Targeted integrin alpha IIb beta 3 fusion protein containing tissue plasminogen activator or variant thereof and application thereof

Abstract

The present invention provides a fusion protein comprising: a tissue plasminogen activator or a variant thereof; a disintegrant or a variant thereof; and a connector; a linker connects a tissue plasminogen activator or a variant thereof and a disintegrant protein or a variant thereof, and includes an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7. The invention also provides a method for treating or preventing diseases related to thrombosis by using the fusion protein.

Description

Fusion protein containing tissue plasminogen activator or its variant targeting integrin αIIbβ3 and application thereof Technical Field

[0001] The present invention relates to a fusion protein, and particularly to a fusion protein containing tissue plasminogen activator or a variant thereof targeting integrin αIIbβ3 and the application of the protein, including a method for treating or preventing diseases related to thrombosis. Background Art

[0002] Thrombosis is a blood clot formed by the abnormal accumulation of platelets and fibrin in blood vessels. It can hinder or block blood flow in the circulatory system, resulting in a lack of blood supply to various parts of the body, which in turn affects normal physiological functions. Myocardial infarction, cerebral embolism, pulmonary embolism, deep vein thrombosis, and peripheral vascular embolism are common diseases caused by thrombosis. They are serious threats to human health, with high morbidity, disability, and mortality rates. According to statistics from the World Health Organization, approximately 26 million people die from thrombosis-related diseases each year worldwide, far more than all other causes of death.

[0003] Medications that reduce blood clots are categorized by their mechanism of action: antiplatelet drugs, anticoagulants, and thrombolytics. Antiplatelet drugs, such as aspirin, clopidogrel, and ticagrelor, prevent platelets from clotting. Anticoagulants, such as warfarin, rivaroxaban, and heparin, interfere with blood proteins, prolonging clot formation. Thrombolytics, such as streptokinase, urokinase, and tissue plasminogen activator (tPA), break down clots. While these drugs can be used to treat blood clots, they can also cause bleeding, potentially leading to other serious bleeding-related conditions, such as cerebral hemorrhage. Therefore, researchers are still working to find a balance between reducing blood clots and addressing bleeding.

[0004] Tissue plasminogen activator (tPA) is a serine protease that binds to fibrin and converts plasminogen into plasmin, one of the primary enzymes involved in dissolving blood clots. Genetic engineering techniques have enabled the in vitro production of tPA, a genetically engineered product also known as recombinant tissue plasminogen activator (rtPA). This recombinant product can be modified in various ways to enhance its pharmacokinetic and pharmacodynamic properties, particularly its half-life in the circulation and its specificity for fibrin, thereby preventing unwanted fibrinolysis. Common recombinant drugs include alteplase, reteplase, and tenecteplase (TNK).

[0005] Alteplase shares a sequence with wild-type tissue plasminogen activator (TPA) produced in human vascular endothelial cells and is expressed in Chinese hamster ovary (CHO) cells. With a half-life of approximately 5 minutes, alteplase is indicated for treatment of 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 human tissue plasminogen activator (TPA). It contains all 355 amino acids of the original protein and is synthesized in Escherichia coli (E. coli). Compared to alteplase, reteplase has a relatively long half-life of approximately 14 to 18 minutes, allowing it to be administered by bolus injection rather than by infusion as is required for alteplase. Reteplase is currently indicated for the treatment of acute myocardial infarction.

[0007] Tenecteplase is a modified form of human tissue plasminogen activator (TPA) expressed in mammalian cells (e.g., CHO cells). Tenecteplase is a 527-amino acid glycoprotein derived from the wild-type human TPA cDNA with the following modifications: threonine at position 103 is substituted with asparagine, asparagine at position 117 is substituted with glutamine, and amino acids 296-299 are replaced with four consecutive alanine residues. The first two modifications occur in the kringle domain, while the last modification occurs in the protease domain. Tenecteplase has a longer half-life of approximately 20 to 24 minutes and is suitable for indications such as acute myocardial infarction and pulmonary embolism.

[0008] Therefore, developing a novel thrombolytic agent that can reduce thrombus formation while lowering the risk of bleeding is indeed one of the issues that people in the technical field of the present invention are actively trying to solve.

[0009] Summary of the Invention

[0010] The present invention is based on the discovery that linking tissue plasminogen activator (TPA) to disintegrin via a linker with different amino acid sequences can reduce the platelet aggregation inhibitory activity of disintegrin while retaining the thrombolytic activity of TPA. This provides a candidate thrombolytic drug that can reduce thrombus formation while lowering the risk of bleeding.

[0011] Accordingly, the present invention provides a fusion protein comprising: tissue plasminogen activator or a variant thereof; a disintegrin or a variant thereof; and a linker connecting the tissue plasminogen activator or a variant thereof and the disintegrin or a variant thereof, and comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 7.

[0012] For example, the C-terminus of tissue plasminogen activator or a variant thereof is connected to the N-terminus of the linker, and the N-terminus of disintegrin or a variant thereof is connected to the C-terminus of the linker.

[0013] For example, the C-terminus of the disintegrin or its variant is connected to the N-terminus of the linker, and the N-terminus of the tissue plasminogen activator or its variant is connected to the C-terminus of the linker.

[0014] Illustratively, the tissue plasminogen activator is alteplase, reteplase, or tenecteplase.

[0015] Illustratively, the tissue plasminogen activator is tenecteplase.

[0016] Illustratively, the tissue plasminogen activator comprises an amino acid sequence as shown in any one of SEQ ID NOs: 8 to 10.

[0017] Illustratively, the tissue plasminogen activator comprises the amino acid sequence shown in SEQ ID NO:10.

[0018] For example, the disintegrin is albolabrin, applagin, basilicin, batroxostatin, bitistatin, cereberin, cerastin, crotatroxin, durissin, eleganttin, eristicophin, flavoridin, flavostatin, halysin, halostatin, and halostatin. ystatin, jararacin, jarastatin, kistrin, lachesin, lutosin, molossin, rhodostomin, salmosin, saxatilin, tergeminin, trimestatin, trimucrin, trimutase, ussuristatin, or viridian.

[0019] Illustratively, the disintegrin is agkistrodon or protospikemin.

[0020] For example, the variant of the disintegrin comprises: a linker region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 11 to 15; an RGD structural motif comprising an amino acid sequence as shown in any one of SEQ ID NOs: 16 to 28; and a C-terminal region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 29 to 33.

[0021] Illustratively, the linking region comprises the amino acid sequence shown in SEQ ID NO: 11 or 15.

[0022] Illustratively, the linker region comprises the amino acid sequence shown in SEQ ID NO:11.

[0023] Illustratively, the RGD structural motif comprises an amino acid sequence as shown in any one of SEQ ID NOs: 17 to 21, 24 to 26.

[0024] Illustratively, the RGD structural motif comprises the amino acid sequence shown in SEQ ID NO:20.

[0025] Illustratively, the C-terminal region comprises the amino acid sequence shown in SEQ ID NO: 31.

[0026] Illustratively, the disintegrin variant comprises the amino acid sequence shown in SEQ ID NO:34.

[0027] Illustratively, the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 35 to 42.

[0028] Illustratively, fusion proteins are used to bind to integrin αIIbβ3 and fibrin.

[0029] Since fibrin is one of the main components of blood clots, the fusion protein of the present invention can bind to fibrin at the thrombus site and convert plasminogen into plasmin, which then degrades fibrin into FDP (fibrin-fibrinogen degradation product) to achieve the effect of dissolving the thrombus. In addition, since integrin αIIbβ3 is expressed in large quantities on platelets and their precursor cells (see J Hematol Oncol. 2019 Mar 7; 12(1): 26), the fusion protein of the present invention can also bind to platelet integrin αIIbβ3 at the thrombus site to inhibit platelet aggregation, thereby preventing the re-formation of large thrombi. In addition, the fusion protein of the present invention has a lower inhibitory activity on platelet aggregation than disintegrin or its variants, thus reducing the risk of bleeding. Thus, the fusion protein of the present invention can reduce thrombus formation while reducing the risk of bleeding, becoming a thrombolytic agent with great clinical application potential.

[0030] The present invention further provides a pharmaceutical composition comprising: the fusion protein described above; and a pharmaceutically acceptable carrier.

[0031] Illustratively, the pharmaceutical composition is an oral administration formulation, an injectable administration formulation, an inhalation administration formulation, or a topical or transdermal administration formulation.

[0032] For example, the volume molar concentration of the fusion protein is 1 to 1400 nM based on the total volume of the pharmaceutical composition.

[0033] The present invention further provides a use of the pharmaceutical composition, which is used to prepare a medicine for treating or preventing thrombosis-related diseases and reducing the risk of bleeding.

[0034] Illustratively, the thrombosis-related disorder is venous thrombosis or arterial thrombosis.

[0035] Illustratively, the venous thrombotic disorder is branch retinal vein occlusion, Budd-Chiari syndrome, cavernous sinus thrombosis, central retinal vein occlusion, cerebral venous sinus thrombosis, deep vein thrombosis, jugular vein thrombosis, mesenteric vein thrombosis, Paget-Schroetter disease, parodoxical embolism, portal vein thrombosis, pulmonary embolism, renal vein thrombosis, or splenic vein thrombosis.

[0036] Illustratively, the arterial thrombotic disorder is hepatic artery thrombosis, limb ischemia, myocardial infarction, or stroke.

[0037] The present invention further provides a method for treating or preventing thrombosis-related diseases, comprising administering the pharmaceutical composition described above to a subject in need thereof, thereby dissolving thrombus and reducing the risk of bleeding.

[0038] Illustratively, the thrombosis-related disorder is a venous thrombotic disorder or an arterial thrombotic disorder.

[0039] Illustratively, the venous thrombotic disorder is branch retinal vein occlusion, Budd-Chiari syndrome, cavernous sinus thrombosis, central retinal vein occlusion, intracranial venous sinus thrombosis, deep vein thrombosis, internal jugular vein thrombosis, mesenteric vein thrombosis, Paget-Schroeder syndrome, paradoxical embolism, portal vein thrombosis, pulmonary embolism, renal vein thrombosis, or splenic vein thrombosis.

[0040] Illustratively, the arterial thrombotic disorder is hepatic artery embolism, limb ischemia, myocardial infarction, or stroke.

[0041] Illustratively, 0.1 to 1000 mg of the fusion protein per kg of body weight of the subject is administered to the subject.

[0042] The present invention further provides a nucleic acid comprising a nucleotide sequence encoding the fusion protein as described above.

[0043] The present invention further provides a host cell comprising the nucleic acid described above.

[0044] Illustratively, the host cell is a prokaryotic cell or a eukaryotic cell.

[0045] Illustratively, the prokaryotic cell is Escherichia coli.

[0046] Illustratively, the eukaryotic cells are CHO cells, COS cells, or HEK293 cells.

[0047] The present invention further provides a method for preparing the fusion protein described above, which comprises culturing the host cell described above to enable it to express the fusion protein described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG1 is a schematic diagram showing the preparation of the expression construct of the protein TNK-G9-RR.

[0049] FIG2A is a liquid chromatography result diagram illustrating the separation and purification results of protein TNK.

[0050] FIG2B is a liquid chromatography result diagram illustrating the separation and purification results of protein TNK-G9-RR.

[0051] FIG2C is a liquid chromatography result diagram illustrating the separation and purification results of the protein TNK-(G4S)3-RR.

[0052] FIG2D is a liquid chromatography result diagram illustrating the separation and purification results of protein TNK-(PA)3-RR.

[0053] FIG2E is a liquid chromatography result diagram illustrating the separation and purification results of protein TNK-(PA)5-RR.

[0054] FIG2F is a liquid chromatography result diagram illustrating the separation and purification results of protein TNK-(PA)7-RR.

[0055] FIG2G is a liquid chromatography result diagram illustrating the separation and purification results of protein TNK-EA3K(G4S)2-RR.

[0056] FIG2H is a liquid chromatography result diagram illustrating the separation and purification results of the protein TNK-(EA3K)3-RR.

[0057] FIG2I is a liquid chromatography result diagram illustrating the separation and purification results of the protein RR-(PA)5-TNK.

[0058] Figures 3A and 3B are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the separation and purification results of the protein TNK; wherein the symbol "M" represents the protein marker, "LS" represents the loaded sample (20 μL), "FT" represents the flow-through sample (250 μL), and "Arabic numerals" represent the fractionation numbers.

[0059] Figures 3C and 3D are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the separation and purification results of the protein TNK-G9-RR; wherein the symbol "M" represents the protein marker, "LS" represents the loaded sample (20 μL), "FT" represents the flow-through sample (250 μL), and "Arabic numerals" represent the fractionation numbers.

[0060] Figures 3E and 3F are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the separation and purification results of the protein TNK-(G4S)3-RR; wherein the symbol "M" represents the protein marker, "LS" represents the loaded sample (20 μL), "FT" represents the flow-through sample (250 μL), and "Arabic numerals" represent the fractionation numbers.

[0061] Figures 3G and 3H are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the separation and purification results of the protein TNK-(PA)3-RR; wherein the symbol "M" represents the protein marker, "LS" represents the loaded sample (20 μL), "FT" represents the flow-through sample (250 μL), and "Arabic numerals" represent the fractionation numbers.

[0062] Figures 3I and 3J are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the separation and purification results of the protein TNK-(PA)5-RR; wherein the symbol "M" represents the protein marker, "LS" represents the loaded sample (20 μL), "FT" represents the flow-through sample (250 μL), and "Arabic numerals" represent the fractionation numbers.

[0063] Figures 3K and 3L are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the separation and purification results of the protein TNK-(PA)7-RR; wherein the symbol "M" represents the protein marker, "LS" represents the loaded sample (20 μL), "FT" represents the flow-through sample (250 μL), and "Arabic numerals" represent the fractionation numbers.

[0064] Figures 3M and 3N are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the separation and purification results of the protein TNK-EA3K(G4S)2-RR; wherein the symbol "M" represents the protein marker, "LS" represents the loaded sample (20 μL), "FT" represents the flow-through sample (250 μL), and the "Arabic numerals" represent the fractionation numbers.

[0065] Figures 3O and 3P are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the separation and purification results of the protein TNK-(EA3K)3-RR; wherein the symbol "M" represents the protein marker, "LS" represents the loaded sample (20 μL), "FT" represents the flow-through sample (250 μL), and "Arabic numerals" represent the fractionation numbers.

[0066] Figures 3Q and 3R are photographs of non-reduced Tris-glycine SDS-PAGE and reduced Tris-glycine SDS-PAGE, respectively, showing the separation and purification results of the protein RR-(PA)5-TNK; wherein the symbol "M" represents the protein marker, "LS" represents the loaded sample (20 μL), "FT" represents the flow-through sample (250 μL), and "Arabic numerals" represent the fractionation numbers.

[0067] FIG4A is a graph showing the thrombolysis results, illustrating the thrombolysis rates of protein TNK at different concentrations.

[0068] FIG4B is a graph showing the thrombolysis results, illustrating the thrombolysis rates of the protein TNK-G9-RR at different concentrations.

[0069] FIG4C is a graph showing the thrombolysis results, illustrating the thrombolysis rates of the protein TNK-(G4S)3-RR at different concentrations.

[0070] FIG4D is a graph showing the thrombolysis results, illustrating the thrombolysis rates of the protein TNK-(PA)3-RR at different concentrations.

[0071] FIG4E is a graph showing the thrombolysis results, illustrating the thrombolysis rates of the protein TNK-(PA)5-RR at different concentrations.

[0072] FIG4F is a graph showing the thrombolysis results, illustrating the thrombolysis rates of the protein TNK-(PA)7-RR at different concentrations.

[0073] FIG4G is a graph showing the thrombolysis results, illustrating the thrombolysis rates of the protein TNK-EA3K(G4S)2-RR at different concentrations.

[0074] FIG4H is a graph showing the thrombolysis results, illustrating the thrombolysis rate of the protein TNK-(EA3K)3-RR at different concentrations.

[0075] FIG4I is a graph showing the thrombolysis results, illustrating the thrombolysis rates of the protein RR-(PA)5-TNK at different concentrations.

[0076] FIG5 is a histogram illustrating the time required for different proteins to dissolve 50% of thrombus at a concentration of 7.0 nM. DETAILED DESCRIPTION

[0077] To make the above and / or other purposes, effects, and features of the present invention more clearly understood, preferred embodiments are described in detail below:

[0078] I. Definition of Terms

[0079] Unless otherwise specified, "protein" herein includes wild-type proteins expressed in natural cells, recombinant proteins expressed using genetic engineering techniques, and synthetic proteins obtained through chemical means. At least one amino acid may be substituted, deleted, and / or inserted into the protein sequence without affecting the original activity.

[0080] Unless otherwise specified, the "amino acids" referred to in this article include D-amino acids or L-amino acids. D- and L- represent the absolute configuration of the amino acid, rather than the specific rotation direction of plane polarization. Unless otherwise specified, this article uses the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission to represent amino acids. Protein sequences are represented by a string of multiple single-letter symbols, and the order of the single-letter symbols corresponds to the order of amino acids from the N-terminus to the C-terminus of the protein. If there is a superscript number before the single-letter symbol, it indicates the position order of the corresponding amino acid in the protein starting from the N-terminus; for example, 67PRNGLYG indicates proline at position 67 of the protein; the rest are analogous and are omitted. If a single-letter symbol is followed by a subscript number, it indicates the number of repeats of the corresponding amino acid or amino acid group. For example, G9 represents nine consecutive glycine residues linked together. Another example, (G4S)3 represents three consecutive amino acid groups consisting of glycine-glycine-glycine-glycine-serine. The rest are analogous and are omitted.

[0081] Protein sequence substitutions, deletions, and / or insertions can occur in non-backbone regions of the protein, which generally do not affect the original activity. Furthermore, protein sequence substitutions can include conservative amino acid substitutions, which are substitutions between amino acids with similar properties or related side chains. Substitutions between amino acids with similar properties, for example: acidic amino acids can substitute for each other, i.e., aspartate and glutamate; basic amino acids can substitute for each other, i.e., lysine, arginine, and histidine; non-polar amino acids can substitute for each other, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and uncharged polar amino acids can substitute for each other, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Substitutions between amino acids with related side chains, for example: aliphatic-hydroxy amino acids can replace each other, i.e., serine and threonine; amide-containing amino acids can replace each other, i.e., asparagine and glutamine; aliphatic amino acids can replace each other, i.e., alanine, valine, leucine, and isoleucine; aromatic amino acids can replace each other, i.e., phenylalanine, tryptophan, and tyrosine.

[0082] Unless otherwise specified, the "tissue plasminogen activator" referred to herein includes wild-type or recombinant tissue plasminogen activator, such as wild-type tissue plasminogen activator produced by human vascular endothelial cells, and recombinant tissue plasminogen activator such as alteplase (SEQ ID NO: 8), reteplase (SEQ ID NO: 9), or tenecteplase (SEQ ID NO: 10).

[0083] Unless otherwise specified, the term "disintegrin" used herein refers to platelet aggregation inhibitors found in the saliva of venomous snakes, typically containing 47 to 84 amino acids and 4 to 7 disulfide bonds, such as: white-lipped bamboo leaf green, piscivorin, Mexican west coast crotalin, large crotalin, flat headed gastropodin, Arizona black crotalin, horned crotalin, western diamond-back crotalin, South American crotalin, Huaconotoxin, flat nosed agkistrodon, etc. Toxin, yellow-green bamboo leaf snake toxin, yellow-green bamboo leaf snake inhibitor, white-browed complex, Harris complex inhibitor, American lancehead complex, American lancehead complex inhibitor, complex snake toxin, Yelin belly toxin, Great Basin Crotalus toxin, Black tail Crotalus toxin, Malayan Agkistrodon venom protein, white-browed complex itaton, Harris complex, Prairie Crotalus toxin, yellow-green bamboo leaf snake toxin, prolancehead complex, prolancehead complex enzyme, Ussuri complex, or Hopi Crotalus toxin.

[0084] Unless otherwise specified, "variants" as used herein refer to modified proteins resulting from substitution, deletion, and / or insertion of at least one amino acid into a reference sequence without affecting the original activity. For example, variants of disintegrin include variants of agkistrodon acutus venom or protospike complex, which can bind to integrin αIIbβ3 like wild-type disintegrin to inhibit platelet aggregation. Variants can have at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, or 60% sequence similarity to the reference sequence. Similarity can be defined as gap-excluded identity, BLAST identity, or gap-compressed identity, depending on the criteria. BLAST similarity can be calculated using the Basic Local Alignment Search Tool provided by the National Center for Biotechnology Information. For example, the protistrin variant RR used herein has at least 95% BLAST similarity to the wild-type protistrin.

[0085] The "RGD structural motif" referred to herein, unless otherwise specified, refers to the top of the flexible loop composed of arginine-glycine-aspartic acid in the disintegrin protein, which is the integrin binding region. For example, the RGD structural motif of wild-type protospike complex contains 50 ARGDNP, the RGD structural motif of wild-type Malayan agkistrodon venom protein contains 48 PRGDMP. Referring to the above, the RGD structural motif of the protospike complex variant can be compared with the RGD structural motif sequence of the wild-type protospike complex. 50ARGDNP has at least one amino acid mutation that does not affect function, such as SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26; the RGD structural motif of the Malayan agkistrodon venom protein variant can be different from the RGD structural motif sequence of the wild-type Malayan agkistrodon venom protein. 48 The PRGDMP has at least one amino acid mutation that does not affect its function, such as SEQ ID NO: 27 or 28.

[0086] The "linker region" referred to herein, unless otherwise specified, refers to the region immediately adjacent to the N-terminus of the RGD structural motif in the disintegrin, generally any continuous stretch of amino acids from position 38 to 49. For example, the wild-type protospike complex linker region comprises 41 KKKRT (SEQ ID NO: 11), the linker region of wild-type Agkistrodon acutus venom contains 39 SRAGK (SEQ ID NO: 12). Referring to the above, the connecting region of the protospike complex variant can be sequenced relative to the connecting region of the wild-type protospike complex. 41 KKKRT has at least one amino acid mutation that does not affect its function, such as SEQ ID NO: 13, 14, or 15.

[0087] The "C-terminal region" referred to herein, unless otherwise specified, refers to the C-terminal region of the disintegrin immediately adjacent to the RGD structural motif. For example, the C-terminal region of the wild-type protospike complex comprises 67 PRNGLYG (SEQ ID NO: 29), the C-terminal region of wild-type Agkistrodon acutus venom contains 65 PRYH (SEQ ID NO: 30). Referring to the above, the C-terminal region of the protistrin complex variant can be relative to the C-terminal region of the wild-type protistrin complex. 67 PRNGLYG has at least one amino acid mutation that does not affect the function, such as SEQ ID NO: 31, 32, or 33.

[0088] Unless otherwise specified, the "disintegrin variant" referred to herein includes sequences obtained by mutating at least one amino acid in the wild-type RGD structural motif, sequences obtained by mutating at least one amino acid in the wild-type linker region, and / or sequences obtained by mutating at least one amino acid in the wild-type C-terminal region, such as SEQ ID NO: 34.

[0089] Unless otherwise specified, the term "treatment" herein refers to therapeutic intervention to cure or improve thrombotic conditions, including complete or partial cure or improvement.

[0090] The term "prevention" as used herein, unless otherwise defined, refers to the complete or near-complete prevention of thrombotic symptoms. For example, when there is no thrombosis or a mild thrombosis that has not yet caused symptoms, preventive intervention can be used to prevent the occurrence of symptoms.

[0091] Unless otherwise specified, the term "pharmaceutically acceptable carrier" as used herein refers to an additive that is suitable for contact with an individual, without excessive toxicity, irritation, allergic reaction, or other problems or complications, and has a reasonable benefit-risk ratio, within the scope of sound medical judgment, such as a filler, diluent, agglutinating agent, binder, lubricant, glidant, stabilizer, colorant, wetting agent, or disintegrant.

[0092] 2. Fusion Protein

[0093] The first embodiment of the present invention provides a fusion protein that can simultaneously bind to fibrin and platelet integrin αIIbβ3 at the site of a thrombus. Binding to fibrin converts plasminogen into plasmin, which then degrades fibrin into FDP, thereby dissolving the thrombus. Furthermore, binding to integrin αIIbβ3 can inhibit platelet aggregation, thereby preventing the re-formation of large thrombi. However, the platelet aggregation inhibitory activity of the fusion protein of this embodiment bound to integrin αIIbβ3 is lower than that of a protein containing only disintegrin or its variants, thereby reducing the risk of bleeding. Based on the above characteristics, the fusion protein of this embodiment can be used to dissolve thrombi and reduce the risk of bleeding; in other words, it can be used to treat thrombotic diseases and reduce the risk of bleeding.

[0094] The fusion protein of this embodiment comprises: tissue plasminogen activator or a variant thereof, a disintegrin or a variant thereof, and a linker; the linker connects the tissue plasminogen activator or a variant thereof and the disintegrin or a 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 may comprise, from N-terminus to C-terminus, the tissue plasminogen activator or a variant thereof, the linker, and the disintegrin or a variant thereof, or may comprise the disintegrin or a variant thereof, the linker, and the tissue plasminogen activator or a variant thereof. Specifically, the C-terminus of the tissue plasminogen activator or a variant thereof may be linked to the N-terminus of the linker, and the N-terminus of the disintegrin or a variant thereof may be linked to the C-terminus of the linker; alternatively, the C-terminus of the disintegrin or a variant thereof may be linked to the N-terminus of the linker, and the N-terminus of the tissue plasminogen activator or a variant thereof may be linked to the C-terminus of the linker. The term "linked" herein is not limited to direct or indirect linkage, meaning that the linked proteins may or may not have additional linking fragments. Preferably, the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, or 42.

[0095] The tissue plasminogen activator may be alteplase, reteplase, or tenecteplase, depending on the type, preferably tenecteplase.

[0096] The tissue plasminogen activator may comprise the amino acid sequence shown in SEQ ID NO: 8, 9, or 10, preferably the amino acid sequence shown in SEQ ID NO: 10.

[0097] The disintegrin can be, depending on its type, white-lipped bamboo leaf green snake, piscivorin, Mexican west coast crotalin, large crotalin, flat headed pectoral snake, Arizona black crotalin, horned crotalin, western diamond-back crotalin, South American crotalin, huaconotoxin, flat nosed agkistrodon, yellow green bamboo leaf green snake, yellow green bamboo leaf green snake inhibin, white eyebrow inhibin, Harris's inhibin, American lancehead inhibin, American lancehead inhibin, inhibin, lycopodin, Great Basin crotalin, black tailed crotalin, Malayan agkistrodon venom, white eyebrow inhibin, Harris's inhibin, prairie crotalin, yellow green bamboo leaf green snake toxin, prolancehead inhibin, prolancehead inhibinase, Ussuri agkistrodon, or hopi crotalin. Preferably, the disintegrin is Malayan agkistrodon venom or prolancehead inhibin.

[0098] The disintegrator variant may comprise, in terms of sequence, a linker region, an RGD structural motif, and a C-terminal region; the linker region comprises the amino acid sequence set forth in SEQ ID NO: 11, 12, 13, 14, or 15; the RGD structural motif comprises the amino acid sequence set forth 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 set forth in SEQ ID NO: 29, 30, 31, 32, or 33.

[0099] Preferably, the linking region comprises the amino acid sequence shown in SEQ ID NO: 11 or 15. More preferably, the linking region comprises the amino acid sequence shown in SEQ ID NO: 11.

[0100] Preferably, the RGD structural motif comprises the amino acid sequence shown in SEQ ID NO: 17, 18, 19, 20, 21, 24, 25, or 26. More preferably, the RGD structural motif comprises the amino acid sequence shown in SEQ ID NO: 20.

[0101] Preferably, the C-terminal region comprises the amino acid sequence shown in SEQ ID NO: 31.

[0102] Preferably, the disintegrin variant comprises the amino acid sequence shown in SEQ ID NO: 34.

[0103] The fusion protein of this embodiment can be prepared through genetic engineering techniques or chemical methods, such as solid-phase synthesis or solution synthesis. The fusion protein of this embodiment can subsequently be isolated or purified using methods such as ammonium sulfate or ethanol precipitation, acid extraction, ion exchange chromatography, affinity chromatography, or lectin chromatography, preferably using high-performance liquid chromatography.

[0104] The fusion protein of this embodiment may further comprise a hydrophilic group to enhance water solubility or circulation half-life. The hydrophilic group may 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 polyglucose. More preferably, the hydrophilic group is polyethylene glycol composed of 2 to 20 repeating units of ethylene glycol.

[0105] The fusion protein of this embodiment may further contain a purification tag to facilitate purification. The purification tag may 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 terminator / anti-terminator protein (NusA-tag), or a small ubiquitin-related modifier tag (SUMO-tag).

[0106] 3. Pharmaceutical Compositions

[0107] A second embodiment of the present invention provides a pharmaceutical composition comprising the fusion protein of the first embodiment. This composition can be administered to individuals requiring thrombolysis to simultaneously dissolve thrombi and reduce the risk of bleeding. The pharmaceutical composition of this embodiment comprises the fusion protein of the first embodiment and a pharmaceutically acceptable carrier.

[0108] In general, a pharmaceutically acceptable carrier can allow the pharmaceutical composition as a whole to present different forms or be suitable for different routes of administration. Preferably, the pharmaceutical composition is an oral formulation, an injectable formulation, an inhaled formulation, or a topical or transdermal formulation, so as to be used for different routes of administration. Preferably, the pharmaceutical composition is a tablet, capsule, granule, powder, solution, syrup, suspension, or emulsion. The pharmaceutical composition of this embodiment can also be applied to an implantable medical device such as a stent or catheter to simultaneously dissolve blood clots while preventing restenosis of the blood vessel or supporting and strengthening the efficacy of the blood vessel. The pharmaceutical composition of this embodiment can further contain other agents that reduce thrombus formation, such as antiplatelet drugs or anticoagulants. Examples of antiplatelet drugs can be aspirin, clopidogrel, or ticagrelor, and examples of anticoagulants can be warfarin, rivaroxaban, or heparin.

[0109] A pharmaceutically acceptable carrier can be an excipient, filler, diluent, agglutinating agent, binder, lubricant, glidant, stabilizer, colorant, wetting agent, or disintegrant. 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, propylene glycol, or glycerol; examples of binders 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 glidants 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 wetting agents include Pluronic F68, Tween 20, or the like. 20), or Tween 80, and examples of disintegrants may be potato starch, tapioca starch, or silicates.

[0110] The volume molar concentration of the fusion protein can be 1 to 1400 nM based on the total volume of the pharmaceutical composition. Preferably, the volume molar concentration of the fusion protein is 7 to 1370.7 nM based on the total volume of the pharmaceutical composition.

[0111] 4. Medical Use

[0112] A third embodiment of the present invention provides a use of the pharmaceutical composition of the second embodiment for preparing a medicament for treating or preventing a thrombosis-related condition and reducing the risk of bleeding. The medicament can be administered to a subject in need of thrombolysis, thereby simultaneously dissolving the thrombus and reducing the risk of bleeding. Specifically, the medicament can be administered to a subject in need of treating or preventing a thrombosis-related condition, thereby simultaneously achieving a therapeutic or preventive effect and reducing the risk of bleeding.

[0113] The drug can be administered in different ways, such as oral administration, injection, inhalation, or topical or transdermal administration. In addition, an effective dose of 0.1 to 1000 mg of the fusion protein per kg of body weight can be administered to the individual.

[0114] Thrombosis-related disorders can be categorized as venous thrombotic disorders or arterial thrombotic disorders. Examples of venous thrombotic disorders include branch retinal vein occlusion, Budd-Chiari syndrome, cavernous sinus thrombosis, central retinal vein occlusion, intracranial venous sinus thrombosis, deep vein thrombosis, internal jugular vein thrombosis, mesenteric vein thrombosis, Paget-Schroeder syndrome, paradoxical embolism, portal vein thrombosis, pulmonary embolism, renal vein thrombosis, or splenic vein thrombosis, and examples of arterial thrombotic disorders include hepatic artery embolism, limb ischemia, myocardial infarction, or stroke.

[0115] A fourth embodiment of the present invention provides a method for treating or preventing a thrombosis-related disorder, comprising administering the pharmaceutical composition of the second embodiment to a subject in need of such treatment or prevention, thereby dissolving thrombus and reducing the risk of bleeding.

[0116] The drug can be administered in different ways, such as oral administration, injection, inhalation, or topical or transdermal administration. In addition, an effective dose of 0.1 to 1000 mg of the fusion protein per kg of body weight can be administered to the individual.

[0117] Thrombosis-related disorders can be categorized as venous thrombotic disorders or arterial thrombotic disorders. Examples of venous thrombotic disorders include branch retinal vein occlusion, Budd-Chiari syndrome, cavernous sinus thrombosis, central retinal vein occlusion, intracranial venous sinus thrombosis, deep vein thrombosis, internal jugular vein thrombosis, mesenteric vein thrombosis, Paget-Schroeder syndrome, paradoxical embolism, portal vein thrombosis, pulmonary embolism, renal vein thrombosis, or splenic vein thrombosis, and examples of arterial thrombotic disorders include hepatic artery embolism, limb ischemia, myocardial infarction, or stroke.

[0118] V. Other matters

[0119] A fifth embodiment of the present invention provides a nucleic acid comprising a nucleotide sequence encoding the fusion protein of the first embodiment. To regulate protein expression, the nucleic acid may further comprise a promoter operably linked to the nucleotide sequence encoding the fusion protein. "Operably linked" herein means that two or more nucleic acid sequences are in a functional relationship with each other.

[0120] A sixth embodiment of the present invention provides a host cell comprising the nucleic acid of the fifth embodiment. Because the host cell of this embodiment contains a nucleotide sequence encoding a fusion protein, the fusion protein can be produced by culturing the host cell. The host cell can 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.

[0121] The seventh embodiment of the present invention provides a method for preparing the fusion protein of the first embodiment, comprising culturing the host cell of the sixth embodiment to express the fusion protein. An appropriate inducer can be selected based on the promoter to induce the cell to express the protein.

[0122] The present invention is illustrated by the following examples:

[0123] Example 1: Preparation of protein

[0124] The expression construct is transfected into CHO cells or yeast cells to express the recombinant protein. The cell culture supernatant is collected and purified by liquid chromatography to obtain the recombinant protein.

[0125] Taking the expression construct for protein TNK-G9-RR shown in Figure 1 as an example, a polymerase chain reaction (PCR) was performed on plasmid pcDNA3.1 containing the TNK nucleotide fragment using primer pair TNK-F and TNK-G9-R to obtain plasmid pcDNA3.1 containing the TNK nucleotide fragment. A PCR was then performed on plasmid pPICZαA containing the RR nucleotide fragment using primer pair RR-G9-F and RR-R 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 were treated with the DpnI restriction enzyme and then ligated to obtain plasmid pcDNA3.1 containing the TNK-G9-RR nucleotide fragment as the expression construct for protein TNK-G9-RR.

[0126] The above process was followed, except that primer TNK-(G4S)3-R was substituted for TNK-G9-R and primer RR-(G4S)3-F was substituted for RR-G9-F, to obtain plasmid pcDNA3.1 containing the TNK-(G4S)3-RR nucleotide fragment as the expression construct of the protein TNK-(G4S)3-RR.

[0127] The above process was followed, except that primer TNK-(PA)3-R was substituted for TNK-G9-R and primer RR-(PA)3-F was substituted for RR-G9-F, to obtain plasmid pcDNA3.1 containing the TNK-(PA)3-RR nucleotide fragment as the expression construct of the protein TNK-(PA)3-RR.

[0128] The above process was followed, except that primer TNK-(PA)5-R was substituted for TNK-G9-R and primer RR-(PA)5-F was substituted for RR-G9-F, to obtain plasmid pcDNA3.1 containing the TNK-(PA)5-RR nucleotide fragment as the expression construct of protein TNK-(PA)5-RR.

[0129] The above process was followed, except that primer TNK-(PA)7-R was substituted for TNK-G9-R and primer RR-(PA)7-F was substituted for RR-G9-F, to obtain plasmid pcDNA3.1 containing the TNK-(PA)7-RR nucleotide fragment as the expression construct of the protein TNK-(PA)7-RR.

[0130] The above process was followed, except that primer TNK-EA3K(G4S)2-R was used to replace TNK-G9-R and primer RR-EA3K(G4S)2-F was used to replace RR-G9-F, to obtain plasmid pcDNA3.1 containing the TNK-EA3K(G4S)2-RR nucleotide fragment as the expression construct of the protein TNK-EA3K(G4S)2-RR.

[0131] The above process was followed, except that primer TNK-(EA3K)3-R was substituted for TNK-G9-R and primer RR-(EA3K)3-F was substituted for RR-G9-F, to obtain plasmid pcDNA3.1 containing the TNK-(EA3K)3-RR nucleotide fragment as the expression construct of the protein TNK-(EA3K)3-RR.

[0132] The nucleotide sequences of the primers mentioned above are listed in Table 1.

[0133] Table 1. Primer sequences

[0134]

[0135]

[0136] The results of liquid chromatography analysis of protein TNK are shown in FIG2A . FIG3A and FIG3B further demonstrate that protein TNK was obtained from fractions 27 , 28 , 29 , 30 , 33 , and 34 .

[0137] The results of liquid chromatography analysis of protein TNK-G9-RR are shown in FIG2B . FIG3C and FIG3D further demonstrate that protein TNK-G9-RR was obtained from fractions 9, 10, and 11. FIG3C and FIG3D further demonstrate that protein TNK-G9-RR was obtained from fractions 9, 10, and 11.

[0138] The results of liquid chromatography analysis of protein TNK-(G4S)3-RR are shown in FIG2C . FIG3E and FIG3F further demonstrate that protein TNK-(G4S)3-RR was obtained from fraction 15 .

[0139] The results of liquid chromatography analysis of protein TNK-(PA)3-RR are shown in FIG2D . FIG3G and 3H further demonstrate that protein TNK-(PA)3-RR was obtained from fractions 12, 13, 21, and 22.

[0140] The results of liquid chromatography analysis of protein TNK-(PA)5-RR are shown in FIG2E . FIG3I and 3J further demonstrate that protein TNK-(PA)5-RR was obtained from fractions 27 , 28 , and 29 .

[0141] The results of liquid chromatography analysis of protein TNK-(PA)7-RR are shown in FIG2F . FIG3K and 3L further demonstrate that protein TNK-(PA)7-RR was obtained from fractions 33 , 34 , 35 , 36 , 37 , and 39 .

[0142] The results of liquid chromatography analysis of protein TNK-EA3K(G4S)2-RR are shown in FIG2G . FIG3M and 3N further demonstrate that protein TNK-EA3K(G4S)2-RR was obtained from fractions 27 , 28 , 29 , 30 , and 31 .

[0143] The results of liquid chromatography analysis of protein TNK-(EA3K)3-RR are shown in FIG2H . FIG3O and FIG3P further demonstrate that protein TNK-(EA3K)3-RR was obtained from fractions 28 , 29 , 30 , 31 , and 32 .

[0144] The results of liquid chromatography analysis of protein RR-(PA)5-TNK are shown in FIG2I . FIG3Q and FIG3R further demonstrate that protein RR-(PA)5-TNK was obtained from fractions 21 , 22 , 23 , and 24 .

[0145] The amino acid sequences of the proteins mentioned above are listed in Table 2, and the yields of the proteins are listed in Table 3. It can be seen that protein TNK, protein RR, and other fusion proteins containing TNK can all achieve a certain yield.

[0146] Table 2. Protein sequences

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153] Note: The letters in the box are connector sequences.

[0154] Table 3. Protein yield

[0155] Protein expression system Liquid chromatography Purification Yield (mg / L) TNK CHO cell zinc chelate gel 88 RR yeast cell cation exchange resin 6 TNK-G9-RR CHO cell zinc chelate gel 62 TNK-(G4S)3-RR CHO cell zinc chelate gel 65 TNK-(PA)3-RR CHO cell zinc chelate gel 60 TNK-(PA)5-RR CHO cell zinc chelate gel 70 TNK-(PA)7-RR CHO cell zinc chelate gel 44 TNK-EA3K(G4S)2-RR CHO cell zinc chelate gel 24 TNK-(EA3K)3-RR CHO cell zinc chelate gel 21 RR-(PA)5-TNK CHO cell zinc chelate gel 14

[0156] Example 2: Thrombolysis test

[0157] Whole blood thrombolysis plate analysis was performed. Blood was collected from healthy individuals and mixed with 3.8% trisodium citrate at a ratio of 9:1. Thrombin (6.25×10 -3 U) and calcium chloride (250 mM) to produce a coagulation mixture. 5 μL of the coagulation mixture was deposited onto the edge of the bottom wells of a 96-well microplate, followed by the addition of 25 μL of the blood mixture. The microplate was sealed and incubated at 37°C for 30 minutes to allow thrombi to form along the bottom well edges.

[0158] Each protein was prepared at a different target concentration in 70 μL of HEPES solution. The protein solution was added to the well containing the thrombus at room temperature and placed in a 37°C thermostatted ELISA reader for 120 minutes. During the reaction, the well was shaken once per minute (200 rpm), and the absorbance at 510 nm was measured every 3 minutes using the ELISA reader. Because the dissolved thrombus will flow over the center of the well during the reaction, the absorbance at 510 nm can be used to determine the time required to dissolve 50% of the thrombus (T0.5, minutes). This can be used to determine the extent of thrombus dissolution.

[0159] 4A to 4I , which show the thrombolytic activity of each protein at different concentrations. In addition, Table 4 shows the time required for each protein to dissolve 50% of the thrombus at different concentrations.

[0160] Table 4. The time required for each protein to dissolve 50% of the thrombus at different concentrations

[0161]

[0162] ND: Indicates that the thrombus cannot be dissolved within 120 minutes and cannot be measured

[0163] 5 and Table 5, which show the time required for each protein to dissolve 50% of the thrombus at a concentration of 7.0 nM. As shown above, the known thrombolytic agent TNK and each fusion protein containing TNK can dissolve thrombus.

[0164] Table 5. Time required for each protein to dissolve 50% of thrombus at a concentration of 7.0 nM

[0165] Protein T0.5: thrombolysis (7.0 nM, min) ratio (TNK / corresponding protein) TNK19.8±3.21 / 1

[0166] TNK-G9-RR25.9±2.41 / 1.31TNK-(G4S)3-RR26.3±2.41 / 1.33TNK-(PA)3-RR25.3±1.61 / 1.28TNK-(PA)5-RR37.4±14.01 / 1.89TNK-( PA)7-RR36.5±14.11 / 1.84TNK-EA3K(G4S)2-RR93.0±9.51 / 4.70TNK-(EA3K)3-RR73.9±5.41 / 3.73RR-(PA)5-TNK32.7±14.11 / 1.65

[0167] Example 3: Platelet aggregation inhibition test

[0168] Ten milliliters of venous blood was collected from individuals who had not received any medication for at least two weeks. The blood was mixed with 3.13% sodium citrate (pH 7.4) at a ratio of 9:1. The blood samples were centrifuged at 1000 rpm for 10 minutes, and the supernatant was collected to obtain platelet-rich plasma (PRP). The remaining fraction was centrifuged again at 4000 rpm for 10 minutes, and the supernatant was collected to obtain platelet-poor plasma (PPP). Separately, proteins were dissolved in R+E buffer (2.5 mM Tris, 1.5 mM sodium chloride, 50 mM arginine, and 50 mM glutamine) at various target concentrations. Then, 190 μL of PRP was mixed with either 10 μL of PBS buffer or 10 μL of the protein solution and incubated at 37°C for 1 minute using an HTracer 601, Nikoh Bioscience, Tokyo, Japan. 10 μL of 200 μM adenosine diphosphate was further added to monitor platelet aggregation reaction by light transmission. The platelet aggregation data obtained represent the mean value of the inhibition percentage relative to the control value.

[0169] Please refer to Table 6, which shows the platelet aggregation inhibitory abilities of different proteins. Compared to the known platelet aggregation inhibitor protistin variant RR, each fusion protein has a lower platelet aggregation inhibitory ability.

[0170] Table 6. Half-inhibitory concentration of each protein on platelet aggregation

[0171] Protein IC 50 Platelet aggregation (nM) ratio (RR / corresponding protein) RR52.5±11.01 / 1TNK-G9-RR567.2±26.81 / 10.80TNK-(G4S)3-RR249.3±57.51 / 4.75

[0172] TNK-(PA)3-RR505.5±49.11 / 9.63TNK-(PA)5-RR379.9±31.91 / 7.24TNK-(PA)7-RR347.4±33.61 / 6.62TNK-E A3K(G4S)2-RR275.5±67.11 / 5.25TNK-(EA3K)3-RR457.5±25.71 / 8.71RR-(PA)5-TNK1370.7±155.61 / 26.11

[0173] In summary, it is confirmed that the fusion protein of the present invention can reduce thrombus formation while lowering the risk of bleeding, and therefore has the potential to be a candidate thrombolytic drug.

[0174] However, the above is only a preferred embodiment of the present invention, and it cannot be used to limit the scope of protection of the patent of the present invention; therefore, any simple equivalent changes and modifications made according to the scope of protection of the patent of the present invention and the content of the invention specification still fall within the scope of protection of the patent of the present invention.

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

Claims

1. A fusion protein, characterized in that include: tissue plasminogen activator or its variants; disintegrin or its variants; as well as The connector connects the tissue plasminogen activator or its variant and the disintegratin or its variant, and comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 7.

2. The fusion protein according to claim 1, characterized in that: The C-terminus of the tissue plasminogen activator or a variant thereof is connected to the N-terminus of the linker, and the N-terminus of the disintegratin or a variant thereof is connected to the C-terminus of the linker; or the C-terminus of the disintegratin or a variant thereof is connected to the N-terminus of the linker, and the N-terminus of the tissue plasminogen activator or a variant thereof is connected to the C-terminus of the linker.

3. The fusion protein according to claim 1, characterized in that: The tissue plasminogen activator is alteplase, reteplase, or tenecteplase.

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

5. The fusion protein according to claim 1, characterized in that: The disintegrins are albolabrin, applagin, basilicin, batroxostatin, bitistatin, cereberin, cerastin, crotatroxin, durissin, elegantin, eristicophin, flavoridin, flavostatin, halysin, halystatin, jararacin, jarastatin, kistrin, lachesin, lutosin, molossin, rhodostomin, salmosin, saxatilin, tergeminin, trimestatin, trimucrin, trimutase, ussuristatin, or viridian.

6. The fusion protein according to claim 1, characterized in that: Variants of this disintegrin include: a linking region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 11 to 15; An RGD structural motif comprising an amino acid sequence as shown in any one of SEQ ID NOs: 16 to 28; and The C-terminal region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 29 to 33.

7. The fusion protein according to claim 1, characterized in that: The disintegratin variant comprises the amino acid sequence shown in SEQ ID NO:

34.

8. The fusion protein according to claim 1, characterized in that: The amino acid sequence comprises any one of SEQ ID NOs: 35 to 42.

9. A pharmaceutical composition, characterized in that: include: The fusion protein according to any one of claims 1 to 8; as well as Pharmaceutically acceptable carrier.

10. Use of the pharmaceutical composition according to claim 9, characterized in that: The invention is used for preparing medicines for treating or preventing diseases related to thrombosis and reducing the risk of bleeding.

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