UA75865C2 - USE OF AGONIST OF TISSUE FACTOR FVII OR FVIIa FOR TREATING PATHOLOGIES ASSOCIATED WITH CELL MIGRATION OR CHEMOTAXIS - Google Patents

Abstract

The invention relates to the use of agonist of the tissue factor FVII or FVIIa or their combination for treating the pathologies associated with cell migration where the specific control of such cell migration or chemotaxis is advantageous for the therapy. The following pathologies may be treated with the agents stated above: atherosclerosis, cancer growth, metastasizing, and invasion, wounds and burns (wound healing and the regeneration of the vascular walls).

Description

Description of the invention

A new cell-regulatory activity of MI coagulation factor has been described! (EMII) or an antagonist of tissue factor 2, for example, inactivated coagulation factor Ma (EMiIai) of cells that express tissue factor (TP). The present invention relates to a method of regulating cell migration or chemotaxis by contacting a cell with EMiia or another TE agonist, or EMiia or another TE antagonist and determining the migration of the aforementioned cell. The invention also relates to the use of EMI or other TE agonist, or EMI or other TE antagonist for the preparation of a medicament for regulating cell migration in a patient. In addition, this 70 invention relates to a method of treatment and a method of detecting the activity of compounds, in particular tested drugs, which interact with cell migration.

The external pathway of blood coagulation begins to act when EMI circulating in the plasma binds to the component protein of the membrane, tissue factor (TE). Many studies have been devoted to the role of TE in blood coagulation. It is believed that the participation of EMI as a proteolytic enzyme in the blood coagulation sequence is limited to the extracellular layer of 72 TE-expressing cells. The intracellular activity of EMIIa was primarily discussed when the sequence

TE showed homology with the superfamily of cytokine/interferon or hematopoietic receptors. A subclass of the hematopoietic receptor family includes receptors for growth hormone, prolactin, interleukins 1 through 7, granulocyte-macrophage colony-stimulating factors, erythropoietin, and thrombopoietin. Subclass II includes TEs and receptors for interferon a and b.

The similarity of TE to this class of receptors was also confirmed by the appearance of the crystal structure.

Characteristic of this class of cytokine receptors, which includes receptors for interferon B and d and 1-10, is that their activation leads to rapid tyrosine phosphorylation of the receptors themselves, as well as a subset of intracellular proteins. Several minutes after the initial tyrosine phosphorylation, a set of mitogen-activated (Zeg/Tig) kinases (MAPKs) is activated. These kinases are located in several parallel c 29 signaling pathways. Careful studies of the potential intracellular signaling capacity of EMIIIa have shown that it induces mobilization of intracellular free calcium (Ca?) in the human bladder carcinoma cell line, U82, which constitutively expresses TE, and in umbilical vein endothelial cells, which were previously treated with interleukin-1 for TE expression, but did not reveal any cytokine-like activation of intracellular tyrosine kinases.It was concluded that EMiIa, in a TE-dependent manner, induces o

"Mobilization of intracellular Ca" through the activation of phospholipase C. The mechanism by which EMITA (Ce) activates phospholipase C is unknown, but the activation of tyrosine kinase is definitely excluded.

Recent reports from many laboratories suggest that TE can affect several important biological functions other than coagulation, such as angiogenesis, embryonic vascularization, and tumor metastasis. However, the role of TE in these biological processes remains unclear. The extracellular domain of TE consists of two fibronectin type II M modules, as in a typical extracellular domain of a class II cytokine receptor, making it possible that TE may play a role in signal transduction, the primary function of the cytokine receptor. However, TE has a very short cytoplasmic domain (only 21 amino acid residues long) and lacks membrane-proximal motifs that mediate the binding of non-receptor Janus kinases (JKs) that are important for cytokine receptor signaling. Despite this, several biochemical findings point to a signal transduction function for TE. Analysis of the protein sequence of human TE indicates a possible phosphorylation site in the cytoplasmic domain, which is conserved in mouse, rat, and rabbit TE. Characteristic serine residues in the cytoplasmic tail of TE are phosphorylated in cells after stimulation with the activator of protein kinase C. The cytoplasmic tail of human TE is phosphorylated at several 415 sites when incubated with lysates of O087-MoO cells. The possible role of the cytoplasmic domain of TE in transduction of the -1 signal is also indicated by the results of studies that showed that the prometastatic function of TE is critically dependent on the cytoplasmic domain of TE. In addition, it was shown that the cytoplasmic domain of TE interacts with actin-binding protein 280 (ABP-280) and supports cell binding and migration through involvement of ABP-280 in TE-mediated adhesion contacts.

However, TE has also been found to be involved in certain types of signaling in cells as a cofactor for (o)physiological ligand EMIIIa in extracellular signaling through a proteolytic mechanism. For example, it was found that the binding of EMIia to the surface of TE cells causes intracellular Ca 2" oscillation in many TE-expressing cells, short-term tyrosine phosphorylation in monocytes, activation of MAP kinase, changes in gene expression in fibroblasts, and increased expression of the urokinase receptor in In tumor cells, catalytically inactive EMiIa (EMiIa) is unable to induce many of the signaling reactions, from Ca 25 oscillation to MAP kinase activation and gene repair, and apparently the catalytic activity of EMiIa may be necessary for at least some iF) mediated TE-EMiIa signal transduction. Currently, little is known about the signaling pathway(s) that are triggered by proteolytically active EMIα and how EMIPA-induced signals may be involved in angiogenesis and tumor metastasis. In order to study the temporal program of transcription , which is the basis of the reaction caused by EMI, in this study, the authors examined the reaction of human fibroblasts to EMI, using a microarray inu cDNA. The data showed that the cellular expression of several genes in fibroblasts was markedly changed under the influence of EMIa. One such gene is Sugb1, a growth factor-induced intermediate early gene whose product promotes cell adhesion, enhances growth factor-induced DNA synthesis, and stimulates cell migration in fibroblasts and 65 endothelial cells.

The present invention relates to the use of EMII and/or EMIIIa and/or another TE agonist and/or EMIiIai and/or another TE antagonist in the therapeutic treatment of pathological conditions which may be associated with cell migration or which may be treated by specific regulation of cell migration or chemotaxis .

In another aspect, the invention relates to the application of EMI! and/or RMI! and/or another TE agonist and/or EMIIIai and/or another TE antagonist in the therapeutic treatment of pathological conditions that may be associated with the regulation of the expression of at least one gene in the cell, for example, the Sugb1 gene.

In another aspect, the invention relates to a method of inducing or enhancing cell migration, which includes the step of contacting the aforementioned cells with a tissue factor agonist.

In one embodiment, the tissue factor agonist is EMII or EMIIa. 70 In another aspect, the invention relates to a method of reducing or inhibiting cell migration, which includes the step of contacting the cell with a tissue factor antagonist.

In one embodiment, the tissue factor antagonist is a modified EMII.

In one embodiment, the cell is a human cell that expresses tissue factor, including fibroblasts, smooth muscle cells, tumor cells, hematopoietic cells, and epithelial cells.

In one of the variants of the embodiment, the modified MII factor is selected from among the MII factor modified with dansyl-RPpe-Pho-Agd-chloromethyl ketone, dansyl-SISH-Siu-Aga-chloromethyl ketone, dansyl-Rpe-Rpe-Agd-chloromethyl ketone, RPE-RPpe- Agd-chloromethyl ketone, dansyl-O-Rpe-Pho-Agd-chloromethyl ketone, dansyl-O-O1y-c1u-Agda-chloromethyl ketone, dansyl-O-Rpe-Rpe-Agd-chloromethyl ketone and O-Rpe-Rpe-Agda-chloromethyl ketone.

In another aspect, the invention relates to a method of inducing or accelerating wound healing in a patient, which comprises administering to said patient an effective amount of a pharmaceutical composition that includes factor Miia or factor MI! or another tissue factor agonist or a combination thereof.

In another aspect, the invention relates to a method of inhibiting or reducing cell migration, invasion, migration, cell proliferation, or angiogenesis in a patient having a disease associated with the undesirable condition Cell migration, invasion, migration, cell proliferation, or angiogenesis, which comprises administering the aforementioned to the patient an effective amount of a pharmaceutical composition, which includes an antagonist of factor i) tissue.

In one embodiment, the disease or condition is the development of a primary tumor, tumor invasion, or metastasis. In another aspect, the invention relates to the use of a tissue factor agonist to provide a medicament for inducing or enhancing cell migration. ise)

In another aspect, the invention relates to the use of a tissue factor antagonist to provide a medicament for reducing or inhibiting cell migration.

In another aspect, the invention relates to a method of regulating the expression of at least one gene in a cell, which includes the step of either contacting the aforementioned cell with a tissue factor agonist, or contacting the aforementioned cell with a tissue factor antagonist.

In one embodiment, the gene is a gene that belongs to the family of SSIM genes.

In another embodiment, the gene is selected from the group consisting of Sugb1, STEREO, dopamine O2 receptor,

EZT Ipsui RO 395116 or P2 nucleotide receptor. "

In one embodiment, the gene is the Sugb1 gene. with c In one embodiment, regulation causes or enhances expression. In another embodiment, the regulation attenuates or inhibits expression. ;" In one embodiment, EMI or EMI or other tissue factor agonist induces or enhances gene expression, and the modified EMI! or another tissue factor antagonist attenuates or inhibits expression

A gene, for example, if the gene is a gene belonging to the family of SCM genes, or the gene is selected from the group consisting of -I from Sugb61, STEREO, dopamine O2 receptor, EZT Ipsui PO 395116 or P2 nucleotide receptor.

In another embodiment, the EMI or EMIa or other tissue factor agonist attenuates or inhibits gene expression, and the modified EMI or other tissue factor antagonist induces or enhances expression

Go! of a gene, for example, if the gene is ЕЗТРОб74714.

Disease states that can be treated include such pathological conditions as, for example,

Me, atherosclerosis, tumor growth, tumor invasion, metastasis or angiogenesis. Other conditions that may be treated are, for example, wound healing, including regeneration of cell walls and the treatment of burns or inflammation, or the regulation of mycocellular migration, such as tissue growth.

DESCRIPTION OF THE FIGURES (ita 6011) 5B FigLA and 1B: Flow cytometric analysis of TE expression in fibroblasts (1A). Cells were stained with either mouse or rat fluorescein isothiocyanate-conjugated (RITS) mouse anti-

F) (open area), which was used as a negative control, or anti-tissue factor (TE) antibody conjugated with RITS ka monoclonal antibody (filled area). Figure 1B shows the procoagulant activity of fibroblasts. Fibroblasts with TE expression caused a 10-fold increase in PCA compared to monocytes without TP expression.

Fig2A: Effect of EMIa and EEC-EMiia on ROSE-BB induced chemotaxis in human fibroblasts. shows the chemotactic reaction of fibroblasts to different concentrations of ROSE-BB. Fibroblasts incubated with L1000nM EMia (ev) or 100NM EREK-RMIIa (o) migrated to a different concentration of ROSE-BB. Results are mean values and SEM of three separate experiments. P-values less than 0.05" were considered statistically significant (Student's test). 65 Fig. A-G. Effect of different concentrations of EMIa or BEK-EMia on ROSB-BB-induced chemotaxis in fibroblasts. Fig. 3 shows the migration of fibroblasts to other concentrations of ROSE-BB. Cells were incubated with 12.5(A),

25(B), 50(B) and 100(H) nM EMIa (g) or REC-EMIa (o) and subjected to analysis in a Boyden chamber with different concentrations of ROSE-BB. The results are the mean values and SEM of three different experiments. "-p" 0.05, thkr" 0.01 and i-p" 0.001 according to Student's test.

Fig4AA: A mixture of three anti-TE monoclonal antibodies blocks the effect of EMI and EEC-EM on induced

ROSRBE-BB chemotaxis in fibroblasts. shows the migration in the direction of ROSE-BB of fibroblasts without anti-antibodies

TE, (6) fibroblasts pre-incubated with anti-TE antibodies and 100 nM EMPa, and (0) fibroblasts pre-incubated with anti-TE antibodies and 100 nM EEC-EMiIIa. Results are mean values and SEM of three separate experiments.

Fig 5A and 5B: Effect of EC on the chemotactic response to ROSE-BB induced by EMIIIa. Fibroblasts were pre-incubated with 200nM TAR (Fig. BA) (w) or with 0.2-2 mM TAR (Fig. 5B) (w) and then with T00nM EMIia (0). TAR was present at all times during the experiments. Chemotaxis was induced by different concentrations of ROSBE-BB (5A) and

O, Tng/ml ROSB-BB (5B). Results are mean values and 5O of two separate experiments.

FigbA: Effect of thrombin on the chemotactic response to ROSE-BB induced by EMI. Fibroblasts were pre-72 incubated with Bod./ml (final concentration) of hirudin, and then with 100nM Emiia. Hirudin was present at all times during the experiments. Chemotaxis was induced by different concentrations of ROSE-BB. of experiments.

Chemotaxis was induced by different concentrations of ROSE-BB. Fig. 1 shows cells incubated with hirudin alone, and cells with hirudin and EMIIia. Results are mean values and 5O of two separate experiments.

Figure 7A: Effect of RiZ'-kinase inhibition on chemotaxis in fibroblasts incubated with EMiia. Cells were pre-incubated with varying concentrations of I U294002 during ZOhv. at 37 C, and then with L00nM

EMI (w) or without EMI (2). The inhibitor was present at all times during the chemotaxis assay. Chemotaxis was induced by 0.Tng/ml ROSE-BB. Results are the mean and 50 of two separate experiments.

Fig. 8A and 8B: Effect of RI C inhibition on chemotaxis in fibroblasts incubated with EMiIa. Cells were incubated with varying concentrations of 073122 (an active RI C inhibitor) (8A) or Ш73343 (inactive control) (8B) for 30 min. at 37 9С before incubation with L00NM EMIa or without it, and then subjected to analysis in the chamber (8)

Boyden with a concentration gradient of O.Tng/ml ROSE-BB. Agents were present at all times during the experiments. shows cells with SHUZ122 or only 073343, there are cells with OUZ3122 or 073343 and EMIP. Results are the mean and SD of two separate experiments. iv)

Fig. 9: Release of inositol triphosphate (IR) from fibroblasts stimulated by EMIa, only EEC-EMiia or in combination with ROSBE-BB. Cells were labeled until the next day with ZNI-inositol, incubated with 100 nM EMi or

EEC-EMiia or without them in the absence or presence of TOng/ml or 10O0ng/ml ROSB-BB. After that, the cells were subjected to analysis for the release in the IR. Unshaded bars show cells without EMIia or yu

EEC-EMIa (control), shaded bars show cells with REC-EMIa, and black bars show cells incubated with EMIa. -

Fig. 10: Tyrosine phosphorylation of RI C-1 in response to only ROSEBE-BB (control), EMPA or EEC-EMiIIa in combination with ROSBE-BB. Cells were incubated with TL00NM EMIP or REC-EMP for one hour, and then with

ROSBE-BB or without it in the indicated concentrations. Cells were subjected to lysis and tyrosine phosphorylation.

RI C-1 was determined as described for methods.

Fig.1. Northern blotting to confirm data obtained in cDNA microarray analyses. Ten g of total RNA (from the same RNA samples that were used to isolate poly-A)-RNA to form probes for cDNA microarray hybridization) was subjected to Northern blotting and probed with 3-labeled Sugb1 (cDNA of partial length from Sepotis Zuzietv). Panel B. Hybridization signals are quantified using

Riozriogitadeg (MoiIesshag bupatisv).

Fig. 2 and 2B. Expression of Sugb1 induced by the time-dependent Miia factor. Immobile monolayers of UMI-38 cells were treated with Miia factor (5g/ml) (2A) or ROSB-BB (1Ong/ml) (2B) for different periods of time. Total RNA (10 g) was subjected to Northern blotting and probed with radioactively labeled Sugb1. Ethidium bromide 285 ribosomal RNA staining of the corresponding blot is shown in the bottom panel as an RNA loading control. with Fig.3. Expression of Sugb1 induced by Miia factor in a dose-dependent manner. Immobile monolayers of UMI-38 cells

F 20 was treated with different doses of Miia factor, 0, 0.1, 0.5, 2.0 and 5.Og/ml for 45 minutes. Total RNA (10 g) was subjected to northern blotting and probed with radiolabeled Sugb1. Ethidium bromide staining of 285 sl ribosomal RNA of the corresponding blot is shown in the lower panel as an RNA loading control.

Fig. 4. The catalytic activity of the Miia factor is necessary for the induction of Sug61 expression. Immobile monolayers

UMI-38 cells were treated with serum-free control medium or serum-free medium containing Miia factor (5 g/ml) or active site inactivated Miia factor (Miiai, 5 g/ml) for 45 min. Complete RNA

HF) (10g) were subjected to northern blotting and probed with radioactively labeled Sugb1. Ethidium bromide 285 ribosomal RNA staining of the corresponding blot is shown in the bottom panel as an RNA loading control. and Fig. 5. Miia factor-induced Sugb1 expression is not abrogated by specific inhibitors of factor Xa and thrombin. Immobile monolayers of UMI-38 cells were treated with control medium or medium containing 60 Miia factor (5 g/ml; 100 NM for 45 min. Cells were pre-incubated with 200 NM recombinant TAR, lane 3) or hirudin (lane 4) for 30 min. before exposure to the Miia factor for 45 minutes. Total RNA (10 g) was subjected to northern blotting and probed with radiolabeled Sugb1. Ethidium bromide 285 ribosomal RNA staining of the corresponding blot is shown in the bottom panel as an RNA loading control.

Fig. 6. Effect of actinomycin-SU and cycloheximide on the level of Sugb1 mRNA induced by Miia factor in the steady state. Immobile monolayers of UMI-38 cells were pre-incubated with a control carrier, actinomycin O (10 g/ml)

or cycloheximide (1Og/ml) during ZOkhv. before exposing the cells to the action of the Ma factor (5g/ml) for 45 minutes. Full

RNA (10g) was subjected to northern blotting and probed with radiolabeled Sugb1. Ethidium bromide 285 ribosomal RNA staining of the corresponding blot is shown in the bottom panel as an RNA loading control.

Fig. 7. The Ma factor induces the expression of STOR.

Immobile monolayers of UUI-38 cells were treated with Miia factor (5 g/ml) for different periods of time. Total RNA (10 g) was subjected to northern blotting and probed with radioactively labeled STOR. Ethidium bromide 285 ribosomal RNA staining of the corresponding blot is shown as an RNA loading control.

The present invention relates to the use of EMIA or EMIa or another TE agonist for the preparation of a 7/0 pharmaceutical composition for inducing or enhancing cell migration.

In another aspect, the present invention relates to the use of EMII, EMIa, or other TE agonist in the preparation of a pharmaceutical composition to induce or accelerate wound healing or angiogenesis.

In yet another aspect, the present invention relates to the use of ERMiiai or another TE antagonist for the preparation of a pharmaceutical composition for inhibiting or preventing cell migration.

In one embodiment, cell migration is the subject of the invention.

In another aspect, the present invention relates to the use of EMiiIai or another TE antagonist for the preparation of a pharmaceutical composition for inhibiting or preventing angiogenesis, metastasis, tumor growth or tumor invasion.

In another aspect, the present invention relates to a method of inducing or enhancing cell migration in a patient, which comprises administering an effective amount of EMI or EMIIIa or other TE agonist to the aforementioned patient.

In yet another aspect, the present invention relates to a method of inhibiting or preventing cell migration in a patient, which comprises administering to said patient an effective amount of EMIA or other TE antagonist.

In a specific embodiment, the effective amount is a daily dose, which is approximately from

Bbmg/kg/day to about 5O0Omg/kg/day.

In another embodiment, the TE antagonist includes a modified EMI, for example, EEC-EMI. (8)

The present invention provides a mechanism for the activity of EMII and/or EMIiA that relates to the stimulation of cell migration. Such a mechanism suggests the introduction of the involvement of EMII! and/or EMIiA in pathological conditions in which TE-expressing cells are involved, such as endothelial cells, epithelial cells, fibroblasts,

From Smooth muscle cells and monocytes/macrophages. The invention also offers a framework for identifying specific pharmacological targets that are used for therapeutic intervention. X,

Thus, the present invention relates to the use of EMI and/or EMIa and/or EMIiia in the therapeutic co-treatment of pathological conditions that may relate to cell migration or be cured by specific regulation of cell migration. at

In another aspect, the present invention relates to a method of identifying possible drugs that regulate cell migration, which includes a) culturing a cell expressing TE; b) measurement of cell migration; c) incubating the cell with the test drug and d) measuring the migration of the incubated cell and determining any change in the level of migration compared to the migration measured in step (b), such change being indicative of the biological activity of the test drug in the aforementioned cell. ;" In general, blood components that participate in the so-called "cascade" of coagulation are proenzymes or zymogens, enzymatically inactive proteins that are converted into proteolytic enzymes under the action of an activator, which is itself an activated coagulation factor. Coagulation factors subjected to such a transformation -I have the general name of "active factors" and are indicated by adding the letter "a" to the name of the coagulation factor (for example, factor Ma). o The term "zinc complex" means a compound that binds to the Miia factor and causes ion replacement

Go! calcium on zinc ions in the Ma factor, thus inhibiting the activity of the Ma factor or the tissue factor-Mit factor complex (TE-EMIPa).

Me. A suitable TE antagonist according to the invention can be a compound that forms complexes with zinc, for example, dihydroxamate or dihydrazide with hydroxamate or hydrazide groups placed relative to each other in such a position that they are able to form a complex with a zinc ion. A compound that forms complexes with zinc acts in combination with EMIa. 7p" ions exert their inhibitory effect in competition with the stimulating effect of Ca" ions. It is assumed that 7p2" ions displace Ca?" ions. from one or more binding sites of calcium with EMPA. Compounds that form complexes with zinc, for example, hydroxamates and hydrazides, are able to act as powerful supporters of the binding of zinc ions in competition with calcium ions. Specific compounds, i.e.) thus enhance the inhibition by zinc of the activity of the Miia factor/tissue factor complex. The activity of Miia factor in a complex with tissue factor can be inhibited through a mechanism in which zinc complexone 60o binds to Miia factor and provides substitution of Ca? at 7p27. This complexon exerts a modulating effect on TE at a normal concentration of free Ca ions. and 7p2" in the blood.

The term RMI!" or "MI factor!" means "single-chain" (zymogenic) MII coagulation factor. The term "Factor

Ma" or "EMIPa" means "double-chain" activated coagulation factor MII, cleaved by specific cleavage in the Aga152-Pe153 peptide bond. EMII and EMIIa are purified from blood or produced by recombinant methods. It is clear that the practical implementation of the methods described by the authors does not depend from the route of obtaining purified factor MPa, and therefore the present invention can be considered to cover the use of any preparations of factor MII or EMIIIa suitable for use. Human EMIIa is preferred. EMII or EMIP also include variants of EMII in which one is substituted or several amino acid residues.

The term "modified factor MI!I", "I(activated EMM" or "EMIa" means an EMIa that has at least one modification in its catalytic center that significantly inhibits the ability of the modified EMIa to activate EX and RIX. The terms can be used interchangeably. Such modifications include amino acid substitution (or replacement) of one or more catalytic trivalent residues Zeg 344, Avr 142 /o and Niv 193, as well as modification of catalytic trivalent residues with serine protease inhibitors, such as organophosphorus compounds, sulfonyl fluoride, peptide halomethyl ketone or azapeptide. is one example of an EMIA derivative that is prepared by blocking the active site of EMIA with an irreversible inhibitor, p-phenylalanine-!-phenylalanine-!-arginine-chloromethyl ketone (BEC stK). Other suitable EMIA derivatives are inactivated EMIA prepared or prepared by blocking the active site 75 I|-phenylalanine-I--phenylalanine-I-arginine-chloromethyl ketone, dansyl-ii-phenylalanine-I-fe nilalanin-i! -arginine-chloromethyl ketone, or dansyl-O-phenylalanine-! -phenylalanine-! -arginine-chloromethyl ketone, and EGEK-EMiIIa (EMiIpa, inactivated REK stk) is preferred.

The term "protein kinase" means an enzyme capable of phosphorylating serine and/or threonine and/or tyrosine in peptides and/or proteins.

The term "test drug" means any sample that has a biological function or exerts a biological effect on a cellular system. This sample may be a sample of biological material, such as a microbial or plant extract, or may be a sample that contains a compound or mixture of compounds obtained by organic synthesis or genetic engineering. high school

The term "TE agonist" encompasses compounds that involve a) signal transduction by direct binding to TEs (e.g., EMIIIa), and) b) stimulation of the MAPK cascade, c) reversal of inhibition of MAPKs (e.g., PTRase inhibitors), agonists of which are the tried and tested drugs identified above. The term "TE antagonist" includes a) reagents that compete with EMI in binding to TE without transmission, e.g., EMI, ise) b) reagents that bind to EMI and interfere with TE binding, e.g. , zinc hydroxamate, so c) reagents that inhibit signal transduction by interfering with the elements of the MARK cascade, d) reagents that bind to EMIIa/TE and interfere with transmission, o e) reagents that bind to EMIIa/TR/ ЕХ and interfere with the transmission, и- f) reagents that block the activation of human factor X, catalyzed by the tissue factor/Ma factor complex, the antagonists of which are the tested drugs, defined above.

The term "pharmacological targets" means a protein that can alter the migration of TE-expressing cells.

The term "reporter gene" refers to a DNA sequence that, when transcribed, produces a protein that can be detected. c c The term "ZKE-promoter element" means a DNA sequence that binds to transcription factors induced by components present in serum. ;" The term "TE-expressing cell" means any mammalian cell expressing a TE.

The term "protein phosphorylation" refers to the phosphorylation of serine and/or threonine and/or tyrosine in peptides and/or proteins. -I Modulation or regulation of cell migration is defined as the ability of EMI or other TE agonist, or

EMiivi or other TE antagonist 1) increase or decrease current, normal or abnormal cell migration, 2) cause normal cell migration, and 3) cause abnormal cell migration.

Go! Modulation or regulation of gene expression is defined as the ability of EMIIia or other TE agonist, or EMIIia or other TE antagonist 1) to increase or decrease current, normal or abnormal cell migration, 2)

Me, cause normal cell migration and 3) cause abnormal cell migration. en In this context, the term "treatment" covers both the prevention of a negative condition and the regulation of an already existing condition in order to inhibit or minimize the condition. Thus, the prophylactic administration of EMiIa or other TE agonist, or PMiIa or other TE antagonist, is covered by the term "treatment".

In this context, the term "one unit" defines the amount of MII factor present in iml of normal plasma, which corresponds to approximately 0.5 mg of protein. After activation, 50 units correspond to approximately mcg of protein.

F) In this context, the term "patient" means any animal, in particular, a mammal, for example, a human. The term "subject" is used interchangeably with the term "patient"

Abbreviation 60

TE tissue factor

MI factor MI! in its single-chain, non-activated form

EMPA factor MI! in its activated form

KEMPA recombinant MI factor! in its activated form, 65 EMiai is a modified (inactivated) MII factor

EEC-RmiYai factor MII, inactivated by reaction with О-РНе-И-РНе-И -Aga-chloromethylketone

Tissue factor (TE) is a cellular receptor for the factor EMiia (EMiIia), and the complex is the main initiator of blood coagulation. The authors investigated the effect of EM binding and TE binding on cell migration and signal transduction of human fibroblasts expressing a large amount of TE. Fibroblasts incubated with EMIa migrated to a ROSE-BB concentration gradient at a concentration approximately one hundred times lower than that required for fibroblasts not cross-linked with EMIIIa. Antibodies against TE inhibited the increase in chemotaxis induced by EMPal/TR.

In addition, a marked suppression of chemotaxis caused by ROSE-BB was observed with EMPa inhibited in the active site (REK-EMiIa). The possibility of causing hyperchemotaxis by the formation of EC and thrombin activity was ruled out. then EMIia caused the production of inositol-1,4,5-triphosphate to the same extent as ROSE-BB; the influence of EMIa and ROSE-BB was additional. EEC-EMIa did not cause any release of inositol-1,4,5-triphosphate. The reaction of cell migration to ROSE-BB and EMIia was completely blocked by the RI C inhibitor, which indicated that the activation of RI C is important for the reaction. Thus, the binding of EMIa to TE, independent of coagulation, can modulate cellular responses such as chemotaxis, and the catalytic activity of EMIa is necessary. t5 It is believed that TE performs its function in the metastasis of tumor cells, but its mechanism is not yet known. However, in a completely new work (ОЙ ей а.), actin-binding protein 280 (АВР-280) was recognized as a ligand for the cytoplasmic domain of TE, which provides a molecular pathway by which TE can support tumor cell metastasis. However, the molecular signals and biological functions transduced by EMIa/TtE are still poorly understood.

Human fibroblasts have constitutive TE expression. These cells also express receptors for platelet-derived growth factor (PGFR). ROS induces mitogenicity, actin reorganization and directed cell migration (chemotaxis) in target cells. The authors previously demonstrated that ROSBE-BB is an effective chemotactic factor for human fibroblasts, and that the chemotactic response is mediated by class B receptors. Thus, these cells were selected to investigate the likely signal transduction and cell migration induced by the binding of EMia to TE. high school

Below, the authors demonstrate for the first time a clear relationship between the signal caused by the binding of EMiIa to Go)

TE and cellular response to growth factor. The authors presented data according to which in human fibroblasts the EMIP/TE complex leads to a hyperchemotactic reaction to ROSE-BB. In addition, it is inhibited in the active area

EMPA (REK-EMIPa) dose-dependently inhibits directional migration to ROSE-BB. By using specific inhibitors of RIS and phosphatidylinositol-3-kinase (RIS'-kinase), it is also demonstrated that the hyperchemotaxic reaction to ROSE-BB, caused by the EMIIIa/TE signal, depends on the activity of phospholipase C (Ce) ( RI C), but does not depend on RIZ-kinase. EMIIA and ROSE-BB caused the production of inositol-1,4,5-triphosphate (IR 5), one of the second mediators released after activation of RI C, in an additive way.

TE is constitutively expressed on the plasma membrane of many extravascular cells, such as IF) stromal fibroblasts in the vascular adventitia and in the fibrous capsules of the liver, spleen, and kidneys. Thus, TE expression is found in places that are physically separated from the circulating blood and provide a hemostatic membrane. Obviously, in case of injury, this barrier protects the body from bleeding. However, TE can be induced in monocytes/macrophages, vascular smooth muscle cells, endothelial cells, and in many tumor cells by various agents, including cytokines and growth factors. Induction at the transcriptional level occurs rapidly after stimulation, indicating TE as a growth-related immediate early gene. -

In this study, the authors studied the role of TE as a signaling receptor. It was shown that human fibroblasts with constitutive expression of TE migrate to the lowest concentration of ROSE-BB after cross-linking of EMii. :z» Only ТР/РМия does not cause an increase in spontaneous migration, i.e. disordered migration. Thus, the combination of intracellular signal transduction through EMIa/TE and the growth factor ROSE-BB was necessary to achieve the motility response. Not only binding to TE, but also catalytic activity is mandatory - 15 TER/EMIa, since EMIa inhibited in the active site did not cause a migration reaction. In addition, inhibitory monoclonal antibodies prevented the enhancement of the chemotactic reaction by EMIL. The authors also excluded 1 the possibility of an indirect signal through EXa or thrombin, since TAP and hirudin have no effect on co-EMIale-induced chemotaxis. Instead, the authors found that increasing the concentration of EEC-EMiI(a) actively inhibited ROSE-BB induced chemotaxis. Fibroblasts incubated with EEC-EMiα showed completely normal (o) 20 disordered migration. The inhibitory effect of REC-EMIa on ROSE-BB induced chemotaxis was not observed in the presence of the anti-TE antibody combination, thus excluding the possibility of EEC-EMIa toxicity.

The results indicate that it is not the cells that express ROSE B receptors and TE, but rather the EMIP/TE complex that is important for the chemotactic response to ROSE-BB.

The authors revealed the fact that EmiIia increases the production of IR with, and previously obtained data on the induced

The EMPale of Ca 2 oscillations, in particular, in MOSC cells, is a reliable confirmation of the idea that RIS (F) is activated by the EMIIIa/TE signal in many cells. In addition, hyperchemotaxic reaction in human fibroblasts

GI on ROSE-BB, caused by EMI/TE, was blocked in a dose-dependent manner by a RIS inhibitor. Previously, the authors revealed a similar hyperchemotactic reaction to ROSE-BB in mutant cells of the ROSE B-receptor U9ZAE, which showed increased phosphorylation and activation of Ri C-1. In these cells, increased phosphorylation of RI C-1 was correlated with a three-fold higher production of IR compared with cells expressing ROSE B of the wild type.

The combination of EMPA/TE and ROSE-BB caused an approximately two-fold increase in IR production in human fibroblasts. However, the EMIIIa/TE production of IR»z was not correlated with the phosphorylation of RI C-1. Tyrosine phosphorylation of PI C-2 caused by EMIIIa/TE cannot be excluded, but it is unlikely, since the expression of 65 PI C-2 in human fibroblasts is very low. In addition, the intracellular part of TE does not have its own protein tyrosine kinase activity. These results indicate that EMIIIa/TE induces the activation of the B and/or YS isomers. In the IR 3 release assay, the cell culture medium was supplemented with 0.190 EB5 containing only approximately 0.1 nM ECa. The authors found that a concentration of more than 20 nm of ExXa is necessary to induce IR production. The mechanism through which B or i RIS isomers are activated remains unclear. It is possible that the activation is related to the interaction between the TE and the impurity protein of the membrane.

Recently, the connection of TE with the cytoskeleton was identified. A molecular interaction between the cytoplasmic domain of TE and the actin-binding filamin protein ABP 280 was demonstrated. In addition, TE was found to be in close contact with actin and actin-binding filamin proteins such as

A-actinin and ABP280 in lamellopodia and corrugated areas of the membrane in spreading epithelial cells. ABP 70 280, a member of the filamin subfamily, is necessary for the normal functioning of lamellopodia, and therefore very important for cell motility. RIZ'-kinase and RI C isoenzymes are involved in chemotactic reactions, such as the mobilization of actin-binding proteins. In previous studies, the authors observed that the RiIZ'-kinase pathway in ROSE-B receptor-induced chemotaxis was apparently less important in cells with overexpression and increased activity of RI C-1. This was also true for cells with TE-related EMIa. This indicates that the /5 value of activation of RIS'-kinase and RIS isozymes determines which of these pathways will dominate. Taken together, these authors' data show that cell migration is an important morphogenic function induced by EMIP/tE signaling.

Chemotaxis plays a critical role in wound healing, angiogenesis, and metastasis. Chemotaxis is also an important component in the formation of atherosclerotic plaques. In these processes, different cells express TEs as well

ROSE and ROSBE receptors.

Restenosis is the main complication that occurs after the intervention procedure in blocked arteries. ROSE is involved in the reaction of vessel walls (neointima formation) to mechanical damage, mediating the migration and proliferation of smooth muscle cells and fibroblasts. The authors demonstrated for the first time that EMIIIa, which binds to TE-expressing cells, reveals an increased chemotactic response to ROSOBE, which is independent of coagulation. with

Currently, little is known about the signaling pathways induced by proteolytically active Miia and how Ma-induced signals may participate in cellular processes. It is possible that EMIa is capable of inducing the expression of growth regulators that act downstream to induce cellular processes. To investigate this possibility, in this paper, the authors studied changes in the transcriptional program in human fibroblasts in response to exposure to Miia using cDNA microarrays containing more than 8,000 individual human genes. oh

The authors chose fibroblasts because these cells are commonly exposed to serum containing growth factors and activated coagulation factors in association with vascular damage due to physical (eg, surgery) and pathophysiological conditions. The temporal program of gene expression, which was observed in response to He) serum, indicates that fibroblasts are programmed to perceive the sharp action of serum not as a general mitogenic stimulus, but as a specific physiological signal. Characterization of the transcription factor

35 activation in response to serum and growth factors also suggests that fibroblasts are active participants in the dialogue between various cells that together control inflammation, angiogenesis, and wound healing.

Analysis of cDNA microarrays with mRNA isolated from fibroblasts exposed to Ma for 9 hours reveals up-regulation of Sugb1. Northern blotting confirmed Miia-induced Sugb1 expression in fibroblasts.

Although not as strongly as in fibroblasts, Ma also enhances the expression of Sugb1 in vascular smooth muscle cells. Inducing the expression of Sugb1 depends on the catalytic activity of EMiIIa, since EMiIIai is not able to cause the expression of Sugb1. Although factor Xa and thrombin can also induce Sugb1 expression (data shown), these compounds are not involved in EMIP-induced Sugb1 expression. The authors did not find any evidence "" of the formation of traces of factor Xa and thrombin in this experimental system. In addition, a specific inhibitor of factor Xa and thrombin had no significant effect on EMiIIa-induced Sugb1 expression.

Sugb1 is an immediate early gene that is transcriptionally activated by serum growth factors in -I fibroblasts. It encodes a cysteine-rich heparin-bound protein with a mass of 40 OkDa, which is associated with the extracellular matrix and cell surfaces. Sugbb! belongs to the emerging gene family of conserved and i-modular proteins, which is characterized by the presence of an M-terminal secretory signal followed by four o-modular structural domains and 38 cysteine residues that are largely conserved among family members.

This family of proteins now consists of six distinct members, including Sugb1, a connective tissue growth factor

Pho (STOR) and the avian proto-oncoprotein Mom (thus, called the SCM-family) (the SCM-family is described in more detail in the work of Gai et al. Ehr. Seiyi Kez 248: 44-57, 19991). It was found that the Sugb1 protein stimulates the attachment and proliferation of endothelial cells in much the same way as fibronectin, and enhances the effect

ЛБЕОЕ and РОСОБЕ on the intensity of DNA synthesis of fibroblasts and vascular endothelial cells, (ii) stimulates cell migration in both fibroblasts and endothelial cells. Recent studies show that Sugb1 acts as a ligand for integrin AB, an adhesion receptor involved in signaling that regulates many cellular processes, including angiogenesis and tumor metastasis. The purified Sugb1 protein showed a stimulating effect on the directional migration of endothelial cells of human microvessels in culture in an ABz-dependent way and caused the formation of new vessels in the cornea of rats. In addition, Sugb1 expression in tumor cells stimulates tumor growth and vascularization.

On the basis of the presented data, which show that EMIPα induces the expression of Sugb1 in fibroblasts, it can be asserted that induced EMIIa Sugb1, acting through integrin AVz, is responsible for EMIIIa-mediated cell migration and tumor metastasis. Thus, Sugb1 links the EMIa-TE proteolytic signal to the integrin signaling pathway. Observations according to which the catalytic activity of Ma is necessary for the migration of B5 smooth muscle cells and tumor cells and tumor metastases do not contradict other observations according to which the catalytic activity of EMiIIa is necessary for the induction of Sugb1.

In addition to Sugb1, Ma can also induce other regulators that may mediate EMI-induced biological responses. It was found that the binding of EMI to cell surface TE in pancreatic cancer cells is capable of selective overexpression of the SHRAK gene. Previously, using differential display technology, the authors demonstrated up-regulation of poly(A) polymerase gene transcription in fibroblasts exposed to EMIP. Although it would be interesting to find out if the filter does not contain PAP cDNA, if the cDNA microarray also shows differential expression of PAP. In addition to Sugb1, this cDNA microarray also reveals differential expression of four other genes (see results), but the differential expression coefficient was very close to the cutoff value. Since in previous experiments the authors could not confirm their differential expression by Northern blotting, and in the absence of any suggestive data regarding the ability of these gene products to mediate the biological reactions caused by EMI, the authors analyzed their expression in more detail. However, since STOR is a molecule structurally related to Sugb1 and exhibits the same biological responses as Sugb1, the authors investigated the expression of STOR even though the relative rate of STOR expression in the EMIIIa-treated sample compared to the control sample was 7/5 The cDNA microarray is 1.8 (2 is a margin of error for the actual value in the test).

The data showed that EMIa also induced STOR expression, and the dynamics of Ma-induced STOR expression was similar to that of Sugb1.

Although STOR behaves very similarly to Sugb1, there are subtle differences between the two. For example, (a) STOE exhibits natural mitogenicity, and Sugb1 has no natural mitogenic activity, but enhances growth factor-induced DNA synthesis, (b) Sug61 stimulates chemotaxis, and STOE stimulates both chemotaxis and chemokinesis, (c) although Sugb1 and CTORs are ECM-associated signaling molecules, CTORs are secreted into the culture medium.

Thus, it is possible that EMiia regulates cellular functions locally through Sugb1, although acting at a distance from its site through secretion of STOR.

The regimen for each patient, who should be treated with EMIIa or another TE agonist or EMIIa or another TE antagonist mentioned by the authors, is determined by specialists. The daily dose to be administered during therapy is determined by the physician and depends on the specific compound used, the route of administration, and the weight and condition of the patient. An effective amount is an appropriate daily dose of approximately Bmg/kg/day to

B500mg/kg/day, optimally - from about 100mg/kg/day to 300mg/kg/day, even better - from about 15mg/kg/day to 200mg/kg/day, best - from about 20mg/kg/day up to 10 Omg/kg/day. zo EMIia or other TE agonist or RMiIai or other TE antagonist is administered as a single dose, but it can also be administered as multiple doses, ideally at intervals of 4-6-12 hours, depending on the dose of CO and the patient's condition. co

EMPA or other TE agonist or EMIiIiai or other TE antagonist is administered intravenously either by continuous or pulsatile infusion, or administered directly to the appropriate site, for example, by injection directly into the tumor. EMI or other TE agonist or EMI or other TE antagonist is optimally administered by intravenous injection and in an amount of approximately 100-100,000 units per kg of body weight, preferably in an amount of approximately 250-25,000 units per kg of body weight, which corresponds to approximately 5-50 Ωkg/kg, a dose that can be re-administered 2-4 times in 24 hours.

Conventional methods for the preparation of pharmaceutical compositions that can be used according to the present invention are described, for example, in Ketipdiopye Rnpaptaseshiis! Zsiepsevs, 1985. z c Compositions used in accordance with the present invention are prepared by methods that are clear to those skilled in the art. . Briefly speaking, the pharmaceutical compositions suitable for use according to the present invention are obtained and? by mixing Ermia, EMIia or other TE agonist or PMiiIai or other TE antagonist, preferably in purified form, with suitable adjuvants and a suitable carrier or diluent. Suitable physiologically acceptable carriers or diluents include sterile water and saline. Suitable adjuvants in this case include calcium, proteins (for example, albumins) or other inert peptides (for example, glycylglycine) or amino acids (for example, glycine or histidine) to stabilize the purified factor Miiia. o Other physiologically acceptable adjuvants are non-reducing sugars, polyalcohols (eg sorbitol, mannitol or glycerol), polysaccharides such as low molecular weight dextrins, detergents (eg polysorbate) and antioxidants (eg bisulfite and ascorbate). Adjuvants are usually present. in

Me. concentrations from 0.001 to 4905 (mass/volume). The pharmaceutical composition may also contain protease inhibitors, such as aprotinin, and preservatives.

The compositions are sterilized, for example, by filtering through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be produced in the form of sterile solid compositions that can be dissolved in sterile water or some other sterile medium suitable for injection before or immediately (F, before use. ka In various aspects, the present invention relates to:

A method of regulating the expression of at least one gene in a cell, which includes the steps of: a) contacting the aforementioned cell with the MI factor! (a) or a tissue factor antagonist, b) determining the expression of the aforementioned gene in the aforementioned cell.

The above-mentioned method, in which the above-mentioned cell is a human vascular cell expressing tissue factor, including fibroblasts and smooth muscle cells.

The method in which the aforementioned gene is selected from the group consisting of Sugb1, STEREO, dopamine receptor 65 02, ET Ipsui PO 395116 or P2) nucleotide receptor.

A method in which the aforementioned tissue factor antagonist is a modified MI factor! (a), known as the Miyai factor.

The method in which the expression of the above-mentioned gene is enhanced.

A method in which the expression of the aforementioned gene is inhibited or minimized.

The method of increasing the expression of the above-mentioned gene, which involves contacting the cell with the Ma factor.

A method of inhibiting the expression of the aforementioned gene, which involves contacting the cell with a modified MII factor known as EMIiai.

A method in which the aforementioned gene is ET ROb74714.

The method of regulating cell migration, which includes the steps: 70 a) contacting the aforementioned cell with the Miia factor or tissue factor antagonist; b) determining the migration of the aforementioned cell.

A method wherein said cell is a human cell that expresses tissue factor, including fibroblasts, smooth muscle cells, tumor cells, hematopoietic cells, and epithelial cells.

A method in which the tissue factor antagonist is a modified Miia factor known as a Miia factor.

A method in which the modified MI factor is selected from among dansyl-Rhne-Pho-Agd-chloromethylketone, dansyl-SISH-Siu-Agda-chloromethylketone, dansyl-Rpe-Rpe-Agd-chloromethylketone and Rpe-Rpe-Agd-chloromethylketone.

A method of enhancing cell migration, which involves contacting the cell with EMIa or a tissue factor agonist.

A method of reducing or inhibiting cell migration, which involves contacting the cell with a tissue factor antagonist.

A method of inducing or accelerating wound healing in a patient, which comprises administering to the aforementioned patient an effective amount of a pharmaceutical composition comprising Miglia factor or a tissue factor agonist.

A method of inhibiting the invasiveness of tumor cells, which includes contacting the aforementioned cells with tissue with an effective amount of a tissue factor antagonist.

A method of inhibiting cell migration, migration-induced invasion, cell proliferation, or angiogenesis in i) a patient having a disease or condition associated with unwanted cell migration, invasion, migration-induced cell proliferation, or angiogenesis, which comprises administering to the aforementioned patient an effective amount of a pharmaceutical a composition that includes a tissue factor antagonist. in a manner in which the disease or condition is the development of a primary tumor, tumor invasion, or metastasis.

A method in which the tissue factor antagonist is a modified MI factor known as EMI. ise)

Use of Miia factor or tissue factor antagonist to produce a drug to regulate cell migration.

The use according to which the factor Miii is used to obtain a medicament for enhancing the migration of cells. uh-

A use in which a tissue factor antagonist is used to provide a medicament for reducing or inhibiting cell migration.

A method in which the tissue factor antagonist is a modified Miia factor known as a Miia factor.

The use according to which the modified MI factor is selected from dansyl-Rpe-Rpe-Agd-chloromethylketone, dansyl-c1y-c1u-Ago-chloromethylketone, c dansyl-Rpe-Rpe-Agd-chloromethylketone and Rpe-Rpe-Agd -chloromethyl ketone.

The present invention is further illustrated by the following examples, which, however, should not be construed as limiting the scope of protection. The features set forth in the above description and in the following examples may, both individually and in combination, be essential for the implementation of the invention in its various forms.

EXAMPLES

- And Example 1

Obtaining the human Myia purified factor, suitable for use in this invention, in the optimal version

Go! obtained by recombinant DNA technology, (for example, as described in the work of Nadep eish ai., bor Rgos.Man.Asai.Zsi. OA 83: 2412-2416, 1986, or as described in European patent Mo200 421 (7utosSepeiisv))|.

Me, Factor Miia obtained by recombinant technology can be an authentic factor Miia or a more or less modified factor Miia, provided that such a factor Ma has practically the same biological activity for blood coagulation as an authentic factor Ma. Such a modified Ma factor can be obtained by modifying the sequence of the nucleic acid encoding the MII factor, or by changing the amino acid codons, or by deleting some amino acid codons in the nucleic acid encoding the native PMII by known methods, for example, by site-specific mutagenesis.

F) The MII factor can also be obtained by methods (described in the works of Vgoge apa Maiegiv, 9U.ViIoII. Spet. ka 255 (4): 1242-1247, 1980, and Nedpeg apa Kiziei, 9U.Siip.Ipmevi. 71: 1836 -1841, 1983). These ways provide the MI factor! without a noticeable amount of other blood coagulation factors. It is possible to obtain an even more purified MIi factor preparation by adding gel filtration as a final purification step. MI factor! then converted to activated EMI by known means, for example, by several different plasma proteins, such as the factor HiIpa, IX a or Xa. Alternatively, as described in Vioet et al. (Kezeagsp Oizsiovyge, 269 Berietreg 1986, pp. 564-565)), the MII factor can be activated by passing it through an ion-exchange chromatography column such as Mopo OZ (Rpagtasia ipe Spetisa!c) or in another similar way. 65 Example 2

Obtaining EMIIiai

A modified MII factor suitable for use in the present invention is obtained, for example, as described in International Publications MoMe 92/15686, 94/27631, 96/12800 and 97/47651 7utosepiiisv/Momo Mogaizk|.

Example C

The influence of EMiIIa and EEC-EMiIIa on the chemotactic reaction of fibroblasts to ROSE-BB

Fibroblasts expressing active TE (Fig. 1A and Fig. 18B) were incubated with T0OnNM RMiga and seeded in the upper part of a modified Boyden chamber; at the same time, media containing 10956 ЕВ5 and РОСБЕ-ВВ in various concentrations were added under the filter with micropores of 150 μm. Cell migration in the conditions of adding to the filter medium containing 1095 EB5 without ROSE-BB was used as a measure of random migration and was calculated as 7/0 10090 migration. A significant migration reaction was registered at a concentration of 0.00ng/ml of ROSBE-BB in cells stimulated with EMiia compared to ing/ml of ROSEBE-BB for cells not cross-linked with EMiIIa, i.e. at a 100-fold difference in concentration (Fig. 2A). At 0.01-0.ing/ml ROSB-BB, the migration response to EMI was enhanced depending on the dose, starting with 25 nm, and with the maximum effect at 50-100 nm of EMI (Fig. 33ZA-O0). No increase in promiscuous migration was observed after activation with EMIa. In order to test whether proteolytically active EMiIa is required for the hyperchemotactic response to ROSE-BB, fibroblasts were also incubated with 100 nM

EEC-RMiga and subjected to analysis in the Boyden chamber in the same way (Fig. 2A). No increase in chemotaxis was observed with EEC-EMiia at low concentrations of ROSE-BB, 0.01-Tng/ml. In contrast to these concentrations, a clear inhibition of chemotaxis caused by 10-BbOng/mml ROSE-BB was achieved with 100NM REC-EMIa (Fig. 2A and ZA-G). When fibroblasts were pre-incubated with a mixture of three different anti-TE antibodies and then with EMia or REK-PMiIg, the migration response to ROSE-BB was identical to that of fibroblasts without the presence of TE-bound ligand (Fig4A). A foreign monoclonal antibody against db did not prevent either EMiIa-induced hyperchemotaxis or REC-EMIa-induced inhibition of the migration response (data not shown). The presence of anti-DS antibodies or three anti-TE antibodies did not alter the disordered migration of fibroblasts (data not shown). with

Example 4

Hyperchemotaxic reaction is not mediated by EC or thrombin and)

Since EmiIa-induced signal transduction leading to the hyperchemotaxic response to ROSBE-BB was dependent on the catalytic activity of EmiIa, it was important to determine whether the signal occurs directly or through

EXa or thrombin generated by the EMI/TE complex. The increased migration reaction transformed by EMPale was blocked by 0.2-10 μM tick anticoagulant peptide (TAP), which specifically blocks the active site of EXa and prevents further activation of the coagulation cascade leading to thrombin generation (Fig. 5A, 5B). Likewise, addition of 5 Bod/ml hirudin, a specific thrombin inhibitor, had no effect on EMiIPa/TE co-induced hyperchemotaxis (FigbA). TAR and hirudin did not affect the migration of fibroblasts in response to ROSPE without the presence of EMIa ligand (Fig. 5A, 58, bA). at

Thus, it seems unlikely that the effect of EMiia on chemotaxis is mediated by the activation of EX- or thrombin.

Example 5

Hyperchemotaxic reaction to ROSE-BB depends on RI C-dependent pathways, but does not depend on P13'-kinase.

Recently, it was found that the activation of RiIb'-kinase is important for ROSE B-receptor-induced chemotaxis. Thus, the authors investigated whether I U294002, a specific inhibitor of RAZ'kinase, is able to block the chemotactic reaction caused by the EMIa/TE signal. Fibroblasts were pretreated with I 294002 in . at the indicated concentrations for 30 minutes at 37 °C before adding T100nM EMiia and subjected to analysis in a Boyden chamber as described. The concentration of ROSE-BB was kept unchanged at the level of 0.Tng/ml throughout the experiment, that is, at a very low level at which EMIPa/TE caused a significant chemotactic reaction.

ІМ294002 was present all the time during the experiments. Fig. 7A shows that the migration reaction to - and ROSBE-BB, mediated by the EmIia/TE signal, was not affected by the inhibition of RIB'-kinase.

In order to investigate whether the EMIIIa/TE-induced chemotactic response is involved in the activation of phosphatidylinositol-specific phospholipase C (PIC), the authors pre-incubated fibroblasts with different concentrations of 073122, a specific inhibitor of PIC, for 30 minutes at 37 2С before adding TOM EMIIIa; at 50 The cells were then subjected to chemotaxis analysis in the presence of the inhibitor. As a negative control, a close analog, 073343, was used, without affecting RI S. The concentration of ROSE-BB was maintained unchanged at the level of O.Tng/ml in these experiments as well. Pretreatment of cells with the active inhibitor RI C 073122 inhibited the hyperchemotactic response to 0.ng/ml ROSE-BB in a dose-dependent manner, with complete inhibition at 1μM (Fig.vaA and 8B). When using the non-activated analog 073343, no effect on chemotaxis was observed.

Example 6

EMI causes the activation of RI S

F, To further investigate the significance of RIS activity for hyperchemotaxic reaction, the authors also analyzed the direct effect of EMIIIa/TE on RIS activity in fibroblasts. Activation of RI C leads to the production of two other messengers, inositol-1,4,5-triphosphate (IR3) and diacylglycerol. Fibroblasts were incubated with 60 myo-IZNI-inositol until the next day, and then with L00NM EMIa or EEC-EMIa for 60 minutes, after which incubation was carried out with ROSE-BB or without it in the indicated concentrations. Treatment with 1L00nM of EMIa itself for 60 minutes caused the release of IR from fibroblasts at the same level as with 1Ong/ml and 10Ong/ml of ROSE-BB itself (Fig. 9). In addition, the combination of 100 nM EMIa and TOng/ml or 1OOng/ml ROSE-BB doubled the release of R. Inhibited in the active site EMIIa did not cause the release of IR from. These results b5 clearly indicate that RI C is activated after the binding of EMiIa to TE.

Example 7

Phosphorylation of RI C-1 is not enhanced by the TE/EMiIa signal in fibroblasts

In order to determine whether the RiC-1 isoform, which is activated by certain tyrosine kinase receptors, is responsible for the increase in RI C activity caused by EMIIIa/TE, tyrosine phosphorylation was investigated

RI S-1. Fibroblasts were incubated in the absence or presence of L00NM EMIa or EREK-EMiIIa for one hour, after which they were stimulated with 0, 2, 10 or 10O0ng/ml ROSE-BB. After 5 minutes of incubation, the cells were subjected to lysis and RIS-1 was subjected to immunoprecipitation, separated by 5ZO5-RACE and subjected to immunoblot analysis with an antibody against phosphotyrosine. If a significant increase in the tyrosine phosphorylation of PI C-1 was observed with an increase in the concentration of ROSE-BB, then the addition of EMiia itself to fibroblasts did not cause 7/0 No phosphorylation of PIS-1 by tyrosine (Fig. 10). In addition, the combination of EMIP and ROSB-BB in different concentrations did not cause any further phosphorylation compared to stimulation by ROSE-BB alone (Fig. 10). REC-EMiIia had no effect on PI C-1 tyrosine phosphorylation (Fig. 10). Thus, other isoforms of RI C, except for RI C-1, are responsible for increasing the activity of RI C after EMIa stimulation.

Example 8

Methods

Cell cultures. Human foreskin fibroblasts, AC1518 and AC1523, were grown to confluence in Eagle MEM from 1096 bovine embryo serum (EB5). Before use, cells were detached by trypsinization (2.5 mg/ml for 1 hour at 37 C), washed in Hank's balanced salt solution and resuspended in 1095 IU Eagle's MEM or in Heme's medium supplemented with 0.195 IU5.

Squirrels Human EMPA (Momo MogaizkK A/5, SepioPe, Denmark) was expressed and purified as described29. EEC-EMIPa (Momo MogaizkK) was obtained by blocking EMIia in the active site with О-РНе-И -Рпе-И -Aga-chloromethylketone.

Recombinant tick anticoagulant peptide (TAR) was kindly provided by Dr. P. Vlasyuk (Og. R.

Miazik), Sogmaz (San Diego, California). Hirudin was purchased from Bidta. I 294002, 073122, and 073343 were obtained from Viotai (Riutocius Meeipo, PA). Monoclonal antibodies against TR, TE8-5659, TR9-587 and su

MTEN-1 (Motizvzeu, ..N., Rai, 0.5., Eddipdip, T.5. Moposiopa! apirodu apaiuziz oi rigyvei oi apa seiI-azzosiaied (izvtse Tasiog. Typgotr. Kev. 52, 247-261 (1988)) was kindly provided by Dr. James H. Fr

Morrissey (Og. datevs N. Mogtizzeu) from the Oklahoma State Medical Research Organization (OKiapota Meaisai!

Kezeagsp Roshpaaiyop). The anti-phosphotyrosine antibody, RUZ99, was obtained from Santa Cruz, California.

Flow cytometry. The surface expression of TE was analyzed by immunofluorescence with a flow cytometer (SocMeg Erisv XI-MSI, VesKtap Soshts(eg, Eshiepop, California, SocMeg Eiesigopisvb, USA).

The instrument was calibrated daily using Ittypo-Snesk or Riom/Spesk calibration pellets (Soceg). For indirect immunofluorescence experiments, AsC1518 or AC1523 fibroblasts were washed twice with PB5 containing He) 0.195 bovine serum albumin (BSA), incubated for 30 min on ice with fluorescein isothiocyanate (RITS)-labeled TE anti-human monoclonal antibody (4508C ), Ategisap Oiadpowiisa, o Sgeepmisp, Si, USA). As a negative control, a monoclonal antibody dS!I against r. For each sample, the average fluorescence intensity (MEI) and the percentage of positive cells were determined.

Determination of TE activity. The procoagulant activity of TE was determined (as described in the work of Ipatagk ei ai. (ipatagk, E., Teppo, T., Spep, 9., Ziedrapyp, A. 1/-10 ippiri I RoB-ipdisei pitap toposuie i(izzce Tasiog 102, 597-604 (1998). Briefly, aliquots containing 0.2105 s of Ab1518 or AsC1523 fibroblasts were washed twice with RV5, placed in wells 9b -well microtitre and plate (Mips, KozvikKiide, Denmark).Procoagulant activity was measured by a two-step amidolytic "" assay, in which the chromogenic substrate, 5-2222 (Spgotodepih, MoiIpa!, Sweden), cleaved ExXa, which in turn activated with EX complex TE/EMIP.The reaction mixture containing the final was added to the wells

The concentration of 0.bmM 5-2222, 2mM CaSi" and coagulation factors from the concentrate of the factor Rgoipgotrieh-i TIMA -i (Vachieg, Vienna, Austria) in the final concentration of Tod./ml EMII and 1.2 units/ml EH and determined the change in optical density at 405 nm after 30-minute incubation at 372C. Measurements were performed three times.

Mn Analysis of chemotaxis. The migratory response of fibroblasts was analyzed using the "Ieadipa(os)iopi" technology in a modified Boyden chamber, (as previously described (Siedrapp, A., Napitasneg, A., UUesiegtagk, V., Neidipy, S-N. OijbegepiaI! ePesiv oi (ye magioiz isoiogtv5 oi riafeIe(-adehimey dhomlj Tasiog op spetoiyakhiv oi b Trgoriaziv, toposuiez, apa dgapytsosufev. 9. Siip. Ipmevi. 85, 916-920 (1990) and Mivieg, M., Nattasneg, A., with Meivigot .

Potoditeg paz ayegepi Topsyopa! asimyevz iot a ROSE AV Peiiegoditeg rigiivy iot Ppitap riagerei5. Sei 52, 791-799 (1988))). The filter with micropores (pore size 8 μm) was kept covered with a type 1 collagen solution at room temperature until the next day. Filters were dried in an air dryer for 30 minutes immediately before use. Fibroblasts of the human foreskin AsC1523 were grown to confluence in MEM i) Igla with the addition of 1095 EV5. Cells were detached by trypsinization (2.5 mg/ml for 10 minutes at IM) 372) and suspended in Igla's MEM with 1095 EV5. Fibroblasts were incubated for 10 minutes with or without EMI or BEEK-EMP before analysis. One hundred microliters of cell suspension 60 (2105 cells/ml) were placed on Boyden chamber filters. ROSE-BB was diluted in the medium for analysis (MEM Igla with 1095 ЕВ5) and added under the filter in the chamber. Cells were incubated for 2 hours at 372C in a humidified chamber containing 9595 air/595 CO.

EMIa or EGEK-RMIIa were present all the time during the experiment. Filters were then removed, fixed in ethanol, stained with Mauegs Netaishp dye and inserted. Migration was measured as the distance between the two furthest displaced fibroblast nuclei of one high-power field (12.524) at the center. The migration distance in each filter was calculated as the average value of the measurements for at least three different parts of the filter.

Experiments were carried out with two to four separate filters for each chemoattractant concentration. For each series of experiments, the migration of fibroblasts into the assay medium was taken as a control.

In cases where monoclonal antibodies against TE or inhibitors of coagulation factor, TAR and hirudin were used, cells were preincubated for 10 minutes with these agents, then with or without EMIiTa or EEC-EMIa before performing the chemotaxis assay. Antibodies, TAP, or hirudin were also present throughout the chemotaxis experiment. In experiments where the effect of different inhibitors, 294002, 073122 or 073343 on the migration response was tested, the cells were pre-incubated for 30 minutes with the inhibitors at the indicated concentrations, and the inhibitors were also present throughout the experiments. 70 Analysis of the release of inositol triphosphate (IR 3). Six-well plates with semiconfluent cultures of AC1518 human fibroblasts were incubated until the next day (approximately 20 hours) with 2 mCi of myo(ZN)-inositol (Ategepat) in 2 ml

Hema's E12 with 0.195 EV5. The medium was changed to Hema's E12 with 0.195 EVZ (containing 2mM Sasig) and 20mM Is Her cells were incubated for 15 minutes at 37 2C. Cells were then incubated in the absence or presence of 100 nM EMiia or 100 nM EREC-EMiIIa for one hour. ROSBE-BB (0, 10 or 10 ng/ml) was added, and 75 incubation continued for 10 minutes at 37 C. IRz analysis was carried out | As described earlier in the work

Erickson et al. (Egikvzop, A., Maprego, E., Koppvzigapa, Y., Epodvigot, M., Neitap, O., Kirr, E., Sagrepivg, b., Neidipi, S-N., Siaezvzop-MUeivy, |. YuOetopzigayop oi Tpsiopayu ayegepi ipiegasiopz /Breimeep rpozroiiraze S- apa (pe ymo (rez oi ria(eei-degimeid dgouli Tasiog geseriogv. 9. Vioiii. Spet. 270, 7773-7781 (1995))).

Analysis of agonist-induced phosphorylation of Ri C-1. Half-confluent cultures of AC1518 were maintained with 20 serum until the next day (approximately 20 hours) in medium containing 0.195 EB5 and then incubated in the absence or presence of 100 nm EMIlia or REK-PMIia for one hour, followed by incubation with 0, 2, or 10 ng/ml ROSE-BB for 5 minutes at 37 2С. Cells were lysed and RI C-1 was precipitated practically as described previously (Napzep, K., Doippei, M., Ziedreip, A., Kogztap, S., Epodvigot, |.,

MUetvividi, S., Neidip, S-N., Koppzigapa, Y. Miiayop oi a Zgs rpozrpoguiaiop zbe ip She ROS V-geseriog Ieeadzi CM 0 ipsteasea ROSBE-viititsageeai spetoiahiv ryi desgeasei tyodepeziz. EMVO 3). 15, 5299-5313 (1996) about the antiserum against RI C-1, obtained by immunization of rabbits with a peptide corresponding to the carboxy-terminus of bovine RI C-1 (Apeda, S.Y., doppzop, M.O., Toddegoa , village, SoPeu, K.), Sagrepieg, village, Rade,

O.І Іемайей сопіепі ой (Ше (угозіпе Кипазе зірзігаіе рпозрпоііразе С-1 ip rgitagu pitap bBgeavi sagsipotav.

Rgos. May). Asad Ba. O5BA 88,10435-10439 (1991)). The samples were separated using 5O05-RACE and subjected to IF) immunoblot analysis with an antibody against phosphotyrosine RUZ99. "co

Statistical analysis. The data were analyzed using 5iaizisa TM for the M/ipdom/v package (Ziaiboii, Tiiza,

Oklahoma, USA). Student's t-test for dependent samples was used to determine the statistical significance of 00 between different data sets. A p-value of 0.05 was considered statistically significant. yu

Squirrels Recombinant human Myia, received as a gift from MOMo MogaizK (Sepijøje, Denmark), was reproduced in sterile water at a concentration of 1 to 1.Zmg/ml. Base solutions of Ma were checked for the presence of traces and - contamination with endotoxin, using lysate of IptyShv amebocytes (Vio MUpibakeg), and they were not detected (level of detection of ZOpg). Recombinant tick anticoagulant protein (TAR) was kindly provided by George

Vlasiuk (Seogde Miazik) (Soguaz, San Diego, CA), and recombinant hirudin was obtained from Ziedt (St. Louis, MO) or SaiYriospet (San Diego, CA). Purified human factor Xa and thrombin were obtained from Epgute Kezeagsi I Aroghaiges (5ociprepa, Indiana). shi s cDNA microarray. UMI-38 cells were cultured to 8095 confluence, and serum was collected for 24 hours to reach a stationary state as described above. The culture medium was replaced with fresh OMEM without serum (with the addition of 5 mM Sasi") and left to stabilize for 2 hours. in the incubator After that, the cells were treated with purified recombinant Miia (5 μg/ml) for 30 min. At the end of 9 Ohv. treatments from untreated (control) and Miia-treated cells, total RNA was extracted using TgIogII (5IVSO VK). - Poly--A)Z-RNA was purified by double passage through a column to isolate Oiido Teh mRNA as described in the manufacturer's instructions (Oiadep). Eight hundred ng (80 ng) of highly purified poly-A)-RNA from control and treated

Ma cells were sent for cDNA microarray analysis (Nitap OpisEem M tisgoatau, Sepote buvietv Ips, (o) St. Louis, MO). bo 50 Northern blotting. Total RNA was obtained using the TKI2OYI reagent. from a fixed monolayer of Miia-treated UMI-38 cells and other materials as described in Results. Northern blotting was performed using the following standard procedure. That is, 10 μg of total RNA was fractionated by size by gel electrophoresis in 195 agarose/695 formaldehyde gels and transferred to a nitrocellulose membrane by capillary blotting.

Northern blots were pre-hybridized at 422C with a solution containing 5095 formamide, 55552, 5 mM Ttgiv. NSI, pH 7.5, 0.195 sodium pyrophosphate, 195 5O5, 195 polyvinylpyrrolidone, 195 Risoi, 25 mM EOTA, 10O0μg/ml. DNA (FI of denatured salmon milk and 195 B5A) and hybridized with the 32P-labeled Sugb1 cDNA probe (10 bert/ml).

The hybridized membranes were exposed to X-ray film Europi MER or Rcii EH. For quantitative determination, the membrane was exposed to a phosphor screen for 1-4 hours. and the exposed screens were analyzed in the RiozPogitdeg device (MoiIestsag bupatisv) using the "image quantization" program. 60 To obtain average unit values (calculation results) of different experiments, they were normalized to the external control (result for the treated control sample).

Chromogenic analysis. U/I-38 cells were cultured in a 96-well plate and immobilized as described above. After washing the cells, EMIIIa (b5μg/ml) in 1.00μg of calcium-containing buffer was added to wells containing cells or wells covered with buffer (without cells). After the appeal After incubation, 25 μg of chromogenic substrates for factor Xa and thrombin were added to the wells, that is, Spgotogut X and Spgotogut TN. After Agreement

the development of the color of the tablets was read in a reader. Cells used as a control were incubated with trace concentrations of factor Xa (from 50 to 00.Mng/ml) or thrombin (from 0.1 to 0.002 units/ml). No difference in optical density at 450 nm was observed between Miia added to cells or Ma added to wells that did not contain cells. The reading was lower than that obtained at the lowest concentration of factor Xa or thrombin and represents the chromogenic activity of Ma.

Example 9

cDNA microarray. Immobilized fibroblasts were exposed to control medium without serum or medium without serum with the addition of Miia (bmkg/ml) for 10 h. (three T-75 flasks for each treatment). 7/0 After processing, total RNA was collected and poly--(A)-RNA was isolated. Six hundred ng of mRNA were labeled with the fluorescence of SuZ or

Sub and then hybridized with Opibet Nytap U containing 8,000 sequence-verified EZTs representing up to 5,000 known human genes (the service was provided free of charge (Zepote Zuziet Yps). A control plate in which a known concentration of control cDNA entered the probe generation reaction, applied to measure the sensitivity and trace the reaction of reverse transcription, purification and determination of hybridization efficiency, and the overall picture of the quality and efficiency of the analysis indicated the success of the hybridization process.

Overall analysis of the experimental data revealed minimal differences in the hybridization signals between the control and treated MYI samples. Only a small number of genes showed moderate differential expression. The authors found up-regulation of 5 genes (3.5-2-fold increase in Miia treatment), while one gene showed down-regulation after Miia treatment (2.4-fold decrease) (7-2 is an overestimated error for determining the minimum value of real coefficients). The identity of the 3.5-fold upregulated gene was not revealed due to its nature. Other genes up-regulated by Ma are Sugb1 (2.5-fold), dopamine 02 receptor (2.2-fold), ET Ipsui PO 395116 (2-fold) and P2O nucleotide receptor (2-fold). It is interesting to note that STOR, a gene that belongs to the family

Sugb1 showed a 1.8-fold increase in Miia-treated cells compared to control cells. The down-regulated transcript in treated Ma cells was EZTROb74714. For further analysis, the authors chose Sugbe1. and)

Example 10

Confirmation of differential expression of Sugb1. To confirm the data obtained for the microarray, the authors subjected a sample of RNA from control and treated Miia cells (the same RNA samples that were used to obtain poly(A)-RNA for probe generation in the microarray) to northern blotting and probed with radioactivity labeled Sugb1 cDNA. The data show that the Sugb1 cDNA probe hybridized with a single ise) transcript (approximately 2.0kb) of RNA isolated from control and treated Miia cells. However, the intensity of the hybridization signal was significantly higher in RNA isolated from treated Miia cells (Fig. 1). Quantification of the hybridization signal revealed that Sugb1i expression was 2.8-fold higher in Mia-treated cells than in control-treated cells. Cha

Example 11

Dynamics of Miia-induced Sugb1 expression. To determine the dynamics of Sugb1 expression, immobile fibroblasts were treated for different periods of time with 5 μg/ml Miia. Total RNA was extracted and subjected to Northern blotting. As shown in Fig.2, the expression of Sugbb1 increased in a time-dependent manner in Miia-treated cells. Expression peaked at approximately 45 min and then declined to baseline at 2 h. Since it was reported that the expression of Sugb1 in mouse fibroblasts after stimulation with serum and growth factor was maintained for several hours (up to 8-10 hours) before inhibition occurred, the authors investigated the effect of serum and ROSE on the dynamics of Sugb1 expression in immobile human fibroblasts, UMI-38. As shown in Fig. 2B,

Sugb1 is expressed only for a short time after ROSE stimulation and is completely suppressed after 2 h. after adding the stimulant. Similar results were obtained for serum-induced expression of Sugb1 (data not shown).

Example 12 1 Sugb1 expression induced by factor Ma in a dose-dependent manner. To determine the dependence on the dose of Ma

Go! immobile fibroblasts were treated with different doses of EMiia (from 0.1 to 100 mg/ml) for 45 minutes, and then samples of total RNA from the cells were subjected to northern blotting. As shown in Fig. 3, treatment of fibroblasts with 0.1 μg/ml EMIa

Me, was sufficient to cause the expression of Sugb1, and the plasma concentration of EMI(a) (0O.5μg/ml, TONM as a result of c provided a significant reaction close to the maximum.

Example 13

The catalytic activity of the Miia factor is necessary for the induction of Sugb1.

In order to test whether the catalytic activity of Ma is necessary for the induction of Sugb1, UMI-38 cells were treated with Miia and inactivated in the active site of EMIT (EMiIai) for 45 min, and by Northern blotting

F) expression of Sugb1 was determined. As shown in Fig. 4, EMIIa did not cause the expression of Sugbb1, which indicated the need for the proteolytic activity of EMIIa. In this context, it is important to note that EMIIa bound to cell surface TEs with the same or higher affinity compared to EMIIa. It is unlikely that Miia-induced expression of Sugb1 in these experiments was the result of the generation of downstream coagulation factors,

EC and thrombin. Due to the use of sensitive chromogenic assays, we did not detect any evidence of factor Xa and thrombin generation in our experimental system (sensitivity of detection 1Opg). In addition, specific inhibitors of factor Xa and thrombin, i.e., mite anticoagulant protein and hirudin, respectively, could not reverse Miia-induced expression of Sugb1 (Fig. 5). 65 Example 14

Involvement of the transcriptional mechanism in the Miia-induced Sugb1 mRNA level in the steady state. In order to investigate whether transcription is involved in the Miia-mediated increase in steady-state Sugb1 mRNA levels, fixed UMI-38 cells were incubated with actinomycin-O (1 µg/ml) for 30 min. before adding Miia for 45 minutes. As shown in Fig. b, actinomycin-O inhibited the stimulating effect of Miia. These data indicate a transcriptional mechanism for Sugb1 induction.

In order to investigate whether de pomo protein synthesis is necessary for the induction of Subb1 pRNA by Mya, UMI-38 cells were pretreated with the protein synthesis inhibitor cycloheximide before exposing the cells to MPa for 45 min. As shown in Fig. b, the stimulating effect of Ma was not blocked by cycloheximide. In fact, cycloheximide markedly increased Miia-induced Sugb1 mRNA level at steady state.

Claims (2)

The formula of the invention 1. Use of a tissue factor agonist that induces signal transduction by direct binding to a TE, wherein said tissue factor agonist is EMII or EMIia, or a combination thereof, for the preparation of a medicinal product for the therapeutic treatment of pathological conditions that may be associated with cell migration or that can be treated by specific regulation of cell migration or chemotaxis, wherein the pathological conditions are selected from the list including atherosclerosis, tumor metastasis, tumor growth, tumor invasion, metastasis, angiogenesis, wound healing, including regeneration of vascular walls, burns and inflammation. 2. Application according to claim 1, characterized in that the cell is a human cell that expresses tissue factor, including fibroblasts, smooth muscle cells, tumor cells, hematopoietic cells, monocytes, macrophages and epithelial cells. 6) IF) (Se) (ee) IF) and - - and? -i 1 (ee) (o) sl ime) 60 b5