Fusion proteins containing tissue plasminogen activators or their variants that target integrin αIIbβ3 and their use

Abstract

A fusion protein is provided. [Solution] The fusion protein of the present invention comprises a tissue plasminogen activator or a variant thereof, a disintegrin or a variant thereof, and a connector that binds the tissue plasminogen activator or a variant thereof and the disintegrin or variant thereof and comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 7. The present invention further provides a method for using the fusion protein in the treatment or prevention of a disease associated with thrombus formation.

Description

[Technical Field]

[0001] The present invention relates to fusion proteins, and more particularly to fusion proteins comprising tissue plasminogen activators or variants thereof that target integrin αIIbβ3, and to methods for treating or preventing thrombosis-related diseases using such proteins. [Background technology]

[0002] A thrombus is a clump of blood formed by the abnormal accumulation of platelets and fibrin in a blood vessel. It obstructs or impedes blood flow in the circulatory system, preventing the normal supply of blood to various parts of the body and affecting normal physiological functions. Myocardial infarction, cerebral embolism, pulmonary embolism, deep vein thrombosis, and embolus of surrounding blood vessels are common diseases caused by thrombosis. When severe, these diseases threaten human health, with very high incidence rates, prognosis of permanent disability, and mortality rates. According to World Health Organization statistics, approximately 26 million people worldwide die each year from thrombosis-related diseases, which is generally much higher than other causes of death.

[0003] Drugs that reduce blood clots can be divided into at least three categories based on their mechanism of action: antiplatelet agents, anticoagulants, and thrombolytic agents. Antiplatelet agents can prevent platelets from clotting into blood clots, and common examples include aspirin, clopidogrel, and ticagrelor. Anticoagulants can prolong the time it takes for blood clots to form by interfering with blood proteins, and common examples include warfarin, rivaroxaban, and heparin. Thrombolytic agents can dissolve blood clots, and common examples include streptokinase, urokinase, and tissue plasminogen activator (tPA). While the drugs mentioned above can be used to treat blood clots, they can have bleeding side effects and can lead to other serious bleeding-related illnesses, such as cerebral hemorrhage. Therefore, researchers are currently exploring balanced approaches that reduce blood clots while also addressing bleeding problems.

[0004] Tissue plasminogen activator (rtPA) belongs to the serine protease group and binds to fibrin, converting plasminogen to plasmin. Plasmin is one of the main enzymes that break down blood clots. In genetic engineering, tissue plasminogen activator is produced in vitro, and such genetically engineered products are called "recombinant tissue plasminogen activator (rtPA)." These genetically engineered products enhance their pharmacodynamic effects through various different sequence modifications, particularly by increasing the half-life in the circulatory system, thereby further increasing their specificity for fibrin and preventing unnecessary fibrinolysis. Common genetically engineered drugs include alteplase, reteplase, and tenecteplase (TNK).

[0005] Alteplase has the same sequence as wild-type tissue plasminogen activator produced by human vascular endothelial cells and is expressed in Chinese hamster ovary (CHO) cells. Alteplase has a half-life of approximately 5 minutes and is applicable to indications such as ischemic stroke, ST-elevation myocardial infarction, and acute extensive pulmonary embolism. It can also be administered to patients with implanted central venous access devices.

[0006] Leteplase is a non-glycosylated form of recombinant tissue plasminogen activator, containing 355 amino acids from the original protein, and is obtained from synthesis within Escherichia coli (E. coli). Compared to alteplase, leteplase has a longer half-life of approximately 14-18 minutes, allowing for bolus injection and eliminating the need for intravenous infusion as with alteplase. Currently, leteplase is applicable to the treatment of acute myocardial infarction.

[0007] Tenecteplase is another modified form of human tissue plasminogen activator, obtained from mammalian cells (e.g., CHO cells). Tenecteplase is a glycoprotein with 527 amino acids and is obtained by modifying the cDNA of wild-type tissue plasminogen activator corresponding to humans as follows: threonine at position 103 is replaced with asparagine, asparagine at position 117 is replaced with glutamine, and four amino acids at positions 296-299 are replaced with tetraalanine. The first two modifications occur in the kringle domain, and the last modification occurs in the protease domain. Tenecteplase has a longer half-life of approximately 20-24 minutes and is applicable to indications such as acute myocardial infarction and pulmonary embolism.

[0008] Therefore, one of the challenges that people in the technical field to which this invention belongs are actively striving to solve is to develop a novel thrombolytic agent that can reduce the risk of bleeding and decrease blood clots. [Overview of the project]

[0009] This invention was completed through the following discovery: By binding tissue plasminogen activator and disintegrin with a binder of different amino acid sequences, the inhibitory activity of disintegrin against platelet aggregation is reduced while the intrinsic thrombolytic activity of tissue plasminogen activator is still maintained. In this way, a candidate thrombolytic drug is provided that can reduce thrombosis while lowering the risk of bleeding.

[0010] Thus, the fusion protein according to the present invention comprises tissue plasminogen activator or a variant thereof, disintegrin or a variant thereof, and a binder that binds tissue plasminogen activator or a variant thereof and disintegrin or a variant thereof, and includes an amino acid sequence shown in any one of SEQ ID NOs: 1 to 7.

[0011] For example, the C-terminus of tissue plasminogen activator or its variants is bound to the N-terminus of the ligator, and the N-terminus of disintegrin or its variants is bound to the C-terminus of the ligator.

[0012] For example, the C-terminus of disintegrin or its variants is bound to the N-terminus of the ligator, and the N-terminus of tissue plasminogen activator or its variants is bound to the C-terminus of the ligator.

[0013] Exemplary tissue plasminogen activators include alteplases, leteplases, or tenecteplases.

[0014] For example, the tissue plasminogen activator is tenecteplase.

[0015] For example, tissue plasminogen activators contain the amino acid sequence shown in one of SEQ ID NOs: 8-10.

[0016] For example, tissue plasminogen activator contains the amino acid sequence shown in SEQ ID NO:10.

[0017] For example, disintegrins include albolabrin, applagin, basilicin, batroxostatin, bitistatin, cereberin, cerastin, crotatroxin, durissin, elegantin, eristicophin, flavoridin, flavostatin, halysin, halistatin ( These include halystatin, jararacin, jarastatin, kistrin, lachesin, lutosin, molossin, rhodostomin, salmosin, saxatilin, tergeminin, trimestatin, trimucrin, trimutase, ussuristatin, or viridian.

[0018] Examples of disintegrins include rhodostomin or trimucrine.

[0019] For example, a disintegrin variant comprises a binding region containing an amino acid sequence shown in any one of SEQ ID NOs: 11-15, an RGD motif containing an amino acid sequence shown in any one of SEQ ID NOs: 16-28, and a C-terminal region containing an amino acid sequence shown in any one of SEQ ID NOs: 29-33.

[0020] For example, the binding region includes the amino acid sequence shown in SEQ ID NO: 11 or 15.

[0021] For example, the binding region includes the amino acid sequence shown in SEQ ID NO:11.

[0022] Exemplarily, the RGD motif includes the amino acid sequence shown in any one of SEQ ID NOs: 17 to 21, 24 to 26.

[0023] Exemplarily, the RGD motif includes the amino acid sequence shown in SEQ ID NO: 20.

[0024] Exemplarily, the C-terminal region includes the amino acid sequence shown in SEQ ID NO: 31.

[0025] Exemplarily, the disintegrin variant includes the amino acid sequence shown in SEQ ID NO: 34.

[0026] Exemplarily, the fusion protein includes the amino acid sequence shown in any one of SEQ ID NOs: 35 to 42.

[0027] Exemplarily, the fusion protein is used to bind to integrin αIIbβ3 and fibrin.

[0028] Since fibrin is one of the main components of coagulated blood clots, the fusion protein according to the present invention can bind to fibrin at the site of thrombus formation and convert plasminogen to plasmin, which then dissolves fibrin into FDP (fibrin-fibrinogen degradation product), thereby achieving a thrombolytic effect. Furthermore, since integrin αIIbβ3 is expressed in large quantities in platelets and their precursor cells (see J Hematol Oncol. 2019 Mar 7;12(1):26), the fusion protein according to the present invention can simultaneously bind to platelet integrin αIIbβ3 at the site of thrombus formation, suppressing platelet aggregation and preventing the formation of large thrombi. Moreover, because the fusion protein according to the present invention has lower inhibitory activity against platelet aggregation than disintegrin or its variants, it can reduce the risk of bleeding. Therefore, the fusion protein according to the present invention is a thrombolytic agent with extremely high potential for clinical use because it reduces thrombosis while lowering the risk of bleeding.

[0029] The present invention further provides a pharmaceutical composition comprising the fusion protein and a pharmaceutically acceptable carrier.

[0030] Exemplary examples of pharmaceutical compositions include oral formulations, injectable formulations, inhaled formulations, or topical or transdermal formulations.

[0031] For example, based on the total volume of the pharmaceutical composition, the molar concentration of the fusion protein is in the range of 1 to 1400 nM.

[0032] The present invention further provides uses for the pharmaceutical composition used in the preparation of pharmaceuticals that treat or prevent diseases related to thrombus formation and reduce the risk of bleeding.

[0033] Examples of diseases associated with the formation of blood clots include venous thrombosis and arterial thrombosis.

[0034] Examples of venous thrombosis include branch retinal vein occlusion, Budd-Chiari syndrome, cavernous sinus thrombosis, central retinal vein occlusion, cerebral venous sinus thrombosis, deep vein thrombosis, jugular vein thrombosis, superior mesenteric vein thrombosis, primary subclavian vein thrombosis (Paget-Schroetter disease), paradoxical embolism, portal vein thrombosis, pulmonary embolism, and renal vein thrombosis. It is thrombosis, or splenic vein thrombosis.

[0035] Examples of arterial thrombosis include hepatic artery thrombosis, limb ischemia, myocardial infarction, or stroke.

[0036] The present invention further provides a method for treating or preventing diseases related to thrombus formation, comprising the step of administering the aforementioned pharmaceutical composition to an individual in need, thereby dissolving the thrombus and reducing the risk of bleeding.

[0037] Examples of diseases associated with the formation of blood clots include venous thrombosis and arterial thrombosis.

[0038] Exemplary examples of venous thrombosis include branch retinal vein occlusion, Budd-Chiari syndrome, cavernous sinus thrombosis, central retinal vein occlusion, cerebral venous sinus thrombosis, deep vein thrombosis, internal jugular vein thrombosis, superior mesenteric vein thrombosis, primary subclavian vein thrombosis, paradoxical embolism, portal vein thrombosis, pulmonary embolism, renal vein thrombosis, or splenic vein thrombosis.

[0039] Examples of arterial thrombosis include hepatic artery thrombosis, lower limb ischemia, myocardial infarction, or stroke.

[0040] For example, administer 0.1 to 1000 mg of the fusion protein per kg of body weight to the individual.

[0041] The present invention further provides nucleic acids comprising a nucleotide sequence for encoding the fusion protein.

[0042] The present invention further provides a host cell containing the nucleic acid.

[0043] For example, the host cell can be a prokaryotic or eukaryotic cell.

[0044] For example, a prokaryotic cell is *E. coli*.

[0045] Exemplary examples of eukaryotic cells include CHO cells, COS cells, or HEK293 cells.

[0046] The present invention further provides a method for preparing the fusion protein, comprising the step of culturing the host cells to express the fusion protein. [Brief explanation of the drawing]

[0047] [Figure 1] This flowchart schematically illustrates the process of producing a DNA construct for expressing the protein TNK-G9-RR. [Figure 2A] This is a liquid chromatogram showing the isolation results of the protein TNK. [Figure 2B]This is a liquid chromatogram showing the isolation results for the protein TNK-G9-RR. [Figure 2C] This is a liquid chromatogram showing the isolation results for the protein TNK-(G4S)3-RR. [Figure 2D] This is a liquid chromatogram showing the isolation results for the protein TNK-(PA)3-RR. [Figure 2E] This is a liquid chromatogram showing the isolation results for the protein TNK-(PA)5-RR. [Figure 2F] This is a liquid chromatogram showing the isolation results for the protein TNK-(PA)7-RR. [Figure 2G] This is a liquid chromatogram showing the isolation results for the protein TNK-EA3K(G4S)2-RR. [Figure 2H] This is a liquid chromatogram showing the isolation results for the protein TNK-(EA3K)3-RR. [Figure 2I] This is a liquid chromatogram showing the isolation results for the protein RR-(PA)5-TNK. [Figure 3A] These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK. The symbols "M" indicate the protein marker, "LS" indicate the load sample (20 μL), "FT" indicate the flow-through sample (250 μL), and the Arabic numerals indicate the separatory number. [Figure 3B] These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK. The symbols "M" indicate the protein marker, "LS" indicate the load sample (20 μL), "FT" indicate the flow-through sample (250 μL), and the Arabic numerals indicate the separatory number. [Figure 3C]These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-G9-RR. The symbols "M" indicate the protein marker, "LS" indicates the load sample (20 μL), "FT" indicates the flow-through sample (250 μL), and the Arabic numerals indicate the separatory number. [Figure 3D] These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-G9-RR. The symbols "M" indicate the protein marker, "LS" indicates the load sample (20 μL), "FT" indicates the flow-through sample (250 μL), and the Arabic numerals indicate the separatory number. [Figure 3E] These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(G4S)3-RR. The symbols "M" indicate the protein marker, "LS" indicates the load sample (20 μL), "FT" indicates the flow-through sample (250 μL), and the Arabic numerals indicate the separatory number. [Figure 3F] These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(G4S)3-RR. The symbols "M" indicate the protein marker, "LS" indicates the load sample (20 μL), "FT" indicates the flow-through sample (250 μL), and the Arabic numerals indicate the separatory number. [Figure 3G] These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(PA)3-RR. The symbols "M" indicate the protein marker, "LS" indicate the load sample (20 μL), "FT" indicate the flow-through sample (250 μL), and the Arabic numerals indicate the separatory number. [Figure 3H] These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(PA)3-RR. The symbols "M" indicate the protein marker, "LS" indicate the load sample (20 μL), "FT" indicate the flow-through sample (250 μL), and the Arabic numerals indicate the separatory number. [Figure 3I] These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(PA)5-RR. The symbols "M" indicate the protein marker, "LS" indicate the load sample (20 μL), "FT" indicate the flow-through sample (250 μL), and "Arabic numerals" indicate the separatory number. [Figure 3J] These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(PA)5-RR. The symbols "M" indicate the protein marker, "LS" indicate the load sample (20 μL), "FT" indicate the flow-through sample (250 μL), and "Arabic numerals" indicate the separatory number. [Figure 3K] These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(PA)7-RR. The symbols "M" indicate the protein marker, "LS" indicate the load sample (20 μL), "FT" indicate the flow-through sample (250 μL), and the Arabic numerals indicate the separatory number. [Figure 3L]These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(PA)7-RR. The symbols "M" indicate the protein marker, "LS" indicate the load sample (20 μL), "FT" indicate the flow-through sample (250 μL), and the Arabic numerals indicate the separatory number. [Figure 3M] These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-EA3K(G4S)2-RR. The symbols "M" indicate the protein marker, "LS" indicate the load sample (20 μL), "FT" indicate the flow-through sample (250 μL), and "Arabic numerals" indicate the separatory number. [Figure 3N] These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-EA3K(G4S)2-RR. The symbols "M" indicate the protein marker, "LS" indicate the load sample (20 μL), "FT" indicate the flow-through sample (250 μL), and "Arabic numerals" indicate the separatory number. [Figure 3O] These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(EA3K)3-RR. The symbols "M" indicate the protein marker, "LS" indicate the load sample (20 μL), "FT" indicate the flow-through sample (250 μL), and the Arabic numerals indicate the separatory number. [Figure 3P]These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(EA3K)3-RR. The symbols "M" indicate the protein marker, "LS" indicate the load sample (20 μL), "FT" indicate the flow-through sample (250 μL), and the Arabic numerals indicate the separatory number. [Figure 3Q] These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein RR-(PA)5-TNK. The symbols "M" indicate the protein marker, "LS" indicate the load sample (20 μL), "FT" indicate the flow-through sample (250 μL), and the Arabic numerals indicate the separatory number. [Figure 3R] These are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein RR-(PA)5-TNK. The symbols "M" indicate the protein marker, "LS" indicate the load sample (20 μL), "FT" indicate the flow-through sample (250 μL), and the Arabic numerals indicate the separatory number. [Figure 4A] A thrombolysis results chart illustrating the thrombolytic rates of protein TNK at different concentrations. [Figure 4B] A thrombolysis results chart illustrating the thrombolytic rates of the protein TNK-G9-RR at different concentrations. [Figure 4C] A thrombolysis results chart illustrating the thrombolytic rates of the protein TNK-(G4S)3-RR at different concentrations. [Figure 4D] A thrombolysis results chart illustrating the thrombolytic rates of the protein TNK-(PA)3-RR at different concentrations. [Figure 4E] A thrombolysis results chart illustrating the thrombolytic rates of the protein TNK-(PA)5-RR at different concentrations. [Figure 4F]A thrombolysis results chart illustrating the thrombolytic rates of the protein TNK-(PA)7-RR at different concentrations. [Figure 4G] A thrombolysis result chart illustrating the thrombolytic rates of the protein TNK-EA3K(G4S)2-RR at different concentrations. [Figure 4H] A thrombolysis results chart illustrating the thrombolytic rates of the protein TNK-(EA3K)3-RR at different concentrations. [Figure 4I] A thrombolysis results chart illustrating the thrombolytic rates of protein RR-(PA)5-TNK at different concentrations. [Figure 5] This bar graph illustrates the time it takes for the thrombolytic rate of different proteins to reach 50% at a concentration of 7.0 nM. [Modes for carrying out the invention]

[0048] To further clarify and facilitate understanding of the above-mentioned objectives, effects, and features of the present invention, and / or other objectives, effects, and features, preferred embodiments are described below in detail. 1. Definition of Terms As used herein, "protein" includes, unless otherwise specified, wild-type proteins expressed in natural cells, genetically modified proteins expressed using genetic engineering techniques, or chemically obtained synthetic proteins. At least one amino acid in the sequence of a protein may be substituted or deleted, and / or at least one amino acid may be inserted, without affecting its original activity.

[0049] As used herein, "amino acids" include D-amino acids or L-amino acids unless otherwise specified. D- and L- indicate the absolute configuration of the amino acid, not a specific rotational direction of plane polarization. Except in special circumstances, this specification uses single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission to represent amino acids. Protein sequences are represented by strings of single-letter symbols, the order of which corresponds to the order of amino acids from the N-end to the C-end of the protein. Superscripts preceding a single-letter symbol indicate the positional order of the corresponding amino acid, starting from the N-end of the protein. For example, 67 PRNGLYG indicates that proline is located at position 67 of the protein, and the rest can be inferred similarly, so the explanation will not be repeated. When a single letter symbol is followed by a subscript number, it indicates the corresponding amino acid or a repeat of the corresponding amino acid group. For example, G9 indicates that nine consecutive glycine molecules are linked. Also, for example, (G4S)3 indicates that three sets of consecutive glycine-glycine-glycine-glycine-serine amino acid groups are linked, and the rest can be inferred similarly, so the explanation will not be repeated.

[0050] Substitutions, deletions, and / or insertions in protein sequences may occur in non-functional regions of the protein, and these usually do not affect its intrinsic activity. Furthermore, protein sequence substitutions may include substitutions of conserved amino acids, which refer to substitutions between amino acids that have similar properties or related side chains. Substitutions between amino acids with similar properties refer to the ability to substitute each other, for example, between acidic amino acids such as aspartate and glutamate; between alkaline amino acids such as lysine, arginine, and histidine; between nonpolar amino acids such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and between uncharged polar amino acids such as glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Substitutions between amino acids with related side chains refer to the ability to mutually substitute aliphatic hydroxyl amino acids such as serine and threonine, amide-containing amino acids such as asparagine and glutamine, aliphatic amino acids such as alanine, valine, leucine, and isoleucine, and aromatic amino acids such as phenylalanine, tryptophan, and tyrosine.

[0051] As used herein, “tissue plasminogen activator” includes, unless otherwise specified, wild-type or recombinant tissue plasminogen activator, where wild-type is, for example, tissue plasminogen activator produced by human vascular endothelial cells, and recombinant is, for example, alteplase (SEQ ID NO:8), leteplase (SEQ ID NO:9), or tenecteplase (SEQ ID NO:10).

[0052] As used herein, "disintegrin" refers, unless otherwise specified, to platelet aggregation inhibitors extracted from the saliva of venomous snakes, which typically consist of 47 to 84 amino acids and 4 to 7 pairs of disulfide bonds, and include, for example, arborabrin, apragine, basilicin, batroxostatin, vitistatin, celeberin, cerastine, crotatoxin, dulcin, elegantin, erythticopine, flavoridine, flavostatin, halisin, halistine, jaralacin, jarastatin, quistrin, ratesin, rutocin, morosin, rhodostomin, salmonsin, saxatirin, tergeminin, trimestatin, trimucrine, trimutase, usulistatin, or viridian.

[0053] As used herein, "mutant" refers to a modified protein obtained by substituting or deleting at least one amino acid in a reference sequence and / or inserting at least one amino acid, without affecting the original activity. For example, disintegrin mutants include rhodostomin mutants and trimucrine mutants, and can inhibit platelet aggregation by binding integrin αIIbβ3 to wild-type disintegrin. The mutants have sequence similarity of at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, or 60% with respect to the reference sequence, and the similarity is defined as gap-excluded identity, BLAST identity, or gap-compressed identity based on different conditions. BLAST identity is obtained by calculating it using the Basic Local Alignment Search Tool provided by the National Center for Biotechnology Information. For example, the trimucrine variant RR described herein has at least 95% BLAST similarity to wild-type trimucrine.

[0054] As used herein, the “RGD motif” refers to the flexible loop portion of disintegrin composed of arginine-glycine-aspartic acid, unless otherwise specified, and is the integrin-binding region. For example, the RGD motif of wild-type trimucrine is 50 Including ARGDNP, the RGD motif of wild-type rhodostomin is 48 It contains PRGDMP. As mentioned above, the RGD motif of the trimucrine mutant is the RGD motif sequence of wild-type trimucrine. 50 ARGDNP has at least one amino acid mutation, such as SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, and does not affect function. The RGD motif of the rhodostomin mutant is the RGD motif sequence of wild-type rhodostomin.48 PRGDMP has at least one amino acid mutation such as SEQ ID NO: 27 or 28 and does not affect its function.

[0055] As used herein, unless otherwise defined, the "binding region" refers to the region adjacent to the N-terminus of the RGD motif in disintegrin, and is usually any continuous fragment of amino acids at positions 38-49. For example, the binding region of wild-type trimclin contains 41 KKKRT (SEQ ID NO: 11), and the binding region of wild-type rhodostomin contains 39 SRAGK (SEQ ID NO: 12). As described above, the binding region of the trimclin mutant has at least one amino acid mutation such as SEQ ID NO: 13, 14, or 15 with respect to the binding region sequence of wild-type trimclin 41 KKKRT and does not affect its function.

[0056] As used herein, unless otherwise defined, the "C-terminal region" refers to the region adjacent to the C-terminus of the RGD motif in disintegrin. For example, the C-terminal region of wild-type trimclin contains 67 PRNGLYG (SEQ ID NO: 29), and the C-terminal region of wild-type rhodostomin contains 65 PRYH (SEQ ID NO: 30). As described above, the C-terminal region of the trimclin mutant has at least one amino acid mutation such as SEQ ID NO: 31, 32, or 33 with respect to the C-terminal region of wild-type trimclin 67 PRNGLYG and does not affect its function.

[0057] As used herein, "disintegrin variants" include, unless otherwise specified, sequences obtained by mutations in at least one amino acid in the wild-type RGD motif, sequences obtained by mutations in at least one amino acid in the wild-type binding region, and / or sequences obtained by mutations in at least one amino acid in the wild-type C-terminal region, including, for example, SEQ ID NO:34.

[0058] As used herein, “treatment” means curing or improving thrombosis by therapeutic intervention, unless otherwise specified, including complete or local cure or improvement.

[0059] As used herein, "prevention" refers to the complete or near-complete prevention of thrombosis, unless otherwise specified. For example, if there is no blood clot, or only a minor blood clot, but the condition has not progressed to disease, preventive intervention can prevent the onset of the disease.

[0060] As used herein, “medically acceptable carrier” means, unless otherwise specified, an additive that is suitable for contact with an individual, within the bounds of sound medical judgment, is free from excessive toxicity, irritation, allergic reactions, or other problems or complications, and has a reasonable risk-benefit ratio, such as fillers, diluents, flocculants, adhesives, lubricants, fluidizers, stabilizers, colorants, humectants, or disintegrants.

[0061] 2. Fusion protein The fusion protein according to the first embodiment of the present invention can simultaneously bind to fibrin and platelet integrin αIIbβ3 at the site of thrombus. Binding to fibrin converts plasminogen to plasmin, and plasmin dissolves fibrin in FDP, thereby achieving a thrombus-dissolving effect. Furthermore, binding to integrin αIIbβ3 inhibits platelet aggregation, preventing the formation of large thrombi. However, the activity of the fusion protein according to this embodiment in inhibiting platelet aggregation through binding to integrin αIIbβ3 is lower than that of proteins containing only disintegrin or its variants, thus reducing the risk of bleeding. Based on these characteristics, the fusion protein according to this embodiment is used to dissolve thrombi and reduce the risk of bleeding. In other words, it can be used for the treatment of thrombosis and to reduce the risk of bleeding.

[0062] The fusion protein according to this embodiment comprises tissue plasminogen activator or a variant thereof, disintegrin or a variant thereof, and a binder. The binder binds tissue plasminogen activator or a variant thereof and disintegrin or a variant thereof, and contains the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7. The fusion protein sequentially contains tissue plasminogen activator or a variant thereof, a binder, and disintegrin or a variant thereof between the N-terminus and the C-terminus, or contains disintegrin or a variant thereof, a binder, and tissue plasminogen activator or a variant thereof. Specifically, the C-terminus of tissue plasminogen activator or a variant thereof is bound to the N-terminus of the binder, and the N-terminus of disintegrin or a variant thereof is bound to the C-terminus of the binder. Alternatively, the C-terminus of disintegrin or a variant thereof is bound to the N-terminus of the binder, and the N-terminus of tissue plasminogen activator or a variant thereof is bound to the C-terminus of the binder. Here, "binding" is not limited to direct or indirect binding; that is, there may or may not be other binding fragments between the bound proteins. Preferably, the fusion protein contains an amino acid sequence such as those shown in SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, or 42.

[0063] Based on their type, tissue plasminogen activators are alteplase, leteplase, or tenecteplase, preferably tenecteplase.

[0064] Based on its sequence, the tissue plasminogen activator contains the amino acid sequence shown in SEQ ID NO: 8, 9, or 10, and preferably the amino acid sequence shown in SEQ ID NO: 10.

[0065] Disintegrins, based on their type, include arbolabrin, apragine, basilicin, batroxostatin, vitistatin, celeberin, cerastine, crotatoxin, dulcin, elegantin, erythticopine, flavoridine, flavostatin, halisin, halistine, jaralacin, jarastatin, quistrin, racesin, rutocin, morosin, rhodostomin, salmonsin, saxatirin, tergeminin, trimestatin, trimucrine, trimutase, usulistatin, or viridian. Preferably, the disintegrin is rhodostomin or trimucrine.

[0066] Based on their sequences, the disintegrin variants comprise a binding region, an RGD motif, and a C-terminal region. The binding region contains the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14, or 15; the RGD motif contains the amino acid sequence shown in SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28; and the C-terminal region contains the amino acid sequence shown in SEQ ID NO: 29, 30, 31, 32, or 33.

[0067] Preferably, the binding region includes the amino acid sequence shown in SEQ ID NO: 11 or 15. More preferably, the binding region includes the amino acid sequence shown in SEQ ID NO: 11.

[0068] Preferably, the RGD motif contains the amino acid sequence shown in SEQ ID NO: 17, 18, 19, 20, 21, 24, 25, or 26. Preferably, the RGD motif contains the amino acid sequence shown in SEQ ID NO: 20.

[0069] Preferably, the C-terminal region contains the amino acid sequence shown in SEQ ID NO:31.

[0070] Preferably, the disintegrin variant contains the amino acid sequence shown in SEQ ID NO:34.

[0071] The fusion protein according to this embodiment is prepared by genetic engineering techniques or chemical methods, such as solid-phase synthesis or liquid-phase synthesis. Subsequently, the fusion protein according to this embodiment is obtained or purified by separation using methods such as ammonium sulfate or ethanol precipitation, acid extraction, ion exchange chromatography, affinity chromatography, or lectin chromatography. High-performance liquid chromatography is preferably employed.

[0072] The fusion protein according to this embodiment has hydrophilic groups, which further enhances its water solubility or cyclic half-life. The hydrophilic groups can be bound to the N-terminus of the fusion protein. Preferably, the hydrophilic groups are polyethylene glycol, polypropylene glycol, polylactic acid, polyglycolic acid, polyvinyl alcohol, or dextran. More preferably, the hydrophilic groups are polyethylene glycol composed of 2 to 20 overlapping ethylene glycol units.

[0073] The fusion protein according to this embodiment further comprises a purification tag to aid in purification. The purification tag can be bound to the N-terminus or C-terminus of the fusion protein. Preferably, the purification tag is a histidine tag (His-tag), a glutathione S-transferase tag (GST-tag), a maltose-binding protein tag (MBP-tag), a transcription termination / anti-termination protein tag (NusA-tag), or a small molecule ubiquitin-like modifier tag (SUMO-tag).

[0074] 3. Pharmaceutical Compositions The pharmaceutical composition according to the second embodiment of the present invention contains the fusion protein according to the first embodiment, and can therefore be administered to individuals requiring thrombolysis, thereby dissolving thrombi and simultaneously reducing the risk of bleeding. The pharmaceutical composition according to this embodiment contains the fusion protein according to the first embodiment and a pharmaceutically acceptable carrier.

[0075] Generally, pharmaceutically acceptable carriers make the entire pharmaceutical composition into different forms or applicable to different routes of administration. Preferably, the pharmaceutical composition is an oral formulation, an injectable formulation, an inhaled formulation, or a topical or transdermal formulation used for different routes of administration. Preferably, the pharmaceutical composition is a tablet, capsule, granule, dispersant, solvent, syrup, suspension, or emulsion. The pharmaceutical composition according to this embodiment can be applied to implantable medical devices such as stents and catheters, and exerts an effect of preventing the implantable device from narrowing blood vessels, or supporting and strengthening blood vessels, while simultaneously dissolving thrombi. The pharmaceutical composition according to this embodiment may further contain other agents that reduce thrombosis, such as antiplatelet agents and anticoagulants. Examples of antiplatelet agents include aspirin, clopidogrel, or ticagrelor, and examples of anticoagulants include warfarin, rivaroxaban, or heparin.

[0076] Medicinally acceptable carriers may be excipients, fillers, diluents, flocculants, adhesives, lubricants, fluidizers, stabilizers, colorants, humectants, or disintegrants. Examples of excipients include sodium citrate, calcium carbonate, or calcium phosphate. Examples of fillers include lactose or high molecular weight polyethylene glycol. Examples of diluents include water, ethanol, propanediol, or glycerin. Examples of adhesives include sucrose, gelatin, or gum arabic. Examples of lubricants include magnesium stearate, calcium stearate, zinc stearate, sodium stearate, stearic acid, aluminum stearate, leucine, glyceryl behenate, or hydrogenated vegetable oil. Examples of fluidizing agents include sodium aluminosilicate, calcium silicate, microcrystalline cellulose, corn starch, sodium benzoate, calcium carbonate, magnesium carbonate, talc, calcium stearate, magnesium stearate, zinc stearate, magnesium lauryl sulfate, or magnesium oxide. Examples of stabilizers include citric acid or ascorbic acid. Examples of colorants include titanium dioxide or iron oxide. Examples of humectants include Pluronic F68, Tween 20, or Tween 80. Examples of disintegrants include potato starch, tapioca starch, or silicates.

[0077] Based on the total volume of the pharmaceutical composition, the molar concentration of the fusion protein is in the range of 1 to 1400 nM. Preferably, based on the total volume of the pharmaceutical composition, the molar concentration of the fusion protein is in the range of 7 to 1370.7 nM.

[0078] IV. Pharmaceutical Uses A third embodiment of the present invention provides an application of the pharmaceutical composition according to the second embodiment for use in preparing a pharmaceutical for treating or preventing diseases associated with thrombus formation and reducing the risk of bleeding. The prepared pharmaceutical can be administered to individuals who require thrombus dissolution, thereby dissolving the thrombus and simultaneously reducing the risk of bleeding. In other words, the prepared pharmaceutical can be administered to individuals who require treatment or prevention of diseases associated with thrombus formation, achieving a therapeutic or preventive effect while simultaneously reducing the risk of bleeding.

[0079] Different administration methods may be used, such as oral administration, injection, inhalation, or topical or transdermal administration. Furthermore, an effective dose of the fusion protein should be administered to the individual at a rate of 0.1 to 1000 mg per kg of body weight.

[0080] Diseases associated with the formation of blood clots can be divided into venous thrombosis and arterial thrombosis. Examples of venous thrombosis include branch retinal vein occlusion, Budd-Chiari syndrome, cavernous sinus thrombosis, central retinal vein occlusion, cerebral venous sinus thrombosis, deep vein thrombosis, internal jugular vein thrombosis, superior mesenteric vein thrombosis, primary subclavian vein thrombosis, paradoxical embolism, portal vein thrombosis, pulmonary embolism, renal vein thrombosis, or splenic vein thrombosis. Examples of arterial thrombosis include hepatic artery thrombosis, lower limb ischemia, myocardial infarction, or stroke.

[0081] A method for treating or preventing a disease related to thrombus formation according to the fourth embodiment of the present invention includes the step of administering the pharmaceutical composition according to the second embodiment to an individual in need of treatment or prevention, thereby dissolving the thrombus and reducing the risk of bleeding.

[0082] Different administration methods may be used, such as oral administration, injection, inhalation, or topical or transdermal administration. Furthermore, an effective dose of the fusion protein should be administered to the individual at a rate of 0.1 to 1000 mg per kg of body weight.

[0083] Diseases associated with the formation of blood clots can be divided into venous thrombosis and arterial thrombosis. Examples of venous thrombosis include branch retinal vein occlusion, Budd-Chiari syndrome, cavernous sinus thrombosis, central retinal vein occlusion, cerebral venous sinus thrombosis, deep vein thrombosis, internal jugular vein thrombosis, superior mesenteric vein thrombosis, primary subclavian vein thrombosis, paradoxical embolism, portal vein thrombosis, pulmonary embolism, renal vein thrombosis, or splenic vein thrombosis. Examples of arterial thrombosis include hepatic artery thrombosis, lower limb ischemia, myocardial infarction, or stroke.

[0084] 5. Other matters A nucleic acid according to a fifth embodiment of the present invention comprises a nucleotide sequence for encoding the fusion protein according to the first embodiment. To regulate protein expression, the nucleic acid further comprises a promoter which is bound to the nucleotide sequence for encoding the fusion protein in an operational manner. Here, "bound in an operational manner" means making the relationship between two or more nucleic acid sequences functional.

[0085] The host cell according to the sixth embodiment of the present invention contains the nucleic acid according to the fifth embodiment. Since the host cell according to this embodiment contains a nucleotide sequence that encodes a fusion protein, the fusion protein can be produced by culturing the host cell. The host cell may be a prokaryotic cell or a eukaryotic cell. Examples of prokaryotic cells include Escherichia coli, and examples of eukaryotic cells include CHO cells, COS cells, or HEK293 cells.

[0086] A method for preparing a fusion protein according to the first embodiment, as described in the seventh embodiment of the present invention, includes the step of expressing the fusion protein by culturing host cells according to the sixth embodiment. The cells are induced to express the protein by selecting an appropriate inducer using a promoter.

[0087] The present invention will be explained with reference to the following examples. Example 1: Protein preparation The expression construct is transfected into CHO cells or yeast cells to express the recombinant protein. The supernatant of the cell culture is obtained and purified by liquid chromatography to obtain the recombinant protein.

[0088] Let's take the TNK-G9-RR protein expression construct shown in Figure 1 as an example. Using the primer pair TNK-F and TNK-G9-R, a polymerase chain reaction is induced on plasmid pcDNA3.1 containing the TNK nucleotide fragment to obtain plasmid pcDNA3.1 containing the TNK-G9 nucleotide fragment. Using the primer pair RR-G9-F and RR-R, a polymerase chain reaction is induced on plasmid pPICZαA containing the RR nucleotide fragment to obtain an insert containing the RR nucleotide fragment. Finally, plasmid pcDNA3.1 containing the TNK-G9 nucleotide fragment and the insert containing the RR nucleotide fragment are treated with DpnI restriction enzyme and adhered to obtain plasmid pcDNA3.1 containing the TNK-G9-RR nucleotide fragment as the TNK-G9-RR protein expression construct.

[0089] Plasmid pcDNA3.1 containing the TNK-(G4S)3-RR nucleotide fragment is obtained as an expression construct for the protein TNK-(G4S)3-RR, except that the TNK-G9-R is replaced with primer TNK-(G4S)3-R and RR-G9-F is replaced with primer RR-(G4S)3-F, by following the flowchart described above.

[0090] By following the flowchart described above, plasmid pcDNA3.1 containing the TNK-(PA)3-RR nucleotide fragment is obtained as an expression construct for the protein TNK-(PA)3-RR, except that TNK-G9-R is replaced with primer TNK-(PA)3-R and RR-G9-F is replaced with primer RR-(PA)3-F.

[0091] Plasmid pcDNA3.1 containing the TNK-(PA)5-RR nucleotide fragment is obtained as an expression construct for the protein TNK-(PA)5-RR, except that by following the flowchart described above, TNK-G9-R is replaced with primer TNK-(PA)5-R and RR-G9-F is replaced with primer RR-(PA)5-F.

[0092] Plasmid pcDNA3.1 containing the TNK-(PA)7-RR nucleotide fragment is obtained as an expression construct for the protein TNK-(PA)7-RR, except that TNK-G9-R is replaced with primer TNK-(PA)7-R and RR-G9-F is replaced with primer RR-(PA)7-F, by following the flowchart described above.

[0093] Plasmid pcDNA3.1 containing the TNK-EA3K(G4S)2-RR nucleotide fragment is obtained as an expression construct for the protein TNK-EA3K(G4S)2-RR, except that TNK-G9-R is replaced with primer TNK-EA3K(G4S)2-R and RR-G9-F is replaced with primer RR-EA3K(G4S)2-F, by following the flowchart described above.

[0094] By following the flowchart described above, plasmid pcDNA3.1 containing the TNK-(EA3K)3-RR nucleotide fragment is obtained as an expression construct for the protein TNK-(EA3K)3-RR, except that TNK-G9-R is replaced with primer TNK-(EA3K)3-R and RR-G9-F is replaced with primer RR-(EA3K)3-F.

[0095] Table 1 lists the nucleotide sequences of the primers. [Table 1] Primer sequences JPEG0007863710000001.jpg170170

[0096] Figure 2A shows the results of liquid chromatography analysis of protein TNK. Figures 3A and 3B further demonstrate that protein TNK was obtained from separatory 27, 28, 29, 30, 33, and 34.

[0097] Figure 2B shows the results of liquid chromatography analysis of the protein TNK-G9-RR. Figures 3C and 3D further demonstrate that the protein TNK-G9-RR was obtained from separatory 9, 10, and 11.

[0098] Figure 2C shows the results of liquid chromatography analysis of the protein TNK-(G4S)3-RR. Figures 3E and 3F further demonstrate that the protein TNK-(G4S)3-RR was obtained from separatory 15.

[0099] Figure 2D shows the results of liquid chromatography analysis of the protein TNK-(PA)3-RR. Figures 3G and 3H further demonstrate that the protein TNK-(PA)3-RR was obtained from separatory 12, 13, 21, and 22.

[0100] Figure 2E shows the results of liquid chromatography analysis of the protein TNK-(PA)5-RR. Figures 3I and 3J further demonstrate that the protein TNK-(PA)5-RR was obtained from separatory 27, 28, and 29.

[0101] Figure 2F shows the results of liquid chromatography analysis of the protein TNK-(PA)7-RR. Figures 3K and 3L further demonstrate that the protein TNK-(PA)7-RR was obtained from separatory 33, 34, 35, 36, 37, and 39.

[0102] Figure 2G shows the results of liquid chromatography analysis of the protein TNK-EA3K(G4S)2-RR. Figures 3M and 3N further demonstrate that the protein TNK-EA3K(G4S)2-RR was obtained from separatory 27, 28, 29, 30, and 31.

[0103] Figure 2H shows the results of liquid chromatography analysis of the protein TNK-(EA3K)3-RR. Figures 3O and 3P further demonstrate that the protein TNK-(EA3K)3-RR was obtained from separatory 28, 29, 30, 31, and 32.

[0104] Figure 2I shows the results of liquid chromatography analysis of the protein RR-(PA)5-TNK. Figures 3Q and 3R further demonstrate that the protein RR-(PA)5-TNK was obtained from separatory 21, 22, 23, and 24.

[0105] Table 2 lists the amino acid sequences of the aforementioned proteins, and Table 3 lists the yields of each protein. It can be seen that a certain yield was achieved for protein TNK, protein RR, and other fusion proteins containing TNK. [Table 2] Protein sequences JPEG0007863710000002.jpg248170 JPEG0007863710000003.jpg255169 JPEG0007863710000004.jpg255169 JPEG0007863710000005.jpg255168 JPEG0007863710000006.jpg255167 JPEG0007863710000007.jpg244170 Note: The characters within the square frame are sequences of connectives. [Table 3] Protein yield JPEG0007863710000008.jpg219170

[0106] Example 2: Thrombolysis Test Perform whole blood thrombolysis plate analysis. Collect blood from a healthy individual and mix it with 3.8% trisodium citrate in a 9:1 ratio. Add thrombin (6.25 × 10⁻¹⁵) to HEPES buffer (HEPES 25 mM, sodium chloride 137 mM). -3Add U and calcium chloride (250 mM) to obtain a blood clot mixture. Deposit 5 μL of the blood clot mixture onto the bottom edge of the wells of a 96-well microplate, then add 25 μL of L blood mixture. Seal the microwell plate and allow it to react at 37°C for 30 minutes to form thrombi at the bottom edge of the wells.

[0107] Each protein is placed in 70 μL of HEPES solution based on different target concentrations. At room temperature, the protein solution is added to the wells containing the thrombus, and the ELISA plate is placed in an ELISA plate reader and reacted at a constant temperature of 37°C for 120 minutes. During the reaction, the plate is vibrated once per minute (200 rpm), and the absorbance at a wavelength of 510 nm is measured at 3-minute intervals using the ELISA plate reader. Since the dissolved thrombus flows to cover the center of the well during the reaction, the time required to dissolve 50% of the thrombus (T0.5 minutes) is determined by the absorbance at 510 nm. In this way, the degree of thrombus dissolution is determined.

[0108] Figures 4A to 4I show the thrombolytic activity of each protein at different concentrations. Table 4 shows the time required for each protein to dissolve 50% of a thrombus at different concentrations. [Table 4] Time required for each protein to dissolve 50% of a thrombus at different concentrations JPEG0007863710000009.jpg78170ND: Blood clot could not be dissolved within 120 minutes, measurement impossible.

[0109] Figure 5 and Table 5 show the time required for each protein to dissolve 50% of the thrombus at a concentration of 7.0 nM. As mentioned above, both the known thrombolytic agent TNK and each fusion protein containing TNK are capable of dissolving thrombi. [Table 5] Time required for each protein to dissolve 50% of a thrombus at a concentration of 7.0 nM JPEG0007863710000010.jpg101170

[0110] Example 3: Inhibition of platelet aggregation Ten ml of venous blood is collected from individuals who have not received any drug treatment for at least two weeks, and mixed with 3.13% sodium citrate (pH 7.4) in a 9:1 ratio. The blood sample is centrifuged at 1000 rpm for 10 minutes, and the supernatant is collected to obtain platelet-rich plasma (PRP). The remaining portion is centrifuged again at 4000 rpm for 10 minutes, and the supernatant is collected to obtain platelet-poor plasma (PPP). Proteins are also dissolved in R+E buffer (Tris 2.5 mM, sodium chloride 1.5 mM, arginine 50 mM, and glutamate 50 mM) based on different target concentrations. Next, 190 μL of PRP is mixed with 10 μL of PBS buffer or 10 μL of protein solution, and the mixture is incubated at 37°C for 1 minute using a platelet agglutinator (HTracer 601, Nikoh Bioscience, Tokyo, Japan). 10 μL of 200 μM adenosine diphosphate is then added, and the platelet aggregation reaction is monitored by light transmission. The obtained platelet aggregation data represents the mean percentage of inhibition relative to the control value.

[0111] Table 6 shows the platelet aggregation inhibitory activity of different proteins. Compared to the known platelet aggregation inhibitor, the trimucrine variant RR, each fusion protein exhibits low platelet aggregation inhibitory activity. [Table 6] Half-inhibitory concentrations of each protein against platelet aggregation JPEG0007863710000011.jpg98170

[0112] In summary, the fusion protein according to the present invention has been confirmed to have the potential to be a candidate thrombolytic drug because it reduces blood clots while lowering the risk of bleeding.

[0113] Although embodiments of the present invention have been described in detail above with reference to the drawings, the present invention is not limited thereto, and various modifications are possible within the scope described. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

Claims

1. Tissue plasminogen activator or its variants, Disintegrin or its variants, A fusion protein comprising a tissue plasminogen activator or a variant thereof, and a conjugate that binds the disintegrin or a variant thereof and contains an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, The fusion protein is characterized by containing an amino acid sequence shown in any one of SEQ ID NOs: 35 to 42.

2. The fusion protein according to claim 1, A pharmaceutical composition characterized by comprising a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2, characterized in that it is used in the preparation of a medicine that treats or prevents diseases related to the formation of blood clots and reduces the risk of bleeding.

Images