UA75037C2 - Inhibitors used for hemostasis and modulation of immune function - Google Patents

Abstract

The invention relates to the use of protein related to adipocyte-complement providing for hemostasis and modulation of immune function. The inhibitorsused belong to the protein family giving the origin to collagen-like domen and globular one. The inhibitors promotes the blood flow due to decreasing thrombogenic activity complement activity and may be advantageous for providing the inert properties to the surface of the biologic material used for prosthesis as well as for modulating the wound healing.

Description

Description of the invention

Blood vessel injury triggers a series of factors to reduce damage and regulate 2 outflow of blood from the vessel. This process is known as hemostasis. Platelets play a role in early hemostasis by forming a thrombus or plug to temporarily repair damaged vessels. Platelets do not normally interact with the endothelial lining of vessel walls, but injury to blood vessels as a result of an accident or during surgery can destroy the endothelial cells. Depending on the degree of injury, such different subendothelial elements as collagen, elastic film or smooth muscle cells in combination with 70 fibrillar collagens will act on the flowing blood.

When the subendothelium is exposed after vascular injury, platelets traveling in the local blood flow interact with the exposed subendothelial matrix, which contains collagen, and slow down. Further, the interaction between receptors on the surface of platelets and the exposed collagen layer leads to the binding and activation of platelets, which leads to a delay in local blood flow. bridges (Mogoi apa dipo, Egopiiege ip Viozsiepse Z: 719-28, 1998; Vagpez ei ai., AiShegozsiegovov Hi, dasoioi ei ai., edv., Eivemieg Zsiepse rr. 299-306, 1998 and Vagpez ei a!., Sit . Orip. Netagyo)., 5: 314-20, 19981.

The hemostatic reaction changes and depends on the degree of wounding of the blood vessel, the exposed components of specific blood vessels and the conditions of the blood flow in the injured area (Kapa ei ai., Tygotroziz apa Naetosiaziv, 78: 445-50, 1997)|. Exposure of the subendothelial matrix (collagen type MI and Willebrand factor), as in moderate vascular injury, contributes to a low degree of adhesion, activation or aggregation in the zone in conditions of weak blood flow (the word is not visible in the text - translation note) which leads to more degree of vascular injury, and exposure of additional vascular components, such as the inner elastic film and elastin-associated microfibrils, will stimulate the formation of stronger platelet aggregates. Severe vascular injury, exposing the fibrillar collagen, provokes a platelet response that protects the victim from excessive blood loss (Kappa ei a!., above). Gee)

Inhibitors of hemostasis could be useful in increasing blood flow after vascular injury and soothing collagen surfaces.

Complement factor Sid consists of six copies of three related polypeptides (chains A, B, and C), each of which is 225 amino acids long with a tightly bound amino-terminal collagen domain and a carboxy-terminal globular region. The six triple helical regions are formed by the collagen co-domains of six A, six B, and six C chains forming a central region and six stems. The globular head part is formed by the combination of the globular carboxy-terminal domain A, B and C of the Seed chain and therefore consists of six globular heads connected by six collagen-like stalks of the central fibrillar region. IZeyPag ei a!., Viospet. 9. 274: 4881-90, 1991). This configuration is often referred to as a bouquet of flowers.

Zo Asgr30O has a bouquet-like structure formed from one type of polypeptide chain. -

It was found that Seed stimulates protective mechanisms, and also initiates the generation of toxic oxygen particles that can cause tissue damage. My. 93: 241-53. 1993). Seed binding sites are found on platelets. In addition, complement and Sid play a role in inflammation. Complement activation is initiated by the attachment of Seed to immunoglobulins. C 50 Inhibitors of Sid and the complement pathway could be useful for anti-inflammatory applications, with inhibition of complement activation and thrombotic activity.

From" The invention provides such polypeptides for this and other uses as those skilled in the art should understand from the information provided herein.

According to one aspect of the invention, a method of stimulating blood flow in the vascular system of a mammal is proposed, which includes administering to said mammal a therapeutically effective amount of adipocyte-complement-related protein 7 in a pharmaceutically acceptable excipient, wherein said adipocyte-complement-related protein reduces thrombogenic activity and complement activity in the indicated vascular system. According to one embodiment of the invention, the adipocyte-complement-related protein includes a polypeptide that has a sequence of amino acid residues that is at least 7595 identical to the amino acid sequence of 20 5ED IU MO:2 at residues 26-281, where the specified sequence includes the repetition of Siu-Haa- Haa or Siu-Haa-Rho, forming a collagen domain where Haa is any amino acid and the carboxy-terminal globular moiety. "m According to a related embodiment of the invention, the polypeptide includes a sequence of amino acid residues that is at least 9095 identical to the amino acid sequence of ZEO IYUO MO:2 at residues 22-281. According to another embodiment of the invention, the polypeptide includes an amino acid sequence that is at least 9095 identical to the 22 amino acid sequence of ZEO IU MO:2 for residues 26-281. According to another embodiment of the invention, any

GF) differences between the specified polypeptide and 5EO IU MO:2 are caused by conservative amino acid substitutions. According to another embodiment of the invention, the collagen domain consists of 13 Siu-Haa-Haa repeats and 1 Siu-Haa-Rgo repeat. According to another embodiment of the invention, the globular domain consists of ten beta-sheets. According to a related embodiment of the invention, beta-sheets are conjugated with amino acid residues corresponding to residues 147-151, 170-172, 178-181, 191-203, 207-214, 219-225, 227-239, 244-250, and 269-274 of the sequence ZE and MO:2. According to another embodiment of the invention, the polypeptide includes residues 1-281 of the sequence ZEO IJU MO:2 or residues 1-281 of the sequence ZEO IJU MO 44.

According to the invention, a polypeptide that is attached to a second polypeptide to form an oligomer is also proposed. According to one embodiment of the invention, the polypeptides are connected by intermolecular disulfide bonds. According to another embodiment of the invention, the oligomer is a trimer. According to another embodiment of the invention, the oligomer is a hexamer. According to another embodiment of the invention, the multimer is an 18-mer.

According to another embodiment of the invention, the polypeptide reduces thrombogenic and complement activity by inhibiting the complement exchange pathway and inhibiting collagen-mediated adhesion, activation or aggregation of platelets. According to another embodiment of the invention, the polypeptide is administered before, during or after an acute injury to the vessels of the specified mammal. According to another embodiment of the invention, the injury is caused by the reconstruction of blood vessels. According to a related embodiment of the invention, vascular reconstruction includes angioplasty, coronary artery bypass grafting, endarterectomy, microvessel repair, or vessel graft anastomosis.

According to another related embodiment of the invention, the injury is due to trauma, stroke or aneurysm. 70 According to another aspect of the invention, a method of restoring damaged collagen tissue of a mammal is proposed, which includes administering to said mammal a therapeutically effective amount of adipocyte-complement-related protein, where said protein renders damaged collagen tissue inert to complement activation, thrombotic activity, or immune activation. According to one embodiment of the invention, collagen tissue damage is caused by injury associated with ischemia and reperfusion.

According to another embodiment of the invention, the injury includes traumatic ischemia, intestinal strangulation, or injury associated with prior and subsequent establishment of blood flow. According to another embodiment of the invention, the polypeptide is administered to a mammal suffering from ischemia during artificial blood circulation and resuscitation (hesis, myocardial infarction, or post-traumatic vasospasm. According to a related embodiment of the invention, post-traumatic vasospasm includes a stroke, percutaneous transluminal anpoplasty, endarterectomy, or accidental injury vessels or vascular injury during surgery.

According to another aspect of the invention, a method of rendering the surface of a prosthetic biomaterial inert for use in combination with an animal is proposed, which includes administering to said mammal a therapeutically effective amount of adipocyte-complement-related protein, and said polypeptide renders the surface of said prosthetic biomaterial inert with respect to complement activation, thrombotic activity or immune activation. According to one embodiment of the invention, the surface of the specified prosthetic biomaterial is covered with collagen or collagen fragments, gelatin, fibrin or i) fibronectin.

According to another aspect of the invention, a method of mediating wound healing in a mammal is proposed, which includes administering to said mammal a therapeutically effective amount of Mzo adipocyte complement-related protein, and said polypeptide enhances progress in wound healing.

Fig. 1 illustrates a multiple overlay of polypeptide 28id37 according to the invention and NOMIORZTO2 1 (Maeeeada ei ai., o

Viospet. Viorpuz., Ke5. Sott. 221(2):286-9, 1996); STLOA LUDYNY (zegpagei a!., Viospet y. 274:481-90, 1991, ("In

Keid, Viospet ..179: 367-71,-1979, and Keid ei aI., Viospet U 203: 559-69, 1982), NR25 TAMAZ (TaKataivy ei a!., Moi. Seiyi. Visi. 13:1516- 21, 1993 and Kopdo 45. Kopado, U.Vioi Spet 267: 473-8, 1992), NR27 TAMAZ (TaKataivy ei Shcheo, a)I., and Kopdo 8: Kopado getegepsei abrome); and SEC! CAT (U/ada 5. Opiapi, Vgaip Kez, Moi. Vgaip. Kezv., 9:71-7, 1991). uh-

Fig. 2 presents a matrix showing the percentage of amino acid identity compared to the six proteins shown in Fig. 1 in a multiple overlay.

Fig. Za shows the attachment of 285id37-RITS to MI type collagen.

Fig.za shows the competition of unlabeled 285id37 with labeled RITS 28id37 in binding to collagen type MI. "

Fig. 4 shows the addition of complement SiId-RITS to 28id37. with c Fig. 5 shows the inhibition of 25id37-activity of human complement.

Fig. 6 shows the percentage of aggregation of platelets by collagen in the presence of 25id37. ;" Fig. 7 shows the proliferation of ZK5 fibroblasts in the presence of 28id37.

For a detailed explanation of the invention, the definition of the following terms may help.

The term "affinity tag" is used to refer to a peptide segment that can be attached to a polypeptide to provide purification or detection of the polypeptide, or to provide sites for attachment of the polypeptide to a substrate. In principle, any peptide or protein for which there is an antibody or other specific binding agent can be used as an affinity tag. o Affine labels include a polypstidine region, protein A (Miyivzop ei ai., EMVO .., 4:1075, 1985; Miyivzop 5o ei ai. Meifoiv Eplutoi. 198.3, 1991), glutathione-Z-transferase (Ztiy apa Odoppvop, Sepe 67 :31, 1988), or substance P, Riadtm peptide (Norri et al., Biol., Biol. 6:1204-10, 1988, available from Eazitap Kodak Co., Ltd., Mem Namep, ST), streptavidin binding peptide, or other antigenic epitope or binding domain See generally Roga ei ai., Rgoivip Ehrgezviop apa Rigiisaiop 2:95-107, 1991 DNAs encoding affinity tags available from commercial suppliers (eg, Riagtasia Vioyesii, Rizzagamau, MU).

The term "complements of polynucleotide molecules" refers to a polynucleotide molecule having a complementary sequence of bases and the reverse orientation compared to the reference sequence, (F, for example, the sequence 5 АТОСАСОСО 3 is complementary to 5 СССОТОСТ 3. co The term "degenerate nucleotide sequence" refers to the sequence of nucleotides that includes one or more degenerate codons (compared to the linker polynucleotide molecule that encodes a polypeptide) because degenerate codons include distinct triplets of nucleotides but code for the same amino acid residue (ie, CA and CAC triplets each code for Agr) .

The term "isolated" when applied to a polynucleotide means that the polynucleotide has been removed from its natural genetic environment and is therefore free of other foreign or unwanted coding sequences and exists in a form suitable for use in a genetically engineered protein production system. 65 Such isolated molecules are those removed from their natural environment, | include cDNA and genomic clones.

Isolated DNA molecules according to the invention are devoid of other genes with which they are usually conjugated but may include naturally occurring 5 and 3 untranslated regions, such as promoters and terminators. Identification of conjugated regions is readily apparent to those of ordinary skill in the art (see, e.g., Yuupap apa Tidap, Maishge 316:774-78 1985). An "isolated" polypeptide or protein is a polypeptide or protein obtained in an environment other than its natural environment, such as outside the blood and tissue of an animal. In a preferred form, the isolated polypeptide is essentially devoid of other polypeptides, in particular other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 9595 purity, preferably greater than 9995 purity. When used in this context, the term "isolated" does not exclude the presence of such polypeptide in alternative physical forms such as dimers or alternatively glycosylated or derivatized forms. 70 The term "ortholog" refers to a polypeptide or protein derived from one species that is a functional analogue of a polypeptide or protein from a different species. Sequence differences among ortholops are the result of speciation.

The term "polynucleotide" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases with readings from the 5 to the 3' end. Polynucleotides include RNA and DNA, and they can be isolated from natural sources, synthesized by humans, or made from a combination of natural and synthetic molecules. The sizes of polynucleotides are expressed as base pairs (abbreviated as "po"), nucleotides ("nc"), or kilobases ("ko"). When the context permits, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to a double-stranded molecule, it is used to refer to the total length and should be understood to be equivalent to the term "base pairs". Those skilled in the art will understand that the two 2o strands of a double-stranded polynucleotide may differ slightly in length and therefore their ends may be staggered as a result of cleavage by enzymes, so not all nucleotides in a double-stranded polynucleotide may be paired. Such unpaired ends usually do not exceed 2Onc in length.

Polypeptide: Is a naturally or synthetically produced polymer of amino acid residues connected by peptide bonds. Polypeptides of less than about 10 amino acid residues are usually referred to as "peptides". "Probes and/or primers" can be RNA or DNA. The DNA can be cDNA or genomic DNA. and)

Polynucleotide probes and primers are single- or double-stranded DNA or RNA, usually synthetic oligonucleotides, but can be created from cloned cDNA or genomic sequence or their complements. Analytical probes are usually at least 20 nucleotides long, although shorter probes (14-17 nucleotides) can sometimes be used. RSK primers have a length of at least 5 nucleotides, preferably 15 or more nucleotides, preferably 20 nucleotides. Short polynucleotides can be used when the goal of analysis is a small region of the gene. For general gene analysis, the polynucleotide probe may include the entire exon or more. Probes can be labeled to provide a detectable signal, such as enzyme, biotin, radionuclide, fluorophore, chemiluminophore, paramagnetic particle, etc., which are commercially available from many sources, such as Moiesciag Rgorevs, Ips. Etsdepe OK, and Ategepat Sogr., Agipdiop Neiidniv, I, i- using methods well known in the state of the art.

Molecular weights and lengths of polymers were determined by approximate analytical methods (eg, gel electrophoresis), and should be understood as approximate. When such a value is designated as "approximately" X or "the value of X determined near X, it should be understood that it was determined with an error of -10905. "

The present invention is particularly based on the discovery that a new homologue of adipocyte-complement-related protein 8c inhibits collagen-mediated activation of platelets and the exchange and complement pathway, including Sid. This protein was designated as 25id37 and fully described in the generally defined "" published (patent application PCT UUO 99/04000)1.

Nucleotide sequence 25i937 (5BO IO MO:1) encodes a polypeptide (ZEO IUO MO:2) having an amino-terminal signal sequence, an adjacent M-terminal region lacking homologues, a shortened collagen domain consisting of repeats (zIu-Xaa -Xaa or Siu-Xaa-Pho, and a carboxy-terminal globular portion. The novel polynucleotide sequence also includes a long C untranslated region. The above general polypeptide structure is shared by Asgr3O and NOMYRBTO2 1, except that the collagen-like domain of each of of these proteins is longer than that of the 28-37 polypeptides. Also, the DNA sequence NIOMYRB5TI2 1 50 is characterized by a long 3 untranslated region. Moreover, Asgr3ZO and all of the sequences connected in Fig. 10, with the exception of SEKI! CAT, use a conserved cysteine residue at position 187 "I of the polypeptide 285id37, as shown in Fig. 1 and in the sequence zEO IU MO: 2. Also, polypeptides 28id37 according to the invention include a putative M-linked glycosylated site on the amino acid 93 (Azp) sequence of ZEO IU

MO:2.

Analysis of the tissue distribution of mRNA corresponding to 25id37 showed that expression was highest in the heart and placenta with relatively weak signals in the kidney, ovary, adrenal gland, and skeletal muscle, and weaker iF) signals for a wide variety of other tissues were present in northern -bloti For 25id37, a homologous relationship with the adipocyte-complement-related protein, Asgr3O (ZEO IYUO MO:3) and the adipocyte-secreted protein armI (NOMORBZTO2 1 in Fig. 1 and 2) was proven. A somewhat more distant homology was also identified for the bo chain of the complement component STO A , two factors observed in the active state of winter hibernating Siberian marmots (NR25 TAMAB and NR27 TAMAbZB), and rat brain protein (SEC! CAT), as shown in Fig. 1 and 2.

Nucleotide sequence 28id37 is described by the sequence ZEO IJU MO, and the amino acid sequence derived from it is described by the sequence 5EO IJU MO:2. The degenerate nucleotide sequence encoding a polypeptide with b5 sequence ZEO IU MO:2 is represented by the sequence BEO IU MO:23. As mentioned above in general, the 28idD37 polypeptide includes a signal sequence extending from amino acid 1 (Me) to the amino acid residue

21 (Su).

The alternative signal sequence extends from amino acid 1 (Me) to amino acid 25 (5eg).

The unfolded polypeptide therefore extends from amino acid 22 (I esh) or 26 (Aga) to amino acid 281 (Pho). IN

The M-terminal region of an unknown homolog was found in the unfolded polypeptide, extending between amino acid residues 22 (I ec) and 98 (I uz). In addition, a shortened collagen domain is found between amino acids 99 (Su) and 140 (Agu). 1 complete Siu-Haa-Rho and 13 incomplete repeats are observed in the shortened collagen domain

SiIu-Haa-Haa. In contrast, Asgr3O comprises 22 complete or incomplete repeats. Polypeptide 25id37 also includes a carboxy-terminal globular domain extending from approximately amino acid 141 (Suv) to 281 (Pgo). 7/0 LPolypeptide 28937, NOMORBZT2 1 and Astr3O are found to be homologous in the collagen domain and in the globular domain, but not in the M-terminal part of the unfolded polypeptide.

The globular Seed domain from ASKRZO was determined to have a "jelly-roll" topology with 10 beta bands (ZParigo et al.

Zspegeg, Sy. Vio!., 8: 35-8, 1998), which indicates a significant structural homology with the TME family, and the sequence 28i9d37, which is represented by the sequence ZEO IU MO:2, includes all 10 beta bands of this structure (amino acid /5 residues 147 -151, 170-172, 178-181 185-188, 191-203, 207-214, 219-225, 227-238, 244-250, and 269-274 of the sequence "EU IU MO:2). These strands are labeled "A", "A", "B" B" MS MOM MET", "o" and "H", respectively. 28idZz7 has two receptor-binding loops on amino acid residues 152-180 and 213-226. Amino acid residues 191 (Su), 193 (Tug), 238 (Hei) and 272, (Su) were found to be conserved within the superfamily including

СО40, TMEA, TMRV ASERZO and 28id37.

Another aspect according to the invention includes the use of fragments of the 28-37 polypeptide as inhibitors of hemostasis and immune functions. Preferred fragments include the collagen-like domain of polypeptides 25id37, extending from amino acid 99 (Su) to amino acid 140 (Agu) of the sequence of "EO IU MO:2, a part of the polypeptide 25id37, which includes a collagen-like domain or a part of a collagen-like domain, capable of dimerization or oligomerization. Other preferred fragments include the globular domain of the 78IDZI polypeptide, extending from amino acid 140 (Ago) or 141 (Suz) to 281 (Rio) of the ZEO IU MOS2 sequence, and part of the 285ID37 polypeptide, which includes the globular-like domain or the active part of the globular -similar i) domain. The second fragment of the 25id37 polypeptide according to the invention includes both a collagen-like domain and a globular domain, extending from amino acid residue 99 (su) to 281 (Pho) of the ZEO IU sequence

MO:2. These fragments are particularly useful for inhibiting collagen-mediated platelet activation and M zo inhibition of complement and Sid.

According to the invention, the use of condensed proteins 285id37 is also proposed, for example, and condensed proteins according to the invention, which include (1) a polypeptide that is selected from the group consisting of (a) polypeptide molecules containing the sequence of amino acid residues, which is shown in 5EO IU MO :2 from amino acid residue 1 (Me) 22 (Hetz) or 26 (Ago) to amino acid residue 281 (Pho), (b) polypeptide molecules extending from amino acid 99 (Su) to amino acid 140 (Agd) of the BEO IU sequence MO:2, the part of the 25-37 polypeptide, which includes a collagen-like domain or a part of a collagen-like domain capable of dimerization or oligomerization, (c) polypeptide molecules extending from amino acid 140 (Ag) or 141 (Suz) to 281 (Pho) of the ZEO IHUO MO:2 sequence, part of the 285id37 polypeptide, which includes the globular-like domain or the active part of the globular-like domain, or (a) polypeptide molecules "extending from amino acid 99 (SU) to 281 (Pho) , ch astins of the 28i937 polypeptide comprising a collagen-like domain and a globular domain, and (2) another polypeptide. Another polypeptide can be an alternative or additional globular domain, an alternative or additional collagen-like domain; domain, a signal peptide to facilitate the secretion of a condensed protein, or the like.

Also useful in the methods of the invention are 25id37 agonists and antagonists. Methods of identifying antagonists are known in the art, for example, antagonists of the 285id37 polypeptide can be identified by providing cells sensitive to the 25id37 polypeptide, culturing the first part of the cells in the presence of the 285id37 polypeptide, culturing the second part of the cells in the presence of the 25id37 polypeptide and the test compound, and determining the decrease in cellular reactions in the second part of the cells in comparison with the first part of the cells. In addition to the assay disclosed herein, samples can be tested for inhibition of 25id37 activity in 5or within a variety of assays designed to measure receptor binding or stimulation/inhibition of 25id37-dependent cellular responses, for example, 28id37-sensitive cell lines can be transfected with the construct

And the reporter gene, which is sensitive to the stimulated 25-37 cellular metabolic pathway. Reporter gene constructs of this type are known in the art, and typically include a 25-37 DNA response segment operably linked to a gene encoding a target protein, such as luciferase. Reaction elements of DNA can include, but are not limited to, reaction elements of cyclic AMP (SKE), reaction elements of hormone (NKE), reaction element of insulin (KE) (Mazgt ei aiYi., Rgos. Mai. Asad. Zsi. OBA, 87:5273 -7 1990) and

F) serum reactive elements (ZKE) (ZPPam ei aiYi., Se! 56:563-72, 1989). Reaction elements of cyclic AMP are described in KoezTseg ei ai., 9). Biol Spec., 263(19):9063-6, 1988 and Nabrepeg, Moises Epadostgipo!, 4(8):1087-94, 1990. Hormone reaction elements are described in Biol, Sci. 56:335-44, 1989. Candidate compounds, solutions, mixtures or extracts are united by their ability to inhibit the activity of 285i937 in target cells, which is evidenced by the reduction of stimulation of 25i937 expression of the reporter gene. Studies of this type will identify compounds that directly block the attachment of 275id37 to cell surface receptors, as well as compounds that block processes in the cellular pathway of exchange as a result of receptor-ligand binding. Alternatively, compounds or other samples can be tested to directly block binding of 25id37 to the receptor using 28id37, 65 labeled with a detectable label (eg, 125), biotin, horseradish peroxidase RITS, etc.). In studies of this type, the ability of the test sample to inhibit binding of labeled 25id37 to the receptor is an indicator of inhibitor activity, which can be confirmed by secondary studies. Receptors used in receptor studies can be cellular receptors or isolated, immobilized receptors.

Also useful in the methods of the invention are antibodies that specifically bind to polypeptide epitopes, peptides or 25-37 polypeptides. Methods of making polyclonal and monoclonal antibodies are well known in the art (see, for example, ZatbgooK ei aiYi., MoiIesshag Siopipd-: A I aroghaiogu Mapiai.

Zesopa Eaiiop, Soii Zrgipd Nagrog, MU, 1989, and Niiteii, 9U.S.K., Ea., Moposiopai Nubpdota Apiirodu

Tesppidcezta Arriisayopz, SKS Rgezv, Ips., Vosa Kap, RI, 1982)

As experts should know, polyclonal antibodies can be created by inoculation of various warm-blooded animals, such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, hamsters, guinea pigs and rats, as well as transgenic animals such as transgenic cows , goats, sheep or pigs Antibodies can also be expressed in yeast and fungi in modified forms, as well as in mammalian and insect cells. The 25iud37 polypeptide or its fragment serves as an antigen (immunogen) for inoculating an animal or detecting an immune response. Suitable antigens could include the 28iud37 polypeptide encoded by the sequence 5EO IYU MO:2 from amino acid residue 22 75 TO amino acid residue 281 of the ZEO IYUO MO:2 sequence, from amino acid residue 26 to amino acid residue 281 of the ZEO IU MO:2 sequence, or their related fragment from amino acid residues 9-281.

The immunogenicity of the 28-37 polypeptide can be enhanced by the use of an adjuvant, such as alum (aluminum hydroxide) or complete or incomplete Freud's adjuvant. Polypeptides useful for immunization also include condensed polypeptides, such as condensates of 28id37 or portions thereof with an immunoglobulin polypeptide or an affinity tag. A polypeptide immunogen can be a full-length molecule or a part thereof. If a portion of the polypeptide is "hapten-like," such portion may be preferentially combined or linked with a macromolecular carrier (such as plate hemocyanin (CIN), bovine serum albumin (BSA), or tetanus toxoid) for immunization.

As used herein, the term "antibodies" includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments such as proteolytic fragments (ab)" and Rar. Also included are genetically engineered whole antibodies or fragments such as chimeric antibodies, Hm-fragments, and) single-chain antibodies, etc., as well as synthetic antigen-binding peptides and polypeptides. Non-human antibodies can be adapted to humans by grafting only the non-human SOC to the human framework and unaltered regions, or by inserting continuous non-human variable domains (alternatively, "coating" them with a human Mz surface by replacing exposed residues, resulting in a "veneered " antibody). In some cases, human-adapted antibodies may retain non-human residues in the framework domains and human variable regions to enhance proper binding characteristics. Human-adapted antibodies can increase the biological half-life and reduce the possibility of harmful immune reactions when applied to humans. Alternative methods of generation or selection of antibodies useful in this regard include treatment of lymphocytes with 285id37 protein or peptide, and selection of detectable antibody libraries in phage or similar vectors (eg, using immobilized or tagged 28id37 protein or peptide).

Antibodies are designated as specifically binding if 1) they exhibit a threshold level of activity prior to binding, and/or 2) they do not exhibit significant cross-reactions with cognate polypeptide molecules. "

First, antibodies bind specifically if they bind to a polypeptide, peptide, or epitope in c 28id37 with binding affinity (Ka) of 10 bmol-! or more, preferably 10'mol" or more, better and 1bmol"! or more, and preferably 109mol- or more. The binding affinity of an antibody can easily be determined by an average specialist, for example, by Scatchard's analysis (Zsaisnaga, App, MU Asaid. Zsi. 51: 660-672, 1949).

Second, antibodies bind specifically if they do not show appreciable cross-reactions with -i molecules of the cognate polypeptide Antibodies do not show appreciable cross-reactions with sl molecules of the cognate polypeptide, for example, if they detect the 25dZ/ polypeptide but no known cognate polypeptides using standard Western analysis -blotting (A!YizyBvei ei aI., above). Examples of known (a) related polypeptides include other members of the protein family, such as Asgr3O (ZEO IUO MO:3), polypeptides that are shown in Fig.1 in combination. They also include, as appropriate, orthologs and mutant human 25-37 polypeptides. In addition, antibodies can be "screened against" known cognate polypeptides to isolate a population that specifically binds to polypeptides of the invention, e.g., antibodies raised to human 25-37 polypeptides adsorb to cognate polypeptides that are attached to an insoluble matrix, antibodies, that are specific for human 25-37 polypeptides will flow through the matrix under proper buffering conditions. Such screening allows the isolation of polyclonal and monoclonal antibodies that do not show cross-reactions with closely related polypeptides (Apiirodiez A I arogaiogu Mapiai, Nagiozhm apa I ape o (edv.), Soii Zrgipd Nagrog Iarogaiogu Rgez5v, 1988; Sitepi RgoiosoYiv ip Ittipoiodo, Soopdap, ей ай. (Eav.) ime) Mayopa! Ipviishevz oi Neatsyi, Ropp UUupeu apa Bopv, Ips., 1995). Screening and isolation of specific antibodies is well known in the art |see Ripdatepia! Ittipoiodu, Ratsi (ed.), Kamep Rgezz, 1993; Seigoii ei bo a, Aim ip Ittipoi 43: 1-98, 1988; Moposiope! Apiirodiev Rgipsirieg apa Rkgasiise. Soadipo, 9.MU. (Edv.).

Asadetis Rgezv Tsa., 1996; Vepiatip ei aiI., App Keu. And so on. 2: 67-101, 1984). Representative examples of such assays include competitive immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blotting or Western blotting, inhibition or competition assays, and sandwich assays. 65 The effect of polypeptides, fragments, condensates, agonists or antagonists of 25id37 in hemostasis, in particular, adhesion and activation of platelets, which leads to platelet aggregation, can be determined by the methods and assays presented here and known in the art. Collagen is a powerful inducer of platelet aggregation. This results in a risk to the patient recovering from vascular injuries. Inhibitors of collagen-induced platelet aggregation could be useful for such a purpose. It was found that 785id37 binds to fibronectin and collagens of type !, II, Ш, M and MI. In particular, 28iZ37 binds to specific domains on MI collagen in a concentration-dependent manner. It was also found that 728id37 inhibits collagen-mediated activation of platelets.

The inhibition induced by 725id37 was selective for collagen activation. 258id37 did not affect platelets activated by known platelet activators AOR or thrombin. These results are described in more detail below in the Examples section. 25id37 polypeptides, fragments, condensates, agonists, or antagonists would be expected to be 7/0 useful in blocking platelet attachment to collagen-coated surfaces and reducing conjugated collagen-induced platelet aggregation.

Seed is a component of the complement pathway and has been found to stimulate defense mechanisms and trigger the formation of toxic oxygen particles that can cause tissue damage (Teppeg, Vepygipa Ipvi Mii 93 241-53, 1993). "Seed binding" sites have been found on platelets.

Cid, independent of the immune binding partner, was found to inhibit platelet aggregation, but not platelet adhesion or reshaping. The amino-terminal region of Sid has homology with collagen (ReegespKe et

OSPPebgeiimei, In Ittipoi. 145: 2984-88, 1990). 78idZz7 joins the Sid complement depending on the concentration.

285id37 was found to be effective in inhibiting the complement exchange pathway involving Seed with sensitized and non-sensitized sheep erythrocytes.

The polypeptides, fragments, fused proteins, antibodies, agonists or antagonists of 28id37 according to the invention can be used in methods of stimulating blood flow in the vascular system of a mammal by reducing the number of platelets that stick together and are activated and the size of platelet aggregates. Such methods would include administering to a mammal in need thereof a therapeutically effective amount of polypeptides, fragments, condensates, antibodies, agonists or antagonists of 25id37, and 275id37 reduces thrombogenic activity and complement activity in the vascular system of the mammal. As described below, polypeptides 285id37 inhibit collagen-mediated activation of platelets and inactivate the binding of fibronectin and collagens of type Y, i)

P, PS, M and MI. The appointment of 7iz3937 reduces thrombogenic activity in the area of vascular injury by reducing the ways of adhesion, activation and aggregation of platelets. Polypeptides, fragments, condensates, M zo antibodies, agonists or antagonists of 28id37, used in such methods, can be prescribed before, during or after an acute injury to the vessels of a mammal. and,

According to the preferred method, vascular injury is due to vascular reconstruction, including without limitation, anpoplasty, endarterectomy, coronary artery bypass grafting, microvascular repair, or vascular graft anastomosis. Also included is injury to blood vessels as a result of trauma, seizure, or aneurysm. о з5 According to other preferred methods, vascular injury is caused by the destruction of plaques, degradation of the vascular system, and complications associated with diabetes and atherosclerosis. Plaque destruction in the coronary artery induces a heart attack, and in the cerebral artery induces a stroke. The use of polypeptides, fragments, fused proteins, antibodies, agonists or antagonists of 25id37 in such methods could also be useful for the relief of all systemic diseases of the vasculature coupled with immune dystemia, such as disseminated intravascular coagulation (DIC) and CVD. Additionally, c complement inhibition activity could be useful in the treatment of immune diseases unrelated to the vascular system, such as arteriosclerosis. ;" A correlation was found between the presence of SID in localized ischemic myocardium and the accumulation of leukocytes after coronary occlusion and reperfusion. The release of cellular components after tissue damage triggers complement activation, which leads to the formation of toxic oxygen products, which can be the root cause of myocardial damage (Kozzep et al., Sigs. Kev. 62: 572-84, 1998 and Teppeg, supra). It was found that blocking the pathway of complement exchange protects ischemic myocardium from reperfusion injury (Viegke ei ai|., o U.Rpagt. Ehr. Tpegr. 286 429-38, 1998). Inhibition of complement and the activity of polypeptides 25id37 in relation to the binding of Sid could be useful for such a purpose.

The potential ability of 25id37 to bind SID and collagen could be useful for the repair of damaged collagen tissues, prevention of adhesion, activation or aggregation of platelets, and activation

And the inflammation process, which leads to the release of toxic oxygen products. By rendering exposed tissue inert to phenomena such as complement activity, thrombotic activity, and immune activation, polypeptides, fragments, condensates, antibodies, agonists, or antagonists of 28i937 could be useful in reducing the deleterious effects of ischemia and reperfusion. In particular, such injuries may include traumatic ischemia, intestinal strangulation, and injuries associated with prior and subsequent occlusion of blood flow.

F) 78iZ37 could be useful in the treatment of ischemia during artificial circulation and resuscitation, myocardial infarction and post-traumatic vasospasm, such as a stroke or percutaneous transluminal angioplasty, as well as accidental vascular injury or vascular injury during surgery. Polypeptides, fragments, condensates, antibodies, agonists or antagonists of 78id37 could also be useful for treating prosthetic biomaterials and surgical equipment to render the surface of the materials inert with respect to complement activation, thrombotic activity or immune activation. Such materials include, but are not limited to, collagen or collagen fragment coated biomaterials, gelatin coated biomaterials, fibrin coated biomaterials, fibronectin coated biomaterials, heparin 65 coated biomaterials, collagen and gel coated stents, arterial grafts, synthetic heart valves, artificial organs or any which prosthetic applications in contact with blood, which attach 25 of 37 with an affinity greater than 1x1089. Coating of such materials can be performed using methods known in the art, see | for example, Kybrepz, US Patent 5272074).

Complement and Sid play a role in inflammation. Complement activation is initiated by the binding of Sid to immunoglobulins (Choppziop, Reayaig. Ipbesi. Riv. U. 12: 933-41, 1993; MUaga apa ONeye, Tpegar. Ittipoi. 2: 77-94, 1995) Sid and complement inhibitors could be useful as anti-inflammatory agents. Such use can be made to prevent infection. Additionally, such inhibitors can be used in individuals suffering from inflammation mediated by complement activation and binding of immune complexes to SID.

Polypeptides, fragments, fused proteins, antibodies, agonists or antagonists of 25id37 could be useful in 70 ways to mediate wound healing, enhancing progress in wound healing by overcoming impaired wound healing. Progress in wound healing could include, for example, elements such as reducing inflammation, restoring fibroblasts, closing the wound, and suppressing infection.

The ability of tumor cells to attach to collagen may promote tumor metastasis Inhibitors of attachment to collagen are also useful in mediating adhesive interactions and the spread of tumor metastases 75 |MoezKe-)ipdriis ei ai., US Patent 57233121.

28id37 was found to induce vasodilatation in norepinephrine-narrowed aortic rings using the method of Oaipiu et al., U. Rnagtasoi 100: 767, 1990 and KNee et al., Mayogioh 16: 179, 1995, as described in more detail below.

Adhesion, activation, and aggregation of platelets can be assessed using methods described herein or known in the art, such as the platelet aggregation assay (Spiapud et al., Tygotroziv Kevz. 37: 605-12, 1985) and the platelet adhesion assay (ReegzspKe and Spebgeipimei , U. Ittipoi. 144: 221-25, 1990). Inhibition of SID and the complement pathway can be determined using methods disclosed herein or known in the art, as described by Zira and Svzako, et al. And so on. 117: 304-9, 1976. Measurement of platelet adhesion to collagen and inhibition of collagen-induced platelet aggregation can be performed using methods described

KayPeg ei ai., 9. VIOYI. Spent 26: 5450-6, 1993, MUakhtap apa SoppoiPuU, 9U. Life Spent 268: 5445-9, 1993; Ha

MoevzKe-Uyparichi eyi ai., U. Vioi. Spent 269: 5050-3, 1994 or OesKtup ei a/!., Viosa, 85: 712-9, 1995.

Various models of ip migo and ip mimo are available to evaluate the effects of polypeptides, fragments, condensed o proteins, antibodies, agonists and antagonists of 25i937 on ischemia and reperfusion injury. See, for example,

Zpapaeiua ei ai., Sigstiatsyop, 38: 2812-26, 1993; MUeivtap ei aiYi., Zsiepse 249: 146-151, 1991; Vyuegke her ai.,

Sigstiaop 91: 393-402, 1995; Nogviisk ei aiYi., Sigstaiop 95 701-8, 1997 and VygKe ei aiYi, 9. RNiag. Ex. rch- Tegr. 286: 429-38, 1998). Analysis of hamster platelet aggregation is described above. Bleeding time in hamsters and baboons can be measured after injection of polypeptides 25 and 37 using the model described in OesKtup ei aiI., above. measured using the hamster femoral vein thrombosis model proposed by YuResKtup ei ai., above. Changes in platelet adhesion under flow conditions after the application of 25-37 can be measured using the method described in Nagzia/Miei A!., Viosi 85: 705-11, 1995. -

Inhibition of complement and wound healing by polypeptides, fragments, condensed proteins, antibodies, agonists or antagonists of 25id37 can be studied alone or in combination with other known inhibitors of collagen-induced platelet activation and aggregation, such as paldipine, moubatin or kaline.

Polypeptides, fragments, condensed proteins, antibodies, agonists or antagonists of 28id37 can be evaluated using the methods described here or by methods known in the art, such as the healing of dermal layers in pigs (Igupsi ei ai., Rgos. Mai. Asad. Zsi. OBA 84: 7696-7000, 1987) and full-thickness skin wounds in i" genetically diabetic mice, for example, (Sgeepnaidn ei ai., At. y). Raiyoi. 136: 1235-46, 1990) Polypeptides according to the invention can be studied alone or in combination with other known complement inhibitors described above. -And in addition, polypeptides, fragments, condensates agonists or antagonists of 25iud37 may be therapeutically useful for antimicrobial applications. For example, the complement component Sid plays a role in the protection and the host organism from infectious agents such as bacteria and viruses Cyd is known to exhibit several highly specialized functions, for example Cyd initiates the complement cascade by interacting with bound antibody or C-reactive protein (SKR). Also, Seed interacts directly with some bacteria, RNA viruses,

Mami mycoplasma, uric acid crystals, lipid-A component of bacterial endotoxin, and membranes of some intracellular organelles. Attachment of Sid to the Sid receptor is thought to enhance phagocytosis. ,

Choppwiop, Reayaig. Ipbesi. Ditch. in. 12(11): 933-41, 1993). Therefore, soluble Sid-like molecules can be

Useful as antimicrobial agents that enhance lysis or phagocytosis of infectious agents.

Positively charged extracellular triple helical collagen domains of Seed and saprophyte receptor

F, macrophages were defined as those that play a role in binding ligands and have, as it was shown, a clear specificity of binding polyanions (Asiop eii ai., U. ViOII. Spet. 268: 3530-37, 1993). Lysophospholipid growth factor (lysophosphatidic acid, I RA) and other mitogenic anions are localized in the area of damaged tissue and help in wound healing. RA causes many biological effects, including platelet activation and upregulation of the matrix ensemble. It can be assumed that IRA shows synergism with other blood coagulation factors and mediates wound healing.

The collagen domains of proteins, such as Sid and the saprophyte macrophage receptor, are known to bind acidic phospholipids, such as the IRA 9-mer region of the collagen domain 78id37, amino acid residues 127-135 65 of the ZEO IU MO:2 sequence, participates in homologous with the collagen domain of the sequence found on the Seed and macrophage saprophyte receptor. The interaction of polypeptides, fragments, condensates, agonists or antagonists of 285id37 with mitogenic anions, such as RA, can be determined using research techniques known at the level, see, for example, Asiop eyi a!., above. Inhibition of the inflammatory process by polypeptides and antibodies according to the invention could also be useful in preventing infection at the wound site.

For pharmaceutical use, the proteins of the invention may be formulated with pharmaceutically acceptable carriers for parenteral, oral, nasal, rectal, topical, transdermal or similar administration by conventional methods. Predominantly, the application is carried out in the area of vascular injury or nearby. In general, pharmaceutical compositions include protein 25id37 in combination with a pharmaceutically acceptable excipient such as saline, buffered saline, 590 µl dextrose in water, and the like. The compositions may also include one or more excipients, preservatives, diluents, solubilizers, buffering agents, albumin to prevent loss of protein on the surface of the glass, etc. Forming methods are well known in the art and disclosed (for example, in Ketipdiop Tpe

Zsiepse and Rgasiise oi Rpapptasu Seppago, ed., Musk Rybiizpipd So Eaviop RA, 1910 ey., 1995). Therapeutic doses are usually determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient characteristics, etc. Determination of the dose is at the level of competence of the average specialist.

As used herein, the term "pPharmaceutically effective amount" of a polypeptide, fragment, condensed protein, agonist or antagonist of 28-37 is an amount sufficient to induce the desired biological result. The result may be alleviation of the symptoms or causes of the disease, or any other need

Changes in the biological system. For example, an effective amount of the 28-37 polypeptide is such as to provide subjective relief of symptoms or objectively detectable improvement as noted by a clinical or other skilled observer. Such an effective amount of the 25id37 polypeptide could provide, for example, inhibition of collagen-activated activation of platelets and the complement exchange pathway, including Sid, an increase in blood flow localized in the patient's vascular system and/or suppression of the harmful effects of ischemia and Ha reperfusion. The effective amount of 28-37 polypeptides can vary greatly depending on the disease or symptom being treated. The amount of polypeptide required for use and its concentration in i9) of the composition depends on the selected filler, the route of application, the power of a certain polypeptide, the patient's clinical condition, side effects and stability of the compound in the composition. Therefore, the clinician will use an acceptable drug with an acceptable concentration in the composition, as well as the amount of applied rch-

Compositions depending on clinical experience with the considered or similar patient. Such amount will depend, in particular, on the specific condition to be treated, the age, body weight, and general health of the patient, as well as other factors understood by the skilled worker. Usually the doses will be in the range of 0.01-10Omg/kg body weight! mass of the subject. For applications such as balloon catheters, typical doses will be in the range of 0.05-5mg/kg subject weight. Doses for certain compounds can be determined from ip migo or ex mimo studies along with experimental animal studies. Concentrations of compounds found to be effective ip migo or ex mimo - provide guidance for animal studies, while doses are calculated to provide similar concentrations at the site of action.

The invention is further illustrated by the following non-limiting examples. "

Example 1 Propagation of the E5T sequence

Polynucleotides according to the invention, encoding 28-37 polypeptides, were first identified by E5T selection from the E5T database, predicting the protein sequence based on n, and searching databases of known sequences and secreted protein that are most homologous to the predicted protein based on E5T. ETs that potentially "encode" proteins with biologically interesting homology to known secreted proteins were identified for further study. The single sequence of E5T was revealed and predicted to be homologous to a specific adipocyte protein. 270 (45): 26746-9, 1995 To identify -I the corresponding cDNA, the considered clone, which probably includes a continuous coding sequence, was used for sequencing. , Zap Oiedo, CA) according to the manufacturer's instructions, bml of the overnight culture in IV B--50 (μg/ml) ampicillin was produced. o The template was sequenced on the DNA sequencer AVIRKISMtm model 377 (RegKt-EIteg Sey5, MogmaikK, SU , syu 50 using the kit AVI RKIZMtm Bue Tepgttipayug Susie Zedcepsipd Keadu Keasiyup Kiyi (Regkt-EIteg Sogr.) according to the manufacturer's instructions Oligonucleotides 22695 (5EO IU MO:5), 22694 (5EO IU MO:6) for promoters ZR and "h T7 on vectors, o includes clones used as sequencing primers. Oligonucleotides 2213210 (5BEO

IO MO:7), 2213588 (5EO IU MO:8), 2213532 (5EO IU MO 9), 2213641 (5EO IU MO 10), 2213586 (ZEO IO MO. 11), 2C13651 (ZEO IU MO:12), 2213622 (5EO IYU MO:13), 2213625 (5EO IYU MO:14), 27213650 (ZEO IYUO MO:15), 2213589 (FAILURE MO:16), 2213624 (5EO IYU MO:17), 2213531 (5EO IYU MO:18 ), 2213587 (5EO IO MO:19) and 27213623 (5EO o IO MO:20) were used to complete the sequence from the clone. Sequencing reactions were performed on the system

NubBbayiy OttoOSepe Tetregaishte Susiipd buvieet (Mayopai Harpei So., MUuoDpade, MU). To analyze the data (name) we used the sequencing software ZEOSHOEMSNEK tm 3.0 (Sepe Sodez Sogrogayop, App

Agrog, MI). The resulting sequence of size 2769 by is revealed in the sequence 5Е0О ИЮО МО:1. Comparison of the 60 sequence of the original EZT with the sequence represented by the 5EO IU MO-1 sequence showed the presence of one undefined base pair (an unknown "M" residue) and no base pair insertions, leading to the identification of leucine upon uncertainty resolution and zero changes backbone between established amino acid sequences.

Example 2 Tissue Distribution 65 Northern blots were performed using Nytap Miyrie Tizzce Wioiv from Siopiesp (Region A, CA). Probe

DNA with a size of 30 bases (2212447; ZEO IO MO:4) for the 5' end of the nucleotide sequence of the developed protein,

which is shown by the sequence of ZEO IU MO 1, was labeled with radioactive ZR using polynucleotide kinase TA and direct reaction buffer (ZIVSO VI, Saipegerigo, MO) according to the manufacturer's instructions. The probe was purified using a MOSTKAR pressure column (5igaiadepe Siopipd Zuvietv, Ia doya, CA) . EHRKEZZNUV solution (Siopiesp, Rayo Amo, CA) was used for prehybridization and as a hybridization solution for Northern blots. Hybridization was performed overnight at 502C, and the blots were further washed in 2X 5552 (sodium chloride and citrate solution) and 0.195 - BO (sodium dodecyl sulfate) at room temperature, followed by 1X 55C and 0.195 505 at 682C (approximately 59 below the melting point ). One transcript size was observed at approximately 2.8 kb. Signal intensity was highest in heart and placenta, with relatively weak signals in kidney, ovary, adrenal gland, and skeletal muscle, with weaker signals for a wide variety of other tissues in the Northern blot.

Additional Northern blotting was performed using (Zci Moghiegp Tizvce Vioi Blot was made using mRNA from human colorectal adenocarcinoma 5MU480 cell line (Siopiesi, Rayu A, SA), human small intestine tissue (Siopiesi), human stomach tissue (Siopiesi), cell line smooth muscle of 75 human intestines (Nizt, ATSS Mo SKI-1692, Ategisap Toure Siyige Society (American Type Culture Collection) 12301 RagKIamp Ogime, KosKmPShe, MO), a normalized human colon cell line (ENS, ATSS Mo

STI -1831, Ategisap Toure Siyige SoiIesiop) and a normalized cell line of the small intestine of a human embryo (ENv74 Ip.; ATOSS Mo SSI 241; Ategisap Toure Siyige SoiIesiop).

Total RNA was isolated from Nizt, ENS and ENv74 Ipi. by the acid-guanidine method (Speotsgupeki ei ai.,

Apai Viospet. 162:156-9, 1987). Polid" RNA was selected by elution of total RNA through a column retaining polyA" RNA (Amim ei a!., Rhos. Mai. Asad. Zsi. 69:1408-12, 1972) 2 μg of poly" RNA from each sample was separated in a 1.596 agarose gel in 2.2M formaldehyde and phosphate buffer, RNA was transferred to a Muigap membrane (Zspivispeg and Zsp!ceili, Keepe, MN) at 20X 55: overnight The blot was processed in OM 5ZigaianpKeg 2400 (5iga(adepe,

І a KhdoPa/ SA) at 0.12 J. The blot was further heated at 802 for one hour. with

The full-length cDNA (shown by the sequence BEO IU MO:1) was amplified using PCR (polymerase (3 chain reaction)) and labeled with radioactive YASTR with Z2R, using the Reairgate kit (Ategtvpat,

Apipdiop Neidnpiv, I) according to the manufacturer's instructions. The blot (hybridized in EHRKEZZNUV (Siopiespy) at 56 C overnight. The blot was washed at room temperature in 2X 55C and 0.195 5O5, further in 2X 35C and 0.196 505 at M 20 -652С, and towards the end at 652C in 01X 5BZS and 0.195 505 The results showed that 28id37 hybridized with all tissues except the NIZM cell line of human intestinal smooth muscle.

Example C Chromosomal mapping of the gene 28idZ37 o

The 28idZz7 gene was mapped to human chromosome 17, region 17425.2, by PCR using a panel

MISM5 Nytap/Yukodepi Zotaiyys Se! Nubgii Marripd Rapei Mitreg 2 (Mayopa! Ipviyshe oi Sepegai Medisai

Zsiepsev, Sopei! Ipziytsshe Oy Medes! Kezeagsp). The panel consists of DNA isolated from 24 human/rodent somatic cell hybrids, each of which retained a specific human chromosome and parental DNA. To map the 28id937 gene, 20 μl of reaction mixtures were placed in a 96-cell microtiter plate (Ziga (adepe,

1A) and used in the thermocycler "KoroSusieg (Zgadiepi 96" |Zigaiadepe). Each of the 27 RSC reaction mixtures consisted of 2 μl of 10X Kieptad RSC reaction buffer (Siopiesny I aroghaiges Ips, Rayo AG, CA), 20 1.6 μl of OMTR mixture (2.5 mM easp, REKKIM-EI MEK, Rosieg SPU, CA) . ), 25 ng of DNA from a specific hybrid clone or control and 4 ngO:z" to a total volume of 20 μl. The reaction mixtures were covered with an equal amount of mineral oil and stoppered.

The conditions of the RSC cycler were as follows: an initial 1 cycle of 5-minute denaturation at 9592С, 35 cycles of 1 minute of denaturation at 9592С, 1 minute of hybridization at 609С and 1.5-minute elongation at 7229С, followed by a final 1 cycle of elongation for 7 minutes at 722C. The reaction mixtures were separated by electrophoresis on a 395 MybZieme STO agarose gel (EM Viorgodisiv, KosKiapa, ME). i-th Example 4 Creation of mammalian expression vectors 25i937MEE/p2PO and 28i937SEE/pPO av! Two expression vectors, 28i937MEE/p2PO and 25i937MEE/p7PO, were made for the 25id37 polypeptide, which were designed for the expression of the 285id37 polypeptide, which has a C- or M-terminal tag SISH-c1M. o 75ид37МЕЕ/р7РО "I The DNA fragment 28i937 created in RSC with a size of 800 p was created using RSC primers 215040 (5EO IUO MO:24) and 2215033 (5EO IUO MO:25) and the template described above in Example 1. The reaction the RSK mixture was incubated at 942C for 3 minutes, and then 5 cycles were performed at 942 for 30s, 302C for 20s, and 729 for 1 minute, followed by 25 cycles at 942 for 30s, 6423 for 20s, and 729C for 1 minute. minute elongation at 722. The resulting PCR product was then run on a 0.995 TWE agarose gel with 1 x TWE buffer. The band of expected size was excised and the DNA was purified from the gel with Oiaex 11 resin (Siadep) according to the manufacturer's instructions. The DNA was digested with the restriction enzymes Waigp NI and Xia I, accompanied extraction and precipitation. 60 The excised, restriction-cleaved DNA fragment 28ид37 was subcloned into the plasmid MEE/p2PO, which was cut with the restriction enzymes Vag NI and Xba I. The expression vector 25i937MEE/p7PO includes li ter sequence of TPA and attaches to the polynucleotide sequence that encodes the M-end of the 28id37 polypeptide, the Sish-sSish label (ZEO IU MO:26). Plasmid MEE/p7RO (stored in Ategisap Toure Siyige SoPesiop, 12301

Ragkiiam/p Ogime, Cosmicie, MO, ATCC Mo 98668) is a mammalian expression vector that includes an expression cassette 65 having the murine metallotunein-1 promoter, a peptide with a TPA leader sequence and a sequence encoding a Sitzch-cSish tag (ZEO 10 MO: 26), a plurality of restriction sites for the insertion of the coding sequence, and the human growth hormone terminator. The plasmid also includes the origin of replication of E. soya, the expression element of a selectable mammalian marker having the 5M40 promoter, the enhancer and the origin of replication, the ONEK gene and the terminator 540. 28i937CEE/rePO

The DNA fragment 28i937 created in RSC with a size of 866 was created according to the method described above, using RSC primers 2C15721 (5BO I MO:27) and 2C15035 (5EBO OO MO:28). Peeled

The RSK fragment was cleaved with restriction enzymes Eso KI and Vat NI, the gel was purified using Oiiaex II resin, as described above. 70 Cut out and cleaved by restriction DNA 285id37 was subcloned into plasmid CEE/p7PO, which was cut with restriction enzymes Eso KI and Vagp NI. The expression vector 258i937CEE/p7PO uses the native signal peptide 25id37, and the epitope of CSH-SHI (ZEO IU MO:26) is attached to the C-terminus for the purpose of plasmid purification

SEE/r2RO (stored in Ategisap Toure Siyige SopPesiop, 12301 RagKIamup Ogime, KosKkmiye, MO, ATSS

Mo.98668) is a mammalian expression vector comprising an expression cassette having a mouse metallotunein-1 promoter, a plurality of restriction sites for the insertion of a coding sequence, a sequence encoding a Cic-Si tag (ZEBEO 10 MO:26), a stop codon and human growth hormone terminator. The plasmid also has an E. soya origin of replication, a mammalian selectable marker expression element having a 5M40 promoter, an enhancer and an origin of replication, the ONEK gene, and a 5M40 terminator.

For M- and C-labeled constructs, approximately 200 ng of the restriction-cleaved inserts and 5 ng of the corresponding 20 vectors were condensed at room temperature for 4 hours. One microliter of each condensation reaction mixture was independently injected by electroporation into competent ONITOV cells (SIVSO VK, Saipegerigo, MO) according to the manufacturer's instructions, applied to IV plates containing 50 mg/ml ampicillin and incubated overnight.

Colonies were screened using RSC as described above. For 285i93/MEE/p2RO and 28idz/CEE/raPO, the screening primers were 2213006 (ZEO IYU MO:29) and 2213007 (5EO IYUO MO:20). The RSK reaction mixture was incubated at 942 for 2.5 minutes, then subjected to 25 cycles at 942С for 10s, 582 for 20s and 722Сb for 1 minute, followed by a 5-minute extension at 72 2С. The sequence of the insert of positive clones, the size of 1013 by for 25id3/MEE and the fragment of size 950 by for 285idz/CEE was confirmed by sequencing. Large-scale production of plasmids was performed using the kit

SIASEMO Mahi rger (Siadep) according to the manufacturer's instructions. -

Example 5 Transfection and expression of 25idZ7MEE and CEE polypeptides

VNK 570 seiYiz cells (ATOS Mo. SI -10314) were plated on 1Osm tissue culture dishes and allowed to grow to approximately 50-7095 confluency overnight at 372, 596 CO", in OMEM/EVZ medium (3 (OMEM, Birso/ VKI. Nidp Siisoze, (Sirso VK, Saipegsrygo, MO), 590 bovine embryo serum (Nussiope, u

Godap, OT), 2μM I-glutamine (KN Viozsiepsez Iepekha K5), 1MμM sodium pyruvate (Sbirso VKI)) Cells were further transfected with the plasmid 25id37MEE/p7RO (M-terminal tag SiIsh-Six) or 25id37SEE/p2RO (C-terminal tag uh-

SiIc-SIU), using IiroiHesiatipetm ((Zirso VKI), in a serum-deprived (ZE) medium composition (OMEM, Birso/VKI. Nidp Sisoze, (Sirso VK, Saipegzrygo, MO), 2mM I-glutamine, 2mM sodium pyruvate, (1 µg/ml of transferrin, 5 µg/ml of insulin, 10 µg/ml of fetuin and 2 ng/ml of selenium). Sixteen micrograms of 25i937MEE/p2RO and 1bmkg of 28i937SEE/p2RO were separately diluted in 15bml tubes to a total final volume of 640Omcl in BE medium. In separate tubes, 35 µl of Irohesiatipetm ((Zirso VKI) was mixed with 605 µl of 5E-medium. C s The mixture of Irohesiatipetm was added to the DNA mixture and allowed to incubate for approximately 30 minutes at room temperature. Five milliliters of 5E-medium was added to the DNA mixture Cells were washed once with BbBml of ZE-medium, aspirated and a mixture of DNA-Y was added. y y y y y -1 overnight and a mixture of DNA And the next day's culture medium was replaced with fresh EVZ/OMEM medium. On day 2 after transfection, the cells were distributed in the selection medium (EBTER Mo13 iM MTX) on 150 mm plates 1 at ratios of 1:50, 1:100 and 1:200. Tablets were fed on day 5 after transfection with fresh breeding medium.

Screening of colonies o Approximately 10-12 days after transfection, one 150-mm plate for the culture of methotrexate-resistant colonies was selected from each transfection, the medium was aspirated, the plates were washed with 1 Oml of serum-free ESTER 2 medium (668.7 g7/5O0l OMEM (birso), 5.5g/5000l of pyruvic acid, sodium salt 9690 (MaiipskgodO, 185.0/50l MansoO»z (MaiipKgoayuU, 5.Omg/ml, 25ml/5Ol insulin, 10.Omg/ml and 25ml/50l transferrin). Washing medium aspirated and replaced with 5 ml of serum-free ESTER 2 Grid

Ziepie Teyop (Zresigit Meadis! Ipdyzigiez, Oz Apdeyez, SA), which was pre-impregnated with (F)-free EZBTER 2 serum, was further placed on top of the cells. A sterile nitrocellulose filter, which was previously

GI impregnated with serum-free EBZTER 2 was placed further on top of the grid. Orientation marks on nitrocellulose were transferred to a culture cup. The tablets were then incubated for 5-6 hours in an incubator at 372С, 590С. After incubation, the filter was removed, and the medium was aspirated replaced with medium

OMEM/596 EV IX RBM (Birso VK). The filter was then placed in a hermetic flask containing 500 ml of buffer (25 mM Tris, 25 mM glycine, 5 mM B-mercaptoethanol) and incubated in a water bath at 65923 for 10 minutes.

Filters were blocked in 10956 skimmed milk powder/buffer M/ezygp A (UUeviegp A 50 mM Tris pH7.4, 5 mM

EDTA, 0.0595 MP-40, 150 mm Mass! and 0.2595 gelatin) for 15 minutes at room temperature on a 65 rpm shaker. The filter was further incubated with the anti-siI-SiI-antibody-NCR conjugate at a dilution of 1:1000 in 2.590 skimmed milk powder/UUviegp buffer (Uueviegp A 50MM Tris pH7.4, 5mM EOTA, 0.0595 MP-40, 150MM

Mass and 0.25965 gelatin) overnight at 49C on a rotary shaker. The filter was then washed three times at room temperature in РВ5 plus 0.195 Tween 20, 5-15 minutes per wash. The filter was developed with ESI reagent. (Ategepat Sogr., Agipdiop Neiidnpiv, I) according to the manufacturer's instructions and exposed to film (Nurepiit ESI, Ategeyat) for about 5 minutes.

The film was combined with a plate containing colonies, using the film as a guide, suitable colonies were selected. Sterile discs according to Zmm for colonies (ROS Zsiepiyis Sogr., Egedegisk, MO) were impregnated with trypsin and placed on the colonies. Twelve colonies for each construct were transferred to 200 μl of selection medium in a 96-well plate. For each colony, sera from seven two-fold dilutions were made. 7/0 cells were grown for one week at 372C, during which time cells receiving the lowest dilution of cells at optimal density at that time were selected, trypsinized, and transferred to a 12-well plate containing selection medium. A 150 mm culture dish was also trypsinized and the remaining cells pooled and subjected to Western analysis and mycoplasma testing. The association was frozen for storage.

Clones were split directly from the 12-cell plate into two T-75 flasks. One flask was kept for 75 cell growth elongation, the second flask was grown in serum-free EBTER 2, which was collected for Western blot analysis. Clones of each of the expression constructs were selected based on the analysis

by Western blotting, pooled and transferred to a high-yielding culture.

Example 7 High-performance expression of 25id37SEE by a mammal

One T-162 flask containing confluent cells expressing 25u937CEE and one containing 28u37MEE expressed as described above were divided into four T-162 flasks each. One of the four resulting flasks was used to freeze four cryovials, and the other three flasks were used to establish Mips cell colonies.

Cells from three T-162 flasks with 28i937CEE and 25i937MEEE were used to independently seed two colonies of Mips cells (10 layers, commercially available from MMUK). Briefly, the cells from the T-162 flasks described above were dissociated using trypsin, pooled and added to 1.5 L of ESTER1 medium (668.77/50 L OMEM (Sirso),), 5 g/50 L of pyruvic acid, sodium salt 9695 (MaiipskgoaO 185.0g/50l Manso" (Maiipkgoayu, about 5.Omg/ml and 25ml/5Ol insulin (KN Viozsiepsev), 10.Omg/ml and 25ml/50l transferrin (KN Viozsiepsev), 25g/50l cow's serum whose embryo has been characterized) (Nussiope), iMm MTX, with adjusted to 7.05-47-0.05рНН), which was pre-heated to 372. The media, including the cells, was further poured into the Mips cell colony through a funnel. Columns of cells were placed in an incubator with 372С/5.095 СО. co

At a confluency of 80-10095, the contents of Mips cell colonies were tested for visual contamination (phenol red color change). As no contamination was observed, supernatant from confluent colonies (α) was poured into small collection containers, sampled and discarded. The attached cells were then washed once with 400 ml of PB5. To separate the cells from the colonies, T00 ml of trypsin was added to each and removed, and the cells were then incubated for 5-10 minutes in the residual trypsin. Cells were collected - accompanied by two washes of 200 ml of E5BTERI medium. 4 Oml of collected cells were added to each of the ten vials containing E5TERI medium (1.5 L each, at 372C). One 1.5 liter bottle was then used to fill one Mips colony. Each colony of cells was placed in an incubator with 37 2C/5.0905 "

So".

At a confluency of 80-100905, the Mips cell colonies were tested for visual contamination (color change from phenol red). Since contamination was observed, the supernatant from the confluent colonies was poured into small "» containers for collection, samples were taken and discarded. The attached cells were then washed once with 400 ml " RVZ5 1.5 L of E5TER2 medium (668.7 g/50 L OMEM (Sirso), 5.5 g/ 50 l of pyruvic acid, sodium salt 9690 (MaiipskgodO, 185.0/50l MansSoO»z (MaiipkKgoayuU, 5.Omg/ml, 25ml/50l insulin, 10.Omg/ml and 25ml/50l transferrin) were added to each column of Myps cells . The cell columns were incubated at 372C/5.095С". - After approximately 48 hours, Mips cell colonies were subjected to a visual staining test ("sl phenol red color change). The supernatant from each colony was poured into small containers for collection. Fresh serum-free medium (1.5L) was poured into each colony of Mips cells, and the colonies were incubated at 372C/5.096 CO". One ml of supernatant collection for each construct was transferred to a microscope slide and (4) 50 microscopically examined for contamination. The contents of small collection containers i for each construct. combined and immediately filtered. A second collection was then carried out, as essentially described above, at the 48th hour, and the cell colonies were then discarded. Aseptically collected equipment from a set of filters was used for aseptically filtering the collected supernatant (conditioned medium).

The assembly was such that the tube was attached with a wire to the Orii-Sar filter (MiiPiroge Sogr., Vejtoga, MA) and 99 filter Seitap Ziregsar 50, (Seitap bsiepsez5, App Agrog MI). The Ziregsar 50 filter was also connected to

GF) of a sterile covered container, placing in the lid, the tube located above the MiiPiroge t Orii-sar filter was inserted into a peristaltic pump, and the free end of the tube was placed in a large collection container. The peristaltic pump was operated at 200-300 rpm until all of the conditioned media passed through the 0.22 µm final filter into the sterile collection container. The filtrate was placed in a cold room with a temperature of 49C until purification. The medium was concentrated 10X through a Miiroge 5BKOA limiting concentrator (Mipiroge Sogr., Weatoga, MA) according to the manufacturer's instructions and subjected to analysis

by Western blotting, using an antibody against RI AcS-label (Kodak). 78idZz7SEE: 5 flasks T-162-0.12mg/l, ZvkDa, 6541 colony, ЕВ5-012mg/l, ZVkDa,

10 colonies, EB5-0.12mg/l, ZvkDa, 10 colonies (21), ZE-1.2mg/l, ZvkDa, and 10 colonies (252), 5E-3.5bmg/l, ZvkDa 28id437MEE 5 flasks T- 162-0.137 mg/l, З5kDa, 1 colony, ЕВ5-0.137mg/l, ZokDa, colonies, ЕВ5-0.137 mg/l, ЗБкDa, 10 colonies ((41), ZE-1.37mg/l, З5kDa, and 70 10 colonies (52), 5E-4.11mg/l, ZBkDa

Example 7 Purification of 25id37 MEE and 285idZz7 CEE

Unless otherwise stated, all operations were performed at 42C. The following procedures were used to purify 25937, which includes an M-terminal or C-terminal IS-SIsh (EE) tag. A total of 25 L of conditioned media from pupal hamster kidney (HKN) cells were sequentially filtered under sterile conditions through a 4-inch 7/5 capsule filter on 0.2MM Miijiroge (Weatoga, MA) OriiSar and 0.2MM Seitap (App Agrog, MI) Z, Iregsar 50.

The material was further concentrated to approximately 1.3L using a Miyiroge tangential flow concentrator

RgoBiikh AZO, which is equipped with a limiting membrane at 30OOkDa Artichoke (Veditoga, MA) 5103.

The concentrated material was refiltered under sterile conditions through a SeyItap filter as described above. A mixture of protease inhibitors was added to the concentrated conditioned media to final concentrations of 2.5 mM ethylenediamine-tetraacetic acid (EDTA, Zidta SNetisai Co. ZI Iotsiv, MO), 0.001 mM leupeptin (Voepppdeg-Mappeit, Ipaiaparoiiv, IM), 0.001 mM opepstatin (Voepppdeg-Mappeit ) and O04mM Regarios (Voepppdeg-Mappieit). A 25.0 mL sample of anti-EE-Sepharose, prepared as described below, was added to the sample for the adsorption portion and the mixture was gently mixed on a UUpeyup culture rotator (MiiymiPe, MU) for 18 hours at 4260.s

The mixture was then poured into a 5.0x20.0 cm Esopo-SoYishtp column (ViO-Kad, I arogyugies Negsciez, CA) and the gel was washed with 30 column volumes of phosphate-buffered saline (RV5). A faction that was not caught and missed was rejected. As soon as the absorbance of the effluent at 280 nm was less than 0.05, the flow through the column was reduced to zero and the anti-EE-Sepharose gel was washed in 2.0 column volumes of PB5 containing 0.4 mg/ml

EE-peptide (Apazres, Zap dose, SA). The peptide used has the sequence (SISH-Tug-Meig-Rho-MaI-Agr, ZEO i- 10 MO:31). After 1.0 hour at 42C, the flow was recovered and eluted protein was collected. This fraction was designated as . - , . with the elution of the peptide, the Anti-EE-sepharose gel was then washed with 2.0 column volumes of 0.1 M glycine, pH 2.5, and the glycine wash was collected separately. The pH of the fraction eluted with glycine was adjusted to 7.0 by adding a small volume of //C2 to him 10X РВ5 and stored at 42С for further analysis, as necessary. yu

The peptide elution was concentrated to 5.0 ml using a 15,000 molecular weight concentrator with a limiting membrane (Miiiroge, Weatyuga, MA) according to the manufacturer's instructions. The concentrated elution of peptide y- was separated from the free peptide by chromatography on a 1.5 xbOsm Zerpadeh 0-50 column (Rpagtasia, Rizsaiaulau, MO) equilibrated with РВ5 at a flow rate of 1.0 ml/min, using VioSide Zrgipi NRI C (Regzeriime

Viobuvietv, Egatipoapat, MA). Two-milliliter fractions were collected and monitored for absorbance at 28 Ohm. "

The first release of material with absorbance at 280 nm and eluting at approximately the volume of the unfilled column was collected. These fractions were pure 28-37 MEE or 28-37 CEE. The pure material was concentrated as described above, analyzed by 5O5Z-RACE (5O5-polyacrylamide gel electrophoresis) and Western blotting with "" anti-EE antibodies, and samples were subjected to amino acid analysis and M-end sequencing. The residue " sample was aliquoted and stored at -802C according to our standard procedure.

Electrophoresis of 25 and 937 MEEE on 5O5-RACE gels in the absence of reducing agents showed one main stained. Coomassie blue showed a band with an apparent molecular weight of 39,000 and several weaker components with molecular weights of 60,000 and 116,000. All bands showed cross-reactivity with anti-EE antibodies on Western blots. In the presence of a reducing agent, only a protein band with a mass of 39,000 OkDa was observed, and the intensity of Coomassie blue staining increased. This band also showed cross-reactivity with anti-EE antibody on Western blots.

Ha 50 For 28 and 937SEEE, electrophoresis on 505-RACE showed one main Coomassie blue-stained band with an apparent molecular weight of 39,000 and several weaker components with molecular weights of 60,000 and 116,000. Western blots showed cross-reactivity with anti-EE antibody only bands of probable molecular weights of 150,000, 116,000, and 60,000. In the presence of a reducing agent, only a band of protein mass

C900OkDa, and this material showed cross-reactivity with the anti-EE antibody on Western blots. Under these conditions, small amounts of cross-reacting material were also observed at 150,000 kDa.

GF) Production of anti-EE-sepharose

The initial volume of protein in 100 ml of s-Sepharose (RNagtasia, Rizsaijau, MO) was washed three times with 1000 ml of PB5 containing 0.0296 sodium azide using a 0.45 micron filter element per 500 ml of Mayidepe. The gel was washed with 6.0 volumes of 200 mM triethanolamine, pH 8.2 (TEA, Zidta, 5. I otsiv, MO), and an equal volume of 60 EE-antibody solution containing 90 Ωg of antibody was added. After overnight incubation at 42C, unbound antibody was removed by washing the resin with 5 volumes of 200 mM TEA as described above. The resin was resuspended in 2 volumes of TEA, transferred to a suitable container, and added to the final concentration of the gel was Zbmg/ml of dimethylpimylimstatate-2HCl dissolved in TEA (Riegse, Koskioga, I). The gel was shaken at room temperature for 45 minutes and the liquid was removed using the filter element described above. Nonspecific areas on the gel were further blocked by incubation for 10 minutes at room temperature with 5 volumes of 20 mM ethanolamine in

200MM TEA. The gel was washed with 5 volumes of PB5, which includes 0.02905 sodium azide and stored in this solution at

Example 8 Study of adhesion and proliferation

The ability of 28id37 to stimulate adhesion and plating of TE-1 cells was analyzed as such. From the labeled C-end

SiIc-SiIsh 28id37 was prepared in a series of dilutions, from 10 to 0.0625 µg/ml, in coating buffer (0.1 M Maso z)

PB5 or EIIZA and each was placed in a 96-well plate (Soviag, Riezapiop, CA) at 100 µl/well.

Tablets were incubated at 372С, 595 СО for 2 hours. The tablets were then washed three times with KRM1/1096 IU (KRMI 1640, 2mM I!-glutamine, 11Okg/ml sodium pyruvate, P5M and heat-inactivated 1095 bovine 70 embryo serum) and blocked for 15 minutes.

TE-1 cells (derived from acute myeloid leukemia cells) were resuspended in KRMI/1095 EVZ and plated at 10,000 cells/cell on 25iD37CEE-coated 96-well plates in a final volume of 120 μl/cell. The tablet was incubated at 372°C at 5956°C for 2 hours. The plates were then washed three times with RVZ5 and 200 Ωcl/cell of growth medium (KRM1/1095 ЕВ5, 5 ng/ml ZM-SZE) was added. Cells were inspected by microscopy before and 75 days after washing.

The color analysis was also used to quantitatively measure the number of attached cell-based colorimetric changes and the amplification of the fluorescent signal. Dye Aiatag Vice tm (AssyMea,

Spisado, I) was added to 96-cell plates and the cells were incubated at 372С and 596 СО» during the night.

The tablets were then scanned using a fluorometer with an excitation wavelength of 544 nm and an emission wavelength of 590 nm. There were more attached cells on 25i937CEE-RV5 coated plates than on 28i937CEE-0.1M MassSo»z coated plates. Addition of soluble 25id37 did not block cell adhesion to attached 28id37.

The color analysis was also used to quantitatively measure the number of attached cell-based colorimetric changes and the amplification of the fluorescent signal. Aliatag Vice tm dye (AssyMea, SM Spisado, I) was added to 96-cell plates and the cells were incubated at 372C and 5956 C overnight. heh)

The tablets were then scanned using a fluorometer with an excitation wavelength of 544 nm and an emission wavelength of 5900 nm. There were more attached cells on the 285i937CEE-RV5 coated plates than on the 28i937CEE-0.1M MassSo»z coated plates. Addition of soluble 25id37 did not block cell adhesion to attached 28id37. -

The second analysis was performed using cell lines TE-1, BA-1 (II-3-dependent cell lines derived from the lymph node of a mouse with B-cell lymphoma by growing in a medium with II-3 (proposed by Og

Keppeii KaizpapezKu, Opimegeyu oi UUazpipdiop, ZeashShe, MUA)), pre-B (p5Z-/- mouse bone marrow cells, o dependent on II-7, 8220, Tnu! Iomu, Zsa-In), and A7Vanvg-3, which described above, at 500 cells/cell. ZNK cells were also applied at 5000 cells/cell. 785idZ7 enhanced the growth of A7-Vakg-3 cells and weakly inhibited the growth of CA-1 cells. 3o Example 9 The sequence of the mouse ortholog -

The new human polypeptide 25id37 encoded by polynucleotides according to the invention was used to screen the mouse E5T database for the mouse homologous sequence. The single sequence of EZT was revealed and predicted with respect to the human 28id37 sequence. To identify the corresponding cDNA for sequencing, a clone was used, which is believed to probably contain a continuous coding sequence, using the Ipiyodep ZMARtm Mtirger kit (Ipmygodep Sogr) according to the manufacturer's instructions, Bml of the overnight Z culture was prepared in YVI-50 mcg/ml ampicillin. The template was sequenced on a DNA sequencer model 377 AVIRKISmtm (Regkip-EIteg Seyiv MogmaiK, ST) using the kit AVI RKISmtm From YuBue Tegtipag Susie Zedcepsipd

Keadu Keasiyup Kii (Regkip-EIteg Sogr.) according to the manufacturer's instructions. Oligonucleotides 72694 (ZEBEO IU MO:6), 2C6768 (ZEO IU MO:32), 2218297 (ZEO IU MO:33), 2218298 (5EO IU MO:34), 2218402 (5EO I MO:35), 2218403 -1 15 (ZEO IU MO:Z6), 2218456 (5EO IU MO:37), 2218457 (5EO IU MO:З8), 2218560, (5EO IU MO:3,9), 2218561 (5EO Ir

MO:40), 2218687 (ZEO IU MO:41) and 2218688 (ZEO IU MO:42) were used to complement the sequence from 1 clone. Sequencing reactions were carried out on the Nubaiiii Otpibepe Tetregaishge Susiipd Zuviet system (Mayopai! o IHarpei So., Muoodrpade, MU). Sequencing software was used for data analysis

ZEOTSOEMSNEK tm 3.0 (Sepe Sodez Sogrogayop, App Agrog, MI). The resulting sequence of size 2559 by (95) 20 was revealed by the sequence ZEO IYU MO:43 and the derived amino acid sequence ZEO IYUO MO:44. Overlay on the nucleotide sequence of human 25 and 937 (ZEO IU MO:1) showed 77,905 identities at the nucleotide level.

The predicted amino acid sequence (BEO IU MO:44) has 7795 identities with the human polypeptide sequence (ZEO IU MO:2). 5 Example 10 Research based on cells

Polypeptides 25idZ37 were analyzed with high productivity in the analysis of IP migo to identify substances that (F) selectively activate cellular reactions in immortal cell lines of osteoblasts. used in the analysis

He developed a cell line of osteoblasts CCC4, derived from p53-/- (deficient) mice, which are transfected with a plasmid that includes allowing the induction of serum reactive elements (SRE), which leads to the expression of luciferase. These cells also express endogenous PTN, ROSE and BESR receptors. Stimulation of 5KE, and thereby the expression of luciferase in SSCA cells, indicates that the chemical object probably stimulates mitogenesis in osteoblasts.

CCC4 lines were trypsinized and grown to Bx10 cells/ml in seeding medium (alpha-MEM, 195 heat-inactivated fetal bovine serum, TMM Ma pyruvate, and 2 mM I!-glutamate) and plated 65 (200 μl/cell) on Oupayesp Microscopy light-opaque white microtiter tablets (Oupayesp, Spapiyu,

MA) and incubated overnight at 372, 596 СО". The growth medium was then aspirated and replaced

5µl/cell with medium for analysis (E-12 NAM, 0.595 bovine serum albumin, 20 mM HEPES, 1 mM Ma pyruvate and 2 mM I-glutamate). Serial dilutions of 25 and 37 were made into the medium for analysis (0.29-100Ong/ml final concentration of the analysis) and added to the cells. Samples 758 and 37 were analyzed in triplicate.

Serum (negative) and BESE (positive) controls were also used. The final concentration of BESE was Zng/ml. Controls were analyzed in quadruplicate. Tablets were incubated for 4 hours at 3720, 596 СО". The assay medium was then aspirated and the plates were washed once with PB5. Next, 25 μl of lysis buffer was added to each cell (I isitegase Apaiuziz Keadepu/ E1501, Rgoteda Sogr., Madizop, UMI).

Tablets were incubated for 15 minutes at room temperature. Fifteen μl/cell of 70 luciferase substrate (I isiyegase Analysis Keadepi, E1501, Rgoteda Sogr.) was added and luciferase activity was determined using I arzuvietv I OMIMOZKAM?U at 2s/cell with a subsequent interval of 1s. The mean basal (uninduced) signal was subtracted from all data presented in Table 5 as a percentage of the maximum induction produced Zng/ml BSE. 78idZ37 stimulates luciferase expression in this assay, indicating that they stimulate osteoblasts 78id37 75 stimulates at 7Z to 7595 from the maximum at 10O0Ong/ml.

A partial growth factor mimetic counter-assay was performed to determine whether 25id37 acts as a growth factor mimetic, specifically the tyrosine kinase receptor ligands ROSE, BACE, and ESE (insulin-K-negative). The analysis used a clonal cell line derived from mice Zm/izz ZTZ, Zm/izz ZTZ, which are transfected with a plasmid that includes allowing the induction of serum reactive elements (SRE), which leads to 20 expression of luciferase. These cells also express endogenous PMA, ESE and BEOR receptors. Stimulation of ZKE and thus the expression of luciferase in cells of Zm/izz PPE indicates that the chemical object probably imitates the activity of growth factors ROSE, BESE and EOBE.

Zmivv ZTZ cells were trypsinized and grown to 5x10 cells/ml in seeding medium, plated and incubated as described above. The growth medium was then aspirated and replaced with Bb5Omcl/cell Ge 25 assay medium (E-12 NAM, 0.595 bovine albumin serum, 20MM HEPES) Serial dilutions of 25937 were made into assay medium (0.29-100Ong/ml final assay concentration) and added to the cells.

Samples 78 and 37 were analyzed in triplicate. Serum (negative) and BESE (positive) controls were also used. The final concentration of BESE was Zng/ml. Controls were analyzed in quadruplicate. Tablets were incubated for 4 hours at 37 C, 5906 СО". The medium for further analysis was aspirated and the plates were washed once with PB5. 25 μl of lysis buffer was then added to each cell (I ositegaze Analysis Keadepi, E1501, Rgoteda Sogr, Madizop, UM). Tablets were incubated for 15 minutes at room temperature. Forty μl/cell of the luciferase substrate (I isiyegase Assay Keadepi, E1501, Rgoteda ("2

Sogr.) was added and the luciferase activity was determined using IOMIMOZKAMU Iarvuvietv at 2 s/cell with a subsequent interval of 1 s. The average basal (non-induced) signal was subtracted from all the data that 32 is presented in Table 5 as a percentage of the maximum induction produced Zng/ml BSE. -

Five-hour treatment of this cell line with BESOE, SOC, ESE or PMA leads to a 25-50-fold induction of ZKE-luciferase expression. 78idZ37 was not found to stimulate the expression of luciferase in this analysis, 78id37 stimulates it at 0.2-0.1965 " maximum at 100Ong/ml. 40 Example 10 Administration of ip mimo 28id37 by delivery of adenoviruses with c Twenty-four male and 24 female C57B16/ mice, approximately 12 weeks old (daskzop I arv, Vag Nagbog, "from ME) were weighed, body temperature was measured, and food intake was monitored every day within four " days to days -4 - -1). On day 0, mice were divided into three groups and they received 0.1 ml of virus (AJIM-empty 1.8x1011 viral particles/0.1ml or Aam-2vid37-CEEEE 5x1011 viral particles/0.1ml) by intravenous injection into the tail. or no injection at all. Injection can lead to infection of the host's liver, and expression of the virus-delivered gene can begin within 24 hours and continue for 1-4 weeks. c Three groups of mice were tested. Group 1, untreated, n-8 each of males and females Group 2, Aam- blank (empty virus), n-8 each of males and females. Group Z Dam-fromZz7 EE n-8 of each of the males and females. o The body weight of the animals, body temperature and the weight of ingested food were monitored during three weeks of the study. No difference was found between (4) 50 groups. . 21 female mice and 22 male mice were euthanized by cervical dislocation per day. Animals were bled and tissues were collected for autopsy.

A standard panel of serum chemistry was done at the time of sacrifice. Liver, kidney, and metabolic parameters were all within normal limits. However, a difference was observed between the 25id37-treated group and the 22 blank virus-treated group. Animals 25 out of 37 had a higher average lipemic index than control animals with blank

HF) virus. The difference is small, but further research is warranted. Total free fatty acids were analyzed on serum remaining from each animal. A statistically significant difference in the level of free serum fatty acids was observed between male mice (p-0.0379) that received the blank virus and those that received the virus encoding 285id37, 25id37 mice had higher levels. The difference, although not statistically significant, was also observed in females (р-0.3357). Liver, spleen, kidney, thymus, heart, and brain were weighed after removal. No difference was found between treatment groups. Histopathological analysis of these tissues and bone marrow revealed no difference between treatment groups.

To confirm the above results, a second screening was performed as above, with the following modifications. Three groups containing 20 C57816/) mice were tested. a) untreated and hungry, B) Aam-null and hungry, c) Aam-28idZ7-SEE and hungry, 10 of each male and female. Mice were fasted overnight and 100 μl of serum was collected to determine the basal level of such parameters as fasting glucose, TP (testosterone propionate), alkaline phosphatase, cholesterol, triglycerides, free fatty acids, and insulin. Body weights were determined three times a week. On day 0, mice were injected into the lateral tail vein with 0.1 ml of a suitable virus solution. Blood was collected on day 17 after an overnight fast. After 3 weeks, mice were killed and whole blood was collected. A portion of the blood was mixed with EDTA for clinical blood analysis and the remainder should be reanalyzed and screened as described above. Organs were harvested and the carcass was reserved for histopathology.

Example 11 Vasodilatation of aortic rings

The effect of 25id37 on the vasodilatation of aortic rings was measured according to the methods |Oaipiu ei ai., / Rpaptasoi! 100: 7/0 757 1990 and КПее ей а), Mashgoyoh 16:179, 1995). Briefly, aortic rings 4 mm in length were taken from Zrgadie rats

Oauleu aged 4 months and placed in a modified Krebs solution (118.5 mM Mass, 46 mM KS, 1.2 mM

MazoO,u.-7NhO, 1.2 mM KNoRO,, 2.5 mM Sasi» 2H20, 24.8 mM Manso» and 10 mM glucose). The rings were then attached to an isometric force transducer (Kaadpoii Ips., Mopgomia, CA), and data were recorded on a physiological platform

Ropetype (Soc! Ipzigitepi zuvietvev Ips., MaMieu Miem,, OH) and placed in a modified Krebs solution in a 75 oxygenated tissue bath at 1 Oml (9595 0", 596 CO"). The tissues were then subjected to a tension of 1 gram at rest and allowed to stabilize for one hour before testing. The rings were tested by adding 5 μl of 1X10-7M norepinephrine (Zidta So., 8. Gotsiv, MO) to a final concentration of approximately 1ХЙО9M and carbachol, a muscarinic acetylcholine agonist (Zidta So.) at a final 2X10-M, to test the integrity of the rings. Next, each test ring was washed three times with fresh buffer, with intervals of 5 minutes between washes, and allowed to rest for one hour. For vasodilation testing, the rings were tightened to two grams and allowed to stabilize for 15 minutes. Next, 75iDZz7 was added to 1, 2, or 3 of the 4 baths without displacement, and the tension on the rings was recorded and compared with the control rings. The rings were further tested for contraction with norepinephrine as described above. Rings were tested at 323, 162, and 8 ng/ml 28idZ7, but a dose response could not be determined. To assess the statistical significance of the results, a limited test was performed on all treated 25-37 and control rings, using stretch as a determinant of 10 of 12 rings, and 25-37 vasodilation was tested as was done with 2 of 7 controls, the Fisher's exact P-value is 0.045.

It was concluded that 25id37 induces vasodilation in the aortic rings shortened by norepinephrine.

Example 12 Binding of 25id37 to matrix proteins

ELISA (enzyme-linked immunosorbent assay) was used to measure the binding of 25id37o to various matrix proteins and Sid complement. The matrix proteins used were bovine type I collagen (Vesiop OisKipzop, Hypsoip Ragk, M) with laminin, vitronectin, fibronectin, human collagen of types II, Sh, M, M, MI (Spetisop Ipiegpaiopa!i, Tetessha, SA). VBA M (bidta So.) was used as (a) negative control. Immediately before use, proteins were diluted in 2 X РВ5 (phosphate-buffered saline, Zidta Co.) to 100 μg/ml and adjusted to pH7.2 with 0.1N Mahon. Each protein sample

Zo was applied in four replicates (1000 μl/cell) on a 96-well plate. The tablet was allowed to dry in overnight in a cabinet with a laminar flow mode and washed 3 times with 400 μl of a solution of 5 mg/ml B5A in 1 Xx РВ5 and blotted dry. 785idZ7 was RITS-labeled according to the manufacturer's instructions (Riegse, Kosktoga, 3. 100 μl of 1.8 μg/ml 28id37-RITS in 595 VBA, RV5 was added to each cell. The plates were incubated for 1.5 hours at room temperature, then washed 3 times 595 В5А, РВ5. 100 μl of diluted 1:400 mouse anti-RITS/biotin (Zidta So.) was then added to each cell. The plate was incubated for 1.5 hours at room temperature and washed three times with 595 В5BA, РВ5. The plate was further incubated with 100 μl of a 1:1000 solution of streptavidin/NKR "" (Atehepat, Rizsa(azhau, M.)) for 1 hour and washed 3 times with 595 VZA, РВ5. The plate was further developed " using Ziregvidpa!? ha (Riegse, Koskoga, I ) according to the manufacturer's instructions.

After the course of the reaction for 1 minute, the excess liquid was removed from the tablets by turning them over and patting them dry. The tablet was exposed to X-ray film (Kodak, Bosnezieg, Mem/Mogk). - The results of this screening show that only fibronectin and collagens I!, II, ISH, M and MI noticeably bind to 275id37-RITS. Such binding was not observed with laminin, vitronectin, IM collagen or

V5ZA control. o Example 13 Specificity of 25idZ7 binding to MI type collagen

Ha 50 The EGISA binding assay described in Example 12 was modified to quantify the binding of 28iZ37-RITS in the range of 0.4-4µg/ml bound to 1µg of collagen type MI (Spetisop Ipiegpaiopai) as 7 described above. The luminescence of the reagent "ZyregzidpaI" was read on a tablet reader Uyas 1420 (Umais,

Saipegzrigd MO), and the intensity was used to quantitatively measure the binding of 25id37-RITS to the EGISA tablet. 25 Joining 25id37 to type M collagen! corresponds to a typical hyperbolic binding curve (Fig. 3a).

GF) Bound 758id37-HITS, which was applied at 0.4μg/ml, can compete with collagen by adding unlabeled 28idz7 in the range of 0.8-8μg/ml (Fig. 3a). These results may indicate that binding to domains on collagen type MI is specific and concentration dependent.

Example 14 Binding of 28id37 to the Sid complement 60 78id37-RITS at 0.2 μg/ml, as was shown by the method described above in Example 13, binds to the Sid complement (Zidta So.) at 0.1-1 Ωkg/ml ( Fig. 4). The degree of binding is concentration-dependent and saturable.

Example 15 Inhibition of complement using 75id37

Complement studies were carried out in 96-cell round-bottom plates. Megapai's gelatin buffer containing magnesium and calcium (141 mM Mg, 1.8 mM sodium barbitol, 3.1 mM barbituric acid, 0.190 bovine gelatin, 0.5 mM MgCl, and 0.15 mM Sac) was used for all serum dilutions and inhibitors, as well as erythrocyte suspensions. Fifty microliters of standardized human complementary serum (Zidta

So.), diluted 1/37.5 (for a final dilution of 1/150), was added to each cell. The inhibitor was added in triplicate, 50 μl/cell. Serum and inhibitor were incubated for 30 minutes at room temperature. The analysis was started by adding 100 μl of 2X10 Zml of non-sensitized sheep erythrocytes (Soiogado

Zegyt So., YuOepmeg, SO) sensitized sheep erythrocytes, sensitized using the protocol of the manufacturer of hemolysin (Viomupichakeg Ips., UuaiIkegemShe, MO) and rabbit erythrocytes containing 16mM EGTA and 4mM Ma 77.

A sickle dilution of human serum from 1/50 to 1/400 was also applied as an active control. Erythrocytes 70 lysed with distilled water and diluted to 100, 75, 50, 25, and 12.5 percent lysis were used to quantify percent complement lysis. The plate was capped and incubated at 372C for 1 hour with agitation every 15 minutes. The reaction was stopped by adding 220 mM EDTA, 2 µL/cell, and the plates were centrifuged at 1500 x for 10 minutes. One hundred microliters of supernatant was removed from each cell and transferred to a 96-well flat-bottomed plate for analysis. The plate was read at 415 nm and 75 percent lysis was calculated. 78id37 was effective in inhibiting the classical exchange pathway with both sensitized and non-sensitized sheep erythrocytes (Fig. 5). No significant inhibition of the alternative metabolic pathway tested by rabbit erythrocytes and EGTA was found. The mechanism of inhibition is uncertain, but since Cyd binds to 25id37, Cyd is the most likely target.

Example 16 Inhibition by means of 758id37 activation of platelet collagen

Blood from healthy volunteers was collected in tubes containing sodium citrate, kept at room temperature and used within four hours of collection. Whole blood was analyzed for platelet activation using a whole blood luminescent aggregometer Spgopo-iI od 560A I iti-Adagedoteyeg (Spgopo-I od Sogr., Namepop, RA) according to the manufacturer's instructions. For each investigated point, 500 μl of blood was added to a reaction tube containing a stirrer and 500 μl of isotonic physiological solution containing 278 of 37 at concentrations of 0-20 μg/ml. The mixture was incubated for four minutes, followed by activation of platelets, which was initiated by adding 5 μl of a solution of mg/ml cross-linked collagen (SPpgopo-ii od) to the mixture

Sogr.). Inhibition of activation by AP (final concentration 1 µM) and thrombin (final concentration 1 µL/ml) was tested similarly.

Inhibition of collagen-mediated platelet activation by 25id37 reveals a dose-dependent relationship between 5 and 20 μg/ml (Fig.ba). Inhibition is selective for collagen activation and does not affect AOR- or thrombin-stimulated activation (Fig.br). Collagen activation was not inhibited by another complement-related (α) Sid protein 25id39 | co-filed US patent application 09/1408041. oh

Example 17 Growth stimulation of 5K5 fibroblasts using 75id37

Human fibronectin (25 μg/ml, ZIVSO VA, Saipegerigd, MO) was applied to 96-well plates (Soviag, their

Rigegazapiop, SA) at 1000 μl/cell and allowed to dry in a laminar cabinet overnight. Human fibroblasts

ZK5 in OMEM (birso), which includes 1095 cow embryo serum - lower endotoxin (Nusivpe, I odap,

OT) were applied at 5BOOO cells/cell on fibronectin-coated tablets and incubated at 379C, 590

CO" for 2-3 days. The number of cells per plate was brought up to a non-confluent state. The cells were then washed twice with serum-deprived UOMEM with glucose (Sirso) and, experiencing a lack of serum ts due to growth in serum-deprived OMEM with glucose for 24 hours, 78 of 37 was added to the cells in triplicate, at concentrations from 312.2 ng/ml to 1000 ng /ml in 100 μl of OMEM. The cells were further incubated "for 48 hours at 372, 5956 СО". Cell proliferation was tested by adding 15 μl of MTT dye solution (Seilighobtm kit, Rgoteda) to each cell. The tablet was incubated for 4 hours at 372С, 5956 СО" and the reaction was stopped with Zoicrpgaiop/Zor solution (SeiTi kit (egob tm, Rgoted) according to the manufacturer's instructions. The tablet was incubated for 1 hour until the formazan crystals dissolved and the absorbance was measured at 57 Ohm with reference to b50 nm , using the EIG IZA tablet reader.

The results (Fig. 7) show a dose-dependent increase in the number of ZK5 fibroblasts within the tested concentrations of 25 and 37. These concentrations were within the limits of the values shown for the mitogenic effect of the fibrinogen chain оз 20 ї (Ogau, ей ай, Ат .) Kezrig SeiІ Mo! Vio! 12, 684, 1995 and Sgau, et al., 9. Vioi. Spent 270, 26602, 1995) and . for the adhesion of fibroblast cells to the applied Seed (Vogaip, and zpebgepimei, 9). Etc. 145:2520, 1990) both of which are thought to interact with cell surface calrecitulin.

Example 18 Production of serum against 28idZ7

Rabbit polyclonal antisera were produced by immunization of two female New Zealand white rabbits with VNK 25id37-CEE cells purified. The protein was condensed with the carrier protein hemocyanin of the plate (KIN) for

GF) using glutaraldehyde. Rabbits each received an intraperitoneal (ir) injection of 200 µg peptide in complete Freund's adjuvant, followed by booster intraperitoneal injections of 10 µg peptide in incomplete Freund's adjuvant every three weeks. 7-10 days after receiving the second booster injection, animals were bled and serum was collected. The animals were then bled every three weeks. 60 Example 19 Detection of labeled RITS protein 78id37 by binding in tissues

Labeled RITS 728idZz7 binding protein in tissues was determined as follows. Paraffin-embedded and slide-cut tissues of human or mouse embryos were obtained from commercial sources (ie, BAKO Sogrogayop,

Sagripiepa, SA, Viobepeh, Zap Katop, SA, Momadep, Madizop, UMI, and Vioteda, Rovzieg Su, SA) or themselves Human tissue sections included adrenal gland, brain, heart, small intestine, large intestine, kidney, liver, because lung , ovary, pancreas, prostate, spleen, stomach, testes, thyroid, and uterus. Sections of mouse embryos were from the 16-day stage.

Tissue sections were deparaffinized using standard conditions - 3x5 minutes in xylene, 4 minutes in 10090 ethanol (EUN), 3 minutes in 10095-EUN, and 2 minutes in 95965 EUN. The tissue sections were then subjected to a 20-minute antigen retrieval at 942C according to the manufacturer's instructions (SAKO Sogrogayop), followed by a 20-minute digestion with 0.0195 pepsin/0.2N NOI. Tissue sections were washed twice in 4H»O and once in buffer РВ5/0.0590.

Tween 20 (Zidta. ZI. I otsiv, MO) and then blocked for 45 minutes with 1 X RVBZ/59085A/5906 skim milk powder (Sagpaiyop, I oz Apdeiez, SA). This was followed by an avidin/biotin blocking step, which was performed according to the manufacturer's instructions (Mesiog I Arogayogiez, PS, Vygipdate, CA). Tissue sections were washed 3 times in buffer 70.1 X RVB/0.0595 Tween 20 and further incubated with the concentration of RITS-labeled protein 28idZz7 in RVB/59085A for 45-60 minutes. After washing the tissue sections 3 times in 1 X РВ5З/0.0595 Tween 20, they were incubated with diluted 1:400 anti-RITS (to) MAB with biotin condensate (Zidta) for 30-60 minutes, washed 3 times in RVB/0 ,05905 Tween and further incubated for 30-60 minutes with diluted 1:500 streptavidin-RITS (MEM I Fe Zsiepse Rhodisiv,

Vozviop, MA), followed by 2 washes in 1 X RVB/0.0595 Tween 20 buffer and 1 wash in 1 X RB5 without Tween 20. 75 Tissue sections were then placed on glass slides with counterstaining medium containing 0.5 µg/ ml of propidium iodide as a counterstain.

Prominent binding was observed with vessel walls and thin fibrous connective tissues such as collagen matrices in most human and embryonic tissue sections examined.

Example 20 78id37 in the rabbit carotid artery injury model 20 78idZ7 was used in a modified rabbit carotid artery injury model |Roiiv et al., Sigsshaiop, 79 116-24, 1989 and Soiip et al., Tpygotroviv and Naetozviavziz 67:302-5, 1992) to determine the degree of protection offered in preventing vascular occlusion following compression injury.

Thirty-four male New Zealand white rabbits, approximately 3-6 months old (KK Karbigu, Ziapzhmosa,

MA) were divided into two groups. Fifteen rabbits received a dose of 28idZ7 from 2 to 13.5 μg/kg, and 19 control Ha rabbits were injected with RBBZ or an equivalent amount of RBB5 or 28idZO, another adipocyte complement-related protein (MU099/10492). Rabbits were anesthetized with ketamine (5Omg/kg, IM) and anesthesia was maintained with halothane inhalations during the study. Hair from the ears and cheeks was shaved and an anpocatheter was placed in a small ear vein for IV support. An incision was made along the midline of the cheek and the carotid artery was exposed. Approximately Bbsm of the common carotid artery proximal to the internal/external bifurcation was bluntly dissected from the surrounding tissue and any visible side branches cauterized. A flow probe (Tgtapzopis Zuzietv Ips., Kasa MU) was placed distal to the intended wound site and baseline blood flow was established. A segment of a vessel 2.5-3.0 cm long was further isolated from the circulation using non-traumatic vascular clamps. After removing blood from the vessel segment, 0.4 ml of 28id37 in 0.995 sodium chloride or

0.04 ml of 0.996 sodium chloride as a control was injected into the empty segment of the vessel using a 300 gauge needle.

The vessel was left intact during the 5-minute treatment of the previous wound. A 1.0 cm compression wound was further applied to the center of the vessel segment using a protected hemostat and left undisturbed for 10 minutes. The vascular clamps were then removed and blood flow was reestablished. Blood flow was continuously monitored for 60 minutes, after which the rabbits were sacrificed and the vessel excised for histological analysis.

No dose dependence was observed at these concentrations. A meta-analysis of all doses of 285id37 led to a significant increase in transit time when compared with the control in an unpaired i-test (Р-0.019). The mean percent transit time for the combined C-group negative control animals determined from blood flow recordings was 13.595 with a standard error of --1.795 The mean percent transit time for the combined treated 25 of 37 groups of animals determined from blood flow recordings was C37.295 with a standard error of -10.395. -i In a second series of experiments, fluorescein 25id37 was used in the carotid artery injury model. sl Male New Zealand white rabbits were anesthetized as above. The carotid artery was exposed through an incision in the cheek and separated approximately 5 cm from the surrounding adjacent tissue. Blood was removed from the selected segment and (c) non-traumatic vessel clamps were applied. Approximately 0.0 bml of fluoresced 28i37 (concentration of 50 00 µg/ml) was injected into the selected segment until the vessel was completely filled using a 309 needle. After holding for 5 minutes, the vessel were damaged and left for another 110 minutes, after which the clamps were removed and dried blood flow was established. Animals were sacrificed as described above at 1, 10, and 60 minutes after establishment of blood flow, and vessels were harvested and fixed in formalin for histological evaluation.

Labeled 25id37 preferentially binds to the receptor in the environment of injured vessels. Marked 25idZ37 do not join the areas of the vessel that are not injured. Blood flow time to vessel sampling was not found to affect the amount of 25i37 remaining bound to the tissue, ie, there was no difference in the amount of labeled 28i37 bound to the tissue at 1 minute compared to the 1-minute sampling time. This may indicate that 28id37 is compactly attached to the wounded vessel and is not washed away again by the established blood flow.

The effect of 285iZ7 on the dynamics of blood flow after vascular injury in the compression/stenosis injury model of the rabbit iliac artery was also evaluated. Young adult male New Zealand White rabbits were anesthetized as described above. Through an abdominal incision, the branches of the iliac artery were exposed, and each iliac artery was freed from the surrounding tissue and the main branches were ligated. Each iliac was equipped with an ultrasonic flow probe to monitor blood flow through the vessel. Based on the blood flow data, one iliac was selected for use in the wound, and the others were catheterized to obtain a test sample. B5 rabbits were divided into dosage groups of b animals/group. Doses of the test sample including 285iZ7 were increased in semi-logarithmic increments from Z to 7100 Ωkg/kg during the selected infusion period. Infusion of the test samples was started followed by the creation of a critical stenosis, which reduced the blood flow through the vessels by approximately 5095. After creating the stenosis and stabilizing the blood flow, the vessels were damaged by squeezing them between the grips of smooth needle holders. The infusion was continued after the injury for 10-20 minutes. Blood flow

Through the injured vessel was monitored for 60 minutes after injury. Animals were killed at the end of the study. The lower section of the abdominal aorta and each iliac was collected and fixed in formalin for histological evaluation.

Blood flow parameters determined from flow recordings included mean flows after stenosis, mean flows after wounding, and time during which vessels remained patent. These results confirm the tendency of 28i937 to promote an increase in the time of the open state with an increase in dose up to ZbOmkg/kg during the 6bO-minute period.

Example 21 Relaxation of serotonin-induced contraction of rat aortic rings

Male Zrgadoe-OaulLeu rats, approximately 3 months old, were lightly anesthetized with CO 5 and decapitated.

The thoracic aorta was then quickly removed and placed in modified Krebs-Henseleit buffer (Massey, 1.18.2mM,

KSI 4.6mM, Sasi", 2.5mM MazO), 7NHO, 12mM, MansSoz, 24.8mM, KNoRO,), 1.2MM, and glucose, 10.0mM). From each rat, four sections of aortic rings of 2-3 mm were cut after discarding the uneven end of the aorta. In some experiments, the subendothelium was exposed before cutting the rings by rubbing the aortic cavity along 21-gauge needles. Exposure of the subendothelium was confirmed by the addition of an analogue of acetylcholine, carbachol, to determine the concentration-dependent reactions of 25 and 37. In the absence of subendothelium did not cause vasorelaxation contraction

Sections of vessel rings.

The rings were fixed and attached to a shear force sensor in an oxygenated (9595 025, 596 СО"), lined glass organ bath at 302С in a modified Krebs-Henseleit buffer, pH 7 4. The tension at rest was set when the rods were loaded and continuously adjusted to g during 1 hour of incubation. Fresh oxygenated modified Krebs-Henseleit buffer was added to the bath every 15 minutes during incubation at rest. After 1 hour of incubation, the sections of the rings were shortened by adding 10 μM serotonin. After reaching the maximum reduction, approximately 15-20 minutes after the addition of serotonin, cumulative concentration response curves of 25 and 37 were constructed. 78idZ7 was added to baths of 5 ml in volumes from 5 to 1500 μl to final concentrations in the range from Ing/ml to 4 Ωkg/ml. The viability of the ring sections was confirmed after the concentration reaction by adding forskolin (2.5 µM or 25 µM) or nitroglycerin (22 µM).

Addition of 25id37 induced concentration-dependent vasorelaxation of serotonin-shortened aortic slices and. rat with or without intact subendothelium. Relaxation in the reaction of 28id37 was first observed at concentrations above 10 ng/ml. Relaxation was observed approximately 30-6 seconds after the addition of each concentration of 25937 to the bath, and the relaxation reactions reached a plateau within 3-5 minutes after the addition of 25id37. The nature of

35 relaxation of the reaction to 28id337 indicates that vasorelaxation is mediated by the receptor-additional messenger of the result. Additionally, the ability of 25id37 to vasorelax with exposed subendothelium of aortic slices suggests that 25937 acts directly on smooth muscle cells to induce a vasorelaxation response.

Example 22 Identification of cells expressing the 25id37 receptor using IP hybridization

Specific human tissues were isolated and screened for the expression of 25id37 by PCR hybridization. Various human tissues prepared, sliced and subjected to ip hybridization included aorta, heart, lymph node, placenta, prostate, salivary glands, skin, and testes. Tissues were fixed in 1095 buffered formalin (Zygairai, and Kisptopa, I) and embedded in X-iga paraplast (Ohioga Zsiepiyis, 5. I otzis, MO) and cut into slides with a thickness of "" BbBmkm with a Keispagi-ypo 2050 microtome (I eisa Ipvigitepiz StrN , Mizzviosi, Segtapu). The fabrics were cut with a thickness of 4-8 μm. Tissues were produced using a standard protocol (Oemeiortepi oi popo-isorois ip

VSH Pubgidigayop" (Development of nono-phsopotic hybridization of ip vii), Garogaigu oi Ehregitepivi! Raipoiodu, -i Myopa! Ipviyshe oi Epmigoptepiai Neaky Zsiepsez, Kezeagsp Ragk Tgtiapdie, MS). Briefly, the tissue sections were deparaffinized using NivioSieag (Mayopa! Oiadpoviiav, ACapis , SA) and further dehydrated with ethanol. Then the sections were digested with proteinase K (5 Ωkg/ml) (Voeigzpdeg Mapppeit, Ipaipaparoyz IM) at 3723 for av) 2-20 minutes. This step was accompanied by acetylation and tissue rehydration.

The three probes created by RSK were designed with respect to the sequence of 25id37 of a person. Two sets

Oligonucleotide primers were designed to create probes for the separation of 25id37 cDNA regions: (1) "I Oligonucleotide 2023689 (ZEO IU MO:45) and 2223694 (5BO I MO:46) were used to create a 414-by probe for 28id37; (2) 7c23703 (5EBO IU MO:47) and 7023697 (5EBO IU MO:48) were used to create a probe of size 896 for 28idZz/; (3) 2024441 (5BO I IU MO:49) and 2024442 (5BO IU MO:50) were used to create a probe of size 290 by 28 by 37. Antisense oligos from each set also contained a working sequence for RNA polymerase 77 promoters to facilitate transcription of antisense RNA probes from these products. The PCR products were purified with Oiadep zvrip columns (Oiadep Ips., Spayovo, CA), co followed by phenol/chloroform extraction and ethanol precipitation.Probes were further labeled with digoxiginin (Voepppdeg) or biotin (Voepppdeg) using the ip migo transcription system (Rgoteda Sogr., Madizop, 60 MI) according to the manufacturer's instructions.

After that, hybridization was performed with digoxigenin- or biotin-labeled probe 28id37, as described above. The probe was added to the slides at a concentration of 1-spmol/ml for 12-16 hours at 50-602С. Slides were further washed in 2

X 5B5C and 001 X 55C at 50-559C. The slides were further washed in 2 X 55C and 0.1 X 55C at 50-5592C. Signals were amplified using tyramide signal amplification (TZA ip zyi indirect kit, MEM, Voviop, 65 MA) and visualized with the Mesiog Kei substrate kit (Mesiog I arogyaugiez, Wygitdate, CA) according to the manufacturer's instructions. The slides were further counterstained with hematoxylin (Mesiog I Arogayogiev).

Positive signals were observed in human aorta, heart, prostate, salivary glands and testes. Positively stained cells were detected as endothelial cells of vessels of small diameter in the adventitia surrounding the aorta, mesothelial cells covering the epicardium, acinoid cells of the salivary glands and scattered mononuclear cells, trophoblasts of the placenta, epithelial cells of the prostate and stratified epithelium of the vas deferens of the testicles.

Example 23 BES-MAI I 5-analysis of 28idZ7 78idZ7 includes an M-terminal collagen-like domain, as well as a C-terminal globular region that is homologous to the tumor necrosis factor family and, like other such proteins, 28id37 would be expected to 7/0 MUmultimerize. Purified 25id37 analyzed using an Eb5I ion trap mass spectrometer (RGippidap Mai, Zap Doze, CA) showed the presence of specific approximation trimers and O9-mers, which were unexpected results when compared to other homologous proteins. Peptide mapping of 28id37 using mass spectrometer I! C-M5/M5 with an EZI-ion trap (Rippidap Mayu), revealed that several cysteine residues are modified with a 5-cysteinyl group. It may be that the modification of the key cysteine residue in the 25id37 protein during fermentation with free cysteine in the medium prevents the possible oligomeric association of these molecules.

For a better study, reduced and unreduced 25id37 were compared using a Vioser 5-300 size exclusion column at 1.00 ml/min (7.8x3000 mm Rpepotepeh Toitapse, SA) on HPLC HP 1050 (NemLei RuskKaga, NeiiaIerego.-Zeptapu) HP 1050 was combined with weakly scattering detectors according to the refractive index, tipi0AMUM and Ortsar OR, (UUau(f Tesppoiodu Zapia Vagraga SA) for ZES-MAG I 5 working online.

One milligram of recombinant 28id37 (1.00 mg/ml) was added to TSER at a ratio of 10:1 mol/mol TSER to 28id37 and incubated at room temperature for 70 minutes. Sixty microliters of the recovered 28id37 was introduced into the analysis of BES-MAIGH HER 5, and the rest of the recovered 25id37 was dialyzed in cassettes of 0.5-3, Oml 5iide-A-I! ugheg, p

LOCK MUUSO (Riegse, Koskooga, I.) with mixing against РВ5, рН7.4 with three buffer changes in this way Tl o

RV5 at room temperature for 4 hours, Tl RVZ at 42C overnight, and Tl RV5Z at room temperature for 4 hours.

After dialysis, oxidation was continued at 42. Oxidation was monitored by ZES-MAG I analysis of 5 aliquots taken at three time points, 1-0 hours, 1-24 hours, and 1-96 hours. Molecular mass values were determined - using the two-detector method and 5-KI.

Analysis of the reconstituted dialyzed recombinant 285id37 initially shows the formation of hexamers and o 18-mers, as determined by ZES-MAIH I 5, which better coincides with the oligomeric states observed in homologues. ("in

These forms were also active in the study of IP miigo.

Example 24 Binding of 28idZ7 to monocytes o

Cr14-positive monocytes were isolated from frozen peripheral blood aphaeresis using the /-Iche method of positive selection with MiMepuy beads (Mikepuyi Vioyes Airrigp, CA). Purified cells were more than 8096 CO14-positive at EGASZ-manifestation. Cells were resuspended at 1 x 1O cells/ml in VRMI1095 bovine embryo serum (EB5) and applied to 100 mm tissue culture dishes, bml/plate. "

Recombinant human interferon G was added at 1OOng/ml and the cells were incubated at 595С, 372С for 48 hours. t c Cells were removed from the plates by an enzyme-free method using EDTA and scraping, concentrated by centrifugation, resuspended in GASZ development buffer and aliquoted at 500,000 cells/tube for development. Non-activated cells were obtained by carrying out another CO14-selection of the same apheresis product after 48 hours of exposure in G-interferon. Cells were incubated in variable concentrations of biotinylated 25id37, followed by PE streptavidin. All blocks with a "cold" 28-37 were performed on the ice within 30 minutes. Unbound protein was removed by washing once in GAS5 buffer. Binding c was quantified using an EAScS5-calibrated instrument (Vesiop OisKipzop, Ipsoip Ragk, M), and expressed as only the signal control of the secondary antibody defined above. The activation of monocytes was confirmed by an increase of about an order of magnitude in the expression of ISAM-1 in G-interferon-treated cells. c 50 78id37 binding was determined in activated and non-activated monocytes with an increase in 28id37 binding observed in G-interferon-treated cells. Binding was determined up to 1.5 μg/ml, the lowest concentration tested. At 15 μg/ml, binding increased approximately 4-fold in activated cells. A slight (approximately 1095) decrease in binding was observed only in activated cells, that were pretreated with a 70-fold excess of "cold" 28id37. The increase in 25id37 binding in activated 255 monocytes confirms the upregulation of monocyte-binding proteins/receptors for 25id37 by inflammatory cytokines. This could

GF! potentially leading to the involvement of 78id37 in monocyte phagocytosis, microbial killing, and cellular cytotoxicity. After two days in the culture, macrophages appear in the culture and 25id37 can be preferentially associated with this subset of cells. There are no good macrophage markers suitable for determining whether this occurs 725id37 also binds to the mouse monocyte/macrophage line KAMU 264.7 (ATSS Mo.SKI -2278), 60 showing macrophage specificity.

It is clear from the foregoing that although specific embodiments of the invention are set forth herein for illustration purposes, various modifications may be made without departing from the spirit and spirit of the invention. Accordingly, the invention is not limited by anything other than the appended claims. b5

Claims (27)

The formula of the invention 1. Use of an adipocyte-complement-related protein, which reduces thrombogenic activity and the activity of complement in the vascular system, for the manufacture of a pharmaceutical composition intended to stimulate blood flow in the vascular system of a mammal. 2. Application according to claim 1, which is characterized by the fact that the indicated polypeptide reduces thrombogenic activity and complement activity due to inhibition of the complement exchange pathway and inhibition of collagen-mediated adhesion, activation or aggregation of platelets. 3. Application according to claims 1 or 2, which differs in that the composition is made to be administered before, during or after acute vascular injury in a mammal. 4. The application according to point C, which differs in that the composition is intended for the therapy of wounds caused by the reconstruction of blood vessels. 5. Application according to claim 4, which differs in that the specified vascular reconstruction includes angioplasty, coronary artery bypass grafting, endarterectomy, microvessel repair or vessel graft anastomosis. 6. Application according to claim 3, which differs in that the specified injury is due to trauma, attack or aneurysm. 7. Use of an adipocyte-complement-related protein, which renders damaged collagen tissue inert to complement activation, thrombotic activity, and immune activation, for the manufacture of drugs designed to repair damaged collagen tissue in a mammal. 8. Application according to claim 7, which is characterized by the fact that the indicated damage to the collagen tissue is due to injury associated with ischemia and reperfusion. 9. Application according to claim 8, which is characterized by the fact that said injury includes ischemia in a traumatic injury, intestinal strangulation, or injuries associated with previous and subsequent restoration of blood flow. with 10. Use according to any one of claims 8, 9, which is characterized by the fact that the indicated polypeptide is prescribed (5) to a mammal suffering from ischemia during artificial blood circulation and resuscitation, myocardial infarction or post-traumatic vasospasm. 11. Application according to claim 10, which is characterized by the fact that said post-traumatic vasospasm includes an attack, percutaneous transluminal angioplasty, endarterectomy, vascular injury in an accident or vascular injury in a surgical operation. with 12. Use of an adipocyte-complement-related protein that renders the surface of a prosthetic biomaterial inert with respect to complement activation, thrombotic activity, or immune activation, for (av) the manufacture of drugs to inert the surface of a prosthetic biomaterial used in mammals. 13. Application according to claim 12, which differs in that the surface of the prosthetic biomaterial is covered with collagen or collagen fragments, gelatin, fibrin or fibronectin. 14. Use of an adipocyte complement-related protein that enhances the progress of wound healing for the manufacture of a medicament for mediating wound healing in a mammal. " 15. Application according to any of claims 1-13, which is characterized by the fact that the specified adipocyte-complement-related protein includes a polypeptide that corresponds to the sequence of amino acid residues, which is at least 7595 identical to the amino acid sequence of ZEO IYUO MO:2 by residues 26-281, where the sequence is indicated and includes the repeats (u-Haa-Haa or Siu-Haa-Rho) forming a collagen domain, where Haa is "any amino acid" and a carboxy-terminal globular part. 16. Application according to claim 14, which is characterized by the fact that the polypeptide includes a sequence of amino acid residues that is at least 9095 identical to the amino acid sequence of ZEO IYUO MO:2 by residues 22-281. - and 17. Application according to claim 14, which is characterized by the fact that the specified polypeptide includes an amino acid sequence that is at least 9095 identical to the amino acid sequence of ZEO IJUO MO:2 at residues 265-281. (av) 18. Application according to any of claims 14-16, which is characterized by the fact that any differences between the specified 50 polypeptide and ES IUO MO:2 are due to conservative amino acid substitutions. 19. Application according to any of claims 14-17, which differs in that the specified collagen domain consists of 13 Siu-Haa-Haa repeats and 1 Siu-Haa-Rgo repeat. 20. The application according to any one of claims 14-18, which is characterized in that said globular domain consists of ten beta-sheets. 21. Use according to any one of claims 14-19, which is characterized by the fact that said beta-plasts are linked to amino acid residues corresponding to residues 147-151, 170-172, 178-181, 191-203, 207 -214, 219-225, 227-239, 244-250 and 269-274 of the sequence of BEO IU MO:2. name) 22. Application according to claim 14, which is characterized by the fact that the specified polypeptide includes residues 1-281 of the ZEO IU MO:2 sequence or residues 1-281 of the ZESO IU MO:44 sequence. 6o0 23. Application according to claim 14, which is characterized by the fact that the indicated polypeptide is combined with the second polypeptide to form an oligomer. 24. Application according to claim 22, which differs in that the specified polypeptides are united by intermolecular disulfide bonds. 25. Application according to any one of claims 22, 23, which differs in that the indicated oligomer is a trimer. 65 26. Application according to any of claims 22, 23, which differs in that the specified oligomer is a hexamer. 27. Application according to any of claims 22, 23, which differs in that the indicated multimer is an 18-mer.