered sequence on the C-terminal side of the hexapeptide. Thus, a series of peptides ranging from the original hexapeptide to a 14-mer were synthesized and purified and tested in a cell-based assay for PAR4 activation. The corresponding murine PAR4 hexapeptide and a mutated human hexapeptide (Tyr mutated to Ala) were also synthesized, purified and tested. Illustrative polypeptides are provided in Table 1. Table 2 shows exemplary results of a biological activity assay using cultured transgenic rat cells that express the human PAR4 receptor.

Table 1

Table 2

¹ Peptides and polypeptides were added to cell culture plates at a final concentration of 156 μM.

As shown in Table 2, the results of the biological activity studies indicated that lengthening the hexapeptide sequence at the C-terminus by just one amino acid results in a large increase in PAR4 activation. However, additional lengthening of the sequence (8-mer to 14-mer) did not result in an increase in PAR4 activation. Furthermore, the murine hexapeptide was virtually equi-active to the human peptide, while the mutated human peptide was inactive. The single amino acid, which upon addition to the C-terminus resulted in a dramatic increase in activity, was Cys, an amino acid that has a highly nucleophilic sulfhydryl side chain. The results of a platelet aggregation response assay verified the higher activity of peptides, Gly-Tyr-Pro-Gly- Gln-Val-Cys-NH₂ and Gly-Tvr-Pro-Gly-Gln-Val-Cys-Ala-NH₂, compared with the peptide, Gly-Tyr-Pro-Gly-Lys-Phe-NH₂. The murine hexapeptide was used as the starting point for subsequent analysis of structure activity relationships and activity optimization. In a preliminary optimization approach, each of the amino acids, except for the N-terminal Gly, were replaced by a structurally similar or analogous amino acid. Accordingly, a series of peptides were synthesized, purified and tested side-by-side in a similar assay format. As shown in Tables 1 and 3, Tyr was replaced by Phe bearing polar (COOH, SO₃H), or nonpolar lipophilic (CN, F, Me) functional groups. Tyr was also replaced by bicyclic amino acids (1-NA, 2-NA, biphenyl), a heterocyclic amino acid (thienyl), truncated Phe (Phg), and a homologated Phe (hPhe). Pro was replaced by a substituted Pro (Hyp), a heterocyclic analog (Thz), and a homo-Pro (Pip). Gly in the center of the sequence was replaced by Ala and β-Ala, which lengthens the distance between the adjacent amino acid by an additional carbon atom. Lys was replaced by Arg and Orn. Phe was replaced by replaced by Phe bearing polar (COOH, SO₃H), and nonpolar lipophilic (CN, F, CI, NO₂, Me) functional groups. Phe was also replaced by bicyclic amino acids (1-NA, 2-NA, biphenyl), heterocyclic amino acids (thienyl), and truncated Phe (Phg). Finally, the C-terminal Cys was replaced by Met, the sterically hindered Pen, the bioisosteric analogs Ser, Dap, and homo C. Additionally, the C-terminal Gly was replaced by an acetyl group and a cinnamyl group.

Table 3

'Abbreviations: β-Ala: β-alanine; Bip: biphenylalanine; Dap: diamino propionic acid; hCys: homocysteine; hPhe: homophenylalanine; Hyp: hydroxyproline; 1-Nal: 1 -napthylalanme; 2-Nal: 2- napthylalanine; Orn: ornithine; Pen: penicillanic acid; Phg: phenylglycine; Pip: pipecolic acid; Thz: thiazolidine carboxylic acid; Thi: thienylalanine; Tyr(Me): tyrosine methylether; Phe(4-"X"): fourth position on the phenyl of phenylalanine is substituted with the indicated moiety.

Table 3 (continued)

'Abbreviations: β-Ala: β-alanine; Bip: biphenylalanine; Dap: diamino propionic acid; hCys: homocysteine; hPhe: homophenylalanine; Hyp: hydroxyproline; 1-Nal: 1-napthylalanine; 2-Nal: 2- napthylalanine; Orn: ornithine; Pen: penicillanic acid; Phg: phenylglycine; Pip: pipecolic acid; Thz: thiazolidine carboxylic acid; Thi: thienylalanine; Tyr(Me): tyrosine methylether; Phe(4-"X"): fourth position on the phenyl of phenylalanine is substituted with the indicated moiety.

The murine 7-mer (PAR4-12) was found to be equi-active to the human 7-mer with an approximate four-fold induction at 15.6 μM. Modifications of the C- terminal Gly resulted in a complete inactivation of the peptides, indicating that the C- terminal Gly was essential for activity perhaps through the N-terminal amino group. Replacement of the Tyr with Phe bearing polar groups also resulted in complete inactivity, while replacement with Phe bearing lipohilic groups resulted in substantial, but not complete, retention of activity. These results indicate that the polar groups probably disrupted a hydrophobic contact with the receptor site. The constrained Phg with a truncated Phe-like side chain and the highly flexible hPhe with the homologated Phe-like side chain, were also completely inactive, further illustrating the importance of an optimally flexible Tyr for binding to and activation of the receptor.

When Tyr was replaced with a heterocyclic amino acid (Thi) similar activity retention was observed, while the bicyclic amino acid provided very interesting results. That is, Bip with the extended biphenyl side chain was equi-active to PAR4-12 as was the 1-Nal with the constrained napthyl ring. However, PAR4-50, which had the less constrained 2-Nal replacing Tyr, showed a dramatic increase in activity with a 16- fold induction at 15.6 μM and an approximate four-fold induction as low as 5 μM. This leads to the conclusion that a flexible bicyclic ring system probably improves the activity substantially, perhaps by providing additional hydrophobic area of contact with the receptor site.

The Pro at position three probably acts as a constrained spacer to orient the adjacent amino acids in an optimal way. This is shown by the fact that even slight modification of Pro, such as substitution with a hydroxyl group (Hyp) or a hetero atom (Thz) or a homologation of the ring size by one carbon (Pip), resulted in substantial losses of activity.

The Gly in the center probably also serves as a an optimally flexible spacer, because its replacement with an extended amino acid spacer, like β-Ala, leads to complete inactivity, while introduction of a branch, as in Ala, leads to substantial reduction in activity.

The Lys at position five presents an interesting scenario, because the only modifications, though both conservative in principle, result in retention of activity. For example, PAR4-30 and PAR4-31, having an Arg with a cationic guanidine side chain, and Orn with a truncated Lys-like side chain, respectively, are equi-active to PAR4-12, which has a Lys at that position. Conceivably, additional modifications at this position are needed to ascertain the importance of Lys at this position.

Replacement of the Phe at position six with Phe bearing polar groups resulted in almost complete inactivity, while replacement with Phe bearing lipohilic groups resulted in substantial, but not complete retention of activity. This indicates that the polar groups probably disrupted another hydrophobic contact with the receptor site. The constrained Phg with a truncated Phe-like side chain showed a reduction of the activity, while the highly flexible hPhe with the homologated Phe-like side chain, surprisingly fully retained the activity. This suggests the importance of an optimally flexible Phe for binding to and activation of the receptor. When Phe was replaced with a heterocyclic amino acid (Thi) or a bicyclic amino acid (1-Nal, 2-Nal and Bip), approximately similar retention of activity was observed, indicating that there was no spatial or steric constraint in the region of the receptor site to which Phe at this position binds. Finally, at the extreme C-terminus, replacement of Cys with the bioisosteric amino acid Ser and Dap resulted in substantial loss of activity, while replacement with Met or homologation to hCys resulted in somewhat less reduction of activity. This indicates that the nucleophilicity of the sulfhydryl side-chain of Cys is essential for activity, because the less nucleophilic Dap and Ser are substantially less active. Furthermore, the nucleophile appears to be in an optimal position to interact with the binding site, because any homologation as in Met or hCys results in reduction of activity. Introduction of a steric constraint around the nucleophilic sulfhydryl side- chain, as in Pen, results in a slightly improved activity. By constraining the flexibility of the side chain, the nucleophile is in a better position to bind to the receptor. This may also prove to be an advantage where stability of the peptide is concerned, because the constraint may also render the peptide less susceptible to air oxidation or heterodimerization

In sum, these studies indicate that the peptide, Gly-2Nal-Pro-Gly-Lys- Phe-Cys-NH₂ (SEQ ID NO:50), is not only several fold more active at lower concentrations in a cell-based assay, but also, at low micromolar concentrations stimulates activation of the platelets in a manner similar to that of thrombin. The results also indicate additional new peptides with modifications to improve activity. An example of such a peptide is Gly-2Nal-Pro-Gly-Lys-Phe~Pen-NH₂ (SEQ ID NO:55). This peptide is predicted to have several fold improved activity. Another example is provided by the formula: Gly-Xaa Pro-Gly-Lys-Xaa₂-Xaa₃-NH₂ (SEQ ID NO:56), wherein Xaaϊ is Tyr, Tyr(Me), Bip, or 2-Nal, Xaa₂ is Phe, hPhe, Phe(4-F), Phe(4-Me), Thi, 1-Nal, 2-Nal, or Bip, and Xaa₃ is Cys or Pen.

In view of the information provided herein, one of skill in the art can devise additional PAR4 peptides and polypeptides. As one example, L-amino acids were used to synthesize the amino acid sequences described above. However, peptides and polypeptides can be produced that comprise one or more D-amino acid residues. The biological activity of such peptides and polypeptides can be tested in a variety of assays. Example 1 illustrates the use of cultured cells that express human PAR4. A platelet aggregation assay, which uses human blood cells, is described in Example 2. Moreover, Kahn et al, J. Clin. Invest. 103:819 (1999), describe activity assays including platelet aggregation, measurement of PAR4 receptor cleavage, and measurement of PAR4 signaling as shown by increases in intracellular calcium levels. In addition, Coughlin et al., U.S. Patent No. 5,925,529, describe a method for characterizing peptide agonists using a tethered ligand for probing receptor binding. Additional assays can be devised by those of skill in the art. 4. Chemical Synthesis and Semi-synthesis of PAR-4 Polypeptides

PAR4 peptides and polypeptides of the present invention can be synthesized using standard techniques, including solid phase synthesis, partial solid phase methods, fragment condensation, or classical solution synthesis. The polypeptides can be prepared by solid phase peptide synthesis, for example as described by Merrifield, J. Am. Chem. Soc. &5:2149 (1963). The synthesis is carried out with amino acids that are protected at the α-amino terminus. Trifunctional amino acids with labile side-chains are also protected with suitable groups to prevent undesired chemical reactions from occurring during the assembly of the polypeptides. The α-amino protecting group is selectively removed to allow subsequent reaction to take place at the arnino-terminus. The conditions for the removal of the α-amino protecting group do not remove the side-chain protecting groups.

The α-amino protecting groups are those known to be useful in the art of stepwise polypeptide synthesis. Included are acyl-type protecting groups (e.g., formyl, trifluoroacetyl, acetyl), aryl type protecting groups (e.g., biotinyl), aromatic urethane type protecting groups (e.g., benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl and 9-fluorenylmethyloxy-carbonyl (Fmoc)), aliphatic urethane protecting groups (e.g., t-butyloxycarbonyl (tBoc), isopropyloxycarbonyl, cyclohexloxycarbonyl) and alkyl type protecting groups (e.g., benzyl, triphenylmethyl). The preferred protecting groups are tBoc and Fmoc, thus the peptides are said to be synthesized by tBoc and Fmoc chemistry, respectively.

The side-chain protecting groups selected must remain intact during coupling and not be removed during the deprotection of the arnino-terminus protecting group or during coupling conditions. The side-chain protecting groups must also be removable upon the completion of synthesis using reaction conditions that will not alter the finished polypeptide. In tBoc chemistry, the side-chain protecting groups for trifunctional amino acids are mostly benzyl based. In Fmoc chemistry, they are mostly tert-butyl or trityl based.

In tBoc chemistry, the preferred side-chain protecting groups are tosyl for arginine, cyclohexyl for aspartic acid, 4-methylbenzyl (and acetamidomethyl) for cysteine, benzyl for glutamic acid, serine and fhreonine, benzyloxymethyl (and dinitrophenyl) for histidine, 2-Cl-benzyloxycarbonyl for lysine, formyl for tryptophan and 2-bromobenzyl for tyrosine. In Fmoc chemistry, the preferred side-chain protecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) or 2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine, trityl for asparagine, cysteine, glutamine and histidine, tert-butyl for aspartic acid, glutamic acid, serine, threonine and tyrosine, tBoc for lysine and tryptophan. For the synthesis of phosphopeptides, either direct or post-assembly incorporation of the phosphate group is used. In the direct incorporation strategy, the phosphate group on serine, threonine or tyrosine may be protected by methyl, benzyl, or tert-butyl in Fmoc chemistry or by methyl, benzyl or phenyl in tBoc chemistry. Direct incorporation of phosphotyrosine without phosphate protection can also be used in Fmoc chemistry. In the post-assembly incorporation strategy, the unprotected hydroxyl groups of serine, threonine or tyrosine are derivatized on solid phase with di-tert-butyl-, dibenzyl- or dimethyl-N,N'-diisopropylphosphoramidite and then oxidized by tert- butylhydroperoxide. Solid phase synthesis is usually carried out from the carboxyl-terminus by coupling the α-amino protected (side-chain protected) amino acid to a suitable solid support. An ester linkage is formed when the attachment is made to a chloromethyl, chlortrityl or hydroxymethyl resin, and the resulting polypeptide will have a free carboxyl group at the C-terminus. Alternatively, when an amide resin such as benzhyό lamine or p-methylbenzhydrylamine resin (for tBoc chemistry) and Rink amide or PAL resin (for Fmoc chemistry) are used, an amide bond is formed and the resulting polypeptide will have a carboxamide group at the C-terminus. These resins, whether polystyrene- or polyamide-based or polyethyleneglycol-grafted, with or without a handle or linker, with or without the first amino acid attached, are commercially available, and their preparations have been described by Stewart et al, "Solid Phase Peptide Synthesis" (2nd Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al, Solid Phase Peptide Synthesis: A Practical Approach (TRL Press 1989), and by Lloyd- Williams et al, Chemical Approaches to the Synthesis of Peptides and Proteins (CRC Press, Inc. 1997). The C-terminal amino acid, protected at the side chain if necessary, and at the α-amino group, is attached to a hydroxylmethyl resin using various activating agents including dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIPCDI) and carbonyldiimidazole (CDI). It can be attached to chloromethyl or chlorotrityl resin directly in its cesium tetramethylammonium salt form or in the presence of triethylamine (TEA) or diisopropylethylamine (DIEA). First amino acid attachment to an amide resin is the same as amide bond formation during coupling reactions.

Following the attachment to the resin support, the α-amino protecting group is removed using various reagents depending on the protecting chemistry (e.g., tBoc, Fmoc). The extent of Fmoc removal can be monitored at 300-320 nm or by a conductivity cell. After removal of the α-amino protecting group, the remaining protected amino acids are coupled stepwise in the required order to obtain the desired sequence.

Various activating agents can be used for the coupling reactions including DCC, DIPCDI, 2-chloro-l,3-dimethylimidium hexafluorophosphate (CIP), benzotriazol-l-yl-oxy-tris-(dimethylamino)-phosphonium hexafluoro-phosphate (BOP) and its pyrrolidine analog (PyBOP), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), O-(benzotriazol- 1 -yl)- 1 , 1 ,3 ,3-tetramethyl-uronium hexafluorophosphate (HBTU) and its tetrafluoroborate analog (TBTU) or its pyrrolidine analog (HBPyU), O-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyl-uronium hexafluorophosphate (HATU) and its tetrafluoroborate analog (TATU) or its pyrrolidine analog (HAPyU). The most common catalytic additives used in coupling reactions include 4-dimethylaminopyridine (DMAP), 3-hydroxy-3,4-dihydro-4-oxo- 1,2,3-benzotriazine (HODhbt), N-hydroxybenzotriazole (HOBt) and l-hydroxy-7- azabenzotriazole (HOAt). Each protected amino acid is used in excess (>2.0 equivalents), and the couplings are usually carried out in N-methylpyrrolidone (NMP) or in DMF, CH2C12 or mixtures thereof. The extent of completion of the coupling reaction can be monitored at each stage, e.g., by the ninhydrin reaction as described by Kaiser et al, Anal Biochem. 34:595 (1970). In cases where incomplete coupling is found, the coupling reaction is extended and repeated and may have chaotropic salts added. The coupling reactions can be performed automatically with commercially available instruments such as ABI model 430A, 431 A and 433 A peptide synthesizers.

After the entire assembly of the desired peptide, the peptide-resin is cleaved with a reagent with proper scavengers. The Fmoc peptides are usually cleaved and deprotected by TFA with scavengers (e.g., water, ethanedithiol, phenol and thioanisole). The tBoc peptides are usually cleaved and deprotected with liquid HF for 1-2 hours at -5 to 0°C, which cleaves the polypeptide from the resin and removes most of the side-chain protecting groups. Scavengers such as anisole, dimethylsulfide and p- thiocresol are usually used with the liquid HF to prevent cations formed during the cleavage from alkylating and acylating the amino acid residues present in the polypeptide. The formyl group of tryptophan and the dinitrophenyl group of histidine need to be removed, respectively by piperidine and thiophenyl in DMF prior to the HF cleavage. The acetamidomethyl group of cysteine can be removed by mercury(H)acetate and alternatively by iodine, thallium(ILT)trifIuoroacetate or silver tetrafluoroborate which simultaneously oxidize cysteine to cystine. Other strong acids used for tBoc peptide cleavage and deprotection include trifluoromethanesulfonic acid (TFMSA) and trimethylsilyltrifluoroacetate (TMSOTf). The "native chemical ligation" approach to producing polypeptides is one variation of total chemical synthesis strategy (see, for example, Dawson et al, Science 266:116 (1994), Hackeng et al, Proc. Nat'l Acad. Sci. USA 94:1845 (1997), and Dawson, Methods Enzymol. 287: 34 (1997)). According to this method, an N- terminal cysteine-containing peptide is chemically ligated to a peptide having a C- terminal thioester group to form a normal peptide bond at the ligation site.

The "expressed protein ligation" method is a semi-synthesis variation of the ligation approach (see, for example, Muir et al, Proc. Nat'l Acad. Sci. USA 95:6105 (1998); Severinov and Muir, 7. Biol. Chem. 273:16205 (1998)). Here, synthetic peptides and protein cleavage fragments are linked to form the desired protein product. This method is particularly useful for the site-specific incorporation of unnatural amino acids (e.g., amino acids comprising biophysical or biochemical probes) into proteins.

In an approach illustrated by Muir et al, Proc. Nat'l Acad. Sci. USA 95:6105 (1998), a gene or gene fragment is cloned into the PCYB2-IMPACT vector (New England Biolabs, Inc.; Beverly, MA) using the Ndeϊ and S l restriction sites. As a result, the gene or gene fragment is expressed in frame fused with a chitin binding domain sequence, and a Pro-Gly is appended to the native C terminus of the protein of interest. The presence of a C-terminal glycine reduces the chance of side reactions, because the glycine residue accelerates native chemical ligation. Affinity chromatography with a chitin resin is used to purify the expressed fusion protein, and the chemical ligation step is initiated by incubating the resin-bound protein with thiophenol and synthetic peptide in buffer. This mixture produces the in situ generation of a highly reactive phenyl ^αthioester derivative of the protein that rapidly ligates with the synthetic peptide to produce the desired semi-synthetic protein. For a review, see Kochendoerfer and Kent, Curr. Opin. Chem. Biol. 3:665 (1999).

In an alternative approach, peptides and polypeptides can be produced using combinatorial chemistry to synthesize a library of analogs for all positions of the desired peptide or polypeptide. See, for example, Gershengorn et al., international publication No. WO 98/34948. The peptides and polypeptides of the present invention can comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4- hydroxyproline, tr rcs-4-hydroxyproline, N-methylglycine, α/ o-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4- methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is typically carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al, J. Am. Chem. Soc. 113:2122 (1991), Εllman et al, Methods Enzymol 202:301 (1991), Chung et al, Science 259:806 (1993), and Chung et al, Proc. Nat'l Acad. Sci. USA 90:10145 (1993).

5. Production of the Human PAR4 Gene

One type of assay that can be used to assess the activity of PAR4 peptides and polypeptides requires cultured recombinant host cells that express the human PAR4 protein. Nucleic acid molecules encoding a human PAR4 gene can be obtained by screening a human cDNA or genomic library using PAR4 polynucleotide probes, described by Xu et αl, international publication No. WO99/50415. PAR4 nucleotide and amino acid sequences are also provided herein as SΕQ ID NO:l and SΕQ ID NO:2, respectively. General techniques for obtaining Par4-encoding sequences are standard and well-established. For example, a nucleic acid molecule that encodes a human PAR4 gene can be isolated from a human cDNA library. In this case, the first step would be to prepare the cDNA library by isolating RNA from tissue using methods well- known to those of skill in the art. In general, RNA isolation techniques must provide a method for breaking cells, a means of inhibiting RNase-directed degradation of RNA, and a method of separating RNA from DNA, protein, and polysaccharide contaminants. For example, total RNA can be isolated by freezing tissue in liquid nitrogen, grinding the frozen tissue with a mortar and pestle to lyse the cells, extracting the ground tissue with a solution of phenol/chloroform to remove proteins, and separating RNA from the remaining impurities by selective precipitation with lithium chloride (see, for example, Ausubel et αl. (eds.), Short Protocols in Molecular Biology, 3^rd Edition, pages 4-1 to 4-6 (John Wiley & Sons 1995) ["Ausubel (1995)"]; Wu et al, Methods in Gene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) ["Wu (1997)"]). Alternatively, total RNA can be isolated from tissue by extracting ground tissue with guanidinium isothiocyanate, extracting with organic solvents, and separating RNA from contaminants using differential centrifugation (see, for example, Chirgwin et al, Biochemistry 18:52 (1979); Ausubel (1995) at pages 4-1 to 4-6; Wu (1997) at pages 33-41).

In order to construct a cDNA library, poly(A)⁺ RNA must be isolated from a total RNA preparation. Poly(A)⁺ RNA can be isolated from total RNA using the standard technique of oligo(dT)-cellulose chromatography (see, for example, Aviv and Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972); Ausubel (1995) at pages 4-11 to 4- 12).

Double-stranded cDNA molecules are synthesized from poly(A)⁺ RNA using techniques well-known to those in the art. (see, for example, Wu (1997) at pages 41-46). Moreover, commercially available kits can be used to synthesize double- stranded cDNA molecules. For example, such kits are available from Life Technologies, Inc. (Gaithersburg, MD), CLONTECH Laboratories, Inc. (Palo Alto, CA), Promega Corporation (Madison, WI) and STRATAGENE (La Jolla, CA).

Various cloning vectors are appropriate for the construction of a cDNA library. For example, a cDNA library can be prepared in a vector derived from bacteriophage, such as a λgtlO vector. See, for example, Huynh et al, "Constructing and Screening cDNA Libraries in λgtlO and λgtl l," in DNA Cloning: A Practical Approach Vol. I, Glover (ed.), page 49 (TRL Press, 1985); Wu (1997) at pages 47-52.

Alternatively, double-stranded cDNA molecules can be inserted into a plasmid vector, such as a PBLUESCRIPT vector (STRATAGENE; La Jolla, CA), a LAMDAGEM-4 (Promega Corp.) or other commercially available vectors. Suitable cloning vectors also can be obtained from the American Type Culture Collection (Manassas, VA).

To amplify the cloned cDNA molecules, the cDNA library is inserted into a prokaryotic host, using standard techniques. For example, a cDNA library can be introduced into competent E. coli DH5 cells, which can be obtained, for example, from Life Technologies, Inc. (Gaithersburg, MD).

A human genomic library can be prepared by means well-known in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327). Genomic DNA can be isolated by lysing tissue with the detergent Sarkosyl, digesting the lysate with proteinase K, clearing insoluble debris from the lysate by centrifugation, precipitating nucleic acid from the lysate using isopropanol, and purifying resuspended DNA on a cesium chloride density gradient.

DNA fragments that are suitable for the production of a genomic library can be obtained by the random shearing of genomic DNA or by the partial digestion of genomic DNA with restriction endonucleases. Genomic DNA fragments can be inserted into a vector, such as a bacteriophage or cosmid vector, in accordance with conventional techniques,, such as the use of restriction enzyme digestion to provide appropriate termini, the use of alkaline phosphatase treatment to avoid undesirable joining of DNA molecules, and ligation with appropriate ligases. Techniques for such manipulation are well-known in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307- 327).

Nucleic acid molecules that encode a human PAR4 gene can also be obtained using the polymerase chain reaction (PCR) with oligonucleotide primers having nucleotide sequences that are based upon the nucleotide sequences of the human PAR4 gene, as described herein. General methods for screening libraries with PCR are provided by, for example, Yu et al, "Use of the Polymerase Chain Reaction to Screen Phage Libraries," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 211-215 (Humana Press, Inc. 1993). Moreover, techniques for using PCR to isolate related genes are described by, for example, Preston, "Use of Degenerate Oligonucleotide Primers and the Polymerase Chain Reaction to Clone Gene Family Members," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 317- 337 (Humana Press, Inc. 1993).

Alternatively, human genomic libraries can be obtained from commercial sources such as Research Genetics (Huntsville, AL) and the American Type Culture Collection (Manassas, VA).

A library containing cDNA or genomic clones can be screened with one or more polynucleotide probes based upon SEQ ID NO:l, using standard methods (see, for example, Ausubel (1995) at pages 6-1 to 6-11).

Anti-PAR4 antibodies, produced as described below, can also be used to isolate DNA sequences that encode human PAR4 genes from cDNA libraries. For example, the antibodies can be used to screen λgtll expression libraries, or the antibodies can be used for immunoscreening following hybrid selection and translation (see, for example, Ausubel (1995) at pages 6-12 to 6-16; Margolis et al, "Screening λ expression libraries with antibody and protein probes," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al (eds.), pages 1-14 (Oxford University Press 1995)).

As an alternative, a PAR4 gene can be obtained by synthesizing nucleic acid molecules using mutually priming long oligonucleotides and the nucleotide sequences described herein (see, for example, Ausubel (1995) at pages 8-8 to 8-9).

Established techniques using the polymerase chain reaction provide the ability to synthesize DNA molecules at least two kilobases in length (Adang et al, Plant Molec. Biol. 27:1131 (1993), Bambot et al, PCR Methods and Applications 2:266 (1993), Dillon et al, "Use of the Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 263-268, (Humana Press, Inc. 1993), and Holowachuk et al, PCR Methods Appl. 4:299 (1995)).

The nucleic acid molecules of the present invention can also be synthesized with "gene machines" using protocols such as the phosphoramidite method. If chemically-synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 base pairs) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer genes (>300 base pairs), however, special strategies may be required, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%. To overcome this problem, synthetic genes (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length. For reviews on polynucleotide synthesis, see, for example, Glick and Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA (ASM Press 1994), Itakura et al, Annu. Rev. Biochem. 53:323 (1984), and Climie et al, Proc. Nat'l Acad. Sci. USA 87:633 (1990).

6. Production of PAR4 Peptides and Polypeptides The peptides and polypeptides of the present can also be produced in recombinant host cells following conventional techniques. To express a PAR4-ρeptide or PAR4-polypeptide encoding sequence, a nucleic acid molecule encoding the peptide or polypeptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then, introduced into a host cell. In addition to transcriptional regulatory sequences, such as promoters and enhancers, expression vectors can include translational regulatory sequences and a marker gene, which is suitable for selection of cells that carry the expression vector.

Expression vectors that are suitable for production of a foreign protein in eukaryotic cells typically contain (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription termination/polyadenylation sequence. As discussed above, expression vectors can also include nucleotide sequences encoding a secretory sequence that directs the heterologous polypeptide into the secretory pathway of a host cell. PAR4 peptides and polypeptides of the present invention may be expressed in mammalian cells. Examples of suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44 [Chasin et al, Som. Cell Molec. Genet. 12:555 1986]), rat pituitary cells (GHl; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-H-E; ATCC CRL 1548) SV40- transformed monkey kidney cells (COS-1; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).

For a mammalian host, the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression. Suitable transcriptional and translational regulatory sequences also can be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis. Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer et al, J. Molec. Appl Genet. 1:213 (1982)), the TK promoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 early promoter (Benoist et al, Nature 290:304 (1981)), the Rous sarcoma virus promoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6111 (1982)), the cytomegalo virus promoter (Foecking et al, Gene 45:101 (1980)), and the mouse mammary tumor virus promoter (see, generally, Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture," in Protein Engineering: Principles and Practice, Cleland et al (eds.), pages 163-181 (John Wiley & Sons, Inc. 1996)).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNA polymerase promoter, can be used to control expression in mammalian cells if the prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et al, Mol. Cell. Biol. 10:4529 (1990), and Kaufman et al, Nucl. Acids Res. 19:4485 (1991)).

An expression vector can be introduced into host cells using a variety of standard techniques including calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, electroporation, and the like. The transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome. Techniques for introducing vectors into eukaryotic cells and techniques for selecting such stable transformants using a dominant selectable marker are described, for example, by Ausubel (1995) and. by Murray (ed.), Gene Transfer and Expression Protocols (Humana Press 1991).

For example, one suitable selectable marker is a gene that provides resistance to the antibiotic neomycin. In this case, selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A suitable amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternatively, markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.

PAR4 peptides and polypeptides can also be produced by cultured mammalian cells using a viral delivery system. Exemplary viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see Becker et al, Meth. Cell Biol. 43:161 (1994), and Douglas and Curiel, Science & Medicine 4:44 (1997)). Advantages of the adenovirus system include the accommodation of relatively large DNA inserts, the ability to grow to high-titer, the ability to infect a broad range of mammalian cell types, and flexibility that allows use with a large number of available vectors containing different promoters.

By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co- transfected plasmid. An option is to delete the essential El gene from the viral vector, which results in the inability to replicate unless the El gene is provided by the host cell. Adenovirus vector-infected human 293 cells (ATCC Nos. CRL-1573, 45504, 45505), for example, can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (see Gamier et al, Cytotechnol 75:145 (1994)).

Nucleic acid molecules encoding PAR4 peptides and polypeptides may also be expressed in other higher eukaryotic cells, such as avian, fungal, insect, yeast, or plant cells. The baculovirus system provides an efficient means to introduce cloned PAR4 genes into insect cells. Suitable expression vectors are based upon the Autographa califomica multiple nuclear polyhedrosis virus (AcMNPV), and contain well-known promoters such as Drosophila heat shock protein (hsp) 70 promoter, Autographa califomica nuclear polyhedrosis virus immediate-early gene promoter (ie- 1) and the delayed early 39K promoter, baculovirus plO promoter, and the Drosophila metallothionein promoter. A second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, et al, J. Virol. 67:4566 (1993)). This system, which utilizes transfer vectors, is sold in the BAC-to- BAC kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, PFASTBAC (Life Technologies) containing a Tn7 transposon to move the DNA encoding the PAR4 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." See, Hill-Perkins and Possee, /. Gen. Virol. 71:911 (1990), Bonning, et al, J. Gen. Virol. 75:1551 (1994), and Chazenbalk, and Rapoport, J. Biol. Chem. 270:1543 (1995). In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed PAR4 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et al, Proc. Nat'l Acad. Sci. 82:1952 (1985)). Using a technique known in the art, a transfer vector containing a PAR4 peptide or polypeptide encoding sequence is transformed into E. coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is then isolated using common techniques.

The illustrative PFASTBAC vector can be modified to a considerable degree. For example, the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcør, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins (see, for example, Hill-Perkins and Possee, J. Gen. Virol. 71:911 (1990), Bonning, et al, J. Gen. Virol 75:1551 (1994), and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). In such transfer vector constructs, a short or long version of the basic protein promoter can be used. Moreover, transfer vectors can be constructed which replace the native PAR4 secretory signal sequences with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from Εcdysteroid Glucosyltransferase (ΕGT), honey bee Melittin (Ihvitrogen Corporation; Carlsbad, CA), or baculovirus gp67 (PharMingen: San Diego, CA) can be used in constructs to replace the native PAR4 secretory signal sequence.

The recombinant virus or bacmid is used to transfect host cells. Suitable insect host cells include cell lines derived from -PLB-Sjf-21, a Spodoptera frugiperda pupal ovarian cell line, such as Sβ (ATCC CRL 1711), 5/ 1 AE, and Sβl (Invitrogen Corporation; San Diego, CA), as well as Drosophila Schneider-2 cells, and the HIGH FINEO cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No. 5,300,435). Commercially available serum-free media can be used to grow and to maintain the cells. Suitable media are Sf900 H™ (Life Technologies) or ESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa, KS) or Express FiveO™ (Life Technologies) for the T. ni cells. When recombinant virus is used, the cells are typically grown up from an inoculation density of approximately 2-5 x 10⁵ cells to a density of 1-2 x 10⁶ cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.

Established techniques for producing recombinant proteins in baculovirus systems are provided by Bailey et al, "Manipulation of Baculovirus Vectors," in Methods in Molecular Biology, Volume 7: Gene Transfer and Expression Protocols, Murray (ed.), pages 147-168 (The Humana Press, Inc. 1991), by Patel et al, "The baculovirus expression system," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages 205-244 (Oxford University Press 1995), by Ausubel (1995) at pages 16-37 to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (The Humana Press, Inc. 1995), and by Lucknow, "Insect Cell Expression Technology," in Protein Engineering: Principles and Practice, Cleland et al (eds.), pages 183-218 (John Wiley & Sons, Inc. 1996).

Fungal cells, including yeast cells, can also be used to express the genes described herein. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Suitable promoters for expression in yeast include promoters from GALl (galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like. Many yeast cloning vectors have been designed and are readily available. These vectors include Yip-based vectors, such as YIp5, YRp vectors, such as YRpl7, YEp vectors such as YEpl3 and YCp vectors, such as YCp 19. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311, Kawasaki et al, U.S. Patent No. 4,931,373, Brake, U.S. Patent No. 4,870,008, Welch et al., U.S. Patent No. 5,037,743, and Murray et al, U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A suitable vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Additional suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311, Kingsman et al, U.S. Patent No. 4,615,974, and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446, 5,063,154, 5,139,936, and 4,661,454.

Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al, J. Gen. Microbiol. 132:3459 (1986), and Gregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al, U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al, U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533. For example, the use of Pichia methanolica as host for the production of recombinant proteins is disclosed by Raymond, U.S. Patent No. 5,716,808, Raymond, U.S. Patent No. 5,736,383, Raymond et al, Yeast 14:11-23 (1998), and in international publication Nos. WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which can be linearized prior to transformation. For polypeptide production in P. methanolica, the promoter and terminator in the plasmid can be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, the entire expression segment of the plasmid can be flanked at both ends by host DNA sequences. A suitable selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AJRC; EC 4.1.1.21), and which allows ade2 host cells to grow in the absence of adenine. For large-scale, industrial processes where it is desirable to minimize the use of methanol, host cells can be used in which both methanol utilization genes (AUG1 and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRB1) can be used. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. P. methanolica cells can be transformed by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.

Expression vectors can also be introduced into plant protoplasts, intact plant tissues, or isolated plant cells. Methods for introducing expression vectors into plant tissue include the direct infection or co-cultivation of plant tissue with Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA injection, electroporation, and the like. See, for example, Horsch et al, Science 227:1229 (1985), Klein et al, Biotechnology 10:268 (1992), and MM et al, "Procedures for Introducing Foreign DNA into Plants," in Methods in Plant Molecular Biology and Biotechnology, Glick et al (eds.), pages 67-88 (CRC Press, 1993).

Alternatively, nucleotide sequence encoding PAR4 peptides and polypeptides can be expressed in prokaryotic host cells. Suitable promoters that can be used to express eukaryotic polypeptides in a prokaryotic host are well-known to those of skill in the art and include promoters capable of recognizing the T4, T3, Sp6 and T7 polymerases, the P_R and P promoters of bacteriophage lambda, the trp, recA, heat shock, lacUV5, tac, Ipp-lacSpr, phoA, and lacZ promoters of E. coli, promoters of B. subtilis, the promoters of the bacteriophages of Bacillus, Streptomyces promoters, the int promoter of bacteriophage lambda, the bla promoter of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene. Prokaryotic promoters have been reviewed by Glick, J. Ind. Microbiol 1:211 (1987), Watson et al, Molecular Biology ofthe Gene, 4th Ed. (Benjamin Cummins 1987), and by Ausubel et al (1995).

Illustrative prokaryotic hosts include E. coli and Bacillus subtilus. Suitable strains of E. coli include BL21(DΕ3), BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF, DH5 CR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), Molecular Biology Labfax (Academic Press 1991)). Suitable strains of Bacillus subtilus include BR151, YB886, Mil 19, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning Methods," in DNA Cloning: A Practical Approach, Glover (ed.) (IRL Press 1985)). When expressing a PAR4 peptide or polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding. Methods for expressing proteins in prokaryotic hosts are well-known to those of skill in the art (see, for example, Williams et al, "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford University Press 1995), Ward et al, "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, page 137 (Wiley-Liss, Inc. 1995), and Georgiou, "Expression of Proteins in Bacteria," in Protein Engineering: Principles and Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc. 1996)).

Standard methods for introducing expression vectors into bacterial, yeast, insect, and plant cells are provided, for example, by Ausubel (1995).

General methods for expressing and recovering foreign protein produced by a mammalian cell system are provided by, for example, Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture," in Protein Engineering: Principles and Practice, Cleland etal. (eds.), pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recovering protein produced by a bacterial system is provided by, for example, Grisshammer et al, "Purification of over-produced proteins from E. coli cells," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages 59-92 (Oxford University Press 1995). Established methods for isolating recombinant proteins from a baculovirus system are described by Richardson (ed.), Baculovirus Expression Protocols (The Humana Press, Inc. 1995).

7. Isolation of PAR4 Polypeptides

The peptides and polypeptides ofthe present invention can be purified to at least about 80% purity, to at least about 90% purity, to at least about 95% purity, or even greater than 95% purity with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. The peptides and polypeptides of the present invention may also be purified to a pharmaceutically pure state, which is greater than 99.9% pure. In certain preparations, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. Fractionation and/or conventional purification methods can be used to obtain preparations of PAR4 peptides and polypeptides purified from recombinant host cells. Numerous methods for purifying proteins are known in the art. In general, ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred. Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.

Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Selection of a particular method for polypeptide isolation and purification is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988), and Doonan, Protein Purification Protocols (The Humana Press 1996).

The peptides and polypeptides of the present invention can also be isolated by exploitation of particular properties. For example, immobilized metal ion adsorption (MAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (M. Deutscher, (ed.), Meth. Enzymol. 182:529 (1990)). Within additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (e.g., maltose-binding protein, an immunoglobulin domain) may be constructed to facilitate purification.

PAR4 polypeptides or fragments thereof may also be prepared through chemical synthesis, as described below. PAR4 polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.

As an example of type of modification of a PAR peptide or polypeptide, a peptide or polypeptide is linked with a polymer. Typically, the polymer is water soluble so that the PAR4 conjugate does not precipitate in an aqueous environment, such as a physiological environment. An example of a suitable polymer is one that has been modified to have a single reactive group, such as an active ester for acylation, or an aldehyde for alkylation, In this way, the degree of polymerization can be controlled. An example of a reactive aldehyde is polyethylene glycol propionaldehyde, or mono- ( - o) alkoxy, or aryloxy derivatives thereof (see, for example, Harris, et al, U.S. Patent No. 5,252,714). The polymer may be branched or unbranched. Moreover, a mixture of polymers can be used to produce PAR4 conjugates.

PAR4 conjugates used for therapy can comprise pharmaceutically acceptable water-soluble polymer moieties. Suitable water-soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, mono-(CrC₁₀)alkoxy-PEG, aryloxy- PEG, poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde, bz^'s-succinirnidyl carbonate PEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or other carbohydrate-based polymers. Suitable PEG may have a molecular weight from about 600 to about 60,000, including, for example, 5,000, 12,000, 20,000 and 25,000. A PAR4 peptide or polypeptide conjugate can also comprise a mixture of such water-soluble polymers.

One example of a PAR4 peptide or polypeptide conjugate comprises a PAR4 moiety and a polyalkyl oxide moiety attached to the N-terminus of the PAR4 moiety. PEG is one suitable polyalkyl oxide. As an illustration, a PAR4 peptide or polypeptide can be modified with PEG, a process known as "PEGylation." PEGylation of peptides and polypeptides can be carried out by any of the PEGylation reactions known in the art (see, for example, EP 0 154 316, Delgado et al, Critical Reviews in Therapeutic Drug Carrier Systems 9:249 (1992), Duncan and Spreafico, Clin. Pharmacokinet. 27:290 (1994), and Francis et al, Int J Hematol 68:1 (1998)). For example, PEGylation can be performed by an acylation reaction or by an alkylation reaction with a reactive polyethylene glycol molecule. In an alternative approach, PAR4 peptide and polypeptide conjugates are formed by condensing activated PEG, in which a terminal hydroxy or amino group of PEG has been replaced by an activated linker (see, for example, Karasiewicz et al, U.S. Patent No. 5,382,657).

PEGylation by acylation typically requires reacting an active ester derivative of PEG with a PAR4 peptide or polypeptide. An example of an activated PEG ester is PEG esterified to N-hydroxysuccinimide. As used herein, the term "acylation" includes the following types of linkages between a PAR4 peptide or polypeptide and a water soluble polymer: amide, carbamate, urethane, and the like. Methods for preparing PEGylated PAR4 peptides or polypeptides by acylation will typically comprise the steps of (a) reacting a PAR4 moiety with PEG (such as a reactive ester of an aldehyde derivative of PEG) under conditions whereby one or more PEG groups attach to the PAR4 moiety, and (b) obtaining the reaction product(s). Generally, the optimal reaction conditions for acylation reactions will be determined based upon known parameters and desired results. For example, the larger the ratio of PEG: PAR4 moiety, the greater the percentage of polyPEGylated PAR4 product.

The product of PEGylation by acylation is typically a polyPEGylated PAR4 product, wherein the lysine ε-amino groups are PEGylated via an acyl linking group. An example of a connecting linkage is an amide. Typically, the resulting PAR4 moiety will be at least 95% mono-, di-, or tri-pegylated, although some species with higher degrees of PEGylation may be formed depending upon the reaction conditions. PEGylated species can be separated from unconjugated PAR4 peptides and polypeptides using standard purification methods, such as dialysis, ultrafiltration, ion exchange chromatography, affinity chromatography, and the like.

PEGylation by alkylation generally involves reacting a terminal aldehyde derivative of PEG with a PAR4 moiety in the presence of a reducing agent. PEG groups can be attached to the polypeptide via a -CH₂-ΝH group.

Derivatization via reductive alkylation to produce a monoPEGylated product takes advantage of the differential reactivity of different types of primary amino groups available for derivatization. Typically, the reaction is performed at a pH that allows one to take advantage of the pKa differences between the ε-amino groups of the lysine residues and the α-amino group of the N-terminal residue of the protein. By such selective derivatization, attachment of a water-soluble polymer that contains a reactive group such as an aldehyde, to a protein is controlled. The conjugation with the polymer occurs predominantly at the N-terminus of the protein without significant modification of other reactive groups such as the lysine side chain amino groups. The present invention provides a substantially homogenous preparation of PAR4 monopolymer conjugates. Reductive alkylation to produce a substantially homogenous population of monopolymer PAR4 conjugate molecule can comprise the steps of: (a) reacting a PAR4 peptide or polypeptide with a reactive PEG under reductive alkylation conditions at a pH suitable to permit selective modification of the α-amino group at the amino terminus of the PAR4 moiety, and (b) obtaining the reaction product(s). The reducing agent used for reductive alkylation should be stable in aqueous solution and able to reduce only the Schiff base formed in the initial process of reductive alkylation. Illustrative reducing agents include sodium borohydride, sodium cyanoborohydride, dimethylamine borane, trimethylamine borane, and pyridine borane. For a substantially homogenous population of monopolymer PAR4 conjugates, the reductive alkylation reaction conditions are those which permit the selective attachment of the water soluble polymer moiety to the N-terminus of the PAR4 moiety. Such reaction conditions generally provide for pKa differences between the lysine amino groups and the α-amino group at the N-terminus. The pH also affects the ratio of polymer to protein to be used. In general, if the pH is lower, a larger excess of polymer to protein will be desired because the less reactive the N-terminal α-group, the more polymer is needed to achieve optimal conditions. If the pH is higher, the polymer: PAR4 moiety need not be as large because more reactive groups are available. Typically, the pH will fall within the range of 3 to 9, or 3 to 6. General methods for producing conjugates comprising a polypeptide and water-soluble polymer moieties are known in the art. See, for example, Karasiewicz et al, U.S. Patent No. 5,382,657, Greenwald et al, U.S. Patent No. 5,738, 846, Nieforth et al, Clin. Pharmacol Ther. 59:636 (1996), Monkarsh et al, Anal. Biochem. 247:434 (1997)). The present invention contemplates compositions comprising a peptide or polypeptide described herein. Such compositions can further comprise a carrier. The carrier can be a conventional organic or inorganic carrier. Examples of carriers include water, buffer solution, alcohol, propylene glycol, macrogol, sesame oil, corn oil, and the like.

8. Therapeutic Uses of PAR4 Peptides and Polypeptides

The PAR4 peptides and polypeptides described herein can be used to stimulate platelet aggregate formation. For example, PAR4 peptides and polypeptides are useful as a localized application to treat an internal bleeding site of a hemophiliac, or to counteract the effect of an anticoagulant. PAR4 peptides and polypeptides can also be used to inhibit proliferation of tumor cells, as illustrated by Example 3. These molecules can be administered to any subject in need of treatment, and the present invention contemplates both veterinary and human therapeutic uses. Illustrative subjects include mammalian subjects, such as farm animals, domestic animals, and human patients. Generally, the dosage of administered polypeptide or peptide will vary depending upon such factors as the subject's age, weight, height, sex, general medical condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of a PAR4 peptide or polypeptide, which is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of subject), although a lower or higher dosage also may be administered as circumstances dictate.

Administration of a PAR4 peptide or polypeptide to a subject can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection. When administering therapeutic proteins by injection, the administration may be by continuous infusion or by single or multiple boluses.

Additional routes of administration include oral, mucosal-membrane, pulmonary, and transcutaneous. Oral delivery is suitable for polyester microspheres, zein microspheres, proteinoid microspheres, polycyanoacrylate microspheres, and lipid- based systems (see, for example, DiBase and Morrel, "Oral Delivery of Microencapsulated Proteins," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). The feasibility of an intranasal delivery is exemplified by such a mode of insulin administration (see, for example, Hinchcliffe and Ilium, Adv. Drug Deliv. Rev. 55:199 (1999)). Dry or liquid particles comprising PAR4 peptides or polypeptides can be prepared and inhaled with the aid of dry-powder dispersers, liquid aerosol generators, or nebulizers (e.g., Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al, Adv. Drug Deliv. Rev. 35:235 (1999)). This approach is illustrated by the AERX diabetes management system, which is a hand-held electronic inhaler that delivers aerosolized insulin into the lungs. Studies have shown that proteins as large as 48,000 kDa have been delivered across skin at therapeutic concentrations with the aid of low-frequency ultrasound, which illustrates the feasibility of trascutaneous administration (Mitragotri et al, Science 269:850 (1995)). Transdermal delivery using electroporation provides another means to administer PAR4 peptides or polypeptides (Potts et al, Pharm. Biotechnol 10:213 (1997)). A pharmaceutical composition comprising a PAR4 peptide or polypeptide can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic proteins are combined in a mixture with a pharmaceutically acceptable carrier. A composition is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient subject. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well-known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).

For purposes of therapy, a PAR4 peptide or polypeptide and a pharmaceutically acceptable carrier are administered to a subject in a therapeutically effective amount. A combination of a PAR4 peptide, or polypeptide, and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient subject. For example, the present invention includes methods of inhibiting the proliferation of tumor cells, comprising the step of administering a composition comprising a PAR4 polypeptide or peptide to the tumor cells. In an in vivo approach, the composition is a pharmaceutical composition, administered in a therapeutically effective amount to a mammalian subject which has a tumor. Such in vivo administration can provide at least one physiological effect selected from the group consisting of decreased number of tumor cells, decreased metastasis, decreased size of a solid tumor, and increased necrosis of a tumor.

A pharmaceutical composition comprising a PAR4 peptide or polypeptide can be furnished in liquid form, in an aerosol, or in solid form. Liquid forms, are illustrated by injectable solutions and oral suspensions. Exemplary solid forms include capsules, tablets, and controlled-release forms. The latter form is illustrated by miniosmotic pumps and implants (Bremer et al, Pharm. Biotechnol 10:239 (1997); Ranade, "Implants in Drug Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al, "Protein Delivery with Infusion Pumps," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997); Yewey et al, "Delivery of Proteins from a Controlled Release Injectable Implant," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).

Liposomes provide one means to deliver therapeutic polypeptides to a subject intravenously, intraperitoneally, intrathecally, intramuscularly, subcutaneously, or via oral administration, inhalation, or intranasal administration. Liposomes are microscopic vesicles that consist of one or more lipid bilayers surrounding aqueous compartments (see, generally, Bakker-Woudenberg et al, Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl 7 :S61 (1993), Kim, Drugs 46:618 (1993), and Ranade, "Site- Specific Drug Delivery Using Liposomes as Carriers," in Drug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRC Press 1995)). Liposomes are similar in composition to cellular membranes and as a result, liposomes can be administered safely and are biodegradable. Depending on the method of preparation, liposomes may be unilamellar or multilamellar, and liposomes can vary in size with diameters ranging from 0.02 μm to greater than 10 μm. A variety of agents can be encapsulated in liposomes: hydrophobic agents partition in the bilayers and hydrophilic agents partition within the inner aqueous space(s) (see, for example, Machy et al, Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and Ostro et al, American J. Hosp. Pharm. 46:1516 (1989)). Moreover, it is possible to control the therapeutic availability of the encapsulated agent by varying liposome size, the number of bilayers, lipid composition, as well as the charge and surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowly release the encapsulated agent. Alternatively, an absorbed liposome may be endocytosed by cells that are phagocytic. Endocytosis is followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents (Scherphof et al, Ann. N.Y. Acad. Sci. 446:368 (1985)). After intravenous administration, small liposomes (0.1 to 1.0 μm) are typically taken up by cells of the reticuloendothelial system, located principally in the liver and spleen, whereas liposomes larger than 3.0 μm are deposited in the lung. This preferential uptake of smaller liposomes by the cells of the reticuloendothelial system has been used to deliver chemotherapeutic agents to macrophages and to tumors of the liver.

The reticuloendothelial system can be circumvented by several methods ^■ including saturation with large doses of liposome particles, or selective macrophage inactivation by pharmacological means (Claassen et al, Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporation of glycolipid- or polyethelene glycol- derivatized phospholipids into liposome membranes has been shown to result in a significantly reduced uptake by the reticuloendothelial system (Allen et al, Biochim. Biophys. Acta 1068:133 (1991); Allen et al, Biochim. Biophys. Acta 1150:9 (1993)). Liposomes can also be prepared to target particular cells or organs by varying phospholipid composition or by inserting receptors or ligands into the liposomes. For example, liposomes, prepared with a high content of a nonionic surfactant, have been used to target the liver (Hayakawa et al, Japanese Patent 04- 244,018; Kato et al, Biol. Pharm. Bull. 16:960 (1993)). These formulations were prepared by mixing soybean phospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castor oil (HCO-60) in methanol, concentrating the mixture under vacuum, and then reconstituting the mixture with water. A liposomal formulation of dipalmitoylphosphatidylcholine (DPPC) with a soybean-derived sterylglucoside mixture (SG) and cholesterol (Ch) has also been shown to target the liver (Shimizu et al, Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface of the liposome, such as antibodies, antibody fragments, carbohydrates, vitamins, and transport proteins. For example, liposomes can be modified with branched type galactosyllipid derivatives to target asialoglycoprotein (galactose) receptors, which are exclusively expressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev. Ther. Drug Carrier Syst. 14:281 (1997); Murahashi et al, Biol. Pharm. Bull.20:259 (1997)). Similarly, Wu et al, Hepatology 27:112 (1998), have shown that labeling liposomes with asialofetuin led to a shortened liposome plasma half-life and greatly enhanced uptake of asialofetuin-labeled liposome by hepatocytes. On the other hand, hepatic accumulation of liposomes comprising branched type galactosyllipid derivatives can be inhibited by preinjection of asialofetuin (Murahashi et al, Biol. Pharm. Bull.20:259 (1997)). Polyaconitylated human serum albumin liposomes provide another approach for targeting liposomes to liver cells (Kamps et al, Proc. Nat'l Acad. Sci. USA 94:11681 (1997)). Moreover, Geho, et al U.S. Patent No. 4,603,044, describe a hepatocyte-directed liposome vesicle delivery system, wliich has specificity for hepatobiliary receptors associated with the specialized metabolic cells of the liver. In a more general approach to tissue targeting, target cells are prelabeled with biotinylated antibodies specific for a ligand expressed by the target cell (Harasym et al, Adv. Drug Deliv. Rev. 32:99 (1998)). After plasma elimination of free antibody, streptavidin-conjugated liposomes are administered. In another approach, targeting antibodies are directly attached to liposomes (Harasym et al, Adv. Drug Deliv. Rev. 32:99 (1998)).

A PAR4 peptide or polypeptide can be encapsulated within liposomes using standard techniques of protein microencapsulation (see, for example, Anderson et al, Infect. Immun. 31:1099 (1981), Anderson et al, Cancer Res. 50:1853 (1990), and Cohen et al, Biochim. Biophys. Acta 1063:95 (1991), Alving et al. "Preparation and Use of Liposomes in Immunological Studies," in Liposome Technology, 2nd Edition, Vol. HI, Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al, Meth. Enzymol. 149:124 (1987)). As noted above, therapeutically useful liposomes may contain a variety of components. For example, liposomes may comprise lipid derivatives of poly(ethylene glycol) (Allen et al, Biochim. Biophys. Acta 1150:9 (1993)). Degradable polymer microspheres have been designed to maintain high systemic levels of therapeutic proteins. Microspheres are prepared from degradable polymers such as poly(lactide-co-glycolide) (PLG), polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetate polymers, in which proteins are entrapped in the polymer (Gombotz and Pettit, Bioconjugate Chem. 6:332 (1995); Ranade, "Role of Polymers in Drug Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.), pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable Controlled Release Systems Useful for Protein Delivery," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92 (Plenum Press 1997); Bartus et al, Science 287:1161 (1998); Putney and Burke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem. Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres can also provide carriers for intravenous administration of therapeutic proteins (see, for example, Gref et al, Pharm. Biotechnol 10:161 (1997)).

Other dosage forms can be devised by those skilled in the art, as shown, for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5^th Edition (Lea & Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences, 19^th Edition (Mack Publishing Company 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRC Press 1996).

As an illustration, pharmaceutical compositions may be supplied as a kit comprising a container that comprises a PAR4 peptide or polypeptide. Therapeutic polypeptides can be provided in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection. Alternatively, such a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a therapeutic polypeptide. Such a kit may further comprise written information on indications and usage of the pharmaceutical composition. Moreover, such information may include a statement that the PAR4 peptide or polypeptide is contraindicated ' in patients with known hypersensitivity to PAR4 peptides or polypeptides.

9. Therapeutic Uses of Nucleotide Sequences Encoding PAR4 Peptides and Polypeptides

The present invention includes the use of nucleotide sequences to provide PAR4 peptides and polypeptides to a subject in need of such treatment. For example, PAR4 peptides and polypeptides can be administered to inhibit proliferation of tumor cells.

There are numerous approaches to introduce a foreign gene to a subject, including the use of recombinant host cells that express PAR4 peptides or polypeptides, delivery of naked nucleic acid encoding PAR4 peptides or polypeptides, use of a cationic lipid carrier with a nucleic acid molecule that encodes PAR4 peptides or polypeptides, and the use of viruses that express PAR4 peptides or polypeptides, such as recombinant retroviruses, recombinant adeno-associated viruses, recombinant adenoviruses, and recombinant Herpes simplex viruses (see, for example, Mulligan, Science 260:926 (1993), Rosenberg et al, Science 242:1515 (1988), LaSalle et al, Science 259:988 (1993), Wolff et al, Science 247: 1465 (1990), Breakfield and Deluca, The New Biologist 3:203 (1991)). In an ex vivo approach, for example, cells are isolated from a subject, transfected with a vector that expresses a PAR4 peptide or polypeptide, and then transplanted into the subject. i order to effect expression of PAR4 peptides or polypeptides, an expression vector is constructed in which a nucleotide sequence encoding these amino acid sequences is operably linked to a core promoter, and optionally a regulatory element, to control gene transcription. The general requirements of an expression vector are described above.

Alternatively, a PAR4 peptide or polypeptide can be delivered using recombinant viral vectors, including for example, adenoviral vectors (e.g., Kass-Eisler et al, Proc. Nat'l Acad. Sci. USA 90:11498 (1993), Kolls et al, Proc. Nat'l Acad. Sci. USA 91:215 (1994), Li et al, Hum. Gene Ther. 4:403 (1993), Vincent et al, Nat. Genet. 5:130 (1993), and Zabner et al, Cell 75:207 (1993)), adenovirus-associated viral vectors (Flotte et al, Proc. Nat'l Acad. Sci. USA 90:10613 (1993)), alphaviruses such as Semliki Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:851 (1992), Raju and Huang, /. Vir. 65:2501 (1991), and Xiong et al, Science 243:1188 (1989)), herpes viral vectors (e.g., U.S. Patent Nos. 4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus vectors (Koering et al, Hum. Gene Therap. 5:457 (1994)), pox virus vectors (Ozaki etal, Biochem. Biophys. Res. Comm. 193:653 (1993), Panicali and Paoletti, Proc. Nat'l Acad. Sci. USA 79:4927 (1982)), pox viruses, such as canary pox virus or vaccinia virus (Fisher-Hoch et al, Proc. Nat'l Acad. Sci. USA 86:311 (1989), and Flexner etal, Ann. N.Y. Acad. Sci. 569:86 (1989)), and retroviruses (e.g., Baba et al, J. Neurosurg 79:129 (1993), Ram et al, Cancer Res. 53:83 (1993), Takamiya et al, J. Neurosci. Res 33:493 (1992), Vile and Hart, Cancer Res. 53:962 (1993), Vile and Hart, Cancer Res. 53:3860 (1993), and Anderson et al, U.S. Patent No. 5,399,346). Within various embodiments, either the viral vector itself, or a viral particle which contains the viral vector may be utilized in the methods and compositions described below.

As an illustration of one system, adenovirus, a double-stranded DNA virus, is a well-characterized gene transfer vector for delivery of a heterologous nucleic acid molecule (for a review, see Becker et al, Meth. Cell Biol. 43:161 (1994); Douglas and Curiel, Science & Medicine 4:44 (1997)). The adenovirus system offers several advantages including: (i) the ability to accommodate relatively large DNA inserts, (ii) the ability to be grown to high-titer, (iii) the ability to infect a broad range of mammalian cell types, and (iv) the ability to be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. In addition, adenoviruses can be administered by intravenous injection, because the viruses are stable in the bloodstream.

Using adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential El gene is deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell. When intravenously administered to intact animals, adenovirus primarily targets the liver. Although an adenoviral delivery system with an El gene deletion cannot replicate in the host cells, the host's tissue will express and process an encoded heterologous protein. Host cells will also secrete the heterologous protein if the corresponding gene includes a secretory signal sequence. Secreted proteins will enter the circulation from tissue that expresses the heterologous gene (e.g., the highly vascularized liver).

Moreover, adenoviral vectors containing various deletions of viral genes can be used to reduce or eliminate immune responses to the vector. Such adenoviruses are El -deleted, and in addition, contain deletions of E2A or E4 (Lusky et al, J. Virol. 72:2022 (1998); Raper et al, Human Gene Therapy 9:671 (1998)). The deletion of E2b has also been reported to reduce immune responses (Amalfitano et al, J. Virol. 72:926 (1998)). By deleting the entire adenovirus genome, very large inserts of heterologous DNA can be accommodated. Generation of so called "gutless" adenoviruses, where all viral genes are deleted, are particularly advantageous for insertion of large inserts of heterologous DNA (for a review, see Yeh. and Perricaudet, FASEB J. 11:615 (1997)).

High titer stocks of recombinant viruses capable of expressing a therapeutic gene can be obtained from infected mammalian cells using standard methods. For example, recombinant HSV can be prepared in Vero cells, as described by Brandt et al, J. Gen. Virol 72:2043 (1991), Herold et al, J. Gen. Virol. 75:1211 (1994), Visalli and Brandt, Virology 185:419 (1991), Grau et al, Invest. Ophthalmol Vis. Sci. 30:2474 (1989), Brandt et al, J. Virol. Meth. 36:209 (1992), and by Brown and MacLean (eds.), HSV Virus Protocols (Humana Press 1997).

Alternatively, an expression vector comprising a nucleic acid molecule that encodes a PAR4 peptide or polypeptide can be introduced into a subject's cells by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al, Proc. Nat'l Acad. Sci. USA 84:1413 (1987); Mackey et al, Proc. Nat'l Acad. Sci. USA 85:8021 (1988)). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Liposomes can be used to direct transfection to particular cell types, which is particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. Targeted peptides (e.g., hormones or neurotransmitters), proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.

Electroporation is another alternative mode of administration of a such PAR4 nucleic acid molecules. For example, Aihara and Miyazaki, Nature Biotechnology 16:861 (1998), have demonstrated the use of in vivo electroporation for gene transfer into muscle.

In general, the dosage of a composition comprising a therapeutic vector having a PAR4 nucleotide acid sequence, such as a recombinant virus, will vary depending upon such factors as the subject's age, weight, height, sex, general medical condition and previous medical history. Suitable routes of administration of therapeutic vectors include intravenous injection, intraarterial injection, intraperitoneal injection, intramuscular injection, intratumoral injection, and injection into a cavity that contains a tumor. A composition comprising viral vectors, non-viral vectors, or a combination of viral and non- viral vectors of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby vectors or viruses are combined in a mixture with a pharmaceutically acceptable carrier. As noted above, a composition, such as phosphate-buffered saline is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient subject. Other suitable carriers are well-known to those in the art (see, for example, Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co. 1995), and Gilman's the Pharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co. 1985)). For purposes of therapy, a therapeutic gene expression vector, or a recombinant virus comprising such a vector, and a pharmaceutically acceptable carrier are administered to a subject in a therapeutically effective amount. A combination of an expression vector (or virus) and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient subject. As described above, the present invention includes methods of inhibiting the proliferation of tumor cells, comprising the step of administering a composition that provides a PAR4 polypeptide or peptide to the tumor cells. In an in vivo approach, the composition is a pharmaceutical composition, administered in a therapeutically effective amount to a mammalian subject, which has a tumor. Such in vivo administration can provide at least one physiological effect selected from the group consisting of decreased number of tumor cells, decreased metastasis, decreased size of a solid tumor, and increased necrosis of a tumor.

When the subject treated with a therapeutic gene expression vector or a recombinant virus is a human, then the therapy is preferably somatic cell gene therapy. That is, the preferred treatment of a human with a therapeutic gene expression vector or a recombinant virus does not entail introducing into cells a nucleic acid molecule that can form part of a human germ line and be passed onto successive generations (i.e., human germ line gene therapy).

The present invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting ofthe present invention.

EXAMPLE 1

Method for Detecting PAR4 Activation In a phosphoinositide hydrolysis assay, COS-7 cells are grown in

Dulbecco's modified Eagle's medium (DMEM; Gibco/BRL, Gaithersburg, MD) with 10% fetal bovine serum (FBS). Cells are plated at 3.5xl0⁵/35-mm plate one day before transfection. To introduce a human PAR4 receptor, about two micrograms of PAR4 DNA are transfected using 12 ml of lipofect AMINE (Gibcσ/BRL) for five hours. The cells are incubated overnight in DMEM with 10% FBS, and then split into triplicate 35- m wells. Forty-eight hours after transfection, the cells are loaded with 2 mCi/ml [ H]myo-inositol (Amersham, Arlington Heights, IL) in serum-free DMEM and incubated overnight at 37°C. Cells are washed and treated with 20 mM LiCl in DMEM, with or without activators added at various concentrations. Cells are then incubated for two hours at 37°C and extracted with 750 ml of 20 mM formic acid for 30 minutes on ice. The inositol mono-, bis-, and trisphosphates are purified through a one milliliter AG 1-X8 anion-exchange resin (Bio-Rad, Hercules, CA) (Nanevicz et al, J. Biol. Chem. 271:102 (1996)), and quantitated by scintillation counting. In each hydrolysis assay, surface expression levels of receptors are determined in triplicate in parallel cultures. PAR4 activation is indicated by phosphatidylinositol 4,5 diphosphate hydrolysis. EXAMPLE 2

Platelet Aggregation Assay

Donors of blood for aggregation tests should be resting, non-smoking, and should avoid taking medications known to affect platelet function for ten days prior to the test. Blood was collected into sterile evacuated tubes, and mixed with 3.2% sodium citrate in the ratio of one part anticoagulant solution to nine parts blood. Testing was performed immediately after collection, or for up to three hours after collection. Blood samples were kept at room temperature (24° to 27°C).

Platelet rich plasma preparations were obtained by collecting whole blood, mixing the sample with sodium citrate by gentle inversion, and centrifuging the sample at about lOOxg for 15 minutes. Platelet rich plasma was removed with a polypropylene transfer pipette, and placed into a polypropylene plastic tube. Samples of platelet poor plasma were obtained by centrifuging a blood sample at approximately 2400xg for 20 minutes. Platelet poor plasma samples were removed from centrifuge tubes and stored in polypropylene plastic tubes, as described above.

Impedance aggregation of whole blood was measured with a Chrono-log 560 Ca Platelet Aggregometer (Chrono-log Corporation; Havertown, PA). Briefly, sample cuvettes were prewarmed to 37° C for at least 10 minutes. One milliliter of sample was pipetted into a prewarmed cuvette and mixed with one milliliter of sterile saline. A stir bar was placed into the sample. The sample was then warmed for five minutes in the incubation well. After transferring the sample into the instrument, the impedance probe was placed into the cuvette, and the sample started spinning (1200 rpm). A baseline impedance was established for two minutes. The test substance (e.g., thrombin or PAR4 peptide) was then introduced into the sample, and platelet aggregation was measured. In this method, aggregation is determined by increased impedance across the probe tip.

Optical aggregation was observed using a Chrono-log 560 Ca Platelet Aggregometer with washed platelets and platelet rich plasma. In these studies, sample cuvettes were prewarmed as described above, 500 μl of platelet rich plasma were introduced into cuvettes, and allowed to equilibrate for five minutes at 37°C. The sample was then introduced into the sample block and a test substance was added. Platelet aggregation in this method was determined by a decrease in light scattering.

EXAMPLE 3 Mitogenesis Assay

Northern analysis revealed that human PAR4 is expressed by the K562 cell line, which is a human chronic myelogenous leukemia cell line. PAR4 peptides and polypeptides were tested for the ability to either inhibit or promote mitogenesis in K562 cells. Briefly, K562 cells (ATCC No. 45506; American Type Culture Collection; Manassas, VA) in log phase were pelleted and resuspended in either DMEM with 0.2% fetal bovine serum (FBS) to evaluate proliferative potential, or DMEM with 5% FBS to evaluate mitogenesis inhibitory potential. Cells were plated at 5,000 cells/well (lxl0⁵/ml) in a 96 well plate in a 50μl volume of either DMEM with 0.2% FBS, or DMEM with 5% FBS. PAR4 peptides were diluted in DMEM serum-free media to twice the final concentration of 100, 10, and lμM (200, 20 and 2μM). Fifty microliters of diluted peptide at three concentrations were added to quadruplicate wells of K562 cells. The final serum concentration was 0.1% or 2.5%. Certain wells received only 50 μl of DMEM to serve as 2.5% and 0.1% controls. Plates were incubated for 48 hours at 37°C in a 5% CO₂ incubator.

Ten microliters of ALAMAR Blue growth indicator reagent (Accumed International, Inc.; Westiake, OH) were added to each well. The plates were then returned to the incubator for another 24 hours. Thirty six hours after plating and 24 hours after ALAMAR Blue addition, absorbance was read at 570 and 600 nm on a Spectromax plate reader. The results showed that the PAR peptides and polypeptides that were most potent in other assays, as described above, inhibited the proliferation of K562 cells. For example, two inhibitory peptides were Gly-Tyr-Pro-Gly-Gln-Val-Cys- Ala-Asn-Asp-Ser-Asp-Thr-Leu-NH₂ (SEQ ID NO: 11), and Gly-Tyr-Pro-Gly-Lys-Phe- Cys-NH₂ (SEQ ID NO: 15). In contrast, the human and murine PAR4 hexapeptides (Gly-Tyr-Pro-Gly-Gln-Val-NH₂ (SEQ ID NO:3), and Gly-Tyr-Pro-Gly-Lys-Phe-NH₂ (SEQ ID NO: 13), respectively) failed to inhibit cell proliferation.

Claims

CLAIMS What is claimed is:

1. A peptide, comprising an amino acid sequence selected from the group consisting of:

(a) Gly-Tyr-Pro-Gly-Gln-Val-Cys-NH₂ (SEQ ID NO:4),

(b) Gly-Tyr-Pro-Gly-Gln-Val-Cys-Ala-NH₂ (SEQ ID NO:5), and

(c) Gly-XaarPro-Gly-Lys-Xaa₂-Xaa₃-NH₂ (SEQ ID NO:56), wherein

Xaa_! is selected from the group consisting of Tyr, Tyr(Me), Bip, and 2- Nal,

Xaa₂ is selected from the group consisting of Phe, hPhe, Phe(4-F), Phe(4-Me), Thi, 1-Nal, 2-Nal, and Bip, and Xaa₃ is Cys or Pen.

2. The peptide of claim 1, wherein peptide (c) is Gly-Tyr-Pro-Gly- Lys-hPhe-Cys-NH₂ (SEQ ID NO:31).

3. The peptide of claim 1, wherein peptide (c) is Gly-Tyr(Me)-Pro-

Gly-Lys-Phe-Cys-NH₂ (SEQ ID NO:37).

4. The peptide of claim 1, wherein peptide (c) is Gly-Tyr-Pro-Gly- Lys-Phe(4-F)-Cys-NH₂ (SEQ ID NO:39).

5. The peptide of claim 1, wherein peptide (c) is Gly-Tyr-Pro-Gly- Lys-Phe(4-Me)-Cys-NH₂ (SEQ ID NO:42).

6. The peptide of claim 1, wherein peptide (c) is Gly-Bip-Pro-Gly- Lys-Phe-Cys-NH₂ (SEQ ID NO:44).

7. The peptide of claim 1, wherein peptide (c) is Gfy-2Nal-Pro-Gly- Lys-Phe-Cys-NH₂ (SEQ ID NO:50).

8. The peptide of claim 1, wherein peptide (c) is Gly-Tyr-Pro-Gly-

Lys-Thi-Cys-NH₂ (SEQ ID NO:53).

9. The peptide of claim 1, wherein peptide (c) is Gly-Tyr-Pro-Gly- Lys-Phe-Pen-NH₂ (SEQ ID NO:54).

10. The peptide of claim 1, wherein peptide (c) is Gly-2Nal-Pro-Gly- Lys-Phe-Pen-NH₂ (SEQ ID NO:55).

11. A composition, comprising a carrier and a peptide of claim 1.

12. A method of stimulating platelet aggregation, comprising administering the composition of claim 11 to platelets.

13. The method of claim 12, wherein the composition is administered to a mammalian subject.

14. A method of inhibiting tumor cell proliferation, comprising administering the composition of claim 11 to tumor cells.

15. The method of claim 14, wherein the composition is administered to a mammalian subject.