UA48149C2 - Method for inhibiting tissue factor activity; method for inhibiting platelet deposition; method for inhibiting vascular restenosis; method for treating acute clogging of coronary artery using modified factor vii - Google Patents

Abstract

The catalytic active site of Factor VII is modified to produce a compound which effectively interrupts the blood coagulation cascade. The modifications render Factor VIla substantially unable to activate plasma Factors X or IX. Pharmaceutical compositions of the modified Factor VII are used to treat a variety of coagulation-related disorders.

Description

Description of the invention

This invention is a partial continuation of application Mo 08/327,690, registered on October 24, 1994, which is a partial continuation of PCT/OZ 94/05779, registered on May 23, 1994, and a partial continuation of application Mo 08/065,725, registered on May 21, 1993, which is a partial continuation of PCT/O592/01636 and application Mo 07/662,920, registered on February 28, 1991 with transfer of rights to application Mo 08/164,666, each of which is specifically incorporated herein in its entirety by reference. 70 Scope of the invention

The present invention relates to proteins used as anticoagulants. In more detail, the present invention relates to modified forms of Factor MII, inhibiting blood coagulation and tissue factor.

Prerequisites for the creation of the invention 19 Blood coagulation is a process involving a complex interaction of various blood components or factors, which ultimately leads to the formation of a fibrin clot.

Generally, blood components that participate in what is defined as the coagulation "cascade" are known as proenzymes or zymogens - enzymatically inactive proteins that turn into proteolytic enzymes under the action of an activator, an activated factor of the coagulation system! blood Factor! svertivaniya, which are subjected to such a transformation, are casually referred to as "active factors" and denoted by the addition of a capital index "a" (for example, Factor Ma).

Activated Factor X ("Xa") is necessary for the conversion of prothrombin into thrombin, which then turns fibrinogen into fibrin at the final stage of fibrin clot formation. There are two systems! or two metabolic pathways that contribute to the activation of Factor X. "Internal metabolic pathway with 29" covers those reactions that lead to the formation of thrombin through the utilization of factors that are present only in plasma. A series of protease-mediated reactions ultimately generates Factor IXa, which, combined with Factor MISHa, cleaves Factor X into Xa. Identical proteolysis in the "external metabolic pathway" is carried out by Miia factor and its cofactor, tissue factor. Tissue factor is a membrane-bound protein and does not normally circulate in the plasma. However, in the case of rupture of a vessel, it can form a complex with Factor Mia to catalyze the activation of Factor X or Factor IX in the presence of San ion and phospholipid (Metetgwop apa Sepiggu, Biospet. 25: 4020 - 4033 (1986)). Despite the fact that the relative importance of the two metabolic pathways of coagulation in hemostasis remains unclear, in the last (7 h! it was found that MI factor and tissue factor play the main role in the regulation of blood coagulation. Ge)

The MI factor is a marker plasma glycoprotein) that circulates in the blood in the form of a 325 single-chain zymogen. Zymogen is catalytically inactive (Mushatv ei ai., y. Vioi. Speth. 264: 7536 - 7543 in (1989); Kao ei ai., Rhos. Mai. Asad. Zsi. OBA. 85: 6687 - 6691 (1988)). Single-chain Factor MI! Ip myo can be converted into double-chain Factor Ma by Factor Xa, Factor Xia, Factor IXa, or thrombin.

Factor Xa is considered the main physiological activator of Factor II. Similar to some other proteins "plasma, included in hemostasis, Factor MIA for activity associated with gamma-carboxylation of numerous glutamic acid residues), concentrated at the amino end of the protein, vitamin C is necessary. This gamma-carboxylation of glutamic acid is necessary! for interaction of Factor MI! with phospholipids in the presence of metal ions.

The transformation of the zymogenic Factor MI into an activated double-chain molecule occurs when the internal peptide bond, located approximately in the middle of the molecule, is split. In human

Factor MY site of activation cleavage is located at Аго 452-Пейз5з (Nadep ei AiY., Rgos. Mai). Asai. is SI. BOTH. 83:24122416 (1986);

Ge | TNIt ei ai., Viospet. 27: 7785 - 7793 (1988), both works! turn it on! here in the form of links). Bichiy Factor MII is activated by cleavage of Ago 452-MPei5z by a similar bond (Takeua ei aI.,0O0VISY Spet. 263: 14868 - - - 14877 (1988)). In the presence of tissue factor, phospholipids, and calcium ions, double-chain Factor Ma "iz" 20 rapidly activates Factor X or Factor IX by limiting proteolysis.

It is often necessary to selectively block the coagulation cascade in the patient. Anticoagulants, such as heparin, coumarin, coumarin derivatives, indandione or other agents, can be used, for example, in renal dialysis or in the treatment of deep vein thrombosis, consumption coagulopathy (IS) and many other medical disorders. For example, treatment with heparin or extracorporeal treatment with 29 citrate ion (0.5. Raiepi 4,500,309) can be used! during dialysis to prevent coagulation in

HF) during treatment. Heparin is also used to prevent deep vein thrombosis in patients undergoing surgery. o However, treatment with heparin and other anticoagulants may have undesirable side effects.

All calcium anticoagulants, as a rule, act throughout the body, and not specifically on the clot. 60 For example, heparin can cause severe bleeding. Moreover, having a half-life of approximately 80 minutes, heparin quickly disappears from the blood, thus necessitating frequent administration. Due to the fact that heparin acts as a cofactor of antithrombin PI! (ATII), and ATII quickly kills with treated BIS, it is very difficult to maintain the proper dosage of heparin, which causes the need for long-term monitoring of ATII and heparin levels. Heparin is also ineffective if the decrease in ATII is extreme. In addition, long-term use of heparin can increase the aggregation of platelets and reduce their number, and is one of the causes of osteoporosis. Indandione products can also have side toxic effects.

It was found that, in addition to the anticoagulants briefly described above, some natural proteins have anticoagulant activity. For example, Ketsieipozregdeg (0.5. Raiepi Mo 4,736,018) isolated anticoagulant proteins from the bovine aorta! and vessels of the human umbilical vein. McKee et al. (0.5. Raiepi Mo 4,732,891) discovered anticoagulant proteins in the human placenta. In addition, ATI was proposed as a therapeutic anticoagulant (ZspiIrreg eii AI., I apsei 1 (8069): 854 - 856 (1978); dogaap, 0.5. Raiepi

Mo 4,386,025; Wax her a!., 0.5. Rayepi Mo 4,517,294). 70 Proliferation of smooth muscle cells (SMS35z) of the vascular wall is an important factor in vascular damage caused by atherosclerosis, after vessel restoration, or in response to other vascular injuries. For example, the treatment of atherosclerosis often includes cleaning blocked vessels with angioplasty, zndarterectomy or reduction atherzectomy, or implantation of shunts - surgical procedures in which atherosclerotic plaques are compressed or removed /5 by means of catheterization (angioplasty), removed from the arterial wall by means of dissection (zndarterzktomy) or shunted with natural or synthetic implants. This is the procedure! they remove the vascular endothelium, disrupt the underlying inner membrane (intima) and lead to the death of the medial 5 MSv. The damage is accompanied by the proliferation of medial ZMS5 and their migration into the inner shell, which is characteristically manifested in the first weeks and up to six months 2o after the damage and stops when the surface endothelial layer is restored.

In humans, these lesions amount to approximately 20,905 cells and 8,095 extracellular matrix.

In approximately 3,095 patients and more who underwent angioplasty, sndarterzktomy or shunting, thrombosis and/or proliferation of 5MSz in the inner lining leads to re-adhesion of the vessel and, as a result, the failure of reconstructive surgery. Such closure of a vessel after surgical intervention is known as restenosis.

There is still a need to create improved compositions with anticoagulant activity, i) which can be administered in relatively low doses and do not give undesirable side effects inherent in traditional anticoagulants. The present invention fulfills this need by providing anticoagulants that act specifically at the sites of damage and additionally provide related benefits.

Essence -

Preparations of a new composition, including modified My Factor, possessing anti-coagulant properties. Make up! with the modified MII factor also inhibits the tissue factor.

The sequence of Factor MI has at least one amino acid modification, which with 5 is combined in such a way that it significantly reduces the ability of activated Factor MI! to catalyze the activation of plasma Factors X or X and, as a result, the ability to inhibit clotting activity. The new MII factor has an active center, modified by replacing at least one amino acid, and in its modified form is able to bind the tissue factor. For the compositions of the modified Factor MI! typical is a largely pure form. "

Make up! Inventions are especially useful for the treatment of patients when they are transformed into pharmaceutical compositions that can be given to patients suffering from various diseases to treat the connection with . state coagulation. Such molecules! modified MII Factor, capable of binding tissue factor, but having a significantly reduced ability to catalyze the activation of other factors in the coagulation cascade, may have a longer half-life from plasma and, as a result, a correspondingly longer period of anticoagulation activity compared to other anticoagulants. Among the medical indications for the compositions of the invention are those that are routinely treated with anticoagulants, such as, for example, deep vein thrombosis, pulmonary embolism, stroke, consumption coagulopathy (0IS), fibrin deposits in the lungs and kidneys, associated with gram-negative zondotoxemia, and - myocardial infarction. Make up! can be used to suppress vascular restenosis that occurs 5 years after mechanical damage to a vessel, such as damage caused by balloon angioplasty, zndarterctomy, reduction atherctomy, stent placement, laser therapy, or excision

He of disintegrating tissue, or arising in addition to vascular implants, stents, shunts or organ transplants. Compositions can be used to suppress the deposition of thrombocytes and their associated disorders. The method of suppressing coagulation, vascular restenosis, or platelet deposition consists in administering to the patient a composition that includes a modified Factor MI, which has at least one substituted amino acid in the catalytic triad Zeg344, Azr242, and Niv193 in (F, in an amount sufficient for effective suppression coagulation, vascular restenosis, or platelet deposition. The method is also used in the treatment of acute coronary artery occlusion in a patient and consists in the introduction of modified Factor MII, which includes OEBESK-Factor MI! in combination with tissue plasminogen activator (RA) or streptokinase and can accelerate IRA-induced thrombolysis.

Characteristically, pharmaceutical compositions for administration to humans will consist of the protein of the modified Factor MI and pharmaceutically acceptable carriers and buffers.

In the present embodiments of the human and bovine Factor MY, the bZeg344 residue in the active site 65 is modified, replaced by Su, Mei, Tig or, preferably, by Aa. Such a replacement can be made separately or in combination with replacement(s) in other sites of the catalytic triad, which includes Azr242 and Nivz193.

In the second aspect, the invention is connected with a polynucleotide molecule consisting of two operatively linked regions of coding sequences, which encode, respectively, the pre-propeptide and the dia-domain of the vitamin K-dependent plasma protein and the remaining dia-domain of the Factor MI protein, when the said polynucleotide encodes the Factor molecule MIA, which does not sufficiently activate plasma Factor X or IX, but is capable of binding tissue factor. Modified molecule

Factor MII, expressed with this polynucleotide, is a biologically active anticoagulant, i.e. capable of suppressing the coagulation cascade and, consequently, the formation of fibrin deposits or clots. C 7/0 Chelyu zxpressy modified Factor MI! the polynucleotide molecule is transfected in a mammalian cell line, such as, for example, VNK, VNK 570 or VNK 293.

A brief description of the drawings.

Fig. 1 illustrates the construction of an expression vector with a DNA sequence for Factor MI with the modification of Zegz44 to Aa. IYUsage of the symbol! 0-1, the sequence of unit mapping 0-1/5 of adenovirus 5; E, ZM40 virus enhancer gene; MI R, major late promoter; 55, a set of splicing sites; and pA, polyadenylation signal from Z5M40 in late orientation.

Fig. 2 shows the effect of jet injection of OESC-Factor Ma on the formation of a thrombus (platelet deposition) in the snarterectomized aorta of a baboon compared to saline-treated controls. Arteries were measured after 60 minutes. In this primate model of acute vascular injury, OEOC-Factor Miia markedly inhibited the development of platelet-enriched thrombi.

Fig. It shows the results obtained during the incubation of smooth muscle cells! baboon with increasing concentrations of either EMIia (unfilled square)) or OEEEOK-EMiIIa in the presence of a constant amount

EMIP (5nM) (shaded square). The level of EX activation was then determined using the chromogenic substrate 5-2222. This data is presented in the form of amidolytic activity, namely as a percentage of the activity generated in the presence of only 57NM EMIP.

Fig. 4 depicts the size of the integument area after baboon carotid artery sndarterctomy and i) treatment with OESK-Factor MiIa for 7 or 30 days compared to control animals.

Fig. 5 shows the ratio of the area of the inner sheath to the sum of the areas of the inner and medial sheath of the femoral artery of a baboon after balloon damage and treatment with OESK-Factor Mia, b z The control group included 5 vessels, with a 7-day treatment examination 11 vessels, with a 3-day treatment examination 2 vessel (n - the number of examined vessels). -

The description of specific embodied. "-

The present invention provides a new modified MII factor with anticoagulant activity. The modified MII factor can be in the form of a zymogen (i.e. a single-chain molecule)) with

Zb MlY can be split in the activation site. Thus, the concept of "modified Factor MI!" "E includes the modified Mii Factor and molecules of the modified Mii Factor, which bind the tissue factor and suppress the activation of Factor IX in IXa and Factor X in Xa. The composition of the modified Factor MI is suitable for administration to various mammals, especially humans, with the aim of inhibiting the coagulation cascade. Modified Factor MI can be administered to the patient in combination with other anticoagulants or instead of them. in c The MII factor plays an important role in the coagulation cascade, especially its inclusion in the external metabolic pathway. Present in the circulating plasma in the form of an inactive single-chain ;" pro-enzyme protein and having undergone activation, Factor Ma in combination with tissue factor and calcium ions activates Factor X in Xa and Factor IX in YXa with the possible formation of a fibrin clot.

The present invention provides an opportunity to suppress this sequence in the 5" coagulation cascade, preventing or otherwise inhibiting the activation of Factors X and IX with the help of

Miia factor. The protein Factor MY of this invention has a catalytic center, which is modified in such a way that! to reduce the catalytic activity of Factor Miia, but to preserve the ability to bind the factor - tissues. The molecule of the modified Factor MI competes with the native Factor Mi and/or Mia for binding to the tissue factor. As a result, the activation of Factors X and IX is suppressed. o In one aspect of the present invention, the catalytic activity of Factor Ma is chemically inhibited

He derivatization of the catalytic center, or triads. Derivatization can be carried out by interaction of Factor MI with an irreversible inhibitor, such as an organophosphorus compound, a sulfonyl chloride, a peptide-containing halomethyl ketone or an azapeptide, or, for example, by acylation. dv Preferred peptides containing halomethyl ketones! include PRASK (0-PHe-Pho-Agd-chloromethyl ketone; see

T.5. Raiepi Mo 4,318,904, incorporated herein by reference), O-Rpe-Rpe-Ago- and Rpe-Rpe-Agod-chloromethylketone; and

Ф) реОСКсК (Bapzu!I-SISH-SiIu-Agd-chloromethylketone). ka In another aspect, the catalytic activity of Factor Ma can also be suppressed by substitution, introduction or deletion of amino acids. In the presented embodiments, amino acid substitutions are made in the amino acid sequence of the catalytic triad! Factor MIA, defined here as a region containing an amino acid that contributes to the activity of the catalytic site of Factor Ma. Substitutions, introductions, or deletions in the catalytic triad are generally made by the amino acid that enters the catalytic site or is located next to it. In the proteins of the human and bovine Factor MY, the amino acids that form the catalytic "triad" are Zeg z44, Avrodo and Nivzchoz (the lower numerical index 65 indicates the position in the amino acid sequence). Catalytic site! MI factor! Other types of mammals can beat definitions! using available technology, including, but not limited to,

Protein isolation and amino acid sequence analysis. Catalytic site! can also be determined by lengthening the sequence in the presence of other serine proteases, especially chymotrypsin, the active center of which has been deciphered earlier (5idieg eii ai., U. Moi. Vioi. 35: 143 - 164 (1968), included here

In the form of a link), establishing with the help of the aforementioned extension, similar amino acid residues of the active center.

Substitutions, introduction or removal of amino acids are made in such a way that! prevent or otherwise suppress the activation of X Factors and/or X Factor Ma. Modified in this way

MI factor! must, however, retain the ability to compete with authentic Factor MI and/or Factor 7/0 Ma for binding to tissue factor in the coagulation cascade. Such competition is easily determined by, for example, the analysis of blood clotting, as described here, or the analysis of competitive binding using, for example, a cell line that has surface-binding tissue factor, such as human carcinoma cells! urinary bladder line 982 (Zakai et al., U. Viol. Spet. 264: 9980 - 9988 (1989), incorporated herein by reference).

Amino acid), which form the catalytic site of Factor MII, such as Zegzly, Avrolo MI Nivchoz human and bull Factor MI, can be either replaced or removed." Within the framework of the present invention, it is preferable to change only one amino acid, thereby minimizing the possibility of increasing the antigenicity of molecules! or suppression of its ability to bind the tissue factor, however, changes of two or more amino acids (substitutions, additions, or deletions) may occur, and combinations of substitutions, additions, and deletions may also be made. In the chosen one, the MI factor is embodied for the human and the bull! it is preferable to replace Zeg with Aa, but it is possible to replace Su, Mei, Tig or other amino acids. It is preferable to replace Azr with Sy and Niz with I uz or Ag. In general, the replacement is selected in such a way that it destroys the tertiary structure of the protein as little as possible. The model by Dykhoff et al. (in Atlas of Protein Structure, 1978, Maj. Wiot. Kez. Goshpa, Washington, DC), incorporated herein by reference, may be used as a guide in the selection of other amino acid substituents. It is possible to make a change, as described above, in the catalytic site of the corresponding sequence of Factor MI i) of a human, bull, or other species and test the resulting protein for suppression of catalytic activity and emerging anticoagulant activity, as described here. For the modified Factor MI, the catalytic activity will be significantly reduced, in general it is less than 595% of the catalytic Ge! from the activity of the corresponding samples of Factor MI! wild type, mostly less than 1905.

Proteins of the present invention can be obtained using recombinant DNA technology. In general, the cloned DNA sequence of Factor MI! The wild type is modified to encode the desired protein. This modified sequence is then inserted into the expression vector, which, in turn, is transformed or introduced into the host's cells. Cells of higher zoukaryotes, especially so z5 Cultivated mammalian cells, are preferred as host cells. For the human Factor MI! lime! complete nucleotide and amino acid sequences. See 0.5. Paradise

No. 4,784,950 (which is incorporated herein by reference), which describes the cloning and expression of human recombinant MIII Factor. Sequence of the bull Factor MI! described in the work TakKeua ei ai., 9. Vioi.

Spent 263: 14868 - 14872 (1988), which is incorporated by reference. "

Changes in the amino acid sequence can be implemented by various methods. Site-specific mutagenesis is used to modify the DNA sequence. Methods! site-specific mutagenesis is well known and described), for example, by Zoller and Smith (DNA 3: 479-488 (1984)). Such;" thus, using the nucleotide and amino acid sequences of Factor MI, it is possible to carry out selective changes.

The MII factor, modified in accordance with the present invention, includes those proteins in which the amino-terminal region (dia-domain) is replaced by the dia-domain of one of the vitamin K-dependent plasma proteins Factor IX, Factor X, prothrombin, protein C, protein 5 or protein 7. dia-Domains of vitamin C K-dependent plasma proteins are characterized by the presence of gamma-carboxyglutamic acid residues - and generally have a length from approximately 30 to 40 amino acids with C-termini, similar to the positions on the zxon-intron boundaries of the corresponding genes . Method! obtaining the MI Factor! with a heterologous dia-domain, reveal! in O.5. Patent No. 4,784,950, incorporated herein by reference.

Ge DNA sequences for use within the framework of the present invention will typically encode a pre-propeptide at the amino terminus of the Factor MI protein to establish proper post-translational processing (for example, gamma-carboxylation of glutamic acid residues) and secretion from two host cells. The pre-propeptide can bind to the same as in Factor II, or to another vitamin K-dependent plasma protein, such as Factor X, Factor X, prothrombin, protein C or protein 5. modifications of the amino acid sequence of the modified Factor MII, which do not significantly weaken the ability of the protein to act as an anticoagulant. For example, the Factor MII, modified by the catalytic triad, can also be modified by the activation cleavage site in order to inhibit the transformation of the zymogenic Factor MI into its activated two-chain form as described in O.5 of Patent 5,288,629, incorporated herein by reference.

Zkspressionnye vector! for use in the implementation of the present invention will include a promoter capable of directing the transcription of a cloned gene or cDNA. Preferred 6v5 promoters for use in mammalian cell culture are viral and cellular promoters. Viral ones include the 5M40 promoter (Z!irgatapi! ei aiYi., Moi. Se Bioi. 1: 854 - 864 (1981); and the promoter ot SMM (cytomegalovirus)) (Vozpai ei ai., Se 41: 521 - 530 (1985) ). A particularly preferred viral promoter is the major late promoter of adenovirus 2 (Katsitap apa ZpNagr, Moi. Seii| Vio!. 2: 1304 - 1319 (1982)). Cellular promoters include the muscle kappa promoter (Vedtanp ei ai., Rgos. Maii Assad.

SI. OBA 81: 7041 - 7045 (1983)) and the muscle promoter (Hoi et al., Seii! 33: 85 - 93 (1983)). A particularly preferred cellular promoter is the muscle metallothionein promoter I (RaiItNeg ei ai.,

Zsiepse 222: 809 - 814 (1983)). The expression vector may also contain a set of RNA splicing sites located upstream of the promoter and upstream of the inclusion site for the MI Factor sequence. Preferential sites of RNA splicing can affect production! from/about adenoviruses and/or immunoglobulin genes. The expression vector also contains a polyadenylation signal located downstream of the inclusion site. Particularly preferred polyadenylation signals include the early or late signal from 5M40 (Kashcitap apa Zpagr, ibid.), the polyadenylation signal from the EI region of adenovirus 5, the terminator of the human growth hormone gene (Oe

Moio eg ai., Mysi. Asiaz Kev. 9: 3719 - 3730 (1981)), the signal of polyadenylation of the human Factor 7/5 gene. Mi or the gene of the bull factor MII. Expression vectors can also include a non-coding viral leader sequence, such as that of adenovirus 2, located between the promoter and RNA splicing sites; znhanserny sequences, such as znhanser ot 5M40.

Cloned DNA sequences are introduced into the culture of mammalian cells, for example, by transfection mediated by calcium phosphate (UMIdieg ei ai., Seii 14: 725 - 732 (1978); Sogzago apa Reagsgop, 2o ZotaiNs Se! Sepeisz /: 603 - 616, 1981; OSgapat apa Map deg EB, Migoiyudu 524: 456 - 467 (1973)) or by electroporation (Metsapp et al., EMVO 3. 1: 841 - 845 (1982)). For the identification and selection of cells expressing xenogeneic DNA, a gene that determines the selectable phenotype (selectable marker) is routinely introduced into the cells, along with the main gene or cDNA. Preferred selectable markers include genes) that determine resistance to such drugs as neomycin, hygromycin, and methotrexate. The selectable marker can be an amplifiable selectable marker.

The sequence of i) dihydrofolate reductase (ONEK) is a preferred amplifiable selective marker. Selective Markers and Considerations of Tilla (Mammal Cell Technology. Vyepmogy Rybriizpege, Stoneham, MA; incorporated herein by reference). The selection of a selectable marker is available to an ordinary specialist. The selectable marker can be inserted into the cell in a separate plasmid at the same time as the main gene or can be inserted into the same plasmid. When the selectable marker and the main gene are located in the same plasmid, they can be controlled by different promoters or by one -/- where the same promoter, the latter scheme gives a dicistronic unit of the genetic code. Constructions from that type of lime! specialists (for example, I emipzop apa Zitopzep, O.5. Raiyepi 4,713,339). Adding additional DNA, known as "carrier DNA," to the mixture that is injected into the cell can also be beneficial. "IS

After the introduction of DNA into the cells, they grow in a suitable growth medium, usually for 1-2 days, until the start of expression of the target gene. The term "suitable growth medium" used here means a medium containing nutrients and other components necessary for cell growth and gene expression of the modified Factor Mi. The environment usually includes sources of carbon, nitrogen, essential amino acids, carbohydrates, vitamins, salts, phospholipids, proteins, and growth factors. For the production of gamma-carboxylated modified Factor MI! Wednesday will contain vitamin K, preferably in a concentration of 0.1 - 5 μg/ml. Then the selection of drugs for the selection of cells that are stable is carried out;" the selectable marker is expressed. For cells that have been transfected with an amplified selectable marker, the concentration of the drug may be increased, so! select a larger number of copies of the cloned sequences, thereby increasing the expression levels. Then, clones of stable transfected cells are screened for the expression of the modified MI factor.

Preferred lines of mammalian cells for use in the present invention include CO5-1 cells (ATSS SKI 1650), newborn hamster kidney cells (VNK) and 293 (ATOS SK. 1573; Stapat ei - aI., U. SiIIp. Migoi. 36: 59 - 72 (1977)). The preferred line of VNK cells is the strain ik 7 and 813 (U/aesnieg apa Vazegde, Rgos. Mai. Asad. si. OA 79: 1106 - 1110 (1982), incorporated here as a link), hereinafter referred to as VNK 570. The line cell VNK 570 is registered in the American Collection of Type Cultures (Che) (ATSS), 12301 Ragkiazp Og., Rockville, Maryland 20852, under the number SKI. 10314. The VNK IC cell line (813 is also known in the ATCC under the number SC! 1632. In addition, other cell lines can be used within the scope of the present invention, including Kainer I cells (rat hepatoma; ATCC SC 1600), cells

Kai Ner II (rat hepatoma; ATOS SK 1548), TSMK cells (ATSS SS. 139), human lung cells (ATSS HB 8065), MSTS 1469 cells (ATSS SS 9.1), CHO cells (ATSS SS 61) and OYUKH cells ( pats apa Spavip, Rgos. o Mic. bos. 5si. OBA 77: 4216 - 4220 (1980). Name) In the framework of the present invention, the technology of transgenic animals can be used to obtain a modified Factor MI. It is preferable to produce proteins in the mammary glands of female mammals. Expression in the mammary gland and subsequent secretion of the target protein into milk overcome many difficulties associated with the release of proteins from other sources. Milk is easily collected, available in large quantities and well characterized biochemically. Moreover, the main proteins of milk are present in it in high concentrations (generally 1 - 15 g/l). From a commercial point of view, it is obvious that it is preferable to use as a host cows that give a large amount of b5 milk. While small animals such as mice and rats may be used (which are preferred in proof of concept), domestic mammals, including but not limited to pigs, goats, sheep, and cattle, are preferred within the scope of the present invention. . Sheep are particularly preferred due to such factors as the previous history of transgenesis in this species, milk yield, cost and high availability of equipment for collecting sheep milk. For a comparison of the factors affecting the host species' behavior, see MMIRO Rybiisayop. U/O 88/00239. In general, it is desirable to select the host animal breed that is bred for milk production, such as Eazi Egiesiapa sheep, or to introduce a dairy breed by breeding a transgenic line at a later stage. In any case, a known animal and in good condition must be used. 70 To obtain expression in the mammary gland, the promoter of the milk protein gene is used. The milk protein gene group consists of genes encoding casein (see O.5. Raiepi Mo 5,304,489, included here in the form of a link), beta-lactoglobulin, alpha-lactoglobulin, and whey acid protein. A beta-lactoglobulin promoter is preferred. In the case of the sheep beta-lactoglobulin gene, as a rule, the proximal 5-linking sequence of 406 nucleotides will be used, although the more significant fragment is /5! 5-flanking sequences, approximately up to 5 thousand. nucleotides, such as a DNA segment of 4.25 thousand. nucleotides surrounding the 5'-linking promoter and the non-coding part of the beta-lactoglobulin gene. See MUPIeiam/ ei ai., Viospet. 9. 286: 31 - 39 (1992). Let's also use a similar fragment! promoter DNA of other species.

Other areas of the beta-globulin gene can also be included in the design, because The entire gene region can be expressed. It is generally accepted that, for example, constructs lacking introns are poorly expressed compared to subjects containing such DNA sequences (see

Rgos. May Asad si. BOTH 85: 836 -8 40 (1988); RaitKeg ei aiYi., Rgos. May Asad si. BOTH 88: 478 - 482 (1991); MUpIeiam ei ai., Tgapzdepis Kev. 1: Z - 13 (1991); MO 89/01343; and MO 91/02318, each incorporated here by reference). In this regard, it is generally preferable to use, where possible, a genomic sequence containing all or some of the native intron! of the gene encoding the target protein or polypeptide, therefore, additional inclusion of at least some introns, for example, from the beta-lactoglobulin gene, is preferable. One of these regions is a segment of DNA that provides intron splicing and polyadenylation of RNA from the 3-noncoding region of the sheep beta-lactoglobulin gene. When replaced with a natural 3-non-coding sequence of the ztot gene (zu z ovine beta-lactoglobulin segment can both enhance and stabilize the levels of expression of the target protein or protein. In the framework of the second, the region surrounding the ATO initiation of the modified -

The MII factor is replaced by the corresponding sequences of the specific milk protein gene. Such a "replacement" provides an assumed tissue-specific initiation environment in order to enhance zxpression. It is convenient to completely replace the pre-pro- and non-coding sequences of the modified co

MI factor! sequences, for example, of the VI C gene, although it is possible to replace even smaller regions. "IS

For the expression of the modified MI Factor in transgenic animals, the DNA segment encoding the modified MI Factor is operatively connected to additional DNA segments necessary for its expression to form expression units. Such an additional segment! include the above-mentioned promoter, as well as sequences that ensure the termination of transcription and polyadenylation of mRNA. Zkspressionnye units! then they will include a segment of DNA encoding a secretory signal sequence operatively connected to a segment encoding a modified Factor Mi. The secretory signal sequence can beat;" by the sequence of native MII Factor or any other protein, such as milk protein. See, for example, mop Neipie, Mysi. Asidv Caves. 14: 4683 - 4690 (1986); Meade ei a, 0.5. Raiepi Mo 4,873,316, Kotorne included here as links. Construction of expression units in transgenic animals is conveniently carried out by including the sequence of the modified Factor MI in a plasmid or phage vector containing an additional segment! DNA, although the expression unit can be constructed with the help of other ligation sequences. It is especially convenient to create a vector containing a segment of DNA encoding a milk protein and to replace the milk coding sequence with a polypeptide sequence of a modified MY Factor, thereby creating a gene mix that includes the expression

Ge control sequence of the milk protein gene. In any case, cloning of expression units in plasmids or other vectors favors amplification of the sequence of the modified

MI factor. Amplification is conveniently carried out in a host bacterial cell (for example, E. soya), thus, the vectors will include a source of replication and a selectable marker in host bacterial cells. (F, The expression unit is then introduced into a fertilized egg (including early embryos) of the selected host species. The introduction of heterologous DNA can be accomplished by one of several methods, including microinjection (e.g., 0.5. Raiepi Mo 4,873,191), retroviral infection of aepivsp, Zsiepse 240: 1468 - 1474 (1988)) or directed integration using embryonic stem cells (E5) (reviewed by Vgadieu et al., Vio/GTespoirdu 10: 534 - 539 (1992)). Oocytes implanted in the fallopian tubes or uterus of pseudopregnant females are allowed to develop to term.

An offspring carrying the introduced DNA in the embryonic state can transmit DNA to its offspring in the normal way, according to Mendel, with the formation of a transgenic herd. 65 Notice to specialists! general procedure for obtaining transgenic animal limes!. See, for example, Naap eyi AI., Manipulation of muscle embryos: a laboratory manual. Soyd Zrgipu Nagrog

And arogayugu, 1986; Zytopez ei ai., BioDesppoiodu 6: 179 - 183 (1988); UMai! ей ай., ВиоЙ. Kerhod. 32: 645 - 651 (1985); Journal of Science and Technology, BioDesppoydu 8: 140 - 143 (1990); Eregi ei aY., BioDesppoiodu 9: 835 - 838 (1991);

Ctitrepiogi ei ai., Vio/Hesppoiodu 9: 844 - 847 (1991); OMG! ей ай., 9. SeiЙ. Viospet. 49: 113 - 120 (1992);

M. Rayepi Mo 4,873,191 and 4,873,316; ULRO - UUO 88/00239, MO 90/05188, MO 92/11757; and OV 87/00458, which included! here in the form of villages. The technique of introducing foreign DNA sequences into mammals and embryonic cells of other species was initially developed in mice. See, for example, Sogaop ei ai!., Rgos. May). Asad 5 OA 77: 7380 - 7384 (1980); Sogdop apa Kyadaye, Zsiepse 214: 1244 - 1246 (1981); RaitKMeg apa Vgypvieg, SeiYi 41: 343 - 345 (1985); 7/0 Vipvieg ei ai., Rhos. May Asai. All together. OZA 82: 4438 - 4442 (1985) and Nodap et al., (ibid.). Then take it! were adapted for use on larger animals, including domestic animals (see, for example, publications UURO - UUO 88/00239, UMO 90/05188 and UMO 92/117577; and ZItopzg ei ai!., BioDesppoiiodo 6: 179 - 183 ( 1988). So, in most effective methods of generating transgenic mice or domestic animals known to date, several hundred linear DNA molecules of interest are injected into one of the pronuclei of the fertilized egg according to the methods developed. DNA injection into the cytoplasm of the zygote. The production of transgenic plants can also be used. The expression is generalized or directed to a separate organ, such as a tuber. See Niai, Maishge 344: 469 - 479 (1990); Edeiract et al., 9). Ipiepegop Kev. 12: 449 - 453 (1992);

Z|topz ei a!., BioDesppoiodu 8: 217 - 221 (1990); and publication of the European Patent Office EP 255,378.

The modified MII factor obtained in accordance with the present invention can be purified by affinity chromatography on a column with an antibody to the anti-MII factor. Particularly preferred is the use of calcium-dependent monoclonal antibodies, as described in U/akarauvzni ei ai., 9. VisII. Spent 261: 11097 - 11108 (1986) and TpIt ei ai!., Viospet. 27: 7785-7793 (1988), incorporated herein by reference.

Additional purification can be carried out by traditional methods of chemical purification, such as high-performance liquid chromatography. Known and other methods! purification, including precipitation with barium citrate, which can be used to purify the modified MII Factor, described i) here (see, as a rule, Zsorez K., Protein Purification, Zrgipdeg-Mepad, M.U., 1982). Preferable is sufficiently pure modified Factor MI, at least 90 - 9595 homogeneity, most preferably - 98 - 9995 homogeneity or more, used for pharmaceutical purposes. Gee! Partially purified or, if desired, to a homogeneous state, the modified MIA factor can be used for therapeutic purposes. -

In the framework of one of the embodiments of the invention, the modification of the MII factor is cleaved at the activation site in order to convert it into a double-stranded form. z5 2853 - 2857 (1972), Tpotaz, OZ Raiepi Mo 4,456,591, Nedpeg apa Kiviei, U Sit Ipmevi 71 1836 - 1841 (1983), «g

Kiiziiyi apa Etsikamla, Veippo Ipvi MIC 73 29 - 42 (1983), which is incorporated herein by reference The resulting molecule is then converted into a dosage form and administered as described below

Molecules! modified Factor MI! the present invention and pharmaceutical compositions based on them are especially convenient! for administration to people treated for various conditions, including intravascular "Coagulation. For example, although deep vein thrombosis and pulmonary embolism can be treated with traditional anticoagulants, the modified MII factor described here can be used to prevent the development of thromboembolic complications in patients at high risk. such as those who have undergone surgery or suffer from congestive heart failure.

In addition, the modified Factor MI can act as an antagonist mediated by the tissue factor

Induction of coagulation, therefore, by blocking the production of thrombin and the subsequent deposition of fibrin, the Modified MI Factor, as such, may be useful in suppressing the activity of the tissue factor, leading, in particular, to the suppression of blood coagulation, thrombosis or deposition of platelets with the Modified MI Factor Molecule of the present invention they can be especially useful! c - treated hyperplasia of the inner membrane or restenosis due to an acute vascular disorder. Acute vascular disorders are those that arise quickly (i.e., from days to months) compared to chronic vascular disorders (for example, atherosclerosis), which develop over the course of

Ge of life. Acute vascular disorders often occur during surgical procedures, such as reconstruction of blood vessels, in which the methods of angioplasty, tendon arterectomy, atherectomy, shunting, etc. are used.

Hyperplasia can also occur as a delayed reaction in response to, for example, shunting or organ transplantation. Since the modified MII factor has a more selective action than heparin, it binds only the tissue factor in the damage sites, and, due to the fact that

F) modified Factor MI! does not destroy other coagulation proteins, it will be more effective than heparin, and less likely to cause unwanted bleeding when prophylactically used to prevent deep vein thrombosis. The dose of modified MI factor for the prevention of deep vein thrombosis is in the range from about 5 Ωkg to 50 Ωg per day, more typically 1 - 200 mg/day, preferably 10 - 175 mg/day for a patient weighing 7 Оkg, administration should begin at least 6 hours before the surgical intervention and will continue until the patient is transferred to the outpatient mode. A dose of modified MI Factor! for the treatment of restenosis will differ for each patient, but, in general, will be within the limits mentioned above. 65 The latest advances in the treatment of coronary vascular diseases include the use of mechanical intervention to either remove or relocate the offending plaque in order to restore adequate blood flow in the coronary arteries. Despite the use of various forms of mechanical intervention, including balloon angioplasty, reduction atherectomy, vascular stenting, laser therapy, or excision of decaying tissue,

The effectiveness of these methods is limited to approximately 4095 restenosis occurring within 6 months after treatment.

It is believed that restenosis is the result of a complex interaction of biological processes, including the deposition of platelets and thrombus formation, the release of chemotaxis and mitogenic factors, and the migration and proliferation of vascular smooth muscle cells into the inner lining of the dilated arterial segment.

Suppression of the accumulation of platelets in the sites of mechanical damage can limit the rate of restenosis in humans. Therapeutic use of a monoclonal antibody to the platelet of SryiBLia is able to reduce the level of restenosis in a person (Sayi a. M. EpaI. U. Mei. 330: 956 - 961 (1994).

The antibody can bind to the SryB/LPa receptor on the surface of platelets and thus suppress the accumulation of platelets. These data suggest that the suppression of platelet accumulation at the site of mechanical damage to the human coronary artery has a positive effect on the final outcome of the treatment. Since the accumulation of thrombocytes occurs in the sites of acute vascular disorders, the generation of thrombin in those sites may be responsible for the activation of platelets and their subsequent accumulation.

As shown in the following examples, the modified Factor MY of the present invention is capable of binding to the tissue factor located on the surface of the cell. For example, OESMK-Factor Miia binds tissue surface factor with the same or higher affinity than wild-type Factor Miia. However,

OESK-Factor Ma does not have enzymatic activity, but binds to tissue factor and acts as a competitive antagonist of wild-type Factor Mia, thereby inhibiting subsequent steps in the metabolic pathway of coagulation that lead to thrombin generation.

Molecules! modified Factor MI! of the present invention, which support the binding of factor i) tissues, suppress the accumulation of platelets in the site of a vascular disorder by blocking the production of thrombin and the subsequent deposition of fibrin.

Thanks to the ability of OESK-Factor MI to block the generation of thrombin and limit the deposition of He! from platelets in the site of an acute vascular disorder, molecules of the modified MII Factor, which have tissue factor binding activity, but only enzymatic activity, can be used to suppress vascular restenosis. "-

Thus, composition and method! the present invention has a wide field of application. For example, they are useful for preventing or suppressing restenosis, which typically accompanies mechanical intervention with the purpose of either removal or replacement! the culprit in the disturbed material of the plaque "g during the treatment of damaged coronary or peripheral vessels, such as accompany and/or follow balloon angioplasty, reduction atherzctomy, installation of vascular stents, laser therapy, excision of disintegrating tissues, etc. Compounds will typically be administered 24 hours before the intervention and for 7 days or more after it. The introduction can be carried out in various ways, as described in the following. The compounds of the present invention can also be administered systemically and locally during the installation of vascular implants (for example, covering artificial or modified natural implants

and arterial vessels) in the sites of anastomosis, surgical zndarterzktomy (typical for zndarterzktomiya and? carotid artery), shunting, etc. Modification Factor! MII and Ma are also used to suppress hyperplasia of the inner choroid, early sclerosis, and venous occlusion associated with organ transplantation, for example, occurring after bone marrow transplantation. 5" In the treatment of established deep vein thrombosis and/or pulmonary embolism, the shock and maintenance dose of the modified MII factor, depending on the patient's weight and the severity of the disease, varies from approximately 5 Ωkg to 50 Ω/day, more typically 1 - 200 mg/day and more preferably 10 - - 175 mg/day for a patient weighing 7 kg. Due to the reduced probability of unwanted bleeding during the infusion of Factor MIA, it is possible to replace heparin with it or reduce the dose of heparin during or after surgical intervention combined with thrombectomy or thrombectomy.

The composition of the modified MI Factor of the present invention is very useful for the prevention of cardiogenic embolism and in the treatment of thrombotic strokes. Due to the low probability of unwanted bleeding and the selectivity of the action, the modified MIA factor can be given to those who have suffered a stroke, and it can prevent the spread of occluding arterial thrombi. The amount of modified MI Factor injected will vary for each patient depending on the nature and severity (F, impact, but the dose will generally be within the limits suggested below. The pharmaceutical compositions of the modified MI Factor, including OBEsSK-Factor Mi and presented here , will be useful in the treatment of acute myocardial infarction, due to the ability of Modified Factor MI! to suppress coagulation of Ip. Modified Factor MI! can be given as an adjuvant with tissue plasminogen activator or streptokinase during the acute phase of myocardial infarction and can accelerate IRA-induced thrombolysis With acute myocardial infarction, the patient is given a shock dose of at least 5O0mcg to 50Omg/day, more typically 1 - 200mg/day and more preferably 10 - 175mg/day for a patient weighing 7Okg as a shock and maintenance dose. 65 Modified The MIA factor is given before, in conjunction with, or soon after the introduction of thrombolytics an agent such as tissue plasminogen activator.

Modified MI Factor! the present invention is useful in the treatment of consumption coagulopathy (PS) and other manifestations associated with gram-negative bacteremia. Patients with BIS are characterized by widespread microcirculatory thrombi and frequent acute bleeding, which arise from the replacement of necessary clotting factors. Thanks to its selective action, the modified MI Factor! it will not be, like traditional coagulants, complicated by bleeding, binding of

OIS, but will delay or suppress the formation of additional microvascular fibrin deposits. Thus, the modified Factor MI of this invention, including OESMK-Factor MII and OESK-Factor Ma, is useful for suppressing fibrin deposits associated with zondotoxemia and zondotoxin shock and, therefore, mitigates the effect caused by gram-negative bacteremia. It is shown that the OEOK-Factor

Mya in mice shows a dose-dependent effect when blocking fibrin deposits in the kidneys and lungs, and in baboons it increases the survival of treated animals. In the case of acute bacteremia, zondotoxemia, or IC, the patient is given a loading dose of at least approximately 5 Ωkg to 50 Ω/day, more typically 1 - 200 mg/day and more preferably 10 - 175 mg/day when the patient weighs 7 Ωkg, then maintenance /5 DdOoZzI in the range from 5Omkg to 50Omg/day, typically 1 - 200mg/day with a patient weight of 7Okg.

Pharmaceutical composition! intended for parenteral, local or local administration during prophylactic and/or therapeutic treatment. Preferably pharmaceutical composition! administered parenterally, i.e. intravenously, as well as subcutaneously or intraarm. Thus, the composition is provided by the invention! for parenteral administration, which includes a solution of molecules of modified Factor MI! in an acceptable carrier, preferably aqueous. Various aqueous carriers can be used, for example, water, buffered water, 0.495 physiological solution, 0.395 glycine, etc. Molecules! modified Factor MI! также могут быть злачунь! in a liposome as a dosage form! for delivery to (or targeting to) the site! damage The liposome preparation, in general, is described, for example, in M.5. 4,837,028, 5. 4,501,728 and I.5. 4,975,282, incorporated herein by reference. The composition can be sterilized by standard, well-known methods. The resulting aqueous solution can be transferred to packaging for use or filtered in aseptic conditions and i) lyophilized, and the preparation is lyophilized! before introduction, they are mixed with a sterile aqueous solution. The compositions may contain pharmaceutically acceptable excipients to approximate physiological conditions, such as pH-supporting and buffering agents, tone-supporting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and etc. The concentration of the modified Factor MI! in these dosage forms - it can vary widely, namely, less than 0.595, generally 195 or, at least, up to 15 - 2095 - by weight and will be selected primarily by the volume of liquid, viscosity, etc. in accordance with the features of the selected method of administration. co

Thus, a typical pharmaceutical composition for intravenous infusion could contain 250 ml of sterile Ringer's solution and 10 mg of modified MII factor. Applied methods of preparation of parenterally administered drugs are available to professionals and are described in more detail, for example, in

Remington's Pharmaceutical Science, 16th ed., Musk, Rybizipud Sotrapu, Easton, PA (1982), which is incorporated herein by reference. "

Composition), containing modified Factor MII, can be administered during prophylactic and/or with therapeutic treatment. When the composition is used therapeutically! administered to the patient, as described above, in an amount sufficient for treatment or, at least, for the purpose that! stop the disease and its complications. The amount adequate to achieve that is defined as "therapeutically effective dose".

In this case, the effective amount depends on the severity of the disease or disorder, as well as on the weight and general condition of the patient, but usually varies within 0.05 - BbOmg of the modified MI Factor! in their day for a patient weighing 7Okg, and the dose is more often used! 1 - 200 mg of modified MI Factor! per day It should be remembered that it is material! the present invention can, as a rule, be used for serious diseases or disorders, i.e. in productive life or potentially threatening situations. In such cases, taking into account the minimization of extraneous substances and the general lack of immunogenicity of the Modified MI Factor in humans, it is possible and may be considered desirable to introduce a significant excess of the modified MI Factor to the patient.

Ge) For prophylactic use, a composition containing a modified MII factor is administered to a patient who is susceptible to the disease, or when there is a risk of a disease or disorder in order to strengthen the patient's own anticoagulation capabilities. This amount is defined as a "prophylactically effective dose". In this case, the exact amount used again depends on the patient's state of health and his weight, but, as a rule, varies within the range of 0.5 - 500 mg when the patient's weight is 7Okg, (F, more often 1 - 200mg/7Okg. It is possible to carry out single or multiple administration of compounds with doses and regimens selected by the attending physician.60 For outpatients requiring daily maintenance doses, modified Factor MI may be administered, for example, by continuous infusion using a portable pump.

Local delivery of the modified MI Factor! can be carried out, for example, by perfusion, with the help of double balloon catheters, stents, hydrogels used to cover balloon catheters, or other well-known methods. In any case, pharmaceutical drugs 65/form! must provide the amount of modified Factor MI! of this invention, sufficient for the effective treatment of the patient.

Example 1.

Zxpression of Factor MI! with mutation Zeg344 -» AIaz44.

In order to generate MI factor! with mutation Zeg344 -» Aia344 in the active center of the plasmid EM!I((565 here 2463UrOH (0.5. Raiepi Mo 4,784,950, included here in the form of ssiIlka; the plasmid is registered in

American collection of type cultures under the number 40205) was split from Hra | and Krp I with the formation of a fragment of 0.6 thousand nucleotides, which includes the serine-344 coding region. This fragment was cloned into Hva I, Krp 1-cleavage M1Ztr19, as shown in fig. This procedure and subsequent steps, described below, are usually performed in accordance with standard protocols (as described, for example, in Mapiai5 7/0 2 ai,, Molecular cloning. Laboratory manual, So Zrgipud Nagrog Iaroghaigu Rgezz,

Cold Spring Harbor, New York (1982), incorporated herein by reference).

Mutagenesis was carried out on the M1Z3 matrix in accordance with the methods of Zoller and Smith, see above, using the mutagenic oligonucleotide 7C1656 (5 To SS SS oS TS SSS st 3) and the "universal" second primer 2087 (5 TSS САС TСА СОА СОТ 3). Product reactions were screened using kinase-treated 7C1656. Positive plaques were collected, template DNA was obtained and sequenced

Rv of the I-site at 1077 to Crp of the I-site at 1213. Sequence analysis confirmed the presence of the desired mutation. The mutant clone was designated 1656.

Then, using clone 1656, an expression vector was constructed. The sequence with the introduced mutation was isolated from the M1IZ vector in the form of a fragment of Rhei I-Krp I with 0.14 ks. nucleotides This fragment was ligated to the fragment of Nipa PP-Hva I with 1.7 thousand. nucleotides from RMIA (565 and 2463UrOH, to the fragment

Hva I-Rvai I s 0.5 thousand. nucleotides from EM1I (565 - 2463UroOX) and a fragment of Krp I-NiIpa 111 with 4.3 thousand Factor MI!

MIZ so oKrp b o Khra |, obtaining southern blots of cleaved DNA and probing blots with radioactively labeled substances 271656.

Newborn hamster kidney cells of the VNK 570 line (registered in the American Collection of Type and Cell Culture under number 10314) were transfected with two isolates (designations 8544 and 8545) of the 1656 expression vector. medium (modified needle medium (OMEM) containing 1096 embryonic bull (two serum and 170 ROM antibiotic mixture (SIVSO | Me Tesppoiodiev, Gaithersburg, Maryland). After 24 hours, when the cells reached 20 - 3096 confluence, they were cotransfected with one isolate of the expression vector, - coding mutation 1656, plasmid ri86 (including adenovirus 5 ogy, ZM40 enhancer, the main -/- de late promoter of adenovirus 2, the leader sequence of adenovirus 2, the 5- and 3'-splicing site,

ONEK'-KkDNA and the signal of polyadenylation from 5M40 in rMi-1 (I izKu apa Voisnap, Mayte 293: 79 - 81 (1981)) and with 10 μg of carrier DNA (fragmented under the action of ultrasound DNA of elk sperm), as shown in " IS

Table 1. DNA was introduced into 15 ml test tubes, then 0.5 ml of NERE5-2X buffer was added (25 g of NTNT5, 40 g of Mass, 1.8 g of Xy, 0.75 g of Ma?2HROZ dihydrate, 5 g of dextrose, diluted to a volume of 2.5 l with distilled water, pH 6.95 - 7.0) and mixed. DNA was precipitated in each test tube by adding 0.5 ml of a 0.25M calcium chloride solution while bubbling air through the DNA solution in NEREZ buffer using a Pasteur pipette. The test tubes were shaken, incubated at room temperature for 15 minutes, shaken again. in c The mixture containing DNA was added drop by drop using a pipette to the tablet! with cells The tablet was shaken and incubated at 37"C for 4-6 hours. After incubation, 2 ml was added to each tablet;" 20906 glycerin in a mixture of Tgiz-buffer and physiological solution (0.375g KSI, 0.71g Ma2HPO4, 8.1g mass, 3.Og

TtiIv-NSII, 0.5 g of sucrose, dilution to a total volume of l, with pH 7.9). Tablet! shaken and kept for 2 minutes at room temperature. Then the medium was removed from the tablets and replaced with 2 ml of a mixture of Tgiz buffer and 15% physiological saline. Tablet! kept for 2 minutes at room temperature, then the mixture of buffer and physiological solution was replaced by 1 Oml of non-selective medium. Tablet! incubated at 377C for two days. - and 2 s oklenvm! wm! -400111111110 NNN NO NI NO WITH WE ABOUT BV TV INCULATION CENTER SLABLE CHELECTION CHILDREN (OMEM, SONGIVACA 1095 BRO: 1: 500 was placed in a maxi-plate. Tablet! were incubated at 37"C for a week. After a week, the medium was removed and replaced with a selection medium, the formation of colonies was monitored in the plates.

After eight days, after the formation of colonies, twelve colonies were randomly selected from plates with a 1:500 dilution of transfection of isolates 2544 and 8545. Each clone was placed in a cell of a 6-well plate and grown in a selection medium. Tablet in 7 days! were merged, and each clone was placed in a ten-centimeter plate with selection medium. Clone), described above, and control cells, transfected to express wild-type Factor Mi, were metabolically labeled with a 358-methionine-cysteine mixture for protein labeling (MEM European Biosuppoiod Society, Wilmington, Dzlaver). Clones were grown and prepared for the experiment with a pulse tag in a selective medium.

Cells were washed with a mixture of physiological saline-phosphate buffer (Zidta, St. Louis, Missouri) and exposed to a pulse of 355-Suvz-355-Mei at 20 μCi/ml for 4 hours. After 4 hours, the supernatant and cells were collected.

Cells were lysed essentially as described in IepK apa Reptap (Seal 16: 289-302 (1979)), and 400 μl of each lysate was tested with Staphylococcus aureus (Zidta, St. Louis, MO). 70 Samples! metabolically labeled cells were subjected to radioimmunoprecipitation (KIR) incubation with BMCL polyclonal anti-hematin anti-Factor MI for 4 hours. To each sample was added bOmcl of washed protein A of staphylococcus, and the samples were shaken at 4"C for 1.5 hours. The sample was centrifuged, the supernatant was separated. The sediments were washed twice with 0.7M KIRA buffer (10mM Ttiv, pH 7.4, 196 deoxycholic acid (CaIriospet Sogr. , La Jolla, CA), 1956 Triton X-100, 0.196 505, 5MM 7/5 EOTA, 0.7M Massey) and once with 0.15M KIRA buffer (10mM Ttiv, pH 7.4, 195 deoxycholic acid (CaIriospet

Sogr., La Jolla, California), 1950 Triton X-100, 0.195 505, 5 mM EOTA, 0.15 M Massi)). A dye was added to each sample in 100 μl of 5O5X1 buffer (50 mM Tgi8-HCl, pH 6.8, 100 mM dithiothreitol, 295 5OB5, 0.190 bromophenol blue, 1095 glycerol), samples! boiled for 5 minutes, then centrifuged to remove protein A. 5 µm of each sample was run in a polyacrylamide gel. The result! showed that 9 out of 10 clones secreted the modified MI factor.

Example II.

Anticoagulant activity of modified Factor MI.

The ability of the modified MI Factor protein to suppress blood clotting was measured in a one-step assay using MI Factor! wild type as a control. Recombinant protein c g was obtained, basically, as described above, from cells cultured in a medium containing 5 μg/ml of vitamin K. Different amounts of modified Factor MI (from clone 544) or recombinant factor i)

The MI factor of the wild type was diluted with 5 MM Tgiz buffer with pH 7.5, 0.1956 VBA to a volume of 100 μl. The mixtures were incubated with 100 μl of Factor II-deficient plasma (Seogde Kipo Vio-Meaisai Ips., Overland Park, Kansas) and 200 μl of thromboplastin C (Oade, Miami, Florida; contains rabbit brain thromboplastin and 11.8 mM Sazhkt). Gee!

Coagulation analysis was performed in an automatic coagulation timer (MIA EIIesiga 800, Meisai I arogaigu

Actiotaiyop Ips., Pleasantville, New York), the rolling time was converted into a unit of Factor activity -

MI using the standard curve obtained for normal pooled human plasma (assuming that it contains one unit of MI Factor activity per ml; obtained by combining citrate-treated sera from healthy donors) with a dilution from 1: 5 to 1: 640. therefore, the preparation of the modified Factor MI! did not have detectable coagulant activity. Table 2 "E shows the result of the analysis, expressed in the time of coagulation, for control (non-transfected), conditioned media for BNK cells (-/- vitamin K), wild-type MII factor and two isolates of cells expressing modified MII factor. The activity of Factor MI is presented as a decrease in clotting time compared to control samples. "- - th" phone 18.00 from the city

The MII factor of the wild type so -

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3e)

In order to determine the influence of the modified Factor MI on the substrate factor of plasmas, preparations of the modified Factor MI and recombinant Factor MI! wild type or native Factor

MII were incubated either with Factor X or with Factor IX, and their activation was monitored by coagulation analysis or electrophoresis in a polyacrylamide gel. (F) Example III. The ability of the modified MI Factor! bind the tissue factor to the ability of the modified MI Factor! to compete with Factor MI! wild-type for tissue factor and suppress its coagulation activity was evaluated by a one-step analysis of coagulation in the presence of a limiting amount of tissue factor (thromboplastin).

Clotting time was determined by a one-step assay similar to that described in Example II. In mixed experiments, a limited amount of tissue factor, a constant amount, was used

Wild-type MI factor and increasing amounts of the MI factor genetic variant. Suppression of procoagulant activity of Factor MI/MIa was manifested as an increase in coagulation time in opiates with increasing amounts of the genetic variant of Factor MI.

The magnitude of MI Factor activity! in the tested samples, they were calculated in percent according to the standard curve,

obtained on the basis of measurements of MI Factor activity in normal pooled plasma. The standard curve of MI Factor activity was obtained using a series of diluted normal pooled plasma in phosphate buffer (PB5) in the range from 1: 5 to 1: 640. For this purpose, it was assumed that normal plasma contains approximately 5 OOng/ml of MI Factor, and that was taken as a unit of activity. A mixture consisting of 100 μl of Factor MII-deficient plasma, 100 μl of diluted plasma! and 200 μl of thromboplastin-C (Uoade, Miami, Florida) were used to measure the coagulation time using an automatic timer MI A EIIesig 800. To obtain a standard curve, the result was presented graphically as a percentage of activity (1 : 5 - 10095 activity) versus time / about revolutions in seconds.

According to the conditions of the analysis, it was necessary that in the composition of the medium containing the wild type and variant of the Factor

MI, included at least one percent of serum. Dilutions in РВ5 were made in such a way that the drop in coagulation time occurred in accordance with the standard curve. A typical minimum dilution was 1:2. The final volume was 100 μl. In the experiments, we used two 1/5 different variants of the human MII factor with the zeg344 -» Aa substitution, designated as "MO" and "Zh6" clones. The result!, shown in the following table, shows that, as the amount of the MiI Factor variant increases, the percentage of Ma Factor activity decreases. » reaction). Mon Thu ET Sat Sun

PA "T I NO NOY on Paul M1r111110010111111111111111111111111111111111111111111111111111111111111111111111n NMIEI, 11111000111

From abroad 00000001 have, "

Experiments have shown that Factor MI! with the replacement of zZegZ44 - » Aa competed with the native Factor MI! pt) with a dose-dependent profile and inhibited the procoagulant activity of the native Factor MI/MIa. It can be concluded that the variant of the human Factor MI with the beg344 -" AIa substitution competes with the native :z" human Factor Miia and, therefore, suppresses the activation of Factor X and/or IX in human plasma.

Example of M. iz Reaction of Factor MI! with RPAsSK.

Recombinant Factor MI! protein is produced in transfected kidney cells of a newborn hamster. Proteins were purified and activated as described by TpIt (Viospetiviga 27: 7785 - 7793 (1988)), VhipKopz ei ai., (Rhos. - Main. Asad. si. OBA 86: 1382 - 1386 (1989)) and Vioegp apa Tit (Kevz. OiIvsi. Mo 269, 564 (1986)), which is incorporated herein by reference. The cell medium was collected, filtered and diluted so that! reduce the concentration of salts. Then the diluted medium was fractionated by anion exchange chromatography,

Ge; using a buffer containing calcium chloride for fusion. The fraction containing MII factor was collected and then purified by immunochromatography using a monoclonal antibody to the calcium-dependent MII antifactor. Additional purification was carried out using double anion exchange chromatography, in which MII factor was eluted with solutions of calcium chloride and sodium chloride, respectively.

Miia factor was received in the last disease. (F) Recombinant Factor Miia (1 µM) in 50 mM Ttiv-HCI, 100 mM sodium chloride and 5 mM calcium chloride, with

GI, pH 7.4, was incubated with 20 µM PRASkK (O-Phenylalanyl-Prolyl-Arginyl-Chloromethylketone; Saliospet, La Jolla,

California) for 5, 20 and 60 minutes. Then a buffer containing the chromogenic substrate 5-2288 was added (p-nitroanilide O-isoleucyl-y-prolyl-y-arginine; Kari Migit AB, Molndal, Sweden) in order to! obtain a 2.5-fold dilution and a final concentration of 0.3MM for 5-2288. The release of p-nitroaniline was measured and compared with the result obtained with untreated Miia Factor as a control. Obtaining this data indicates that under the conditions studied, Factor Ma is completely inactivated after approximately 60 minutes. 65 Example U.

Generation of OEOK-Factor MiIa.

Recombinant Human Factor Ma was obtained as described in Example IM. Recombinant human Factor Ma was diluted to a concentration of 1.5 mg/ml in 10 mM glycine buffer with pH 8.0, 10 mM calcium chloride, and 50 mM sodium chloride. A 10-fold excess was added to Factor Ma

Rrapzui!-I-c1IcSh-cC1Iu-Agd- chloromethylketone (CaIriospet, La Jolla, CA 92037), which was dissolved in distilled water. After a two-hour incubation at 37"С, a second 10-fold molar excess of ОЕСКсСК was added to the mixture and incubated for another 2 hours at 37"С. Then, a third 10-fold molar excess of OESCsSK was added to Factor Ma and incubated for approximately 16 hours at 4°C. To remove free UESCsC, the sample of OESC-Factor Mi was carefully dialyzed against Tgiz-buffered 7/0 physiological solution (0.05 M Tgiv-HCI, 0 M mass, pH 7.5) at 4"C. By introducing free OESKSC into the mixture containing Factor Xa, a standard curve was obtained for measuring the amount of free OESKSC in the solution as a measure of the inhibition of the chromogenic activity of Factor Xa. The analysis of the mixture with OESK-Factor MiIIa showed that after extensive dialysis the ratio of free OESK-Factor to CESK-Factor Ma was less than 0.595, thereby confirming that the inhibition observed for OESK-Factor Miia in various /5 test systems, given below, is not is connected with the presence of free OBOKSK.

An example of MI.

Generation of Factor Xa in the cells of the smooth muscle of the mouse.

Cells of vascular smooth muscle! were analyzed for the presence of a surface factor by measuring the ability of cells to stimulate the conversion of Factor X into Factor Xa using a chromogenic substrate specific for Factor Xa.

Chrysin vascular smooth muscle cells (Siomez et al., U. Sp. Ipmevi. 93: 644 - 561 (1994)) were placed in a 96-well plate (Ategisap Zsiepiis Rgodisiv, Chicago, Illinois), 8000 cells per cell in a growth medium ( Table 4). cm and

Logan, Utah)

F what

After 48 hours of incubation at 37"C, the medium was replaced with serum-free medium (Table 5).

The cells were incubated for 72 hours at 37"C. After incubation, either 1 ng/ml of ROSE-BB (human recombinant platelet growth factor) or 1095 embryonic calf serum was added to the cells to stimulate the expression of the tissue factor (Tabtap et al., U. Sp. Ipmevi. 91: 547 - 552 (1993)). In a parallel series of cells, neither ROS nor serum was added in order to control the true activity of unstimulated cells. After a 2-hour incubation, recombinant human Ma factor with the final concentration was added to the cells 1 ONM. One series of cells did not receive Miia Factor as their 20 negative control. Cells were incubated for 2 hours at 37"C, washed with NERE5 buffer (10 mM NEREB, 137 mM Masi, 5 mM Sasig2, 11 mM glucose, 0.195 VBA). After washing, the cells were incubated for 5 minutes with 5 Ωcl per cell of 200 nM purified Factor X plasma! human in Tgiz-buffered physiological solution with 5MM

Sasi2. 25 μl of 0.5 M EOTA and 25 μl of 800 μM solution of chromogenic substrate 5-2222 (Kari Riapgtasia, Franklin, Ohio) were added to each cell. Plates were incubated for 40 minutes at room temperature, then analyzed at 40 ppm using a TNEKMOMAH device (Moiiesshag Zegmisev, Menlo Park,

GF! California).

Table 6 shows the increase in absorption for cells treated with Miia Factor compared to control cells (without the addition of Miia Factor). The increase in absorption is a direct measurement of the level of Factor Xa, generated in the cells, and its subsequent cleavage of the chromogenic substrate, 60, which releases the chromophore. These data also demonstrate that the level of chromogenic activity in cells pretreated with either ROSB-BB or 1095 embryonic calf serum (EC) was higher than in unstimulated cells. b5

Control -043

In - -

Here is the result! clearly show that on the surface of the cells of the rat vascular smooth muscle! Factor MIIa-dependent activation of Factor X into Factor Xa is observed.

An example of MI.

Inhibition of surface cell chromogenic activity by OESK-Factor Ma. 70 Rat vascular smooth muscle cells were placed in a 96-well plate as described above.

Cells were cultured for 72 hours in serum-free medium as described above; then treated with 1096 embryonic calf serum for 6 hours to stimulate tissue factor expression. After stimulation, only buffer (control), LONM Factor Ma or 100 mM Factor Ma mixed with 100 nM OESK-Factor Ma was added to each cell. Cells were incubated for 2 hours at 37"C, then washed with HERE5 buffer. After 1/5 washing, the cells were incubated for 5 minutes with 5 μl per cell of 200 nm of Factor X in Tgiz-buffered physiological solution with the addition of 5 mM calcium chloride. 25 μl of 0.5 M was added to each cell EOTA and 25 μl of chromogenic substrate 5-2222 (VOmMm). Cells were incubated at room temperature for 40 minutes. Chromogenic activity was analyzed at 405 nm as described above.

Table 7 shows the chromogenic activity in cells treated only with Miia Factor, and inhibition of stimulation when OESC-Miia Factor was incubated with Miia Factor. These results demonstrate that OEBEOK-Factor Miia acts as a competitive antagonist of Factor Miia binding, thereby suppressing the activation of Factor X into Factor Xa and the subsequent cleavage of chromogen 5-2222. cm 5 o

An example of MIP. at

Dose-dependent inhibition by UESK-Factor Ma of the surface chromogenic activity on the cells of "F" smooth muscle.

Rat vascular smooth muscle cells were placed in a plate with 96 cells at 4,000 cells per cell in a growth medium with the addition of 195 fetal calf serum (as in Table 4, but without 1090 fetal calf serum). After 5 days, the medium was removed and growing cells were added to the cells

From the concentration of only Factor Miia, or TONM Factor Miia with increasing concentrations of OESK-Factor Ma. M

The cells were incubated for hours at 377"C. After incubation, the cells were washed, then incubated with

BOmcl 200nM Factor X in Tgiz-buffered saline for 5 minutes at room temperature. 25 μl of 0.5 M EOTA and 25 μl of 800 μM 5-2222 (Kari RNagtasia) were added to each cell, "tablet! incubated for 40 minutes at room temperature. Chromogenic activity was analyzed at C 70 doBNM with a reading device for icroplates, as described above. Table 8 shows a dose-dependent increase in chromogenic activity as the amount of Miia factor added to the cells increases. A dose-dependent inhibition of chromogenic activity was observed when a mixture of OESK-Factor Miia and L00NM of Factor Ma (Table 9) was added to the cells. At a molar ratio of rESC-Factor Miia to Factor Miia equal to 1:1, approximately 9595 chromogenic activity was inhibited. 745 These data allow us to assume that in this experiment, in the culture of smooth muscle cells, Miia Factor OESCs had a significantly higher affinity for tissue surface factor than native (ee) Miia Factor. If CESK-Factor Miia and Factor Ma had equal affinity to the tissue factor, then in the case when the two molecules were added to the cells in an equal molar ratio, the observed level of inhibition would not be so high. drive 50

Table 9 shows the dose-dependent inhibition of the chromogenic activity of the factor by the OBOK-Factor.

Ha on the cells of krysina smooth muscle. Increasing concentrations of OESC-Factor Miia were co-incubated with 1O0NM Factor Miia, the chromogenic activity of Factor Xa was determined using the chromogenic substrate 5-2222. b5

Miia factor concentration (nm) 00405

Example IX 70 Inhibition of the formation of Factor Xa by OESK-Factor Miia in the analysis of soluble tissue factor.

The transformation of Factor X into Factor Xa using a purified recombinant solution of tissue factor was established using a chromogenic analysis. Tissue factor was expressed and isolated from

Zasspagotuses segemizaee (ZNPIDetaizy ei ai!., y. Vioi. Spet. 267: 21329 - 21337 (1992)). Soluble tissue factor was purified and characterized by Dr. MU. Kiziee! (University of New Eximo). The reaction mixture contained 6–5.9 μl of soluble tissue factor (2.2 μM), 29.0 μl of PCR (1MM, Zidt, St. Louis, Missouri), 29.5 μl of human Factor X (4.1 μM), 2.77 ml of Hanks buffer (25 mM Tiz pH 7.4, 150 mM Masi, 2.7mM KSI, 5mM

Sasi2, 0.195 B5A). 40 μl of a mixture of tissue factor and Factor X, 25 μl were added to each of the 96 cells of the tablet

Factor Miia, diluted with TV5, and 25 μl of OESK-Factor Miia, diluted with TV5. For control, 4 μl of a mixture of tissue factor and Factor X were used; the control included 400 μl of a mixture of tissue factor and Factor X, 20 μl

Factor Miia, diluted with TV5, and 25 μl of only TV5. 1 µl of chromogenic substrate 5-2222 (4 mM) was added to the reaction mixture in the cells, the mixture was incubated for 2-10 minutes at room temperature.

The results were analyzed at 405 nm with a microplate reader as described above.

The standard curve was obtained using increasing concentrations of Miia factor added to the serum Absence of RESC- Miia factor. The results, presented in Table 10, show that there is a dose-dependent enhancement of chromogenic activity as the amount of Factor Ma added to (8) of the reaction mixture increases. Simultaneous addition of varying amounts of OBOK-Factor Miia and L00nNM Factor

Miia led to a decrease in chromogenic activity (Table 11). These data demonstrate that OESK-Factor

Miia acts as a competitive antagonist of the binding of native Miia Factor to soluble tissue factor, thereby inhibiting the formation of Factor Xa, which is determined by the decrease in chromogenic activity in relation to the chromogenic substrate 5-2222. "

Table 10. "-

Stimulation of the chromogenic activity of Factor Xa by increasing concentrations of Factor Miia added to the soluble tissue factor. The change in optical density was measured using 00 zV Chromogenic Substrate 5-2222. « « и Z - и» 15 и" (ee) Table 11. - Suppression of the chromogenic activity of Factor Xa when added OESK-Factor Mak to the soluble tissue factor in the presence of native Factor Mia. The change in optical density was measured using the 5-2222 chromogenic substrate.

Fo tu o ovo yu o

Example X.

Suppression of coagulation by OBOK-Factor Ma.

Standard analysis! rolling for the purpose of studying the influence of the OESK-Factor MI! at the time of coagulation, it was carried out as follows: 100 μl of normal plasma! baboon, obtained by treatment with sodium citrate as an anticoagulant, was added to 100 μl of increasing concentrations of ЮОЕСК-Factor Miia, diluted with TV5 (20 mM Tgtgiv, pH 7.4 and 150 mM mass). The samples were mixed and briefly incubated at 37-7C.

Then, EiPesiga 800 was added to the automatic coagulation time counter (Meadisai!

Pleasantville, New York). After incubation, 200 μl of the tissue factor preparation containing 20 mM calcium chloride was added to the preparations of CESK-Factor Ma. The preparation of tissue factor was prepared in the form of an extract of freshly frozen baboon brain tissue in physiological solution and characterized by its ability to initiate plasma coagulation! baboon White vibrated the concentration of the tissue factor, which ensured a clotting time of about 40 seconds. 70 Dannewe, presented in Table 12, demonstrate a dose-dependent increase in time with added OESK-Factor Miia. A dose as low as 1 μg/ml showed a significant increase in clotting time. etc

Example XI. high school

Suppression of the accumulation of platelets by OESK-Factor Ma The studied ability of OESK-Factor Ma to suppress the accumulation of platelets in the sites of arterial thrombosis in mechanically damaged nonhuman primates. They used a model of aortic zndarterzectomy! in baboons, mostly as described

Gytvzaep eyi ai., Viosa 81: 1762 - 1770 (1993). A part of the baboon's artery 1-2 cm long was removed, inverted and scraped to remove the inner and approximately 50,956 middle layers. The artery was again inverted, Fo zr Cannulated at both ends and placed in the baboon's extracorporeal shunt, thereby freeing the mechanically damaged baboon's artery from blood flow by means of the shunt. Before starting blood, through the shunt Z, the animal was injected intravenously with its own platelets, labeled 111ip. The level of accumulation of platelets in the damaged artery was determined using a gamma camera video monitor.

In studies of the ability of UESK-Factor Ma to inhibit the accumulation of platelets, d) injection of OESK-Factor Mia or a physiological solution as a control was used before opening the shunt. Measurements of damaged arteries were carried out continuously for 60 minutes. OEOK-Factor Miia in a dose of 0.005 mg/kg inhibited the accumulation of platelets. When injected mg/kg, suppression of platelet accumulation amounted to approximately 9095 1 hour after administration of the drug. Here is the result! representations in fig. 2. « 20 Example of KHII. w-in

OESK-Factor Ma suppresses vascular restenosis occurring after balloon angioplasty in rabbits with atherosclerosis. :з» The investigated ability of OBOK-EMIa to modulate the development of pathological changes after balloon angioplasty in New Zealand White rabbits (MAM/) with atherosclerosis. This model is well characterized and is considered suitable for evaluating the effect of compounds with antithrombotic properties on the development of vascular disorders

Sci. Codovia et al., Sigshayop 89: 1262 - 1271 (1994)). (o) The animal model used to evaluate OESK-EMIa is basically the same as described by Kodovzia, ibid. - Rabbits were anesthetized by intraarm injection of Bbmg/kg of xylazine and 35Bmg/kg of ketamine.

The proximal femoral artery was subjected to venosection below the inguinal ligament with the imposition of proximal and distal ligatures. The isolated segment was cannulated with needles of size 27. Exit holes were created by piercing a needle. The separated segments were washed with physiological solution from the remaining blood, dried with air passing at a speed of 8 Oml/min for 8 minutes. After air-drying, remove the segment! again washed with physiological solution and removed ligatures. Intravenous hemostasis was maintained by nonobstructive local pressure. The segments were limited by metal clamps.

Local spasm was relieved by local treatment with 195 xylocaine. On the next day after surgery, animals (F) were put on a diet containing 195 g of cholesterol and 695 g of peanut oil for one month before balloon angioplasty. To relieve postoperative pain, 10 mg/kg Tylenol was given orally for 3-5 days.

After the surgical procedure! 1 cm7 of Ambipen was given for 3 - 5 days. 60 Delivery of the test drug to animals included an initial jet injection immediately before balloon angioplasty, followed by continuous systemic infusion through the internal jugular vein using an osmotic pump. The duration of infusion of the drug was 3 days. Control animals received 150 IU/kg of heparin by injection before balloon angioplasty, followed by infusion of physiological solution. Animals receiving BESK-EMIa were given a jet injection of 65 mg/kg, followed by an infusion of 5 Ωkg/kg/hour.

When installing osmotic pumps for continuous systemic infusion in animals, anesthesia was performed, as described above, and it was maintained during the entire procedure! with additional 1M injections of ketamine and xylazine. Through an incision along the midline of the neck, the right internal jugular vein was isolated by peeling, and the distal end was ligated. A silicone tube (RE-160) was inserted into the right internal jugular vein. A subcutaneous tunnel was created for the passage of a silicone tube. This tube was connected to an osmotic pump. An osmotic pump was implanted subcutaneously on the back of a rabbit. The right common carotid artery was isolated by peeling, the distal end was ligated. Using a vascular scalpel, a 5E intubator was installed and the ego was moved to the connection with the aortic arch. Blood was taken to determine hemostatic parameters, the content of drugs and cholesterol. Intra-arterially injected. 20 mg 7/0 Xylocaine. Control angiography of the cervical artery was performed by means of a Berman 5BE catheter located under the aortic bifurcation with the use of 3 - 4 ml of renografin, introduced manually after 3 seconds.

After removing the Berman catheter, a 0.014-inch wire guide was inserted into the descending aorta and placed over the aortic bifurcation. Under fluoroscopic control, a balloon /5 catheter for angioplasty of the appropriate size from 2.0 to 2.5 mm was inserted, it was advanced over the wire guide and inserted into the stenosis. The cylinder was pumped up to batm. within 60 seconds manually. Three pumps were carried out with intervals of 60 seconds. This procedure was performed in both femoral arteries in each animal.

After balloon dilatation, the angioplasty catheter was removed and the Berman catheter was re-introduced in the third position cm above the aortic bifurcation. To reduce the spasm, 20 mg of lidocaine was injected intraarterially. A postoperative angiogram was obtained as described above. A grid (1 cm) was placed at the level of the femoral artery in order to! calculate the actual diameter. Then the catheter was removed. The right carotid artery was ligated with Z-0 silk, the wound was sutured in layers. Ambipen and acetaminophen were given as described above. with

Prothrombin time and concentration of OESK-EMIa in the blood were determined immediately before the injection of the drug, 1 hour after the injection and three days later at the end of continuous administration. and)

We received 1 - 2 ml of plasma treated with citrate! and prothrombin time and antigen levels were determined.

The standard coagulation analysis was used to control the prothrombin time in control and treated OBOK-EMIA animals as follows: 25 μl of tested plasma! rabbit, Gee! treated with sodium citrate as an anticoagulant, was added to 150 μl of TV (20 mM Ttiv, pH 7.4, 150 mM mass). The samples were mixed and connected to an automatic coagulation timer EIIesig 800 - (Medisa! and Arogagiev Atsiotaiop, Pleasantville, New York). After incubation, 200 μl of thromboplastin preparation (Zidta Spetisai) containing 25 mM calcium chloride was added to the plasma. The concentration of thromboplastin at which the coagulation time of control rabbit plasma was measured! was approximately 20 seconds. "Mr

The EGPI5ZA method (enzyme immunoassay) was used to determine the concentrations of OESC-EMIa in plasma samples of control and treated OESC-EMIa mice. The analysis included the dilution of a monoclonal antibody to human anti-EMI! (Og. MU. KizIei, University of New Mexico) up to 20mcg/ml in 0.1

M carbonate buffer, pH 9.6 and adding 100 μl per cell in the tablet! with 96 cells. Then the tablet was incubated at 4"C overnight, then washed twice with hydrogen buffer (РВ5, pH 7.4, c) containing 0.0595 Tu'eep 20). Suppression of non-specific binding sites was achieved when the

And 200 μl of blocking buffer per cell (PB5, pH 7.4, containing 0.059565 Tmeep 20 and 195 OA), incubation at 37"C for 2 hours and subsequent treatment with washing buffer.

After blocking, a series of standard diluted OBOMK-EMIIa was added, varying in the range of 20 - 9.027ng/ml, along with a series of diluted tested rabbit plasma! (1 : 100 to 1 : 4000 in blocking buffer) 100 μl per cell. The plasma of non-immunized rabbits was used as a negative control. Then the plates were incubated for 1 hour at 37"C, after which they were washed with a buffer. - Determined OESC-EMIIa by adding O0Omcl to the cell diluted 1 : 1000 rabbit 5r polyclonal antibody to human anti-RMI! (O. Kiviei, University New Mexico) in blocking buffer. The plate was incubated for 14 hours at 37"C, then treated five times with washing buffer.

Ge) Specific antibody binding was determined using 100 μl per cell of a 1:2000 dilution of goat antibody to rabbit dO-peroxidase conjugate (Tado, Ips.). Tablet! incubated 1x4 at 37"C and treated six times with washing buffer. Finally, 10O0μl of a solution of substrate two (0.42mg/ml o-phenylenediamine dihydrochloride (OPO) in 0.2M citrate buffer with pH 5.0, containing 0.390 hydrogen peroxide) was added. After 1 - With minutes at room temperature, the color reaction was stopped

F) by adding 100 μl to cell 1 n. sulfuric acid and the contents of tablets were analyzed at 490 nm using a microplate spectrophotometer. Concentrations of OESK-EMIa in plasma samples! determined by comparing the obtained values of A490 with the values of the standard curve for OBOK-EMIa. in Result! analysis of plasma samples! on prothrombin time and levels of OBOMK-EMIIa antigen are shown in Table 13 and Table 14, respectively. These representations are for each animal separately. IN

Table 15 shows a summary of the average values of the time of contraction. In all cases, the processing of

RESC-EMIa animals had an increased value of prothrombin time at the point corresponding to 1 time after injection; at the point corresponding to the third postoperative day, it almost reached 65 pretreatment levels. The analysis of the antigen OBSC-ERMIa also revealed its high level at the point corresponding to 1 time, varying within 2 - bmkg/ml, and the levels are much lower at the point

corresponding to the third day. Levels of OESK-EMIa, measured after 1 hour, corresponded to the presumed increase in prothrombin time, established by processing normal rabbit plasma with OEOC-EMiia Ip migo and determination of prothrombin time by analysis of standard dilution of thromboplastin.

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Three weeks after angioplasty, immediately before euthanasia, a control angiogram was repeated, as described above, using the left carotid artery. Through a vertical abdominal incision, the distal aorta was isolated and a perfusion cannula was inserted above the aortic bifurcation.

The distal aorta was perfused with 5 Oml of physiological solution, then Ip was fixed by injecting it for 15 minutes at 120 mm Hg. Art. 5 ml of Niviospoise solution (AMKEZSO, Solon, Ohio). As soon as the perfusion began, the animals were euthanized with an overdose of nembutal (3ml (b5mg/ml) of pentobarbital solution intravenously). A segment (5 cm) of the femoral artery was excised bilaterally. For optical microscopy, the tissue was preserved in Nizviospo!se solution.

In order to detect the appearance of damage at the site of balloon angioplasty, a ZMM section was cut from the excised femoral arteries, filled with paraffin, and sections of the areas of the artery with multiple lesions were taken. Preparations of sections were prepared on glass slides, stained with Tematoxylin, Zosin and Map Sietwop dye. With the use of programs! Bioquant performed a morphometric analysis to obtain data on the lumen, internal and middle sheath of arteries. With the help of morphometric analysis of sections from damaged arteries, measurements of the entire area of the lumen were carried out; the area of the inner shell, obtained by measuring the area in the inner elastic layer and reading the corresponding area of the lumen of each section; the area of the middle shell, determined by the measurement of the area inside the outer elastic layer and the reading of the area inside the inner elastic layer. Measurements of damage to the inner lining (intima) of femoral arteries in control (8) and treated OBOMK-EMIIa animals showed that a significant decrease in the size of the intima was observed! in animals treated with OESK-EMIIa (Table 16). In contrast, measurements of the medial region did not show significant differences between the two groups. b zo « - so zv besvtia. 0060000oyaz;0le? "

Group Average (mm?) Standard. off Probability (2-sided test)

M ny ionic NA pooiniy rvsvyamta| loozgaolo 71 h

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The data of angiographic measurements are presented in Table 17 in the form of the values of the average diameter of the lumen (MI 0) and/or standard deviation for control and treated animals for all three time points: immediately before angioplasty, immediately after and 21 days after angioplasty. There were no significant differences in MI values between controls

In animals treated with CESK-EMIIa before and immediately after angioplasty, however, a noticeable increase in MI values was observed in animals treated with OBOK-EMIIa on the 21st day after angioplasty. so - that" so

West 00loizvz) 00019000 5 (2-sided test) o more West 00018323)

Group Koly-chestvo MI O|Stand. off The probability in OM 00 of bodenyut is negative! vine omg!

An example of HIPPIES. because suppression of the generation of surface Factor X by the OESK-Factor on the baboon's ZMS.

A surface cell chromogenic analysis was conducted, mainly as described above in the MIA Example, to determine the effectiveness of OEEOK-EMiIa in suppressing the binding of EMIia to the surface cell factor of the tissue and the subsequent transformation of Factor X into Factor Xa on monolayer smooth muscle cells! (ZMS5) baboon. The method is a modification of the methods, descriptions of its causes., 9. VioI. Spent 264: 9980 - 9988 (1989);

UUPadoose eyi ai., Rhos. May Asad 5 BOTH 87: 7290 - 7294 (1990). Babuinovne ZMO" received from the University

Washington (Sistle, Washington) and cultivated from Babuinovie aorta implants 5MSv5 were introduced into culture dishes with 96 cells at a concentration of 8000 cells per cell in 200 μl per cell of OMEM medium with the addition of 1095 fetal calf serum and kept in this medium for 4 days at 37" C in an atmosphere of 595 CO2. During the analysis, 1100 μl of the culture medium was removed and increasing 7/0 concentrations of PMIa in combination with OESK-EMiIIa were added to the cells. A standard curve was obtained for the concentration of EMIIa, varying in the range of 5 - 0.4NM. To evaluate the inhibitory effect OESC-EMIa for the activity of EMIIIa was added to the test cell in increasing concentrations of OESC-EMIa in the presence of a constant amount (5NM) of EMIIa. Both EMIa and OEESC-EMIa were diluted with NERE5 buffer (10 mM NEREZ5, 137 mM Mass, 4 mM

KSI, 5 mM Sasi?2, 11 mM glucose, 0.195 VZA) and 1 µl of 10x initial solutions added to the cells. /5 cells were incubated with test compounds for 2 hours at 37"C, then washed three times with buffer

STAINLESS STEEL Then 500 μl of a 200 nM solution of Factor X in Tgiz buffer (25 mM Tgtgiv, pH 7.4, 150 mM Masi, 2.7 mM KSI, 5 mM Sasig, 0.1956 VA) was added to each cell. In 4 minutes! at room temperature, 25 μCl of 0.5 M EOTA was added to stop the conversion of Factor X to Xa. 25 μl of 0.8 M Factor was added to the cell

Xa-specific chromogenic substrate 5-2222 in Tgiz buffer and after 60 minutes the absorbance at 405 nm was measured in a microplate reader device TPegtots (Moiesshag UOemisez Sogr., Menlo Park,

California).

The results, presented in fig. C, demonstrate a dose-dependent increase in amidolytic activity in cells treated with EMIIIa (unshaded squares). The increase in absorption is a direct measurement of the level of Factor Xa, generated in the cells, and the subsequent splitting of the chromogenic substrate by them. Addition of increasing amounts of OBOMK-EMIa at a constant amount of EMIa (5NM) caused a dose-dependent decrease in amidolytic activity (colored square!). Equimolar i) amounts of UESK-EMIIa and EMPA capable of inhibiting more than 9095 chromogenic activity. Even with a 10-fold reduced level of OESK-EMIa, inhibition of chromogenic activity is still observed 4095

Xa factor. Here is the result! confirm the conclusion that OBEEOK-EMiIIa is extremely strong Ge! with the antagonist of the activation of Factor X in Xa in the presence of HMiIIa on the surface of an intact cell monolayer

ZMSV An example of CHEM. The influence of ХОЕСК-Factor Ma on the occurrence of vascular thrombosis and vascular - impaired in baboons. "-

The ability of human UESK-Factor Ma to inhibit tissue factor (TE) and the formation of vascular lesions (MIR) mediated by MI factor activation (EMIPa) induced by mechanical damage to vessels in non-human primates was investigated. "Mr

RESC-Factor Miia was administered intravenously for 7 days (5 animals) or 30 days (1 animal), starting immediately before the onset of mechanical vascular damage. Measurements of the formation of vascular damage were carried out on the 30th day. The results obtained in 5 treated animals were compared with the data for 5 parallel controls with infusion of the filler in the buffer. "

Basic values were obtained when determining in animals: a) the number of platelets, neutrophils, monocytes and erythrocytes; b) level of plasma fibrinogen; c) levels of activity of the plasma coagulation factors MIII, Miia, X and M, along with the antigenicity of RMII; d) a basic sample of plasma for the determination of antibodies;" anti-Ma Factor.

Under halothane anesthesia and sterile operating conditions, the animal, marked with autologous 111Ip-platelets, received intravenous infusions using all systems of continuous intravenous administration (initial jet injection of 1 mg/kg, followed by continuous intravenous infusion of 5 BOmkg/kg/hour). Animals were subjected to surgical carotid arterectomy, with bilateral angioplasty of the brachial or femoral artery using a Fogarty catheter. - OESC-EMIP was administered for 7 or 30 days by continuous infusion through a venous catheter with the use of all systems. 30 days after the surgical intervention, the animal was given halothane anesthesia and fixed at 85 by the method of operfusion under pressure with 495

Ge) with paraformaldehyde containing 0.19 glutaraldehyde for 30 minutes. Then the segment (containing previously damaged sites) was selected using the methods of Nagkeg et al., SigshineNop 83: 41 - 44 (1991);

Napsop ei ai., Nurepepviop 18: 1170 - 1176 (1991). The samples were subjected to secondary fixation of Ip mIgo (490 paraformaldehyde containing 0.175 glutaraldehyde), frozen and prepared for morphometric analysis of the lesion area.

F) 11 normal mature baboons (Raio apibriv) were followed. Six animals received infusions of ka (5 Ωkg/kg/h), the remaining five served as controls that did not receive OESC-EMIa. Animals were dewormed! and were under observation to avoid diseases for three months before use. All procedures were approved by the Institutional Animal Care and Use Committee and were in accordance with the procedures and methods outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and similar institutional regulations. Invasive procedures were performed under halothane anesthesia after induction with ketamine (1mg/kg intravenously) and valium (0.5mg/kg intravenously). Ketamine hydrochloride (5 - 20 mg/kg intramuscularly) was used for subsequent 65 short-term immobilization during postoperative experimental procedures.

Zndarterzktomy of the carotid aorta was performed by means of an incision along the midline of the neck using the methods described by Napvop ei ai., Nuregiepviop 18: 1170 - 1176 (1991); KgyreKiI ei ai., SisshShaiNop 84: 1749 - 1757 (1991), included here as references. Zndarterzktomy was used as a model of vascular Ndisruption due to its clinical importance and because, as it was shown, MIR caused by zndarterzktomy of normal arteries is reproducible. Briefly, the common carotid artery was excised from the surrounding tissues from the clavicles! proximal to the bifurcation of the carotid artery distally. The common artery was clamped using atraumatic vascular clamps at each end of the isolated vessel after three minutes! after the injection of heparin sulfate (100 IU/kg intravenously; EICIPz-Zitt Ips., 7/0 Cherry Hill, New Jersey) and the cm proximal to the distal constriction was separated. Then the proximal arterial segment was inverted over the bent clamp. After maximum eversion was achieved, one pair of polypropylene sutures (7 - 0) was placed on either side proximally and the second pair distally in the segment highlighted in the lumen. Then Zndarterzktomy was performed, starting 1 cm from the separated end of the inverted vascular segment and continuing for a measured distance of 3/5 cm. The procedure includes mechanical removal of the normal inner membrane and partial sealing of the middle membrane using a clamp and a surgical microscope (magnification 32x). After zndarterzectomy, the vessel was returned to its normal configuration and an end-to-end anastomosis was imposed with the help of a polypropylene suture (TO) and a constant technique at 2.5-fold magnification, the wound was sutured layer by layer.

During the morphometric analysis, sections embedded in paraffin and treated with a dye for connective tissue components (collagen, zlastin) and a hematoxylin-zosin mixture were examined using a photoscope (7eiI55) connected to an image processing system (Tpotaz Oriis! Meazigetepi Zuvietv,

Columbus, Georgia), consisting of high-resolution SSO microscope cameras (580 lines), connected to a high-resolution monitor (700 lines), an IBM 386, 80 MB computer with a HIGH-resolution graphic INPUT for image acquisition and storage. Quantitative image processing was carried out using orthometric software (Oritavz, Viozsap IPS., Zdmonds, etc.)

Washington). In arterial cross-sections, the total area of proliferative neointimal damage was analyzed! and the corresponding area of the arterial middle membrane. For statistical analysis, the comparison between groups was performed using the Student's i-test (two-sided for paired and M odd data).

The result! showed that the square is intimate! significantly reduced in animals treated with OESK-Factorm -

Ma within 7 days and observed up to 30 days, compared to control animals that did not receive UESK-Factor Ma (Fig. 4). Similar results were obtained in animals treated with OESK-

Miia factor within 30 days and observed up to 30 days. co

Preliminary studies with a model of balloon angiography of the brachial artery did not suggest a noticeable advantage of therapy with OESK-Factor Miia. However, it was not shown on grandmothers that this model is prothrombotic, in which the tissue factor plays a key role.

Studies of balloon injury of the femoral artery in baboons indeed showed a statistically significant advantage of OEEC-Factor Miia compared to the control, as shown in fig. 5. "

An example of KM with c Influence of OESK-Factor Miia on IRA-induced thrombolysis.

The permanent formation of coronary thrombi in acute myocardial infarction is mediated, primarily;" in turn, tissue factor (TE) in a complex with Factor Ma through the external coagulation metabolic pathway. White studied the effect of additional inhibition of the coagulation cascade at various points

External metabolic pathway on the effectiveness of thrombolysis induced by tissue plasminogen activator (IRA).

Thirty-six dogs with coronary thrombi induced by electric current in the course of thrombolysis with IRA (mg/kg, longer than 20 minutes) were prescribed one of four additional treatments: 9 received tick-borne anticoagulant peptide (TAR), a selective inhibitor of Factor Xa, at a dose of ZOmkg/ kg/min for 90 5 years Min. The TE-Factor Ma complex was inhibited by a recombinant inhibitor of tissue factor metabolism (TEKI) in ve (100 - 15O0μg/kg/min for 90 minutes) in 9 dogs and OBOMK-EMIIa (1 - 2mg/kg by jet) as a competitive

Ge) antagonist of the activated Miia factor in 9 dogs. As a control, 9 dogs received physiological solution. Dogs were monitored for reocclusion for 120 minutes after thrombolysis.

Data on the influence of these agents on the effectiveness of thrombolysis are given below in Table 18 (data on presentations in the form of average values of h/- ZO). about yu 111 rerestoriresyama| tet tav 5 x The value is different from the control with physiological solution with the level of significance alpha - 0.05.

From these data it follows that inhibition of the external metabolic pathway either by Factor Xa or by 65 blockade of the TE-Factor Ma complex with OESK-EMPA or TERI accelerated (RA-induced thrombolysis.

Selective inhibition of Factor Xa more effectively supports the opened state of the artery after successful reperfusion.

An example of HM.

Modified Factor Ma inhibits the formation of intravascular thrombi without having an effect on systemic coagulation.

In order to determine whether the inhibition of binding of MI factor! with TE to antithrombotic effects, changes in blood flow cycles (SEM5) were initiated, which were observed by recurrent thrombosis by placing an external clamp around rabbit arteries with 7/0 Zndothelium damage (Fotts method). After placing a clamp around the arteries, CEM8 developed with an average frequency of 11 z4u- 2 cycles/hour in b and b rabbits, while the blood flow velocity in the carotid artery was on average 5 17/- 295 of the baseline values at the lowest level of CEM5. After observation of SEM" within 30 minutes, the animals received an infusion of human recombinant blocked in the active center (Rpe-Rpe-Agd-chlormethylketone) Factor MI! (EMiIIaY) (0O.1mg/kg/min for 10 minutes). The Ma factor! completely 7/5 bnymal SEM" in 6 out of 6 animals (frequency of SEM - 0 cycles/hour; p « 0.05; velocity of blood flow through the carotid artery is equal to 106.995 of the baseline values; p - M5 against the baseline). 30 minutes after CEM5 inhibition, human recombinant EMiia was administered at a dose of 0.1 mg/kg/min for 10 minutes. Infusion of Factor MiIIa restored SEMe in all animals, thus showing that binding of Factor Ma to TE was competitive. Values of prothrombin time, activation time and platelet aggregation time in response to 2o AOR (adenosine diphosphate) and thrombin after the infusion of EMiiaI did not differ when compared with baseline values. Thus, EMII-MIia plays an important role in the initiation of thromboembolism.

The introduction of MiiaI factor exerts stronger antithrombotic effects on this model, without having an effect on systemic coagulation.

From all of the above, it is clear that the production of Factor MIA or Ma with modified catalytic sites is capable of binding the tissue factor, but is not capable of significantly activating it

Factories! X and THEM. Because the MI Factor is modified! specifically interrupts the cascade of coagulation, not i) disrupting or removing the factor! coagulation, introduction of drugs of the modified Factor MI! will be accompanied by less undesirable side effects than what is mentioned! wholesale modern therapy. Further, the modified MY Factor, described here, can easily be obtained by Ge! with recombinant methods. Thus, efficiency, convenience and economic benefit from low doses and reduced frequency of administration, as well as a relative loss of toxicity, are advantages - provided by the compositions of the present invention. "-

Although the above-mentioned invention is described in sufficient detail with the involvement of illustrations and examples to improve understanding, it is obvious that they can do it! some changes and so on 5 Modifications within the scope of the attached claims. "Mr

Claims (1)

The formula of the invention " 1. The method of inhibiting the activity of the tissue factor in the patient, which consists in administering to the patient a therapeutically effective dose of the composition, characterized by the fact that the mentioned composition consists of the MII factor, which has at least one modification in its active center, which modification: z" significantly suppresses the ability of modified factor MII to activate plasma factor X or IX. 2. The method according to claim 1, characterized by the fact that the modification includes the reaction of the MI factor! with serine protease inhibitor. C. The method according to claim 2, characterized by the fact that the protease inhibitor is an organophosphorus compound, a sulfonyl fluoride, a peptide-containing halomethyl ketone or an azapeptide. - 4. The method according to item 3, characterized by the fact that the protease inhibitor is a peptide containing 5p halomethyl ketone, selected from OapzuI-RPie-Pho-Agd-chloromethylketone, Uapzu!-SISH-Siu-Agd-chloromethylketone, - BapvuI-Rpe-Rpe -Agd-chloromethyl ketone and RPE-RPe-Agd-chloromethyl ketone. Ge 5. A method of suppressing deposited platelets in a patient, which consists in administering to the patient a therapeutically effective dose of the composition, characterized by the fact that the mentioned composition consists of the MII factor, which has at least one modification in its active center, the modification of which essentially suppresses the ability of the modified factor MI activate plasma factor X or IX. (F) 6. The method according to claim 5, characterized by the fact that the modification includes the reaction of the MI factor! with GI serine protease inhibitor.. 7. The method according to claim 6, characterized by the fact that the protease inhibitor is an organophosphorus compound, a sulfonyl fluoride, a peptide-containing halomethyl ketone or an azapeptide. 8. The method according to claim 7, characterized by the fact that the protease inhibitor is a peptide-containing halomethyl ketone, selected from YuapvuI-RPne-Pho-Agda-chloromethyl ketone, YuapzuI-SISH-c1Iu-Agd-chloromethyl ketone, BapvuI-Ppe-Ppe-Agd-chloromethyl ketone and RPE-RPe-Agd-chloromethylketone. 9. The method according to claim 5, characterized by the fact that the modification of the MI factor! includes replacement, inclusion or deletion of at least one amino acid! in the catalytic triad Zeg, Avr, Niv. 10. A method of suppressing vascular restenosis in a patient, which consists in administering to the patient a therapeutically effective amount of the composition, characterized by the fact that the mentioned composition includes a pharmacologically acceptable MI factor, having at least one modification in its catalytic center, which significantly suppresses the ability of the modified factor MI to activate plasma factor X or IX. 11. The method according to claim 10, characterized by the fact that the modification of the MI factor is carried out by the reaction of the MI factor with a serine protease inhibitor. 12. The method according to claim 11, characterized by the fact that the protease inhibitor is an organophosphorus compound, a sulfonyl fluoride, a peptide-containing halomethyl ketone or an azapeptide. 70 13. The method according to claim 12, characterized by the fact that it is a protease inhibitor! is a peptide-containing halomethyl ketone selected from YuapzuI-RPNe-Pho-Agda-chloromethylketone, YuapzuI-SISH-c1Iu-Agd-chloromethylketone, BapvuI-Ppe-Ppe-Agd-chloromethylketone and RPe-RPe-Agd-chloromethylketone. 14. The method according to claim 10, characterized by the fact that the modification of the MI factor! includes the replacement, inclusion or deletion of at least one amino acid! in the catalytic triad Zeg, Agvr, Niv. 15. The method according to claim 10, characterized by the fact that it is used for restenosis, which is secondary to mechanical damage to the artery. 16. The method according to claim 15, characterized by the fact that the mentioned mechanical damage is treated by balloon angioplasty, zndarterectomy, reduction atherzectomy, stent placement, laser therapy or excision of decaying tissue. 17. The method according to claim 15, characterized by the fact that the composition is administered to the patient within 24 hours before mechanical damage to the mentioned artery. 18. The method according to claim 15, characterized by the fact that the composition is administered to the patient after mechanical damage to the mentioned artery. 19. The method according to claim 10, characterized by the fact that it is used for restenosis observed in a vascular SC implant, stent, shunt or organ transplant. with and about 20. The method according to claim 19, characterized by the fact that the composition is administered to the patient within 24 hours before the installation of the mentioned implant, stent, shunt and organ transplant. 21. The method according to claim 19, characterized by the fact that the composition is administered to the patient after installation of the mentioned implant, stent, shunt and organ transplant. b z 22. The method according to claim 10, characterized by the fact that it includes the further introduction of the mentioned activator of tissue plasminogen or streptokinase. - 23. A method of treating acute coronary artery occlusion in a patient, which consists in administering to the patient a composition characterized by the fact that the mentioned composition includes a pharmacologically acceptable MiII factor having at least one modification in its catalytic center, which substantially suppresses the ability of the modified MI factor to activate the plasma factor X «E or IX in combination with tissue plasminogen activator or streptokinase. - . and? sh» (ee) - sh» iChe) ime) 60 b5