Fusion proteins containing tissue plasminogen activator or variants thereof targeting integrin alpha IIb beta 3 and uses thereof

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, in particular to fusion proteins comprising tissue plasminogen activator or variants thereof that target integrin αIIbβ3, and uses of the proteins, including methods for treating or preventing diseases associated with thrombosis. [Background technology]

[0002] A thrombus is a coagulated blood clot formed by the abnormal accumulation of platelets and fibrin in blood vessels. It obstructs or blocks blood flow in the circulatory system, preventing normal blood supply to various parts of the body and affecting normal physiological functions. Myocardial infarction, cerebral embolism, pulmonary embolism, deep vein thrombosis, and embolism of surrounding blood vessels are common diseases caused by thrombi. When severe, they threaten human health, with extremely high incidence rates, sequelae, and mortality rates. According to statistics from the World Health Organization, approximately 26 million people worldwide die each year from thrombus-related diseases, a rate significantly higher than other causes of death.

[0003] Drugs that reduce blood clots can be divided into at least three categories based on their mechanisms: antiplatelet drugs, anticoagulants, and thrombolytics. Antiplatelet drugs can prevent platelets from clotting and forming blood clots, and common examples include aspirin, clopidogrel, and ticagrelor. Anticoagulants can prolong the time it takes for a clot to form by interfering with blood proteins, and common examples include warfarin, rivaroxaban, and heparin. Thrombolytics can dissolve clots, and common examples include streptokinase, urokinase, and tissue plasminogen activator (tPA). Although these drugs can be used to treat blood clots, they can cause bleeding side effects and can lead to other serious bleeding-related illnesses, such as cerebral hemorrhage. Therefore, researchers are currently seeking a balanced approach that can reduce blood clots while also addressing the bleeding problem.

[0004] Tissue plasminogen activator (TPA) is a serine protease that binds to fibrin and converts plasminogen to plasmin. Plasmin is one of the main enzymes responsible for breaking down clots in blood. Genetic engineering allows the production of TPA in vitro, and these recombinant products are called recombinant tissue plasminogen activators (rtPAs). These recombinant products have been modified in various sequences to enhance their pharmacodynamic effects, particularly their circulatory half-life, and to further enhance their specificity for fibrin, thereby preventing excessive fibrinolysis. Common recombinant drugs include alteplase, reteplase, and tenecteplase (TNK).

[0005] Alteplase has the same sequence as wild-type tissue plasminogen activator (TPA) 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-segment elevation myocardial infarction, and acute massive pulmonary embolism. It can also be administered to patients with central venous access devices.

[0006] Reteplase is a non-glycosylated form of recombinant tissue plasminogen activator (TPA), containing 355 amino acids from the original protein and synthesized in Escherichia coli (E. coli). Compared to alteplase, reteplase has a longer half-life of approximately 14-18 minutes, allowing for bolus injection rather than the need for intravenous infusion. Reteplase is currently available for use in the treatment of acute myocardial infarction.

[0007] Tenecteplase is another modified form of human tissue plasminogen activator (TPA), obtained by expression in mammalian cells (e.g., CHO cells). Tenecteplase is a 527-amino acid glycoprotein obtained by modifying the cDNA of the wild-type human TPA by the following modifications: a threonine-to-asparagine substitution at position 103, a glutamine-to-asparagine substitution at position 117, and four tetraalanine substitutions at positions 296-299. The first two modifications occur in the kringle domain, and the final 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, achieving the goal of developing a new thrombolytic agent that can reduce the risk of bleeding and reduce thrombus formation is one of the challenges that people in the technical field to which this invention pertains are actively striving to solve. Summary of the Invention

[0009] The present invention was made based on the following discovery: By linking tissue plasminogen activator and disintegrin with a linker having a different amino acid sequence, the inhibitory activity of disintegrin on platelet aggregation is reduced while the inherent thrombolytic activity of tissue plasminogen activator is maintained. In this way, a candidate thrombolytic drug capable of reducing thrombus formation while lowering the risk of bleeding is provided.

[0010] Thus, 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 links the tissue plasminogen activator or a variant thereof with the disintegrin or variant thereof and that includes an amino acid sequence shown in any one of SEQ ID NOs:1 to 7.

[0011] Illustratively, the C-terminus of the tissue plasminogen activator or a variant thereof is bound to the N-terminus of the binder, and the N-terminus of the disintegrin or a variant thereof is bound to the C-terminus of the binder.

[0012] Illustratively, the C-terminus of the disintegrin or variant thereof is bound to the N-terminus of the linker, and the N-terminus of the tissue plasminogen activator or variant thereof is bound to the C-terminus of the linker.

[0013] Exemplary tissue plasminogen activators are alteplase, reteplase, or tenecteplase.

[0014] Exemplary, the tissue plasminogen activator is tenecteplase.

[0015] Illustratively, the tissue plasminogen activator comprises an amino acid sequence set forth in any one of SEQ ID NOs: 8-10.

[0016] Exemplary, the tissue plasminogen activator comprises the amino acid sequence shown in SEQ ID NO:10.

[0017] Exemplary disintegrins include albolabrin, aplagin, basilicin, batroxostatin, bitistatin, cereberin, cerastin, crotatroxin, durissin, elegantin, eristicophin, flavoridin, flavostatin, halysin, halistatin, and the like. halystatin, jararacin, jarastatin, kistrin, lachesin, lutosin, molossin, rhodostomin, salmosin, saxatilin, tergeminin, trimestatin, trimucrin, trimutase, ussuristatin, or viridian.

[0018] Exemplary disintegrins are rhodostomin or trimuculin.

[0019] Exemplarily, the disintegrin mutant comprises a binding region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 to 15, an RGD motif comprising an amino acid sequence set forth in any one of SEQ ID NOs: 16 to 28, and a C-terminal region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 29 to 33.

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

[0021] Exemplarily, the binding region comprises the amino acid sequence shown in SEQ ID NO:11.

[0022] Exemplarily, the RGD motif comprises an amino acid sequence set forth in any one of SEQ ID NOs: 17-21, 24-26.

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

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

[0025] Exemplary disintegrin mutants include the amino acid sequence shown in SEQ ID NO:34.

[0026] Illustratively, the fusion protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 35-42.

[0027] Illustratively, fusion proteins have been used to bind to integrin αIIbβ3 and fibrin.

[0028] Because fibrin is one of the major components of a clot, the fusion protein of the present invention can bind to fibrin at the site of a thrombus and convert plasminogen to plasmin, which then dissolves fibrin into FDP (fibrin-fibrinogen degradation product), thereby achieving a thrombolytic effect. Furthermore, because integrin αIIbβ3 is abundantly expressed on platelets and their precursor cells (see J Hematol Oncol. 2019 Mar 7;12(1):26), the fusion protein of the present invention can simultaneously bind to platelet integrin αIIbβ3 at the site of a thrombus, inhibiting platelet aggregation and preventing the formation of large thrombi. Furthermore, because the fusion protein of the present invention has a lower inhibitory activity against platelet aggregation than disintegrins or their variants, it can reduce the risk of bleeding. Therefore, the fusion protein of the present invention reduces thrombus formation while reducing the risk of bleeding, making it a thrombolytic agent with extremely high potential clinical utility.

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

[0030] Illustratively, the pharmaceutical composition is an oral, injectable, inhaled, or topical or transdermal formulation.

[0031] Illustratively, the molar concentration of the fusion protein ranges from 1 to 1400 nM based on the total volume of the pharmaceutical composition.

[0032] The present invention further provides the use of the pharmaceutical composition in the preparation of a medicament for treating or preventing a disease associated with thrombus formation and reducing the risk of bleeding events.

[0033] Exemplarily, diseases associated with the formation of thrombi are venous thrombosis or arterial thrombosis.

[0034] Exemplary venous thrombosis includes 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 (Paget-Schroetter disease), parodoxical embolism, portal vein thrombosis, pulmonary embolism, and renal vein thrombosis. thrombosis, or splenic vein thrombosis.

[0035] Exemplarily, arterial thrombosis is hepatic artery thrombosis, limb ischemia, myocardial infarction, or stroke.

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

[0037] Exemplarily, diseases associated with the formation of thrombi are venous thrombosis or arterial thrombosis.

[0038] Exemplary venous thrombosis is 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] Exemplarily, the arterial thrombosis is hepatic artery thrombosis, lower limb ischemia, myocardial infarction, or stroke.

[0040] Illustratively, 0.1 to 1000 mg of the fusion protein per kg of the individual's body weight is administered to the individual.

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

[0042] The present invention further provides a host cell comprising said nucleic acid.

[0043] Illustratively, the host cell is a prokaryotic or eukaryotic cell.

[0044] Illustratively, the prokaryotic cell is E. coli.

[0045] Exemplary eukaryotic cells are CHO cells, COS cells, or HEK293 cells.

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

[0047] [Figure 1] 1 is a flow chart showing a schematic diagram of the production of a DNA construct for expressing the protein TNK-G9-RR. [Figure 2A] 1 is a liquid chromatogram showing the isolation results of the protein TNK. [Figure 2B]1 is a liquid chromatogram showing the isolation results of the protein TNK-G9-RR. [Figure 2C] 1 is a liquid chromatogram showing the isolation results of the protein TNK-(G4S)3-RR. [Figure 2D] 1 is a liquid chromatogram showing the isolation results of the protein TNK-(PA)3-RR. [Figure 2E] 1 is a liquid chromatogram showing the isolation results of the protein TNK-(PA)5-RR. [Figure 2F] 1 is a liquid chromatogram showing the isolation results of the protein TNK-(PA)7-RR. [Figure 2G] 1 is a liquid chromatogram showing the isolation results of the protein TNK-EA3K(G4S)2-RR. [Figure 2H] 1 is a liquid chromatogram showing the isolation results of the protein TNK-(EA3K)3-RR. [Figure 2I] 1 is a liquid chromatogram showing the isolation results of the protein RR-(PA)5-TNK. [Figure 3A] These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE showing the isolation results of the protein TNK, respectively. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3B] These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE showing the isolation results of the protein TNK, respectively. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3C]These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-G9-RR. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3D] These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-G9-RR. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3E] These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE showing the isolation results of the protein TNK-(G4S)3-RR, respectively. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3F] These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE showing the isolation results of the protein TNK-(G4S)3-RR, respectively. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3G] These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(PA)3-RR. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3H] These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(PA)3-RR. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3I] These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE showing the isolation results of the protein TNK-(PA)5-RR, respectively. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3J] These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE showing the isolation results of the protein TNK-(PA)5-RR, respectively. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3K] These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(PA)7-RR. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3L]These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(PA)7-RR. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3M] These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE showing the isolation results of the protein TNK-EA3K(G4S)2-RR, respectively. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3N] These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE showing the isolation results of the protein TNK-EA3K(G4S)2-RR, respectively. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3O] These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(EA3K)3-RR. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3P]These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE, respectively, showing the isolation results of the protein TNK-(EA3K)3-RR. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3Q] These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE showing the isolation results of the protein RR-(PA)5-TNK, respectively. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 3R] These are photographs of non-reducing Tris-glycine SDS-PAGE and reducing Tris-glycine SDS-PAGE showing the isolation results of the protein RR-(PA)5-TNK, respectively. The symbol "M" indicates a protein marker, "LS" indicates a load sample (20 μL), "FT" indicates a flow-through sample (250 μL), and "Arabic numerals" indicate the aliquot numbers. [Figure 4A] Thrombolysis results chart illustrating the thrombolysis rate of protein TNK at different concentrations. [Figure 4B] Thrombolysis result chart illustrating the thrombolysis rate of protein TNK-G9-RR at different concentrations. [Figure 4C] Thrombolysis result chart illustrating the thrombolysis rate of the protein TNK-(G4S)3-RR at different concentrations. [Figure 4D] Thrombolysis result chart illustrating the thrombolysis rate of protein TNK-(PA)3-RR at different concentrations. [Figure 4E] Thrombolysis result chart illustrating the thrombolysis rate of protein TNK-(PA)5-RR at different concentrations. [Figure 4F]Thrombolysis result chart illustrating the thrombolysis rate of protein TNK-(PA)7-RR at different concentrations. [Figure 4G] Thrombolysis result chart illustrating the thrombolysis rate of protein TNK-EA3K(G4S)2-RR at different concentrations. [Figure 4H] Thrombolysis result chart illustrating the thrombolysis rate of the protein TNK-(EA3K)3-RR at different concentrations. [Figure 4I] Thrombolysis result chart illustrating the thrombolysis rate of protein RR-(PA)5-TNK at different concentrations. [Figure 5] 1 is a bar graph illustrating the time to 50% thrombolysis for different proteins at a concentration of 7.0 nM. DETAILED DESCRIPTION OF THE INVENTION

[0048] In order to clarify and facilitate understanding of the above-mentioned and / or other objects, advantages, and features of the present invention, preferred embodiments are given below and described in detail. 1. Definitions Unless otherwise specified, the term "protein" as used herein includes wild-type proteins expressed in natural cells, recombinant proteins expressed using genetic engineering techniques, and synthetic proteins obtained chemically. It is also possible to substitute or delete at least one amino acid in the protein sequence and / or insert at least one amino acid without affecting the original activity.

[0049] The term "amino acid" used herein includes D-amino acids and L-amino acids unless otherwise defined. D- and L- refer to the absolute configuration of an amino acid, not a specific rotational direction of plane-polarized light. Except in special circumstances, this specification uses the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission to represent amino acids. Protein sequences are represented by a string of one-letter symbols, and the order of the one-letter symbols corresponds to the order of amino acids from the N-terminus to the C-terminus of the protein. When a superscript number precedes a one-letter symbol, it indicates the order of the position of the corresponding amino acid, counting from the N-terminus of the protein. For example, 67 PRNGLYG indicates that a proline is located at position 67 of the protein, and the rest can be inferred in a similar manner, so the explanation will not be repeated. A subscript number after a single-letter symbol indicates the number of overlaps of the corresponding amino acid or corresponding amino acid group. For example, G9 indicates that nine consecutive glycines are linked. For another example, (G4S)3 indicates that an amino acid group consisting of three consecutive glycine-glycine-glycine-glycine-serine pairs is linked, and the rest can be inferred in a similar manner, so the explanation will not be repeated.

[0050] Substitutions, deletions, and / or insertions in the protein sequence may occur in non-functional regions of the protein and generally do not affect the original activity. Substitutions in the protein sequence may also include conservative amino acid substitutions, which refer to substitutions between amino acids having similar properties or related side chains. Substitutions between amino acids with similar properties refer to, for example, the mutual substitution of acidic amino acids such as aspartic acid and glutamic acid, the mutual substitution of alkaline amino acids such as lysine, arginine, and histidine, the mutual substitution of nonpolar amino acids such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan, and the mutual substitution of uncharged polar amino acids such as glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Substitutions between amino acids having related side chains refer to, for example, aliphatic hydroxy amino acids such as serine and threonine, which can be substituted for each other; amide-containing amino acids such as asparagine and glutamine, which can be substituted for each other; aliphatic amino acids such as alanine, valine, leucine, and isoleucine, which can be substituted for each other; and aromatic amino acids such as phenylalanine, tryptophan, and tyrosine, which can be substituted for each other.

[0051] Unless otherwise specified, the term "tissue plasminogen activator" as used herein includes wild-type or recombinant tissue plasminogen activator, where the wild-type is, for example, tissue plasminogen activator produced by human vascular endothelial cells, and the recombinant is, for example, alteplase (SEQ ID NO:8), reteplase (SEQ ID NO:9), or tenecteplase (SEQ ID NO:10).

[0052] Unless otherwise specified, the term "disintegrin" as used herein refers to a platelet aggregation inhibitor extracted from the saliva of venomous snakes, and typically contains 47 to 84 amino acids and 4 to 7 pairs of disulfide bonds, such as alborabulin, aplagin, bacilysin, batroxostatin, bitistatin, cerebellin, cerastin, crotatoxin, dulicin, elegantin, erythicopin, flavoridin, flavostatin, halicin, halistatin, jaralacin, jarastatin, kistrin, lachesin, lutosin, morosin, rhodostomin, salmosin, saxatilin, tergeminin, trimestatin, trimuculin, trimutase, usuristatin, or viridian.

[0053] Unless otherwise specified, the term "variant" as used herein refers to a modified protein obtained by substituting or deleting at least one amino acid and / or inserting at least one amino acid in a reference sequence without affecting the original activity. For example, disintegrin mutants include rhodostomin mutants and trimuculin mutants, which inhibit platelet aggregation by binding integrin αIIbβ3 to wild-type disintegrins. The variants share at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, or 60% sequence similarity with the reference sequence, where similarity is defined as gap-excluded identity, BLAST identity, or gap-compressed identity, depending on different criteria. BLAST similarity was calculated using the Basic Local Alignment Search Tool provided by the National Center for Biotechnology Information. In one example, there is at least 95% BLAST similarity between the trimuculin mutant RR described herein and wild-type trimuculin.

[0054] Unless otherwise defined, the term "RGD motif" as used herein refers to a flexible loop region in a disintegrin that is composed of arginine-glycine-aspartic acid, which is an integrin-binding region. For example, the RGD motif of wild-type trimucrin is 50 ARGDNP, and the RGD motif of wild-type rhodostomin 48 As mentioned above, the RGD motif of the trimucrine mutant contains the RGD motif sequence of wild-type trimucrine. 50 The RGD motif of the rhodostomin variant has at least one amino acid mutation with respect to ARGDNP, such as SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, which does not affect function.48 It has at least one amino acid mutation relative to PRGDMP, such as SEQ ID NO: 27 or 28, and does not affect function.

[0055] Unless otherwise defined, the "binding region" used herein refers to the region adjacent to the N-terminus of the RGD motif in a disintegrin, and is usually any continuous fragment of amino acids from positions 38 to 49. For example, the binding region of wild-type trimucrin is 41 KKKRT (SEQ ID NO: 11), and the binding domain of wild-type rhodostomin 39 As mentioned above, the binding region of the trimuculin mutant contains the binding region sequence of wild-type trimuculin. 41 KKKRT, having at least one amino acid mutation, such as SEQ ID NO: 13, 14, or 15, and not affecting function.

[0056] Unless otherwise specified, the "C-terminal region" used herein refers to the region adjacent to the C-terminus of the RGD motif in a disintegrin. For example, the C-terminal region of wild-type trimucrin is 67 PRNGLYG (SEQ ID NO: 29), and the C-terminal region of wild-type rhodostomin is 65 PRYH (SEQ ID NO: 30). As mentioned above, the C-terminal region of the trimuculin mutant is the same as the C-terminal region of wild-type trimuculin. 67 It has at least one amino acid mutation relative to PRNGLYG, such as SEQ ID NO: 31, 32, or 33, and does not affect function.

[0057] Unless otherwise defined, the term "disintegrin mutant" as used herein includes sequences obtained by mutation of at least one amino acid in the wild-type RGD motif, sequences obtained by mutation of at least one amino acid in the wild-type binding region, and / or sequences obtained by mutation of at least one amino acid in the wild-type C-terminal region, for example, SEQ ID NO:34.

[0058] Unless otherwise defined, the term "treatment" as used herein refers to curing or ameliorating thrombosis through therapeutic intervention, i.e., including complete or local cure or amelioration.

[0059] Unless otherwise defined, the term "prevention" as used herein refers to complete or almost complete prevention of thrombosis. For example, when there is no thrombus or there is only minor thrombus but it has not progressed to the point of causing disease, preventive intervention can be performed to prevent the onset of disease.

[0060] As used herein, unless otherwise defined, the term "pharmaceutically acceptable carrier" refers to an excipient that, within the scope of sound medical judgment, is suitable for contact with individuals and is not excessively toxic, irritating, allergic, or presents other problems or complications, and has a reasonable risk-benefit ratio, such as a filler, diluent, flocculating agent, adhesive, lubricant, flow agent, stabilizer, colorant, humectant, or disintegrant.

[0061] II. Fusion proteins 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 a thrombus. By binding to fibrin, it converts plasmin into plasmin, which then dissolves the fibrin into FDP, thereby achieving the effect of dissolving the thrombus. Furthermore, by binding to integrin αIIbβ3, it inhibits platelet aggregation, thereby 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 a protein containing only a disintegrin or its variant, thereby reducing the risk of bleeding. Based on these properties, the fusion protein according to this embodiment can be used to dissolve thrombi and reduce the risk of bleeding. That is, it can be used to treat thrombosis and reduce the risk of bleeding.

[0062] The fusion protein according to this embodiment comprises a tissue plasminogen activator or a variant thereof, a disintegrin or a variant thereof, and a binder. The binder connects the tissue plasminogen activator or a variant thereof and the disintegrin or variant thereof and comprises the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7. The fusion protein sequentially comprises, from the N-terminus to the C-terminus, a tissue plasminogen activator or a variant thereof, a binder, and a disintegrin or a variant thereof, or a disintegrin or a variant thereof, a binder, and a tissue plasminogen activator or a variant thereof. Specifically, the C-terminus of the tissue plasminogen activator or a variant thereof is bound to the N-terminus of the binder, and the N-terminus of the disintegrin or a variant thereof is bound to the C-terminus of the binder. Alternatively, the C-terminus of the disintegrin or a variant thereof is bound to the N-terminus of the binder, and the N-terminus of the tissue plasminogen activator or a variant thereof is bound to the C-terminus of the binder. As used herein, "linkage" is not limited to direct or indirect linkage, i.e., there may or may not be other linking fragments between the linked proteins. Preferably, the fusion protein comprises an amino acid sequence as set forth in SEQ ID NO:35, 36, 37, 38, 39, 40, 41, or 42.

[0063] The tissue plasminogen activator, based on type, is alteplase, reteplase, or tenecteplase, preferably tenecteplase.

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

[0065] Disintegrins, based on type, include alborabulin, aplagin, bacilysin, batroxostatin, bitistatin, cerebellin, selastin, crotatoxin, dulicin, elegantin, erythicopin, flavoridin, flavostatin, halicin, halistatin, jararasin, jarastatin, kistrin, lachesin, lutosin, morosin, rhodostomin, salmosin, saxatilin, tergeminin, trimestatin, trimuculin, trimutase, usuristatin, and viridian. Preferably, the disintegrin is rhodostomin or trimuculin.

[0066] Based on the sequences, the disintegrin mutant comprises a binding region, an RGD motif, and a C-terminal region: the binding region comprises the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14, or 15; the RGD motif comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 29, 30, 31, 32, or 33.

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

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

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

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

[0071] The fusion protein of this embodiment is prepared by genetic engineering or chemical methods, such as solid-phase synthesis or liquid-phase synthesis, followed by isolation or purification using methods such as ammonium sulfate or ethanol precipitation, acid extraction, ion exchange chromatography, affinity chromatography, or lectin chromatography. High-performance liquid chromatography is preferred.

[0072] The fusion protein of this embodiment further comprises a hydrophilic group, thereby increasing its water solubility or circulating half-life. The hydrophilic group can be attached to the N-terminus of the fusion protein. Preferably, the hydrophilic group is polyethylene glycol, polypropylene glycol, polylactic acid, polyglycolic acid, polyvinyl alcohol, or dextran. More preferably, the hydrophilic group is polyethylene glycol composed of 2 to 20 overlapping ethylene glycol units.

[0073] The fusion protein of this embodiment further comprises a purification tag to facilitate purification. The purification tag can be attached 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 / antitermination protein tag (NusA-tag), or a small ubiquitin-like modifier tag (SUMO-tag).

[0074] 3. Pharmaceutical Compositions The pharmaceutical composition according to the second embodiment of the present invention comprises the fusion protein according to the first embodiment, and can be administered to an individual in need of thrombolysis, dissolving thrombi while simultaneously reducing the risk of bleeding. The pharmaceutical composition according to this embodiment comprises the fusion protein according to the first embodiment and a pharmaceutically acceptable carrier.

[0075] Generally, the pharmaceutically acceptable carrier allows the pharmaceutical composition to be in different forms or to be administered via different routes. Preferably, the pharmaceutical composition is an oral, injectable, inhalant, or topical or transdermal formulation, and is used for different routes of administration. Preferably, the pharmaceutical composition is a tablet, capsule, granule, dispersion, solution, syrup, suspension, or emulsion. The pharmaceutical composition according to this embodiment can be applied to implantable medical devices such as stents and catheters, preventing the implantable device from narrowing blood vessels or supporting and strengthening blood vessels while simultaneously dissolving thrombus. The pharmaceutical composition according to this embodiment may further contain other drugs that reduce thrombus formation, such as antiplatelet drugs or anticoagulants. Examples of antiplatelet drugs include aspirin, clopidogrel, or ticagrelor, and examples of anticoagulants include warfarin, rivaroxaban, or heparin.

[0076] The pharmaceutically acceptable carrier may be an excipient, filler, diluent, flocculating agent, adhesive, lubricant, flow agent, stabilizer, colorant, moisturizer, or disintegrant. Examples of excipients include sodium citrate, calcium carbonate, or calcium phosphate. Examples of fillers include lactose and 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 the fluidizing agent may be 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 the stabilizer may be citric acid or ascorbic acid. Examples of the colorant may be titanium dioxide or iron oxide. Examples of the humectant may be Pluronic F68, TWEEN 20, or TWEEN 80. Examples of the disintegrant may be potato starch, tapioca starch, or silicates.

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

[0078] 4. Medicinal uses A third embodiment of the present invention provides a use of the pharmaceutical composition according to the second embodiment for preparing a medicament for treating or preventing a disease associated with thrombus formation and reducing the risk of bleeding. The prepared medicament can be administered to an individual in need of thrombus dissolution, and dissolves the thrombus while simultaneously reducing the risk of bleeding. That is, the prepared medicament can be administered to an individual in need of treatment or prevention of a disease associated with thrombus formation, and achieves the therapeutic or preventive effect while simultaneously reducing the risk of bleeding.

[0079] Different administration routes may be used, such as oral administration, injection administration, inhalation administration, or topical or transdermal administration, and an effective amount of the fusion protein is administered to an individual in the range of 0.1 to 1000 mg per kg of body weight of the individual.

[0080] Diseases associated with thrombus formation 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 associated with thrombus formation according to a fourth embodiment of the present invention includes a step of administering the pharmaceutical composition according to the second embodiment to an individual in need of treatment or prevention, thereby dissolving thrombus and reducing the risk of bleeding.

[0082] Different administration routes may be used, such as oral administration, injection administration, inhalation administration, or topical or transdermal administration, and an effective amount of the fusion protein is administered to an individual in the range of 0.1 to 1000 mg per kg of body weight of the individual.

[0083] Diseases associated with thrombus formation 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] V. Other matters A nucleic acid according to a fifth embodiment of the present invention comprises a nucleotide sequence encoding the fusion protein according to the first embodiment. The nucleic acid further comprises a promoter operably linked to the nucleotide sequence encoding the fusion protein to regulate expression of the protein. As used herein, "operably linked" refers to a functional relationship between two or more nucleic acid sequences.

[0085] A host cell according to a sixth embodiment of the present invention includes the nucleic acid according to the fifth embodiment. Because the host cell according to this embodiment includes a nucleotide sequence encoding 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. An example of a prokaryotic cell is E. coli, and an example of a eukaryotic cell is CHO cells, COS cells, or HEK293 cells.

[0086] A seventh embodiment of the present invention provides a method for preparing the fusion protein according to the first embodiment, comprising the step of expressing the fusion protein by culturing the host cell according to the sixth embodiment. The cells are induced to express the protein by selecting an appropriate inducer depending on the promoter.

[0087] The following examples are provided to illustrate and explain the present invention. Example 1: Protein preparation The expression construct is transfected into CHO cells or yeast cells to express the recombinant protein, and the cell culture supernatant is collected and purified by liquid chromatography to obtain the recombinant protein.

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

[0089] By following the flow chart, except that TNK-G9-R is replaced by primer TNK-(G4S)3-R and RR-G9-F is replaced by primer RR-(G4S)3-F, the plasmid pcDNA3.1 containing the TNK-(G4S)3-RR nucleotide fragment is obtained as the expression construct for the protein TNK-(G4S)3-RR.

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

[0091] By following the flow chart, except that TNK-G9-R is replaced by primer TNK-(PA)5-R and RR-G9-F is replaced by primer RR-(PA)5-F, the plasmid pcDNA3.1 containing the TNK-(PA)5-RR nucleotide fragment is obtained as the expression construct for the protein TNK-(PA)5-RR.

[0092] By following the flow chart, except that TNK-G9-R is replaced by primer TNK-(PA)7-R and RR-G9-F is replaced by primer RR-(PA)7-F, the plasmid pcDNA3.1 containing the TNK-(PA)7-RR nucleotide fragment is obtained as the expression construct for the protein TNK-(PA)7-RR.

[0093] By following the flow chart, except that TNK-G9-R is replaced by primer TNK-EA3K(G4S)2-R and RR-G9-F is replaced by primer RR-EA3K(G4S)2-F, the plasmid pcDNA3.1 containing the TNK-EA3K(G4S)2-RR nucleotide fragment is obtained as the expression construct for the protein TNK-EA3K(G4S)2-RR.

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

[0095] Table 1 lists the nucleotide sequences of the primers. [Table 1] Primer sequences JPEG2025532642000002.jpg170170

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

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

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

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

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

[0101] Figure 2F shows the results of liquid chromatography analysis of protein TNK-(PA)7-RR. Figures 3K and 3L further demonstrate that protein TNK-(PA)7-RR was obtained from fractions 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 fractions 27, 28, 29, 30, and 31.

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

[0104] Figure 2I shows the 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 fractions 21, 22, 23, and 24.

[0105] Table 2 lists the amino acid sequences of the proteins, and Table 3 lists the yields of each protein. It can be seen that consistent yields were achieved for protein TNK, protein RR, and other fusion proteins containing TNK. [Table 2] Protein sequence JPEG2025532642000003.jpg248170JPEG2025532642000004.jpg255169JPEG2025532642000005.jpg255169JPEG2025532642000006.jpg255168JPEG2025532642000007.jpg255167JPEG2025532642000008.jpg244170Note: The character symbols in the square frames are an array of combinators [Table 3] Protein yield JPEG2025532642000009.jpg219170

[0106] Example 2: Thrombolysis test A whole blood thrombolysis plate assay is performed. Blood is drawn from a healthy individual and mixed with 3.8% trisodium citrate in a 9:1 ratio. Thrombin (6.25 x 10) is added to a HEPES buffer (25 mM HEPES, 137 mM sodium chloride). -3Add 250 mM of HCl (1000 μL) and calcium chloride (250 mM) to obtain a clotting mixture. 5 μL of the clotting mixture is deposited on the bottom edge of each well of a 96-well microplate, and then 25 μL of the blood mixture is added. The microplate is sealed and incubated at 37°C for 30 minutes to form a clot on the bottom edge of the well.

[0107] Each protein was prepared in 70 μL of HEPES solution at different target concentrations. The protein solution was added to the wells containing the clots at room temperature and placed in an ELISA plate reader for 120 minutes at a constant temperature of 37°C. During the reaction, the plate was vibrated once per minute (200 rpm), and the absorbance at 510 nm was measured every 3 minutes using the ELISA plate reader. During the reaction, the dissolved clots flowed to cover the center of the well, and the time required for 50% dissolution of the clots was determined by the absorbance at 510 nm (T0.5, minutes). This determined the degree of clot dissolution.

[0108] Figures 4A to 4I show the thrombolytic activity of each protein at different concentrations, and Table 4 shows the time required for each protein to dissolve 50% of the thrombus at different concentrations. Table 4: Time required for each protein to dissolve 50% of the clot at different concentrations JPEG2025532642000010.jpg78170ND: The clot was not dissolved within 120 minutes, making it impossible to measure.

[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 can dissolve the thrombus. [Table 5] Time required for each protein to dissolve 50% of the thrombus at a concentration of 7.0 nM JPEG2025532642000011.jpg101170

[0110] Example 3: Platelet aggregation inhibition test Ten milliliters of venous blood was collected from individuals who had not received any drug treatment for at least two weeks and mixed with 3.13% sodium citrate (pH 7.4) at a ratio of 9:1. The blood sample was centrifuged at 1,000 rpm for 10 minutes, and the supernatant was collected to obtain platelet-rich plasma (PRP). The remaining portion was centrifuged again at 4,000 rpm for 10 minutes, and the supernatant was collected to obtain platelet-poor plasma (PPP). Proteins were also dissolved in R+E buffer (Tris 2.5 mM, sodium chloride 1.5 mM, arginine 50 mM, and glutamic acid 50 mM) based on different target concentrations. Then, 190 μL of PRP was mixed with 10 μL of PBS buffer or 10 μL of protein solution and incubated at 37°C for 1 minute using an aggregometer (HTracer 601, Nikoh Bioscience, Tokyo, Japan). 10 μL of 200 μM adenosine diphosphate was added, and the platelet aggregation reaction was monitored by light transmission. The acquired platelet aggregation data represent the mean percent inhibition relative to the control value.

[0111] Table 6 shows the platelet aggregation inhibitory ability of different proteins. Compared to the known platelet aggregation inhibitor trimucrine mutant RR, each fusion protein has low platelet aggregation inhibitory ability. [Table 6] Median inhibitory concentration of each protein for platelet aggregation JPEG2025532642000012.jpg98170

[0112] Taken together, the fusion protein of the present invention is established to have potential as a candidate thrombolytic drug, since it reduces thrombus formation while lowering the risk of bleeding events.

[0113] Although the embodiments of the present invention have been described in detail above with reference to the drawings, the present invention is not limited to these, and various modifications are possible within the scope of the description. 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 a variant thereof; a disintegrin or a mutant thereof; A fusion protein comprising the tissue plasminogen activator or a variant thereof and a connector that binds the disintegrin or a variant thereof and that comprises an amino acid sequence shown in any one of SEQ ID NOs: 1 to 7.

2. The fusion protein of claim 1, wherein the C-terminus of the tissue plasminogen activator or its variant is bound to the N-terminus of the linker, and the N-terminus of the disintegrin or its variant is bound to the C-terminus of the linker, or the C-terminus of the disintegrin or its variant is bound to the N-terminus of the linker, and the N-terminus of the tissue plasminogen activator or its variant is bound to the C-terminus of the linker.

3. 2. The fusion protein of claim 1, wherein the tissue plasminogen activator is alteplase, reteplase, or tenecteplase.

4. The fusion protein according to claim 1, wherein the tissue plasminogen activator comprises an amino acid sequence shown in any one of SEQ ID NOs: 8 to 10.

5. The disintegrins include albolabrin, aplagin, basilicin, batroxostatin, bitistatin, cereberin, cerastin, crotatroxin, durissin, elegantin, eristicophin, flavoridin, flavostatin, halysin, halystatin, diaminidine, and phenotype.

2. The fusion protein according to claim 1, which is jararacin, jarastatin, kistrin, lachesin, lutosin, molossin, rhodostomin, salmosin, saxatilin, tergeminin, trimestatin, trimucrin, trimutase, ussuristatin, or viridian.

6. The disintegrin mutant is a binding region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 to 15; an RGD motif comprising an amino acid sequence shown in any one of SEQ ID NOs: 16 to 28; The fusion protein according to claim 1, further comprising a C-terminal region comprising an amino acid sequence shown in any one of SEQ ID NOs: 29 to 33.

7. The fusion protein of claim 1, wherein the disintegrin mutant comprises the amino acid sequence shown in SEQ ID NO:

34.

8. The fusion protein according to claim 1, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 35 to 42.

9. A fusion protein according to any one of claims 1 to 8; and a pharmaceutically acceptable carrier.

10. 10. The pharmaceutical composition according to claim 9, characterized in that it is used for preparing a medicament for treating or preventing diseases associated with the formation of thrombi and reducing the risk of bleeding.

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