WO2017101866A1 - 一种用于预防或治疗急性及慢性血栓的方法 - Google Patents

一种用于预防或治疗急性及慢性血栓的方法 Download PDF

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WO2017101866A1
WO2017101866A1 PCT/CN2016/110448 CN2016110448W WO2017101866A1 WO 2017101866 A1 WO2017101866 A1 WO 2017101866A1 CN 2016110448 W CN2016110448 W CN 2016110448W WO 2017101866 A1 WO2017101866 A1 WO 2017101866A1
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Prior art keywords
plasminogen
thrombus
thrombosis
tpa
disease
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PCT/CN2016/110448
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English (en)
French (fr)
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李季男
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深圳瑞健生命科学研究院有限公司
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Priority to DK16874923.2T priority Critical patent/DK3395359T3/da
Priority to CA3008185A priority patent/CA3008185C/en
Priority to JP2018550633A priority patent/JP2019500422A/ja
Priority to CN202211684331.2A priority patent/CN115845037A/zh
Priority to EP16874923.2A priority patent/EP3395359B1/en
Priority to CN201680073669.0A priority patent/CN108472342B/zh
Priority to US16/063,534 priority patent/US10864257B2/en
Priority to ES16874923T priority patent/ES2968254T3/es
Publication of WO2017101866A1 publication Critical patent/WO2017101866A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin

Definitions

  • the present invention relates to a novel method of preventing and/or treating thrombosis using plasminogen.
  • Plasminogen specifically dissolves thrombus without causing side effects such as bleeding.
  • the medicament of the invention also has the advantage of dissolving fresh and old thrombus and having long half-life and controllable thrombus-intensity. Therefore, plasminogen may become a new strategy for lysing thrombus in vivo.
  • a thrombus is a blood clot that occurs when a human or animal is alive due to certain causes, blood forms an abnormality in the circulating blood, or blood deposits occur on the inner wall of the heart or the wall of the blood vessel. It includes myocardial infarction, cerebral embolism, pulmonary thrombosis, deep vein thrombosis and peripheral vascular embolism, etc., which are serious diseases that endanger human health, and its incidence, morbidity and mortality are high. According to the World Health Organization, the number of people dying from thromboembolic diseases in the world is about 26 million per year, which is much higher than other causes of death and has become the number one enemy of human health [1] .
  • Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease that hydrolyzes several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycans [2] . In addition, plasmin activates some metalloproteinase precursors (pro-MMP) to form active metalloproteinases (MMPs). Therefore, plasmin is considered to be an important upstream regulator of extracellular proteolysis [3,4] . Plasmin is formed by proteolytic plasminogen by two physiological PA: tissue plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA).
  • tPA tissue plasminogen activator
  • uPA urokinase-type plasminogen activator
  • plasminogen Due to the relatively high levels of plasminogen in plasma and other body fluids, it has been traditionally believed that the regulation of the PA system is primarily achieved by the synthesis and activity levels of PA.
  • the synthesis of components of the PA system is tightly regulated by various factors such as hormones, growth factors and cytokines.
  • specific physiological inhibitors of plasmin and PA are also present.
  • the main inhibitor of plasmin is ⁇ 2-antiplasmin.
  • Some cell surface has direct hydrolysis activity of uPA-specific cell surface receptors (uPAR) [5,6] .
  • Plasminogen is a single-chain glycoprotein with a molecular weight of approximately 92 kDa [7,8] . Plasminogen is mainly synthesized in the liver and is abundantly present in the extracellular fluid. The plasma plasminogen content is approximately 2 ⁇ M. Therefore, plasminogen is a huge potential source of proteolytic activity in tissues and body fluids [9,10] . Plasminogen exists in two molecular forms: glutamate-plasminogen and Lys-plasminogen. The naturally secreted and uncleaved forms of plasminogen have an amino terminal (N-terminal) glutamate and are therefore referred to as glutamate-plasminogen.
  • plasminogen in the presence of plasmin, glutamate-plasminogen is hydrolyzed to Lys-Lysinogen at Lys76-Lys77. Compared to glutamate-plasminogen, lysine-plasminogen has a higher affinity for fibrin and can be activated by PA at a higher rate.
  • the Arg560-Val561 peptide bond of these two forms of plasminogen can be cleaved by uPA or tPA, resulting in the formation of a disulfide-linked double-chain protease plasmin [11] .
  • the amino terminal portion of plasminogen contains five homologous tricycles, the so-called kringle, which contains a protease domain.
  • Some kringles contain a lysine binding site that mediates the specific interaction of plasminogen with fibrin and its inhibitor alpha2-AP.
  • a newly discovered 38 kDa plasminogen fragment, including kringle 1-4, is a potent inhibitor of angiogenesis. This fragment was named angiostatin and can be produced by hydrolysis of plasminogen by several proteases.
  • Plasmin also has substrate specificity for several components of ECM, including laminin, fibronectin, proteoglycans and gelatin, suggesting that plasmin also plays an important role in ECM reconstruction [8,13, 14] .
  • plasmin can also degrade other components of ECM, including MMP-1, MMP-2, MMP-3, and MMP-9, by converting certain protease precursors into active proteases. Therefore, it has been suggested that plasmin may be an important upstream regulator of extracellular proteolysis [15] .
  • plasmin has the ability to activate certain potential forms of growth factors [16-18] . In vitro, plasmin also hydrolyzes components of the complement system and releases chemotactic complement fragments.
  • thrombolytic therapy Current drug treatments that reduce thrombosis are common non-surgical treatments including thrombolytic therapy, anticoagulant therapy, antiplatelet drugs, and vasodilator drugs.
  • the most commonly used and most effective method is thrombolytic therapy.
  • the commonly used thrombolytic drugs have three generations: the first generation is represented by streptokinase (SK) and urokinase (UK), which has strong thrombolytic ability but no thrombolytic specificity. It is prone to systemic thrombolysis and lead to bleeding [19,20] .
  • the second generation is represented by tissue plasminogen activator tPA, which is better than SK and UK in thrombolysis, but has a short half-life in vivo [21] .
  • the third generation uses genetic engineering technology and monoclonal technology to transform the first generation and second generation drugs, but they are basically in the experimental stage. These drugs are based on increasing the activator in the fibrinolysis equilibrium, producing plasmin (Plm) to promote fibrinolysis, thereby achieving thrombolytic goals [22] .
  • thrombolytic drugs fall into two categories: most thrombolytic drugs use plasminogen activators, including natural and different recombinant forms of tPA, uPA, and streptokinase.
  • the plasminogen activator itself does not dissolve the thrombus, and the plasminogen molecule near the thrombus must be activated into active plasmin to effect thrombolysis.
  • active plasmin has been approved for direct local thrombolysis by locally releasing active plasmin in the presence of a catheter to the thrombus site for direct thrombolysis.
  • Plasminogen is an inactive form of plasmin (Plm), which is traditionally considered to be excessive and inert in vivo.
  • the body's thrombolytic process is activated only by plasminogen under its action.
  • the active plasmin further functions to dissolve fibrin clots. It has traditionally been thought that plasminogen itself does not act to dissolve thrombus.
  • natural plasminogen has a good function of dissolving fresh and old thrombus, and has the advantages of good safety, easy adjustment of thrombus strength, and good specificity.
  • the thrombolytic mechanism of the present invention is completely different from the currently known thrombolytic strategies.
  • Prior art methods of dissolving thrombi are achieved by a catalyst that increases the thrombolytic response, ie, a plasminogen activator, including tPA, uPA, streptokinase and its derivatives, or the product of a thrombolytic reaction, active plasmin.
  • the method of lysing thrombus of the present invention is achieved by a strategy for modulating the substrate plasminogen of the thrombolytic reaction.
  • the plasminogen thrombolysis of the present invention has at least the following advantages over the thrombolytic drugs of the prior art.
  • the thrombolytic drugs of the prior art are less effective [23-25 ], while the present invention is the use of plasminogen or plasminogen in combination with PA, the above-described thrombus achieve a good thrombolytic effect. Therefore, the present invention can effectively solve the above problems of tPA, uPA.
  • the half-life in vivo is too short.
  • the in vivo half-life of natural uPA is 5-10 minutes
  • the in vivo half-life of natural tPA is 3-5 minutes
  • the in vivo half-life of natural plasmin is extremely short.
  • genetic engineering is currently being used to extend the half-life of these substances, the effect is not ideal, and the half-life is too short, which greatly limits the application of these substances.
  • the in vivo half-life of plasminogen is as long as 53 hours, which indicates that the combination of plasminogen or plasminogen and PA can significantly prolong the period of thrombolysis in vivo, achieving sustained and stable thrombolysis. purpose.
  • active plasmin since it is a highly active protease, thrombolysis by using active plasmin must be a very rapid reaction process, which leads to the direct use of the catheter to the thrombus in the current use. .
  • plasminogen activator Because it is in the position of the catalyst in the thrombolytic reaction, adding a small amount of plasminogen activator will rapidly form a large amount of active plasmin in a short time, which is a violent enzyme. reaction process.
  • the experiments of the present invention demonstrate that the process of dissolving thrombus by lysing the substrate plasminogen is more mild, and that the plasminogen thrombolysis rate is found by studies of different plasminogen use and thrombolytic effects. It can also be regulated by the dose of plasminogen.
  • plasminogen activator as a thrombolytic drug is bleeding, particularly in the intestines and brain. Since normal plasminogen is widely present in all body fluids, physiological fibrin deposition is normally present in the body, and the increase in plasminogen activator often occurs in special cases such as trauma, hemorrhage, and strenuous exercise. Therefore, once the plasminogen activator is injected, the action of activating plasminogen to form active plasmin is non-specifically widespread in the body, thereby causing dissolution and bleeding of the original normal fibrin deposition. Clinically, the risk of intracranial hemorrhage is a major risk of bleeding. It has been reported that the incidence of intracranial hemorrhage is 1% to 2% during continuous administration for 2-24 hours. There is no better way to avoid the risk of bleeding.
  • plasminogen is not an active enzyme
  • the reaction of activating plasminogen to form active plasmin is not performed non-specifically after injection of plasminogen.
  • the location of this reaction depends on where the plasminogen activator is expressed, which is where the thrombus occurs.
  • the experiment of the present invention proves that plasminogen can specifically adsorb to the thrombus site, has thrombolytic specificity, and the experiment proves that no side effects of bleeding occur.
  • thrombolytic effect of current thrombolytic drugs is concentrated in the early stage of thrombosis, that is, "fresh thrombosis.”
  • injection of recombinant tPA within 3 hours of thrombosis can effectively dissolve thrombus.
  • recombinant tPA can dissolve thrombus up to 4.5 hours after thrombosis, and if it exceeds 4.5 hours, the risk of injection of recombinant tPA may be more than effective. Therefore, in the case of current drugs, recombinant tPA should be injected as early as possible in the early stage of thrombosis (less than 4.5 hours) [26,27] . In other words, there is an urgent need in the art to find a strong old thrombolytic drug.
  • plasminogen alone and physiological level of tPA
  • plasminogen and plasminogen activator tPA or uPA
  • tPA or uPA plasminogen activator
  • plasmin has been expected to become a new and more advantageous new thrombolytic drug.
  • the invention relates to a method of preventing and/or eliminating arterial and venous thrombosis in a subject, comprising administering to the subject a plasminogen.
  • the invention also encompasses the use of plasminogen for preventing and/or eliminating arterial and venous thrombosis in a subject.
  • the thrombus comprises fresh thrombus and stale thrombus.
  • the thrombus is a thrombus caused by a blood system disease, a circulatory disease, an autoimmune disease, a metabolic disorder disease, or an infectious disease.
  • the thrombus is a large, small blood vessel, microvascular thrombus secondary to diabetes.
  • the thrombus is a thrombus caused by a large vascular lesion.
  • the present invention relates to a novel method of preventing and/or treating a thrombosis-related disease, which comprises administering an effective amount of plasminogen to a subject.
  • the invention also relates to the use of plasminogen for the prevention and/or treatment of thrombosis-related diseases.
  • the present invention relates to a novel method of preventing and/or eliminating a pathological thrombus in a subject, which method dissolves the thrombus by systemic or local administration of plasmin.
  • the thrombus described above is a fresh thrombus and/or an old thrombus, which is a disease induced or caused by fresh thrombus and/or old thrombus.
  • the subject is a mammal, preferably a human.
  • the subject is low in plasmin or plasminogen.
  • the low is congenital, secondary and/or local.
  • the thrombus of the invention is a venous thrombosis and/or an arterial thrombus.
  • the thrombosis-related diseases include pancreatitis caused by portal vein thrombosis, cirrhosis of the liver, renal embolism caused by renal vein thrombosis, systemic sepsis caused by internal jugular vein thrombosis, pulmonary embolism, cerebral thrombosis, and organ infarction caused by arterial thrombosis, including Not limited to: cerebral infarction, myocardial infarction, thrombotic stroke, atrial fibrillation, Unstable angina, refractory angina pectoris, transient ischemic attack, pulmonary embolism, diabetic vascular embolism, etc.
  • the thrombosis-related disease is diabetic nephropathy, diabetic retinopathy, diabetic liver disease, diabetic heart disease, diabetic enteropathy, diabetic neuropathy including diabetic neuropathic pain, and the like.
  • the thrombus is a secondary and/or local thrombus; the thrombus-related disease is a secondary and/or local thrombotic disorder.
  • plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2, 6, 8, 10 or 12. And still have plasminogen activity.
  • plasminogen is added, deleted and/or substituted on the basis of sequence 2, 6, 8, 10 or 12, 1-100, 1-90, 1-80, 1-70, 1 -60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3 , 1-2, 1 amino acid, and still have plasminogen activity protein.
  • plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
  • the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, delta-plasminogen, or any combination thereof. In one embodiment, the plasminogen is a conservative substitution variant selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, delta-plasminogen or microfibrinolysis Proenzyme.
  • the plasminogen is a human natural plasminogen, such as an ortholog of plasminogen as shown in SEQ ID NO: 2, for example, a plasminogen from a primate or a rodent To homologs, such as plasminogens from gorillas, rhesus monkeys, rats, cattle, horses, dogs, straight homologs.
  • the amino acid sequence of plasminogen of the invention is shown as sequence 2, 6, 8, 10 or 12.
  • the plasminogen is administered systemically or locally, preferably by: surface, intravenous, intramuscular, subcutaneous, inhalation, intraspinal, local injection, intra-articular injection or through the rectum .
  • the topical administration is by applying a dressing and/or catheter containing plasminogen in the thrombus area.
  • the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer.
  • the plasminogen is 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg/kg per day, 10-100mg / kg (calculated per kg body weight) or 0.0001-2000mg / cm 2, 0.001-800mg / cm 2, 0.01-600mg / cm 2, 0.1-400mg / cm 2, 1-200mg / cm 2, 1- Dosage administration of 100 mg/cm 2 , 10-100 mg/cm 2 (calculated per square centimeter of body surface area) is preferably repeated at least once, preferably at least daily. In the case of topical application, the above dosages may be further adjusted as appropriate.
  • the above plasminogen may be administered alone or in combination with other drugs to prevent and/or treat other diseases associated with pathological thrombosis including, for example, cardiovascular disease therapeutic drugs, arrhythmia therapeutic drugs. , diabetes treatment drugs, etc.
  • the invention relates to the use of plasminogen for the manufacture of a medicament, article, kit for preventing and/or eliminating arterial and venous thrombosis in a subject.
  • the invention further relates to a pharmaceutical method comprising preparing a drug, an article, a kit for preventing and/or eliminating arterial and venous thrombosis in a subject by combining plasminogen with a pharmaceutically acceptable carrier.
  • the thrombus comprises a fresh thrombus (acute thrombus) and an old thrombus (chronic thrombus).
  • the thrombus is a thrombus caused by a blood system disease, a circulatory disease, an autoimmune disease, a metabolic disorder disease, or an infectious disease.
  • the thrombus is a large, small blood vessel, microvascular thrombus secondary to diabetes.
  • the thrombus is a thrombus caused by a large vascular lesion.
  • the present invention relates to the use of plasminogen for the preparation of a medicament, an article, a kit for preventing and/or eliminating a pathological thrombus in a subject, and the preparation of a plasminogen for preventing and/or treating a thrombus in a subject
  • the present invention also relates to a method of preparing a medicament comprising preparing a plasminogen together with a pharmaceutically acceptable carrier to prevent and/or eliminate a pathological thrombus of a subject, a medicament, an article, a kit, or a prophylactic and/or therapeutic treatment Drugs, products, and kits for thrombosis-related diseases in subjects.
  • the thrombus is a fresh thrombus and/or an old thrombus, which is a disease caused by fresh blood clots and/or old thrombus.
  • the subject is a mammal, preferably a human.
  • the subject is low in plasmin or plasminogen.
  • the low is congenital, secondary and/or local.
  • the thrombus is a venous thrombus and/or an arterial thrombus.
  • the thrombosis-related diseases include pancreatitis caused by portal vein thrombosis, cirrhosis of the liver, renal embolism caused by renal vein thrombosis, systemic sepsis caused by internal jugular vein thrombosis, pulmonary embolism, cerebral thrombosis, and organ infarction caused by arterial thrombosis, including Not limited to: cerebral infarction, myocardial infarction, thrombotic stroke, atrial fibrillation, unstable angina pectoris, refractory angina pectoris, transient ischemic attack, pulmonary embolism, diabetic vascular embolism.
  • the thrombosis-related disease is diabetic nephropathy, diabetic retinopathy, diabetic liver disease, diabetic heart disease, diabetic enteropathy, diabetic neuropathy including diabetic neuropathic pain, and the like.
  • the thrombus is a secondary and/or local thrombus; the thrombus-related disease is a secondary and/or local thrombotic disorder.
  • plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2, 6, 8, 10 or 12. And still have plasminogen activity.
  • plasminogen is added, deleted and/or substituted on the basis of sequence 2, 6, 8, 10 or 12, 1-100, 1-90, 1-80, 1-70, 1 -60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3 , 1-2, 1 amino acid, and still have plasminogen activity protein.
  • plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
  • the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, delta-plasminogen, or any combination thereof. In one embodiment, the plasminogen is a conservative substitution variant selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, delta-plasminogen or microfibrinolysis Proenzyme.
  • the plasminogen is a human natural plasminogen, such as an ortholog of plasminogen as shown in SEQ ID NO: 2, for example, a plasminogen from a primate or a rodent To homologs, such as plasminogen from gorillas, rhesus monkeys, rats, cattle, horses, dogs, straight homologs.
  • the amino acid sequence of plasminogen of the invention is shown as sequence 2, 6, 8, 10 or 12.
  • the plasminogen is administered systemically or locally, preferably by: surface, intravenous, intramuscular, subcutaneous, inhalation, intraspinal, local injection, intra-articular injection or through the rectum .
  • the topical administration is by applying a dressing and/or catheter containing plasminogen in the thrombus area.
  • the above plasminogen may be administered alone or in combination with other drugs to treat other diseases associated with pathological thrombosis, including, for example, cardiovascular disease treatment drugs, arrhythmia treatment drugs, diabetes treatment drugs. Wait.
  • the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer.
  • the plasminogen is 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg/kg per day, 10-100mg / kg (calculated per kg body weight) or 0.0001-2000mg / cm 2, 0.001-800mg / cm 2, 0.01-600mg / cm 2, 0.1-400mg / cm 2, 1-200mg / cm 2, 1- 100mg / cm 2, 10-100mg / cm 2 ( calculated per square centimeter of body surface area) of the dose administered, preferably repeated at least once, preferably at least daily administration.
  • the above dosages may be further adjusted as appropriate.
  • the present invention relates to plasminogen for preventing and/or eliminating arterial and venous thrombosis in a subject, and plasminogen comprising plasminogen for preventing and/or eliminating arterial and venous thrombosis in a subject Pharmaceutical composition.
  • the thrombus comprises fresh thrombus and stale thrombus.
  • the thrombus is a thrombus caused by a blood system disease, a circulatory disease, an autoimmune disease, a metabolic disorder disease, or an infectious disease.
  • the thrombus is a large, small blood vessel, microvascular thrombus secondary to diabetes.
  • the thrombus is a thrombus caused by a large vascular lesion.
  • the present invention relates to plasminogen for preventing and/or treating a thrombus-related disease, and a pharmaceutical composition comprising plasminogen for preventing and/or treating a thrombus-related disease.
  • the thrombus is a fresh thrombus and/or an old thrombus, which is a disease caused by fresh blood clots and/or old thrombus.
  • the thrombus is a venous thrombus and/or an arterial thrombus.
  • the thrombosis-related diseases include pancreatitis caused by portal vein thrombosis, cirrhosis of the liver, renal embolism caused by renal vein thrombosis, systemic sepsis caused by internal jugular vein thrombosis, pulmonary embolism, cerebral thrombosis, and organ infarction caused by arterial thrombosis, including Not limited to: cerebral infarction, myocardial infarction, thrombotic stroke, atrial fibrillation, unstable angina pectoris, refractory angina pectoris, transient ischemic attack, pulmonary embolism, diabetic vascular embolism.
  • the thrombosis-related disease is diabetic nephropathy, diabetic retinopathy, diabetic liver disease, diabetic heart disease, diabetic enteropathy, diabetic neuropathy including diabetic neuropathic pain, and the like.
  • the thrombus is an innate, secondary, and/or local thrombus; the thrombotic-related disorder is an innate, secondary, and/or local thrombotic-related disorder.
  • plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2, 6, 8, 10 or 12. And still have plasminogen activity.
  • plasminogen is added, deleted and/or substituted on the basis of sequence 2, 6, 8, 10 or 12, 1-100, 1-90, 1-80, 1-70, 1 -60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3 , 1-2, 1 amino acid, and still have plasminogen activity protein.
  • plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
  • the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, delta-plasminogen, or any combination thereof. In one embodiment, the plasminogen is a conservative substitution variant selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, delta-plasminogen or microfibrinolysis Proenzyme.
  • the plasminogen is a human natural plasminogen, such as an ortholog of plasminogen as shown in SEQ ID NO: 2, for example, a plasminogen from a primate or a rodent To homologs, such as plasminogens from gorillas, rhesus monkeys, rats, cattle, horses, dogs, straight homologs.
  • the amino acid sequence of plasminogen of the invention is shown as sequence 2, 6, 8, 10 or 12.
  • the plasminogen is administered systemically or locally, preferably by: surface, intravenous, intramuscular, subcutaneous, inhalation, intraspinal, local injection, intra-articular injection or through the rectum .
  • the topical administration is by applying a dressing and/or catheter containing plasminogen in the thrombus area.
  • the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer.
  • the plasminogen is 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg/kg per day, 10-100mg / kg (calculated per kg body weight) or 0.0001-2000mg / cm 2, 0.001-800mg / cm 2, 0.01-600mg / cm 2, 0.1-400mg / cm 2, 1-200mg / cm 2, 1- 100mg / cm 2, 10-100mg / cm 2 ( calculated per square centimeter of body surface area) of the dose administered, preferably repeated at least once, preferably at least daily administration.
  • the above dosages may be further adjusted as appropriate.
  • the invention relates to an article or kit comprising plasminogen for use in preventing and/or eliminating arterial and venous thrombosis in a subject.
  • the thrombus comprises fresh thrombus and stale thrombus.
  • the thrombus is a thrombus caused by a blood system disease, a circulatory disease, an autoimmune disease, a metabolic disorder disease, or an infectious disease.
  • the thrombus is a large, small blood vessel, microvascular thrombus secondary to diabetes.
  • the thrombus is a thrombus caused by a large vascular lesion.
  • the article or kit comprises a container containing an effective amount of plasminogen.
  • the article or kit further comprises a container comprising one or more other drugs, wherein the other drug is a therapeutic agent for other diseases associated with thrombosis.
  • the kit may further comprise instructions for use, wherein the plasminogen may be used for preventing and/or treating the arteriovenous, venous thrombosis, or thrombosis-related diseases, and may further illustrate that the plasminogen may be in other Before the drug is administered, at the same time, And/or after administration.
  • the other drug may be a cardiovascular disease therapeutic drug, an arrhythmia therapeutic drug, a diabetes therapeutic drug, or the like to treat other diseases associated with pathological thrombosis.
  • the thrombus is a fresh thrombus and/or an old thrombus
  • the thrombosis-related disease is a disease caused by fresh blood clots and/or old thrombus.
  • the thrombus is a venous thrombus and/or an arterial thrombus.
  • the thrombosis-related diseases include pancreatitis caused by portal vein thrombosis, cirrhosis of the liver, renal embolism caused by renal vein thrombosis, systemic sepsis caused by internal jugular vein thrombosis, pulmonary embolism, cerebral thrombosis, and organ infarction caused by arterial thrombosis, including Not limited to: cerebral infarction, myocardial infarction, thrombotic stroke, atrial fibrillation, unstable angina pectoris, refractory angina pectoris, transient ischemic attack, pulmonary embolism, diabetic vascular embolism.
  • the thrombosis-related disease is diabetic nephropathy, diabetic retinopathy, diabetic liver disease, diabetic heart disease, diabetic enteropathy, diabetic neuropathy including diabetic neuropathic pain, and the like.
  • the thrombus is an innate, secondary, and/or local thrombus; the thrombotic-related disorder is an innate, secondary, and/or local thrombotic-related disorder.
  • plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2, 6, 8, 10 or 12. And still have plasminogen activity.
  • plasminogen is added, deleted and/or substituted on the basis of sequence 2, 6, 8, 10 or 12, 1-100, 1-90, 1-80, 1-70, 1 -60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3 , 1-2, 1 amino acid, and still have plasminogen activity protein.
  • plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
  • the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, delta-plasminogen, or any combination thereof. In one embodiment, the plasminogen is a conservative substitution variant selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, delta-plasminogen or microfibrinolysis Proenzyme.
  • the plasminogen is a human natural plasminogen, such as an ortholog of plasminogen as shown in SEQ ID NO: 2, for example, a plasminogen from a primate or a rodent To homologs, such as plasminogen from gorillas, rhesus monkeys, rats, cattle, horses, dogs, straight homologs.
  • the amino acid sequence of plasminogen of the invention is shown as sequence 2, 6, 8, 10 or 12.
  • the subject is low in plasmin or plasminogen.
  • the low is congenital, secondary and/or local.
  • the present invention expressly covers all combinations of the technical features between the embodiments of the present invention, and these combined technical solutions are explicitly disclosed in the present application, just as the above technical solutions have been separately and explicitly disclosed.
  • the present invention also expressly encompasses all subcombinations of the various embodiments and elements thereof, and is disclosed herein as if each such subcombination is separately and explicitly disclosed herein.
  • Thrombus is a product formed during coagulation.
  • the coagulation process is a defense mechanism for the body to maintain the integrity of the closed high pressure circulation system. Under normal conditions, the process should remain inactive, but when the tissue is damaged, the mechanism needs to be activated immediately to reduce extravasation.
  • the fibrinogen dissolved in the plasma under the action of thrombin will eventually transform into a water-insoluble fibrin multimer, which is interwoven into a net and the blood cells are netted.
  • a blood clot is formed and the blood coagulation process is completed. In this process, the ratio of the size of the blood clot to the injury is critical.
  • thrombus thrombus
  • blood clots can occur anywhere with blood flow, and currently they are mainly divided into two categories: venous thrombosis and arterial thrombosis. Venous thrombosis is caused by blood clots that are produced in the veins.
  • venous thrombosis The most common type of venous thrombosis: deep vein thrombosis (DVT), which usually affects the limb veins, such as the femoral vein, causing pain and redness in the affected area; portal vein thrombosis, which can affect the hepatic portal vein, leading to pancreatitis, cirrhosis, Diverticulitis or cholangiocarcinoma; renal vein thrombosis, leading to renal embolism; internal jugular vein thrombosis, which can cause systemic sepsis, pulmonary embolism and other complications; cerebral venous thrombosis, resulting in patients with headache, visual abnormal stroke and other symptoms.
  • DVT deep vein thrombosis
  • portal vein thrombosis which can affect the hepatic portal vein, leading to pancreatitis, cirrhosis, Diverticulitis or cholangiocarcinoma
  • renal vein thrombosis leading to renal embolism
  • Arterial thrombosis may lead to infarction of almost any organ in the body, including but not limited to: cerebral infarction, myocardial infarction, thrombotic stroke, atherosclerotic disease, unstable angina, refractory angina, transient brain Ischemic attack, pulmonary embolism, etc.
  • thrombous-related diseases are diseases caused by two pathological processes of thrombosis and thromboembolism.
  • thrombotic-associated disease of the present invention specifically encompasses all diseases caused by thrombosis and thromboembolism.
  • Thrombosis refers to a pathological process in which blood forms a embolus in a blood vessel (mostly a small blood vessel) under certain conditions, causing partial or complete occlusion of blood vessels, and blood supply disorders at corresponding sites.
  • the composition of the thrombus it can be divided into platelet thrombus, erythrocyte thrombosis, fibrin thrombus, mixed thrombus and the like.
  • the type of blood vessels can be divided into arterial thrombosis, venous thrombosis and capillary thrombosis.
  • Thromboembolism is the thrombus that falls off from the site of formation. Some or all of the blood vessels are blocked during the movement of the bloodstream, causing ischemia, hypoxia, necrosis (arterial thrombosis) and congestion of the corresponding tissues and/or organs. Pathological process of edema (venous thrombosis).
  • Venous thrombosis is most common in deep vein thrombosis of the lower extremities, which are common in deep veins such as the iliac vein, femoral vein, mesenteric vein and portal vein. Usually red blood cell thrombosis or fibrin thrombosis.
  • the main manifestations are: (1) local swelling and pain of thrombosis; (2) distal blood flow obstruction of thrombus: such as distal edema, pain, skin color change, ascites, etc.; (3) embolization of blood vessels after thrombus detachment Organ dysfunction, such as symptoms and signs of pulmonary infarction.
  • Thrombosis is more common in coronary arteries, cerebral arteries, mesenteric arteries, and limb arteries. Thrombosis is mostly platelet thrombosis in the early stage, followed by fibrin thrombosis.
  • Clinical manifestations include: (1) The incidence is more abrupt, and there may be local severe pain, such as angina pectoris, abdominal pain, severe limb pain, etc.; (2) Organ, tissue structure and dysfunction caused by tissue ischemia and hypoxia in the relevant blood supply site Such as myocardial infarction, heart failure, heart, sexual shock, arrhythmia, disturbance of consciousness and hemiplegia; (3) thrombosis caused by cerebral embolism, renal embolism, splenic embolism and other related symptoms and signs; (4) blood supply ischemia Clinical manifestations caused by sexual necrosis, such as fever. Capillary thrombosis is common in DIC, TTP, and hemolytic uremic syndrome (HUS). Clinical manifestations often lack specificity, mainly embolism necrosis of the skin and mucous membranes, microcirculatory failure and organ dysfunction.
  • Diabetes is a sugar, protein, and fat caused by various factors such as genetic factors, immune dysfunction, microbial infections and their toxins, free radical toxins, and mental factors, which cause the islet dysfunction and insulin resistance.
  • a series of metabolic disorders such as water and electrolytes are clinically characterized by hyperglycemia.
  • Diabetes complications are damage or dysfunction of other organs or tissues of the body caused by poor glycemic control during diabetes, including damage to the liver, kidneys, heart, retina, nervous system, or dysfunction. According to the World Health Organization, there are more than 100 complications of diabetes, which is the most common complication known. The complications of these diabetes are mainly due to the damage of large blood vessels, small blood vessels and microvessels in various organs of the patient.
  • Diabetes macroangiopathy mainly refers to atherosclerosis occurring in the aorta and various organ arteries.
  • the pathogenesis includes the following aspects: (1) persistent hyperglycemia increases blood viscosity and coagulability, which in turn causes weakening or even loss of arterial vascular elasticity; (2) abnormal lipid metabolism, which promotes cholesterol and cholesterol lipids in cells Internal accumulation, leading to the occurrence and development of atherosclerosis; (3) endothelial cell damage of the arterial wall, hemodynamic changes cause the blood to mechanically and long-term impact on the vascular endothelium, causing endothelial damage, which leads to platelets, fibrin, etc.
  • Diabetes microangiopathy refers to microvascular disease caused by abnormalities of microcirculation of various organs or tissues of a diabetic patient.
  • the process of microvascular disease formation is roughly: microcirculatory functional changes, endothelial damage, thickening of the basement membrane, increased blood viscosity, red blood cell aggregation, platelet adhesion and aggregation, and finally microthrombotic formation and/or microvascular occlusion.
  • diabetes angiopathy lead to local vascular damage in tissues or organs, poor blood flow, hypoxia, formation of blood clots, thrombosis and inflammation, and further affect peripheral tissues and organ functions, leading to diabetes Symptoms, for example, diabetic heart disease, diabetic bowel disease, diabetic nephropathy, diabetic retinopathy, diabetic liver disease, diabetic neuropathy.
  • Diabetes nephropathy is a microvascular complication of diabetes, mainly referred to as diabetic glomerulosclerosis, a glomerular lesion characterized by vascular damage, characterized by proteinuria, hypertension, edema, glomerulosclerosis. , vascular structural changes and tubulointerstitial disease.
  • the first clinical evidence for diabetic nephropathy is usually the presence of albuminuria in the urine, such as microalbuminuria or macroalbuminuria.
  • Diabetes neuropathy or “diabetic neuropathy” is caused by damage to the nervous system caused by diabetes, including sensory nerve damage, motor nerve damage, and autonomic nerve damage. Symptoms of sensory nerves are usually more severe. Common symptoms include, but are not limited to, limb pain, hypoesthesia, numbness, burning, cold, and diabetic neuropathic pain, including but not limited to spontaneous pain caused by diabetic complications, and hypoalgesia ( Hypoalgesia), hyperalgesia, etc.
  • Diabetes neuralgia is the most common form of diabetic neuropathy and is usually caused by damage to the sensory nerves of diabetes. The main pain is usually accompanied by temperature and tactile loss, which occurs in the lower limbs and also in the upper limbs and trunk. Generally can be divided into peripheral and central nervous system pain. Peripheral nerve pain is caused by damage to the peripheral nerves, while central nervous system pain is caused by the central nervous system and/or spinal cord injury.
  • Diabetes liver injury refers to a lesion of liver histology and functional changes caused by diabetes. It is mainly caused by large blood vessels and microvascular diseases caused by diabetes. It is known that liver damage caused by diabetes includes: liver enzyme abnormalities, which can cause carbon dioxide accumulation in liver cells, acidosis, reduction of oxygen supply, increase of oxygen consumption, increase of liver transaminase activity, bilirubin metabolism disorder, and severe Cause liver cell necrosis; fatty liver, in all causes of fatty liver, diabetes accounted for the third place, of which 21% to 78% of diabetic patients with fatty liver; hepatitis, cirrhosis and liver cancer, including viral hepatitis in diabetic patients The prevalence rate is about 2-4 times that of normal people, and the incidence of primary liver cancer is about 4 times that of normal people.
  • liver diseases caused by diabetes and related symptoms include, but are not limited to, liver enzyme abnormalities, liver discomfort and tenderness, hepatomegaly, splenomegaly, hepatosplenomegaly, hepatitis, fatty liver, cholangitis , cirrhosis, liver necrosis and liver cancer.
  • Diabetes cardiomyopathy refers to a pathological and functional change of the cardiovascular system caused by diabetes, which is one of the most common diabetic complications, mainly caused by macrovascular and microvascular diseases caused by diabetes. Among them, patients with clinical manifestations of abnormal ECG, heart enlargement, arrhythmia, angina pectoris, painless myocardial infarction, heart failure. According to statistics, about 70% to 80% of diabetic patients eventually die from cardiovascular complications.
  • Diabetes retinopathy also known as “diabetic retinopathy” refers to lesions of the retinal histology and function caused by diabetes, mainly caused by macrovascular and microvascular diseases caused by diabetes. Diabetic retinopathy is the most common form of diabetic eye disease, often resulting in vision loss or blindness. According to statistics, 50% of diabetic patients will develop the disease in the course of 10 years, and 80% in 15 years or more. The heavier the diabetes, the older the age, the higher the incidence.
  • the lesion is fresh or old.
  • the first lesion was a fresh lesion in the acute attack period, and the ischemic center of the lesion was partially necrotic, and some recovered, and the surrounding area was not affected.
  • the purpose of treatment at this time should mainly be to prevent the expansion of the "central infarct zone".
  • the old thrombus is the complete necrosis of the tissue ischemic center, and the purpose of treatment should be to improve the function of tissues around the infarct area.
  • Older thrombi have a higher risk of recurrence, so treatment and prevention are equally important for patients with old thrombosis, and the high recurrence rate should be reduced while reducing the degree of symptoms.
  • the current variety of thrombolytic drugs for the treatment of acute thrombosis in the acute phase is acceptable, but the treatment of old thrombosis is less effective.
  • Plasmid is a very important enzyme found in the blood that hydrolyzes fibrin clots into fibrin degradation products and D-dimers.
  • Plasminogen is a zymogen form of plasmin. According to the sequence in swiss prot, it is composed of 810 amino acids based on the amino acid sequence of the natural human plasminogen (sequence 4) containing the signal peptide. The molecular weight is about 92kD, mainly A glycoprotein synthesized in the liver and capable of circulating in the blood, the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO:3. The full-length PLG contains seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAp) domain at the N-terminus, and five Kringle domains (Kringle 1-5).
  • PAp Pan Apple
  • the signal peptide includes the residue Met1-Gly19
  • PAp includes the residue Glu20-Val98
  • Kringle1 includes the residue Cys103-Cys181
  • Kring1e2 includes the residue Glu184-Cys262
  • Kring1e3 includes the residue Cys275-Cys352
  • Kring1e4 The residue Cys377-Cys454 is included
  • Kring1e5 includes the residue Cys481-Cys560.
  • the serine protease domain includes the residues Val581-Arg804.
  • Glu-plasminogen is a natural full-length plasminogen consisting of 791 amino acids (not containing a 19 amino acid signal peptide), and the cDNA sequence encoding the sequence is shown in SEQ ID NO: 1, and its amino acid sequence is sequence 2. Shown. In vivo, there is also a Lys-plasminogen which is hydrolyzed from amino acids 76-77 of Glu-plasminogen, and as shown in SEQ ID NO: 6, the cDNA sequence encoding the amino acid sequence is as shown in SEQ ID NO: 5 Shown.
  • ⁇ -plasminogen is a fragment of full-length plasminogen deleted from Kringle2-Kringle5 structure, containing only Kringle1 and serine protease domains [28, 29] .
  • the amino acid sequence of ⁇ -plasminogen has been reported in the literature (sequence 8) [30] , the cDNA sequence encoding the amino acid sequence is SEQ ID NO: 7.
  • Mini-plasminogen consists of Kringle5 and a serine protease domain, which has been reported to include the residue Val443-Asn791 (the Glu residue of the Glu-plg sequence containing no signal peptide is the starting amino acid) [31] , and its amino acid sequence is as sequence As shown in Figure 10, the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 9.
  • Micro-plasminogen contains only the serine protease domain, and its amino acid sequence has been reported to include the residue Ala543-Asn791 (the Glu residue of the Glu-plg sequence containing no signal peptide is the starting amino acid) [32] , and the patent CN102154253A It is reported that the sequence includes the residue Lys531-Asn791 (the Glu residue of the Glu-plg sequence not containing the signal peptide is the starting amino acid), and the amino acid sequence of this patent is shown in the sequence 12, and the amino acid sequence is encoded.
  • the cDNA sequence is shown in SEQ ID NO: 11.
  • Plasin of the present invention is used interchangeably with “fibrinolytic enzyme” and “fibrinolytic enzyme”, and has the same meaning; “plasminogen” and “plasminogen”, “fibrinolytic enzyme” "Original” is used interchangeably and has the same meaning.
  • fresh thrombus and "acute thrombosis” of the present invention are used interchangeably; "old thrombus” and “chronic thrombus” are used interchangeably.
  • plasminogen adopts a closed inactive conformation, but when bound to the surface of a thrombus or cell, it is converted to openness mediated by plasminogen activator (PA).
  • PA plasminogen activator
  • Conformational active plasmin The active plasmin further hydrolyzes the fibrin clot into a fibrin degradation product and a D-dimer, thereby dissolving the thrombus.
  • the PAp domain of plasminogen contains an important determinant that maintains plasminogen in an inactive blocking conformation, while the KR domain is capable of binding to lysine residues present on the receptor and substrate.
  • plasminogen activators include tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hag Mann factor) and so on.
  • a "plasminogen active fragment” refers to an active fragment in a plasminogen protein that binds to a target sequence in a substrate and exerts a proteolytic function.
  • the technical solution of the present invention relating to plasminogen covers the technical solution of replacing plasminogen with a plasminogen active fragment.
  • the plasminogen active fragment of the present invention is a protein comprising a serine protease domain of plasminogen.
  • the plasminogen active fragment of the present invention comprises the sequence 14, and the sequence 14 has at least 80%, 90.
  • the plasminogen of the present invention comprises a protein comprising the plasminogen active fragment and still retaining the plasminogen activity.
  • methods for measuring plasminogen and its activity in blood include: detection of tissue plasminogen activator activity (t-PAA), detection of plasma tissue plasminogen activator antigen (t-PAAg) Detection of plasma tissue plasminogen activity (plgA), detection of plasma tissue plasminogen antigen (plgAg), detection of plasma tissue plasminogen activator inhibitor activity, plasma tissue plasminogen activator Detection of inhibitor antigen, plasma plasmin-antiplasmin complex assay (PAP).
  • t-PAA tissue plasminogen activator activity
  • t-PAAg plasma tissue plasminogen activator antigen
  • plgA plasma tissue plasminogen activator antigen
  • PAP plasma plasmin-antiplasmin complex assay
  • the most commonly used detection method is the chromogenic substrate method: adding streptokinase (SK) and chromogenic substrate to the plasma to be tested, and the PLG in the tested plasma is converted into PLM under the action of SK, and the latter acts on The chromogenic substrate is then measured spectrophotometrically and the increase in absorbance is directly proportional to the plasminogen activity.
  • plasminogen activity in blood can also be measured by immunochemical method, gel electrophoresis, immunoturbidimetry, or radioimmunoassay.
  • ortholog or ortholog refers to homologs between different species, including both protein homologs and DNA homologs, also known as orthologs, orthologs. It specifically refers to a protein or gene that has evolved from the same ancestral gene in different species.
  • the plasminogen of the present invention includes human natural plasminogen, and also includes plasminogen orthologs or orthologs of plasminogen activity derived from different species.
  • Constant substitution variant refers to a change in one of the given amino acid residues without altering the overall conformation and function of the protein or enzyme, including but not limited to similar properties (eg, acidic, basic, hydrophobic, etc.)
  • the amino acid replaces the amino acid in the amino acid sequence of the parent protein.
  • Amino acids having similar properties are well known. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and are interchangeable.
  • isoleucine is a hydrophobic amino acid that can be replaced by leucine, methionine or valine. Therefore, the similarity of two protein or amino acid sequences of similar function may be different.
  • Constant substitution variants also includes determining polypeptides or enzymes having more than 60% amino acid identity by BLAST or FASTA algorithm. If it is more than 75%, preferably more than 85%, or even more than 90%. Optimal and have the same or substantially similar properties or functions as the native or parent protein or enzyme.
  • Isolated plasminogen refers to a plasminogen protein that is isolated and/or recovered from its natural environment.
  • the plasminogen will purify (1) to a purity greater than 90%, greater than 95%, or greater than 98% by weight, as determined by the Lowry method, eg, over 99% (by weight), (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotating cup sequence analyzer, or (3) to homogeneity, which is by use Coomassie blue or silver staining was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions.
  • Isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering techniques and isolated by at least one purification step.
  • polypeptide peptide
  • protein protein
  • fusion proteins including, but not limited to, fusion proteins having a heterologous amino acid sequence, fusions having heterologous and homologous leader sequences (with or without an N-terminal methionine residue);
  • percent amino acid sequence identity with respect to a reference polypeptide sequence is defined as the introduction of a gap as necessary to achieve maximum percent sequence identity, and does not treat any conservative substitution as a sequence
  • Comparisons for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art will be able to determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum contrast over the full length of the sequences being compared. However, for the purposes of the present invention, amino acid sequence identity percent values are generated using the sequence comparison computer program ALIGN-2.
  • amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or may be expressed as having or comprising relative to, and, or for a given amino acid sequence)
  • a given amino acid sequence A of a certain % amino acid sequence identity of B is calculated as follows:
  • X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 in the A and B alignments of the program, and wherein Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A relative to B will not be equal to the % amino acid sequence identity of B relative to A. All % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph, unless explicitly stated otherwise.
  • the terms “treating,” “treating,” and “eliminating” refer to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be to completely or partially prevent the disease or its symptoms, and/or to partially or completely cure the disease and/or its symptoms, and includes: (1) preventing the disease from occurring in the subject, the subject may have The cause of the disease, but not yet diagnosed as having a disease; (2) inhibiting the disease, ie, retarding its formation; and (3) reducing the disease and/or its symptoms, ie causing the disease and/or its symptoms to subside.
  • the terms "individual”, “subject” and “patient” are used interchangeably herein to refer to a mammal, including but not limited to a mouse (rat, mouse), a non-human primate, a human, a dog, a cat. Hoofed animals (such as horses, cattle, sheep, pigs, goats).
  • “Therapeutically effective amount” or “effective amount” refers to an amount of plasminogen sufficient to effect such prevention and/or treatment of a disease when administered to a mammal or other subject to treat the disease.
  • the “therapeutically effective amount” will vary depending on the plasminogen used, the severity of the disease and/or its symptoms of the subject to be treated, and the age, weight, and the like.
  • Plasminogen can be isolated and purified from nature for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When the polypeptide is chemically synthesized, it can be synthesized in a liquid phase or a solid phase.
  • Solid phase polypeptide synthesis SPPS
  • Fmoc and Boc Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize plasminogen.
  • the attached solid phase free N-terminal amine is coupled to a single N-protected amino acid unit. This unit is then deprotected to reveal a new N-terminal amine that can be attached to other amino acids.
  • the peptide remains immobilized on the solid phase and then cut off.
  • the plasminogen of the present invention can be produced using standard recombinant methods.
  • a nucleic acid encoding plasminogen is inserted into an expression vector operably linked to a regulatory sequence in an expression vector.
  • Expression control sequences include, but are not limited to, promoters (eg, naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • Expression regulation can be a eukaryotic promoter system in a vector that is capable of transforming or transfecting eukaryotic host cells (eg, COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of plasminogen.
  • Suitable expression vectors are typically replicated as an episome in the host organism or as an integral part of the host chromosomal DNA.
  • expression vectors typically contain a selection marker (eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate transformation of the desired DNA sequence with foreign sources. Those cells are tested.
  • a selection marker eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance
  • Escherichia coli is an example of a prokaryotic host cell that can be used to clone a subject antibody-encoding polynucleotide.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Genus (Pseudomonas) kind.
  • expression vectors can also be generated which will typically contain expression control sequences (e.g., origins of replication) that are compatible with the host cell.
  • promoters such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from phage lambda. Promoters typically control expression, optionally in the context of manipulating a gene sequence, and have a ribosome binding site sequence, etc., to initiate and complete transcription and translation.
  • yeast can also be used for expression.
  • Yeast e.g., S. cerevisiae
  • Pichia are examples of suitable yeast host cells in which a suitable vector has expression control sequences (e.g., a promoter), an origin of replication, a termination sequence, and the like, as desired.
  • a typical promoter comprises 3-phosphoglycerate kinase and other saccharolytic enzymes.
  • Inducible yeast is initiated by a promoter specifically comprising an alcohol dehydrogenase, an isocytochrome C, and an enzyme responsible for the utilization of maltose and galactose.
  • mammalian cells e.g., mammalian cells cultured in in vitro cell culture
  • plasminogen of the invention e.g., a polynucleotide encoding a subject anti-Tau antibody.
  • Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas.
  • Expression vectors for these cells may contain expression control sequences such as origins of replication, promoters and enhancers (Queen et al, Immunol. Rev.
  • RNA splice sites sites that are ribosome binding.
  • RNA splice sites sites that are ribosome binding.
  • polyadenylation sites sites that are ribosome binding sites.
  • transcription terminator sequences sites that are ribosome binding sites.
  • suitable expression control sequences are promoters derived from the white immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al, J. Immunol. 148: 1149 (1992).
  • the invention may be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, and the like.
  • Plasminogen is substantially pure, such as at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure. Or more pure, for example, free of contaminants, such as cellular debris, macromolecules other than the subject antibody, and the like.
  • Frozen can be formed by mixing plasminogen of the desired purity with an optional pharmaceutical carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980))
  • the therapeutic formulation is prepared as a dry formulation or as an aqueous solution.
  • Acceptable carriers, excipients, stabilizers It is non-toxic to the recipient at the dosages and concentrations used, and includes buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl chloride Ammonium; chlorinated hexane diamine; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl p-hydroxybenzoate such as methyl or propyl p-hydroxybenzoate Acid ester; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol; low molecular weight polypeptide (less than about 10 residues); protein such as serum albumin, gelatin or immunoglobulin Protein; hydrophilic polymer such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspara
  • the formulations of the invention may also contain more than one active compound as required for the particular condition being treated, preferably those having complementary activities and no side effects to each other.
  • active compound for example, antihypertensive drugs, antiarrhythmic drugs, drugs for treating diabetes, and the like.
  • the plasminogen of the present invention may be encapsulated in microcapsules prepared by, for example, coacervation techniques or interfacial polymerization, for example, may be placed in a glial drug delivery system (eg, liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules are placed in hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a macroemulsion.
  • glial drug delivery system eg, liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules are placed in hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a macroemulsion.
  • the plasminogen of the invention for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filter before or after lyophilization and reconstitution.
  • the plasminogen of the present invention can prepare a sustained release preparation.
  • sustained release formulations include solid hydrophobic polymeric semi-permeable matrices having a shape and containing glycoproteins, such as films or microcapsules.
  • sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) (Langer et al, J. Biomed. Mater. Res., 15: 167-277 (1981); Langer, Chem .Tech., 12: 98-105 (1982)) or poly(vinyl alcohol), polylactide (U.S.
  • Patent 3,739,919, EP 58,481 L-glutamic acid and ⁇ -ethyl-L-glutamic acid Copolymer (Sidman, et al, Biopolymers 22: 547 (1983)), non-degradable ethylene-vinyl acetate (Langer, et al, supra), or degradable lactic acid-glycolic acid copolymer Such as Lupron DepotTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly D-(-)-3- Hydroxybutyric acid.
  • Lupron DepotTM injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
  • poly D-(-)-3- Hydroxybutyric acid poly D-(-)-3- Hydroxybutyric acid.
  • Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can sustain release molecules for more than 100 days, while some hydrogels release proteins for shorter periods of time.
  • a rational strategy for stabilizing proteins can be designed based on relevant mechanisms. For example, if the mechanism of aggregation is found to be an intermolecular SS bond by thiodisulfide bond exchange, it can be modified by modifying the thiol residue, lyophilizing from an acidic solution, controlling humidity, using suitable additives, and developing specific The polymer matrix composition is used to achieve stability.
  • the invention may be practiced in various ways, for example by intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (for example via carotid), intramuscular, intranasal, topical or intradermal administration or spinal or brain delivery.
  • Administration of the pharmaceutical composition
  • Aerosol formulations such as nasal spray formulations comprise purified aqueous or other solutions of the active agents and preservatives and isotonic agents. Such formulations are adjusted to a pH and isotonic state compatible with the nasal mucosa.
  • the plasminogen pharmaceutical compositions of the invention may be modified or formulated in such a manner as to provide their ability to cross the blood brain barrier.
  • Compositions of such plasminogen can be administered to individuals suffering from thrombotic and/or thrombotic related diseases by a variety of enteral and parenteral routes of administration, including oral, intravenous, and the like.
  • Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffering media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils.
  • Intravenous vehicles contain liquid and nutritional supplements, electrolyte supplements, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, and the like.
  • the plasminogen of the invention is formulated with an agent that promotes across the blood brain barrier.
  • the plasminogen of the invention is fused directly or via a linker to a carrier molecule, peptide or protein that facilitates crossing the blood brain barrier.
  • the plasminogen of the invention is fused to a polypeptide that binds to an endogenous blood brain barrier (BBB) receptor. Linking plasminogen to a polypeptide that binds to the endogenous BBB receptor facilitates passage through the BBB.
  • BBB blood brain barrier
  • Suitable polypeptides that bind to an endogenous BBB receptor include antibodies, such as monoclonal antibodies, or antigen-binding fragments thereof that specifically bind to an endogenous BBB receptor.
  • Suitable endogenous BBB receptors include, but are not limited to, insulin.
  • antibodies are encapsulated in liposomes. Body, transferrin receptor, lipoprotein receptor, and insulin-like growth factor receptor. See, for example, U.S. Patent Publication No. 2009/0156498.
  • the medical staff will determine the dosage regimen based on various clinical factors. As is well known in the medical arts, the dosage of any patient depends on a variety of factors, including the patient's size, body surface area, age, specific compound to be administered, sex, number and route of administration, overall health, and other medications administered simultaneously. .
  • the pharmaceutical composition of the present invention comprising plasminogen may have a dose ranging, for example, from about 0.0001 to 2000 mg/kg per day, or from about 0.001 to 500 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75). Mg/kg, 10 mg/kg, 50 mg/kg, etc.) Subject weight.
  • the dose can be 1 mg/kg body weight or 50 mg/kg body weight or in the range of 1-50 mg/kg, or at least 1 mg/kg. Dosages above or below this exemplary range are also contemplated, particularly in view of the above factors. Intermediate doses in the above ranges are also included in the scope of the present invention.
  • the subject can administer such doses daily, every other day, every week, or according to any other schedule determined by empirical analysis.
  • An exemplary dosage schedule includes 1 to 10 mg/kg for several days.
  • the therapeutic effect and safety of thrombus and thrombosis-related diseases need to be evaluated and periodically evaluated in the drug administration process of the present invention.
  • the evaluation of the therapeutic efficacy of plasminogen is mainly carried out by monitoring the following indicators:
  • Thrombolysis rate after 1 week of treatment For example, a contrast agent can be injected through a catheter, and thrombolysis can be evaluated daily. Each vascular region is scored, with a total open score of 0, a partial occlusion of 1 point, and a complete occlusion of 2 points. According to the total score after thrombolysis minus the total score after thrombolysis, divided by the ratio of the total score before thrombolysis, the different thrombolytic grades were divided, the first level was ⁇ 50%, the second level was 50%-90%, and the third level was thrombus. completely dissolved.
  • Vascular patency rate after 6 months for example, vascular patency rate can be evaluated by endoscopic, CT angiographic analysis, color Doppler ultrasound, and the like. The effectiveness of the treatment was judged by the percentage of vascular patency after treatment and whether there was a statistically significant increase before treatment.
  • Vascular occlusion and/or venous return rate after 6 months The improvement of the thrombolytic rate of the agent was judged by statistically reducing the vascular occlusion and/or the decrease in venous return rate after treatment.
  • vascular echo changes for example, blood vessel wall thickness comparison, and incidence of thrombotic sequelae after 2 years.
  • vessel wall thickness and intraluminal echo can be assessed by gray-scale ultrasound, while iliac, femoral venous flow and femoral valvular insufficiency can be assessed by Doppler ultrasound in a standing position.
  • the safety of plasminogen drugs after thrombus treatment is evaluated, which mainly includes monitoring the incidence of adverse events after treatment. Severe bleeding, embolism, stroke, and death were generally classified as serious adverse events, while secondary bleeding and other minor symptom complications were classified as secondary adverse events.
  • the most common adverse event is bleeding, such as intracranial hemorrhage (also known as hemorrhagic stroke, including subarachnoid hemorrhage, subdural hemorrhage, etc.).
  • Severe bleeding according to the present invention generally refers to intracranial hemorrhage or bleeding events that are severe enough to cause death, surgery, cessation of treatment, or blood transfusion, including "major hemorrhage events” and "life-threatening bleeding events.”
  • the secondary bleeding is to direct bleeding from the sheath side and/or to change the dose of the thrombolytic agent, anticoagulant or antiplatelet agent, or to stop bleeding by compression.
  • major bleeding and “major bleeding event” specifically refers to blood having a hemoglobin content reduced by at least 2.0 g/L or at least 2 units of blood transfusion, or symptomatic bleeding in a critical site or organ.
  • a hemorrhagic event that is more severe than "big bleeding”, a subtype of major bleeding events, is called a "life-threatening bleeding event,” including fatal bleeding, symptomatic intracranial hemorrhage, hypoglycemic reduction of at least 5.0 g/L, or blood transfusion. More than 4 units of blood require bleeding from a myocardial contractor or surgery.
  • the doses are fine-tuned according to the severity of the disease and follow-up monitoring of adverse events after administration is performed for at least 3 months, preferably 6 months or more.
  • the risk of major bleeding includes, but is not limited to, (1) age 75 years and older, (2) a history of previous bleeding events, and (3) reduced creatinine clearance, which is less than 80 mL/min or less than 50 mL/min.
  • One embodiment of the invention relates to an article or kit comprising a plasminogen of the invention useful for treating a thrombus.
  • the article preferably includes a container, label or package insert. Suitable containers are bottles, vials, syringes, and the like.
  • the container can be made of various materials such as glass or plastic.
  • the container contains a composition that is effective to treat a disease or condition of the invention and has a sterile access port (eg, the container can be an intravenous solution or vial containing a stopper that can be penetrated by a hypodermic needle) of). At least one active agent in the composition is plasminogen.
  • the label on or attached to the container indicates that the composition is used to treat the thrombotic and thrombotic related diseases of the present invention.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
  • the article comprises a package insert with instructions for use, including, for example, a user instructing the composition to administer the plasminogen composition and other drugs to treat the accompanying disease.
  • Figure 1 shows the effect of different doses of plasminogen on 20 hours of old thrombolysis in the presence of 125 ng of tPA at 37 °C for 1 hour.
  • Figure 2 shows the dissolution of different doses of plasminogen for 20 hours of old thrombus in the presence of 125 ng of tPA at 37 °C for 2 hours.
  • Figure 3 shows the effect of different doses of plasminogen on 20 hours old thrombolytic thrombolysis at 10 ° tPA for 2 hours at 37 °C.
  • Figure 4 shows the thrombolytic effect of different doses of plasminogen on 72 hours old thrombus under incubation at 125 ng tPA for 2 hours at 125 ng tPA.
  • Figure 5 shows the thrombolytic effect of different doses of plasminogen on 72 hours old thrombus under incubation at 10 ng tPA for 2 hours at 37 °C.
  • Figure 6 shows the change in thrombolysis rate over time after the addition of 10 ng of tPA and 1 mg of plg or 5 ⁇ g of tPA alone.
  • Figure 7 shows the effect of different doses of plasminogen on a 20 hour old thrombolysis effect at 100 ° uPA for 1 hour at 37 °C.
  • Figure 8 shows the effect of different doses of plasminogen on a 20 hour old thrombolytic effect when incubated at 37 °C for 2 hours under 1 ng uPA conditions.
  • Figure 9 shows the results of specific adsorption experiments of plasminogen for thrombus in vivo.
  • Figure 10 shows the thrombolytic effect of different concentrations of plasminogen on fresh thrombus for 30 minutes at 125 ng tPA for 2 hours at 125 ng tPA.
  • Figure 11 shows the results of detecting the concentration of D-dimer in serum after 15 days of administration of plasminogen in 24-25-week-old diabetic mice.
  • Figure 12 shows the results of immunohistochemical staining of cardiac fibrin for 31 days of PBS (A) or plasminogen (B) in 24-25 week diabetic mice.
  • Figure 13 shows the results of immunohistochemical staining of kidney fibrin 31 days after administration of PBS (A) or plasminogen (B) in 24-25 week diabetic mice.
  • Figure 14 24-25 shows the results of immunohistochemical staining of liver fibrin 31 days after administration of PBS (A) or plasminogen (B) in diabetic mice.
  • FIG. 15 24-25 weeks post-diabetes nerve injury Mice were given PBS (A) or plasminogen (B) for 15 days after sciatic nerve fibrin immunohistochemical staining.
  • mice (6-8 weeks old) were purchased from the Experimental Animal Center of Southern Medical University. The purchased mice were housed in a barrier environment animal house. The db/db mice were purchased from the Nanjing Institute of Biomedical Research.
  • the unilateral carotid artery thrombosis was performed by external application of filter paper containing 10% FeCl3 for 5 min. Intravenous injection of plasminogen was started within 1 h after the model was established, and the control group was intravenously injected. Equal volume PBS is used. After 3 hours, the corresponding jugular vein thrombus and the muscles near the contralateral vein were removed. The thrombus and the muscles in the vicinity of the contralateral vein were homogenized using a grinder, and the supernatant was taken after centrifugation.
  • the supernatant was assayed for total protein by BCA method, and the plasminogen content in the homogenate was determined by enzyme-linked immunosorbent assay to calculate a certain amount.
  • the plasminogen content in the total amount of protein was determined by enzyme-linked immunosorbent assay to calculate a certain amount.
  • mice 24-25 week old db/db mice were used as control and experimental animals, and vehicle PBS or plasminogen was administered to the tail vein, respectively. After 31 days, the eyeballs were taken for blood D-dimer detection, and fibrin immunohistochemical staining was performed on nerve, liver, kidney and heart to study the thrombolytic effect of plasminogen.
  • mice were harvested from the eyeball to obtain blood, and the experiment was performed according to the D-dimer kit (Wuhan USCN, China). After the test was completed, the reading was performed at 450 nm using a microplate reader (Biotek USA) for data analysis. .
  • the fixed tissue was dehydrated by ethanol gradient, embedded in paraffin, and sliced with paraffin. The thickness of the slice was 5 ⁇ m. The slice was dewaxed to water and washed once with water, and then the tissue was circled with a PAP pen. Incubate with hydrogen peroxide diluted in 0.3% methanol for 15 minutes and wash 3 times with water. 10% of the normal serum homologous to the secondary antibody was blocked for 10 minutes, and excess serum was aspirated. The primary antibody was incubated at room temperature for 30 minutes or 4 degrees overnight, and TBS was washed 3 times.
  • the HRP-labeled secondary antibody was incubated for 30 minutes at room temperature and 3 times with TBS. Press DAB kit (vector Laboratories, Inc., USA) Color development, hematoxylin counterstaining for 30 seconds, running water back to blue for 5 minutes, then TBS wash 1 time. The gradient is dehydrated and sealed.
  • the antibodies used are: the marker antibody has Fibrin (ogen) (Abcam). Sections were observed under an optical microscope (Olympus, BX43).
  • Healthy human plasma was taken in an ELISA 96-well plate, and a fixed amount of thrombin (Sigma, USA) was added to form a thrombus, and then the following different experiments were performed.
  • a fixed amount of tPA, uPA (sigma, USA) and varying amounts of plasminogen, fixed amounts of plasminogen and varying amounts of tPA, uPA, streptokinase (sigma, USA) were added and the control group was added to PBS. Incubate for different times until thrombolysis was performed and the absorbance readings were recorded at the wavelength of OD405 on a microplate reader (Biotek, USA) and the time of each measurement. Analyze the data.
  • Example 1 20-hour old thrombus was incubated at 37 ° C for 1 hour under 125 ng tPA, and the thrombolytic effect of different doses of plasminogen
  • thrombus Two SD rat whole blood samples were collected into Eppendorf (EP) tubes, and the supernatant was discarded after incubation at 37 ° C for 20 hours to form old thrombus [33,34] .
  • the thrombus was divided into PBS blank control group, 125 ng tPA control group, 20 ⁇ g tPA control group, 0.2 mg plasminogen group, 1 mg plasminogen group and 2 mg plasminogen group, 3 tubes in each group.
  • PBS blank control group was added with 1 mL PBS; 125 ng tPA control group was added with 1 mL PBS and 125 ng tPA; 20 ⁇ g tPA control group was added with 1 mL PBS and 20 ⁇ g tPA; 0.2 mg plasminogen group was added with 1 mL PBS and 125 ng tPA and 0.2 mg plasminogen.
  • 1 mg plasminogen group was added with 1 mL PBS and 125 ng tPA and 1 mg plasminogen; 2 mg plasminogen group was added with 1 mL PBS and 125 ng tPA and 2 mg plasminogen. All reactions were carried out in a 37 ° C incubator. After incubating for 1 hour, the supernatant was aspirated, the blotting paper was as much as possible to absorb the thrombus, and the weight of the thrombus was weighed, and the thrombolysis rate was calculated.
  • the content of tPA is 5-10 ng/mL under normal physiological conditions [35] , and in the case of strenuous exercise or venous congestion, the content of tPA in the body will increase from 20 to 100 times, that is, 100 ng/mL. Above [36] . Therefore, the tPA dose used in this experiment was 125 ng/mL to mimic the naturally occurring tPA content in the presence of thrombus in vivo.
  • the addition of plasminogen 1 mg can achieve an in vivo injection dose of 20 ⁇ g tPA (according to the dosage of the atropin for injection prepared by Boehringer Ingelheim), the dose converted to thrombus in vivo is converted into rats.
  • the required injection dose the same thrombolytic effect. That is, the same rate of thrombolysis is achieved. If 1 mg of plasminogen is present in the body, the amount of tPA required can be reduced to 1/160 of the original.
  • Example 2 20-hour old thrombus was incubated at 37 ° C for 2 hours under 125 ng tPA, and the thrombolytic effect of different doses of plasminogen
  • PBS blank control group was started with 1 mL PBS; 125 ng tPA control group was added with 1 mL PBS and 125 ng tPA; 20 ⁇ g tPA control group was added with 1 mL PBS and 20 ⁇ g tPA; 0.2 mg plasminogen group was added with 1 mL PBS and 125 ng tPA and 0.2 mg plasmin.
  • Original; 1 mg plasminogen group was added with 1 mL PBS and 125 ng tPA and 1 mg plasminogen; 2 mg plasminogen group was added with 1 mL PBS and 125 ng tPA and 2 mg plasminogen. All reactions were carried out in a 37 ° C incubator. After 2 hours of incubation, the supernatant was aspirated, the blotting paper was as much as possible to absorb the thrombus, and the weight of the thrombus was weighed to calculate the thrombolytic rate.
  • the content of tPA is 5-10 ng/mL under normal physiological conditions [35] , and in the case of strenuous exercise or venous congestion, the content of tPA in the body will increase to 20-100 times, that is, 100 ng/ Above mL [36] . Therefore, the tPA dose used in this experiment was 125 ng/mL to mimic the naturally occurring tPA content in the presence of thrombus in vivo.
  • the thrombolytic effect of the 1 mg, 2 mg plasminogen group was better than that of the normal injection dose of 20 ⁇ g tPA in the body (the thrombolysis in the case of thrombosis in vivo according to the instructions for injection of aptase produced by Boehringer Ingelheim)
  • the dose to be used is converted to the required injection dose in rats, that is, the same rate of thrombolysis is achieved. If 1 mg of plasminogen is present in the system, the amount of tPA required can be reduced to 1 mg of plasminogen in the system. Less than 1/160 of the amount of tPA (20 ⁇ g) is required (Fig. 2).
  • 1 mL PBS was added at the beginning of the PBS blank control group; 1 mL PBS and 10 ng tPA were added to the 10 ng tPA control group; 1 mL PBS and 0.2 mg plasminogen were added to the 0.2 mg plasminogen control group; 1 mL PBS was added to the 0.2 mg plasminogen group. And 10 ng of tPA and 0.2 mg of plasminogen; 1 mg of plasminogen group was added with 1 mL of PBS and 10 ng of tPA and 1 mg of plasminogen; 2 mg of plasminogen group was added with 1 mL of PBS and 10 ng of tPA and 2 mg of plasminogen.
  • the content of tPA is 5-10 ng/mL under normal physiological conditions [35] . Therefore, the dose of tPA used in this experiment is 10 ng/mL to mimic the naturally occurring tPA content in normal physiological conditions in vivo.
  • Example 4 72-hour old thrombus thrombolysis rate increased with plasminogen dose at 125 ng tPA
  • thrombus Two SD rats were collected from whole blood into the EP tube, and after 72 hours of incubation at 37 ° C, the supernatant was discarded to form old thrombus [36] .
  • the thrombus was divided into PBS blank control group, 125 ng tPA control group, 0.2 mg plasminogen control group, 0.2 mg plasminogen group, 1 mg plasminogen group and 2 mg plasminogen group, 3 tubes in each group.
  • PBS blank control group was started with 1 mL PBS; 125 ng tPA control group was added with 1 mL PBS and 125 ng tPA; 0.2 mg Plg control group was added with 1 mL PBS and 0.2 mg Plg; 0.2 mg plasminogen group was added with 1 mL PBS and 125 ng tPA and 0.2 mg fiber. Lysozyme; 1 mg plasminogen group was added with 1 mL PBS and 125 ng tPA and 1 mg plasminogen; 2 mg plasminogen group was added with 1 mL PBS and 125 ng tPA and 2 mg plasminogen. All reactions were carried out in a 37 ° C incubator. After 2 hours of incubation, the supernatant was aspirated, the blotting paper was as much as possible to absorb the thrombus, and the weight of the thrombus was weighed to calculate the thrombolytic rate.
  • the content of tPA is 5-10 ng/mL under normal physiological conditions [35] , and in the case of strenuous exercise or venous congestion, the content of tPA in the body will increase to 20-100 times, that is, 100 ng/ Ml above [36] . Therefore, the tPA dose used in this experiment was 125 ng/ml to mimic the naturally occurring tPA content in the case of thrombus in vivo.
  • the thrombolytic rate of plasminogen was higher than that of 125 ng tPA alone under the condition of 125 ng tPA, and the difference was extremely significant, indicating that thrombosis occurred in vivo.
  • the addition of 0.2 mg or more of plasminogen for 2 hours significantly promoted the dissolution of the old thrombus for 72 hours.
  • the thrombolysis rate also showed a gradient increasing trend, indicating that the rate of dissolution of old thrombus can be adjusted by adjusting the dose of plasminogen.
  • the thrombolytic rate of 4 mg of plasminogen was added in this experiment to exceed the normal injection dose of 20 ⁇ g tPA in vivo (the dose required for thrombolysis in vivo in the case of thrombosis in accordance with the instructions for injection of aptase from Boehringer Ingelheim)
  • the thrombolytic rate of the injected dose of the rat which indicates the amount of tPA (125 ng) naturally produced when a thrombus occurs in the body, only the plasminogen is added to dissolve the old blood.
  • the effect of the plug is better than that of the existing thrombolytic drugs (Fig. 4), indicating that plasminogen can be a better thrombolytic substance.
  • Example 2 the addition of 125 ng of tPA alone significantly increased the ability to lyse 20 hours of thrombus compared to the control PBS group.
  • the thrombolysis effect of the 125 ng tPA group and the control PBS group was almost the same, indicating that the natural tendency was caused by the aging of the thrombus and the physiological condition.
  • the thrombolytic capacity of tPA is gradually reduced, and it is explained from the side that the model used in the examples can mimic the in vivo situation to some extent.
  • Example 5 72 hours old stab thrombosis under 10 ng tPA conditions thrombolysis increased with increasing plasminogen dose
  • the thrombus was divided into PBS blank control group, 10 ng tPA control group, 20 ⁇ g tPA control group, 0.2 mg plasminogen control group, 0.2 mg plasminogen group, 1 mg plasminogen group, 2 mg plasminogen group and 4 mg plasminogen group, 3 tubes per group.
  • PBS blank control group was added with 1 mL PBS; 10 ng tPA control group was added with 1 mL PBS and 10 ng tPA; 20 ⁇ g tPA control group was added with 1 mL PBS and 20 ⁇ g tPA; 0.2 mg Plg control group was added with 1 mL PBS and 0.2 mg Plg; 0.2 mg plasminogen group Add 1 mL PBS and 10 ng tPA and 0.2 mg plasminogen; 1 mg plasminogen group added 1 mL PBS and 10 ng tPA and 1 mg plasminogen; 2 mg plasminogen group added 1 mL PBS and 10 ng tPA and 2 mg plasmin Original; 4 mg plasminogen group was added with 1 mL PBS and 10 ng tPA and 4 mg plasminogen.
  • the content of tPA is 5-10 ng/mL under normal physiological conditions [35] . Therefore, the dose of tPA used in this experiment is 10 ng/mL to mimic the naturally occurring tPA content in normal physiological conditions in vivo.
  • the thrombolytic rate of the 4 mg plasminogen group was close to that of the normal tPA injection (the injection required for the thrombolysis in the case of thrombolysis in vivo according to the instructions for injection of atropinase produced by Boehringer Ingelheim)
  • the rate of thrombolysis indicates that at the physiological level of tPA (10 ng), the effect of dissolving the old thrombus after adding only plasminogen can reach the effect of the existing thrombolytic drugs. In this sense, plasmin originally hoped to become a new old thrombolytic drug.
  • Example 6 plasminogen mildly dissolved for 20 hours old thrombus
  • the first group was the tPA control group, 1 mL PBS and 5 ⁇ g tPA were added; the second group was the plasminogen group, and 1 mL PBS, 10 ng tPA, and 1 mg plasminogen were added.
  • All reactions were carried out in a 37 ° C incubator, sampling at 0.5 h, 1 h, 1.5 h, 2 h, respectively. At each time point, three samples were taken from each group, and the supernatant was aspirated. The blotting paper was used to absorb the thrombus as much as possible. The weight of the thrombus was calculated and the thrombolysis rate was calculated.
  • the content of tPA is 5-10 ng/mL under normal physiological conditions [35] . Therefore, the dose of tPA used in this experiment is 10 ng/mL to mimic the naturally occurring tPA content in normal physiological conditions in vivo.
  • Table 1 Changes in the efficiency of 1 mg plasminogen thrombolysis and the 5 ug tPA thrombolysis efficiency over time in the presence of 10 ng of tPA.
  • Example 7 plasminogen promotes 20 hours old thrombolytic in 100 ng uPA
  • 1 mL PBS was added at the beginning of the PBS blank control group; 1 mL PBS and 100 ng uPA were added to the 100 nguPA control group; 1 mL PBS and 0.2 mg plasminogen were added to the 0.2 mg plasminogen control group; 1 mL PBS and 100 ng were added to the 0.2 mg plasminogen group.
  • the tPA Michaelis constant in the enzymatic reaction with plasminogen as substrate is 0.18 ⁇ 10 -7 mol/L [37]
  • the Michaelis constant of uPA is 2.43 ⁇ 10-7 mol/L.
  • the affinity of tPA is about 10 times that of uPA in the same reaction time, so in this experiment, the use of uPA was estimated according to 10 ng tPA/ml used in Example 3. The dose is 100 ng/ml.
  • Example 8 plasminogen promotes 20 hours old thrombolytic in 1 ng uPA
  • PBS blank control group was added with 1 mL PBS; 1 ng uPA control group was added with 1 mL PBS and 1 ng uPA; 0.2 mg Plg control group was added with 1 mL PBS and 0.2 mg Plg; 0.2 mg plasminogen group was added with 1 mL PBS and 1 ng uPA and 0.2 mg fibrinolysis.
  • Proenzyme 1 mg plasminogen group was added with 1 mL PBS and 1 ng uPA and 1 mg plasminogen; 2 mg plasminogen group was added with 1 mL PBS and 1 ng uPA and 2 mg plasminogen. All reactions were carried out in a 37 ° C incubator. After 2 hours of incubation, the supernatant was aspirated, the blotting paper was as much as possible to absorb the thrombus, and the weight of the thrombus was weighed to calculate the thrombolytic rate.
  • the content of uPA is 1 ng/mL under normal physiological conditions [35] . Therefore, the dose of uPA used in this experiment is 1 ng/mL to mimic the naturally occurring uPA content in normal physiological conditions in vivo.
  • mice Fifty-five-year-old C57 wild-type male mice were selected for general anesthesia with 3% pentobarbital, and the tail was trimmed 3 mm, and the tail was placed in warm water at 37 °C to observe the tail bleeding [39] . After hemostasis, they were randomly divided into two groups, 5 in the tPA group and 50 in the plasminogen group. In the tPA group, tPA 400 ⁇ g/0.05mL/line was injected into the orbital vein; plasminogen group was injected with 1mg/0.05mL/plasminogen in the orbital vein. The tail vein of the mouse was always placed in warm water at 37°C during the experiment. The bleeding status was 20 minutes and recorded.
  • mice injected intravenously with 1 mg of plasminogen did not have such side effects (Table 2), indicating that plasminogen is safer than tPA.
  • mice Nine wild-type male mice were randomly divided into 3 groups: vehicle PBS control group, 0.2 mg plasminogen group and 1 mg plasminogen group, with 3 rats in each group. General anesthesia was performed using 3% pentobarbital, and the jugular vein of the mouse was separated, and a venous thrombus was formed by applying a paper (3 mm ⁇ 5 mm) impregnated with a 10% FeCl 3 solution to the jugular vein for 5 minutes. Plasminogen or vehicle PBS was administered immediately after thrombus formation.
  • the vehicle PBS control group was injected with 100 ul of PBS in the tail vein, and 1 mg and 0.2 mg of plasminogen were administered by tail vein injection in the 1 mg plasminogen group and the 0.2 mg plasminogen group, respectively.
  • the corresponding jugular vein thrombus and the muscles near the contralateral vein were removed.
  • the thrombus and the muscles in the vicinity of the contralateral vein were homogenized using a grinder, and the supernatant was taken after centrifugation.
  • the supernatant was assayed for total protein by BCA method, and the plasminogen content in the homogenate was determined by enzyme-linked immunosorbent assay to calculate a certain amount.
  • the plasminogen content in the total amount of protein was determined by enzyme-linked immunosorbent assay to calculate a certain amount.
  • Example 11 30 minutes of fresh thrombus significantly increased thrombolysis rate after addition of plasminogen
  • PBS blank control group was added with 1 mL PBS; tPA control group was added with 1 mL PBS and 125 ng tPA; 0.2 mg plasminogen group was added with 1 mL PBS and 125 ng tPA and 0.2 mg plasminogen; 1 mg plasminogen group was added with 1 mL PBS and 125 ng. tPA and 1 mg plasminogen; 2 mg plasminogen group was added with 1 mL PBS and 125 ng tPA and 2 mg plasminogen. All reactions were carried out in a 37 ° C incubator. After 2 hours of incubation, the supernatant was aspirated, the blotting paper was as much as possible to absorb the thrombus, and the weight of the thrombus was weighed to calculate the thrombolytic rate.
  • the content of tPA is 5-10 ng/mL under normal physiological conditions [35] , and in the case of strenuous exercise or venous congestion, the content of tPA in the body will increase to 20-100 times, that is, 100 ng/ Above mL [36] . Therefore, the tPA dose used in this experiment was 125 ng/mL to mimic the naturally occurring tPA content in the presence of thrombus in vivo.
  • Ten male db/db rats aged 24-25 weeks were randomly divided into two groups, the vehicle PBS control group and the plasminogen group, with 5 rats in each group.
  • the group On the day of the start of the experiment, the group was weighed on the 0th day, and the day after the start of the experiment, plasminogen or PBS was given and recorded as the first day, and the administration was continued for 15 days.
  • the plasminogen group was injected with plasminogen at 2 mg/0.2 mL/day/day in the tail vein, and the same volume of PBS was administered to the vehicle PBS control group. Blood was taken from the eyeball on the 16th day, and the whole blood was allowed to stand for serum to detect the D-dimer content in the blood.
  • mice Ten male db/db rats aged 24-25 weeks were randomly divided into two groups, and the vehicle PBS control group and the plasminogen group were each given 5 rats. On the day of the start of the experiment, the group was weighed on the 0th day, and the day after the start of the experiment, plasminogen or PBS was given and recorded as the first day, and the drug was administered continuously for 31 days. The plasminogen group was injected with plasminogen at 2 mg/0.2 mL/day/day in the tail vein, and the same volume was given to the vehicle PBS control group. PBS. Mice were sacrificed on day 32 and hearts were fixed in 10% neutral formalin fixative for 24 hours.
  • the fixed heart tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 5 ⁇ m, the sections were dewaxed and rehydrated, washed once with water, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 5 minutes each time.
  • 10% of normal sheep serum (Vectorlaboratories, Inc., USA) was blocked for 1 hour; then the serum of the sheep was discarded and the tissue was circled with a PAP pen.
  • Rabbit anti-mouse fibrin (pro) antibody (Abeam) was incubated overnight at 4 ° C and washed twice with TBS for 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and twice with TBS for 5 minutes each time.
  • the color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes.
  • the gradient was dehydrated and sealed, and the sections were observed under a microscope at 400 times.
  • Fibrinogen is a precursor of fibrin. In the case of tissue damage, as a stress response to the body, fibrinogen is hydrolyzed to fibrin [40-42] , so fibrin levels can be used as damage. A sign of the degree. Fibrin is also a major component of thrombosis after tissue damage, so fibrin levels can also be used as a marker of thrombosis.
  • Example 14 Plasminogen promotes thrombosis of kidney tissue in mice with advanced diabetes
  • Twenty-two male db/db rats aged 24-25 weeks were randomly divided into two groups, 10 rats in the vehicle control group and 10 cells in the plasminogen group.
  • the group On the day of the start of the experiment, the group was weighed on the 0th day, and the day after the start of the experiment, plasminogen or PBS was given and recorded as the first day, and the drug was administered continuously for 31 days.
  • the plasminogen group was injected with plasminogen at 2 mg/0.2 mL/day/day in the tail vein, and the same volume of PBS was administered to the vehicle PBS control group. Mice were sacrificed on day 32 and kidneys were fixed in 10% neutral formalin fixative for 24 hours.
  • the fixed kidney tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 5 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. 10% of normal sheep serum (Vector laboratories, Inc., USA) was blocked for 1 hour; after the time was over, the sheep serum was discarded and the tissue was circled with a PAP pen.
  • Rabbit anti-mouse fibrin (pro) antibody (Abeam) was incubated overnight at 4 ° C and washed twice with TBS for 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and twice with TBS for 5 minutes each time. Color was developed according to DAB kit (Vector laboratories, Inc., USA), and washed with water for 3 times. 30 seconds, rinse with running water for 5 minutes. The gradient was dehydrated and sealed, and the sections were observed under a microscope at 200 times.
  • Fibrinogen is a precursor of fibrin. In the case of tissue damage, as a stress response to the body, fibrinogen is hydrolyzed to fibrin [40-42] , so fibrin levels can be used as damage. A sign of the degree. Fibrin is also a major component of thrombosis after tissue damage, so fibrin levels can also be used as a marker of thrombosis.
  • plasminogen positive staining was lighter for the plasminogen group (Fig. 13B) than for the vehicle PBS control group (Fig. 13A).
  • the injection of plasminogen can significantly reduce the renal fibrin deposition in diabetic mice, reflecting the significant repair effect of plasminogen on kidney injury in diabetic mice. It also indicates that plasminogen can promote the dissolution of renal tissue thrombosis.
  • Example 15 Plasminogen promotes thrombosis of advanced liver tissue in diabetes
  • mice Ten male db/db rats aged 24-25 weeks were randomly divided into two groups, and the vehicle PBS control group and the plasminogen group were each given 5 rats. On the day of the start of the experiment, the group was weighed on the 0th day, and the day after the start of the experiment, plasminogen or PBS was given and recorded as the first day, and the drug was administered continuously for 31 days. The plasminogen group was injected with plasminogen at 2 mg/0.2 mL/day/day in the tail vein, and the same volume of PBS was administered to the vehicle PBS control group. Mice were sacrificed on day 32 and liver tissues were fixed in 10% neutral formalin fixative for 24 hours.
  • the fixed liver tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 5 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. 10% of normal sheep serum (Vector laboratories, Inc., USA) was blocked for 1 hour; after the time was over, the sheep serum was discarded and the tissue was circled with a PAP pen.
  • Rabbit anti-mouse fibrin (pro) antibody (Abeam) was incubated overnight at 4 ° C and washed twice with TBS for 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and twice with TBS for 5 minutes each time.
  • the color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient was dehydrated and sealed, and the sections were observed under a microscope at 200 times.
  • Fibrinogen is a precursor of fibrin. In the case of tissue damage, as a stress response to the body, fibrinogen is hydrolyzed to fibrin [40-42] , so fibrin levels can be used as damage. A sign of the degree. Fibrin is also a major component of thrombosis after tissue damage, so fibrin levels can also be used as a marker of thrombosis.
  • Example 16 Plasminogen promotes neurological thrombosis in mice with late diabetic nerve injury
  • mice Ten male db/db rats aged 24-25 weeks were randomly divided into two groups, the vehicle PBS control group and the plasminogen group, with 5 rats in each group. On the day of the start of the experiment, the group was weighed on the 0th day, and the day after the start of the experiment, plasminogen or PBS was given and recorded as the first day, and the administration was continued for 15 days. The plasminogen group was injected with plasminogen at 2 mg/0.2 mL/day/day in the tail vein, and the same volume of PBS was administered to the vehicle PBS control group. Mice were sacrificed on day 16 and the sciatic nerve was fixed in 10% neutral formalin fixative for 24 hours.
  • the fixed sciatic nerve was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 5 ⁇ m, and the sections were dehydrated and rehydrated, washed once with water, and then the tissue was circled with a PAP pen.
  • 10% normal goat serum (Vector laboratories, Inc., USA) was blocked for 1 hour and excess serum was aspirated.
  • Rabbit anti-mouse fibrin (pro) antibody (Abeam) was incubated for 1 hour at room temperature or overnight at 4 ° C and washed 3 times with TBS.
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and 3 times with TBS. The color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient was dehydrated and sealed, and the sections were observed under a microscope at 400 times.
  • Fibrinogen is a precursor of fibrin. In the case of tissue damage, as a stress response to the body, fibrinogen is hydrolyzed to fibrin [40-42] , so fibrin levels can be used as damage. A sign of the degree. Fibrin is also a major component of thrombosis after tissue damage, so fibrin levels can also be used as a marker of thrombosis.
  • mice in the plasminogen group had reduced levels of sciatic nerve fibrin compared to the vehicle PBS control group (Fig. 15A), indicating that plasminogen has a function to degrade fibrin levels. The damage was repaired to a certain extent, indicating that plasminogen can promote the dissolution of thrombus around the nerve tissue.
  • the experiments of the examples of the invention include two parts of plasminogen in vitro thrombolysis and in vivo thrombolysis.
  • In vitro thrombolysis was used to simulate thrombolytic conditions in vivo. 10 ng/mL tPA was selected to mimic the amount of tPA naturally produced in normal physiological conditions. 125 ng/mL tPA mimicked the amount of tPA naturally produced in the case of thrombus in vivo to study the thrombolysis of plasminogen. ability.
  • Plasminogen has a strong ability to dissolve fresh blood clots, and the thrombolytic rate can reach more than 80% after two hours of incubation.
  • Plasminogen also has a very good thrombolytic effect under 1 ng/mL uPA or 100 ng/mL uPA, and the thrombolytic efficiency increases with increasing plasminogen dose.
  • mice in the late stage of diabetes were given 2 mg of plasminogen daily for 15 consecutive days, and the content of D-dimer in the serum was significantly increased.
  • the levels of fibrin in the heart, liver, kidney and nerve tissue decreased significantly, indicating that plasminogen can significantly promote the dissolution of thrombus induced by diabetic tissue damage in these tissues, fibrin degradation, and prove the administration of plasminogen in experimental animals.
  • a significant thrombolytic effect can also be achieved.
  • plasminogen has a very good thrombolytic capacity, especially for old thrombosis, and has the characteristics of high specificity, mildness, quick effect and no bleeding side effects.

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Abstract

本发明公开了纤溶酶原在溶解新鲜和陈旧血栓方面的作用。

Description

一种用于预防或治疗急性及慢性血栓的方法 技术领域
本发明涉及一种新的使用纤溶酶原预防和/或治疗血栓的方法。纤溶酶原可特异性溶解血栓而不会造成出血等副作用。本发明的药物还具有可溶解新鲜和陈旧血栓且具有半衰期长和溶血栓强度可控的优势，因此，纤溶酶原可能成为全新的溶解体内血栓的策略。
背景技术
血栓的形成及危害
血栓是指人体或动物在存活期间因某些诱因，血液有形成分在循环血中异常发生的血凝块，或者在心脏内壁或血管壁上发生的血液沉积物。其包括心肌梗死、脑栓塞、肺血栓、深部静脉血栓和周围血管栓塞等，是严重危害人类健康的疾病，其发病率、致残率、致死率都很高。据世界卫生组织统计，世界上每年死于血栓塞疾病的人数约为2600万，远高于其他死亡原因，成为危害人类健康头号敌人[1]。
纤溶酶是纤溶酶原激活系统(PA系统)的关键组分。它是一种广谱的蛋白酶，能够水解细胞外基质(ECM)的几个组分，包括纤维蛋白、明胶、纤连蛋白、层粘连蛋白和蛋白聚糖[2]。此外，纤溶酶能将一些金属蛋白酶前体(pro-MMP)激活形成具有活性的金属蛋白酶(MMP)。因此纤溶酶被认为是胞外蛋白水解作用的一个重要的上游调节物[3，4]。纤溶酶是由纤溶酶原通过两种生理性的PA：组织型纤溶酶原激活剂(tPA)或尿激酶型纤溶酶原激活剂(uPA)蛋白水解形成的。由于纤溶酶原在血浆和其他体液中相对水平较高，传统上认为PA系统的调节主要通过PA的合成和活性水平实现。PA系统组分的合成受不同因素严格调节，如激素、生长因子和细胞因子。此外，还存在纤溶酶和PA的特定生理抑制剂。纤溶酶的主要抑制剂是α2-抗纤溶酶(α2-antiplasmin)。某些细胞表面具有直接水解活性的uPA特异性细胞表面受体(uPAR)[5，6]。
纤溶酶原(plasminogen，plg)是一个单链糖蛋白，分子量约为92kDa[7，8]。纤溶酶原主要在肝脏合成，大量存在于胞外液中。血浆中纤溶酶原含量约为2μM。因此纤溶酶原是组织和体液中蛋白质水解活性的一个巨 大的潜在来源[9，10]。纤溶酶原存在两种分子形式：谷氨酸-纤溶酶原(Glu-plasminogen)和赖氨酸-纤溶酶原(Lys-plasminogen)。天然分泌和未裂解形式的纤溶酶原具有一个氨基末端(N-末端)谷氨酸，因此被称为谷氨酸-纤溶酶原。然而，在纤溶酶存在时，谷氨酸-纤溶酶原在Lys76-Lys77处水解成为赖氨酸-纤溶酶原。与谷氨酸-纤溶酶原相比，赖氨酸-纤溶酶原与纤维蛋白具有更高的亲和力，并可以更高的速率被PA激活。这两种形式的纤溶酶原的Arg560-Val561肽键可被uPA或tPA切割，导致二硫键连接的双链蛋白酶纤溶酶的形成[11]。纤溶酶原的氨基末端部分包含五个同源三环，即所谓的kringle，羧基末端部分包含蛋白酶结构域。一些kringle含有介导纤溶酶原与纤维蛋白及其抑制剂α2-AP特异性相互作用的赖氨酸结合位点。最新发现一个为38kDa的纤溶酶原片段，其中包括kringle1-4，是血管生成的有效抑制剂。这个片段被命名为血管抑素，可通过几个蛋白酶水解纤溶酶原产生。
纤溶酶的主要底物是纤维蛋白，纤维蛋白的溶解是预防病理性血栓形成的关键[12]。纤溶酶还具有对ECM几个组分的底物特异性，包括层粘连蛋白、纤连蛋白、蛋白聚糖和明胶，表明纤溶酶在ECM重建中也起着重要作用[8，13，14]。间接地，纤溶酶还可以通过将某些蛋白酶前体转化为活性蛋白酶来降解ECM的其他组分，包括MMP-1、MMP-2、MMP-3和MMP-9。因此，有人提出，纤溶酶可能是细胞外蛋白水解的一个重要的上游调节器[15]。此外，纤溶酶具有激活某些潜在形式的生长因子的能力[16-18]。在体外，纤溶酶还能水解补体系统的组分并释放趋化补体片段。
现有溶血栓治疗方法
目前减少血栓的相关药物治疗是常见的非手术治疗方法包括溶栓疗法、抗凝疗法、抗血小板药物和血管扩张药物。现在最常用最有效的方法就是采用溶栓疗法，常用的溶血栓药物有三代：第一代以链激酶(SK)和尿激酶(UK)为代表，溶栓能力强，但没有溶栓特异性，容易出现全身溶栓亢进而导致出血[19，20]。第二代以组织型纤溶酶原激活剂tPA为代表，其溶栓作用优于SK、UK，但在体内半衰期短[21]。第三代以基因工程技术，单克隆技术对第一代，第二代药物进行改造，但基本都在试验阶段。这些药物都基于增加纤溶平衡中的激活剂，产生纤溶酶(Plm)促进纤溶，从而达到溶栓目的 [22]。
目前已批准的溶栓药物分为两类：绝大多数溶栓药物使用的是纤溶酶原激活剂，包括天然和不同重组形式的tPA、uPA，以及链激酶(streptokinase)。纤溶酶原激活剂自身不能溶解血栓，必须将血栓附近的纤溶酶原分子激活成活性的纤溶酶后才能进行溶栓作用。最近几年，活性纤溶酶获得批准用于直接局部溶栓，具体方法是将导管通到血栓部位的情况下局部释放活性纤溶酶从而直接溶栓。
纤溶酶原(plg)是纤溶酶(Plm)的非活性形式，传统上认为其在体内是过量和惰性的，机体的溶解血栓过程只有通过纤溶酶原在其激活剂的作用下激活为有活性的纤溶酶，活性纤溶酶进而行使溶解纤维蛋白血凝块(fibrin clot)的功能。传统上认为纤溶酶原本身不起溶解血栓的作用。
然而，本发明惊奇地发现天然纤溶酶原具有良好的溶解新鲜和陈旧血栓的功能，且具有安全性好，溶解血栓强度便于调节，特异性好等优点。
本发明的溶栓机理与目前已知的溶栓策略完全不同。现有技术溶解血栓的方法是通过增加溶栓反应的催化剂，即纤溶酶原激活剂，包括tPA、uPA、链激酶及其衍生物或者溶栓反应的产物，即活性纤溶酶来实现。本发明溶解血栓的方法是通过调节溶栓反应的底物纤溶酶原的策略来实现的。
相对于现有技术中的溶栓药物，本发明的纤溶酶原溶栓至少具备以下优点。
1.良好的溶栓效果
外周动脉堵塞(peripheral arterial occlusion，PAO)和深静脉血栓(deep vein thrombosis，DVT)的情况下形成的长的血栓和逐步收缩的陈旧血栓，现有技术的溶栓药物效果较差[23-25]，而本发明使用纤溶酶原或者纤溶酶原与PA的组合，对上述血栓实现了良好的溶栓效果。因此，本发明可以有效解决tPA，uPA的上述问题。
2.半衰期长
目前的溶栓物质一个重要特点是体内半衰期过短，如天然uPA的体内半衰期为5-10分钟，天然tPA的体内半衰期为3-5分钟，天然纤溶酶的体内半衰期更是极为短暂。即使目前通过基因工程改造以图延长这些物质的半衰期，但是效果并不理想，半衰期过短仍然极大地限制了这些物质的应用。
然而，纤溶酶原的体内半衰期长达53小时，这说明使用纤溶酶原或者纤溶酶原与PA的组合，均能显著的延长体内溶血栓的作用期，达到持续、稳定溶栓的目的。
3.更具温和性和可调控性
对于活性纤溶酶来讲，由于其是一个高活性的蛋白酶，因此通过使用活性纤溶酶来溶栓必须是一个非常快速的反应过程，从而导致目前的使用中必须通过导管直接引到血栓部位。
对于纤溶酶原激活剂来讲，由于其在溶栓反应中处于催化剂的位置，添加少量的纤溶酶原激活剂会在很短时间里迅速形成大量活性纤溶酶，是一个剧烈的酶反应过程。
然而，本发明实验证明通过调节溶栓反应的底物纤溶酶原溶解血栓的过程更温和，而且，通过不同纤溶酶原使用量和溶栓效果的研究发现，纤溶酶原溶栓速率也可以通过纤溶酶原的剂量来调控。
4.特异性和副作用低
现有技术使用纤溶酶原激活剂作为溶栓药物的一个主要副作用是出血，特别是肠道和脑部的出血。由于正常体内纤溶酶原广泛存在于所有的体液，正常情况下体内存在生理性的纤维蛋白沉积，而纤溶酶原激活剂的增加往往发生于创伤、出血、剧烈运动等特殊情况下。因此，一旦注射纤溶酶原激活剂，体内会非特异性地广泛发生激活纤溶酶原形成活性纤溶酶这一反应，从而造成原有正常的纤维蛋白沉积的溶解并发生出血。在临床上，颅内出血风险是一个主要的出血风险。有报道指出，在持续2-24小时的持续给药过程中，颅内出血的发生率为1％-2％，目前还没有更好的办法避免出血这种风险。
在本发明中，由于纤溶酶原不是活性酶，因此注射纤溶酶原后不会非特异性地广泛发生激活纤溶酶原形成活性纤溶酶这一反应。这一反应发生的部位取决于哪里表达纤溶酶原激活剂，也就是发生血栓的部位。本发明实验证明纤溶酶原可以特异性地吸附于血栓部位，具有溶栓特异性，并且实验证明未发生出血的副作用。
5.有效溶解陈旧血栓
目前的溶栓药物的溶栓作用集中在血栓形成的初期，即“新鲜血栓”。如在缺血性中风中的研究中证实，在血栓形成3个小时内注射重组tPA可以 有效的溶解血栓。后续的研究证明，重组tPA最多可以在血栓形成4.5小时内溶解血栓，如果超过4.5小时的话，注射重组tPA的风险可能要超过有效作用。因此，在目前药物的情况下，要尽可能的在血栓形成的早期(小于4.5小时)注射重组tPA[26，27]。换句话说，目前本领域急需找到一种强有力的陈旧血栓溶栓药物。
在本发明中，单独使用纤溶酶原(以及生理水平的tPA)或者使用纤溶酶原和纤溶酶原激活剂(tPA或uPA)，均可有效地溶解新鲜血栓(血栓形成0.5小时)，或者陈旧血栓(20小时)，甚至极其陈旧血栓(72小时)。这些数据清楚地表明了纤溶酶原在溶解陈旧血栓上的巨大优势。
因此，纤溶酶原有望成为一种新的更具优势的溶栓新药。
发明简述
一方面，本发明涉及预防和/或消除受试者动、静脉血栓的方法，包括给药受试者纤溶酶原。本发明还包括纤溶酶原用于预防和/或消除受试者动、静脉血栓的用途。在一个实施方案中，其中所述血栓包括新鲜血栓和陈旧血栓。在一个实施方案中，所述血栓为血液系统疾病、循环系统疾病、自身免疫疾病、代谢紊乱疾病或感染性疾病导致的血栓。在一个实施方案中，所述血栓为继发于糖尿病的大、小血管、微血管血栓。在一个实施方案中，所述血栓为大小血管病变导致的血栓。
同时，本发明涉及一种新的预防和/或治疗血栓相关疾病的方法，该方法包括向受试者体内给药有效量的纤溶酶原。本发明还涉及纤溶酶原用于预防和/或治疗血栓相关疾病的用途。本发明涉及一种新的预防和/或消除受试者病理性血栓的方法，该方法通过全身或局部给药纤溶酶原来溶解所述血栓。以上所述血栓为新鲜血栓和/或陈旧血栓，所述血栓相关疾病为新鲜血栓和/或陈旧血栓诱导或导致的疾病。所述受试者为哺乳动物，优选为人。
在一个实施方案中，所述受试者纤溶酶或者纤溶酶原低下。具体地，所述低下是先天的、继发的和/或局部的。
在一个实施方案中，本发明的血栓为静脉血栓和/或动脉血栓。所述血栓相关疾病包括门静脉血栓导致的胰腺炎、肝硬化；肾静脉血栓导致的肾栓塞；颈内静脉血栓引起的全身性败血症、肺栓塞、脑血栓；动脉血栓导致的器官的梗死，包括但不限于：脑梗塞、心肌梗死、血栓性中风、房颤、 不稳定性心绞痛、顽固性心绞痛、短暂性脑缺血发作、肺栓塞、糖尿病大小血管栓塞等。
在一个实施方案中，所述血栓相关疾病为糖尿病性肾病、糖尿病性视网膜病、糖尿病性肝病、糖尿病性心脏病、糖尿病性肠病、包括糖尿病性神经痛等的糖尿病性神经病变。
在一个实施方案中，上述血栓是继发的和/或局部的血栓；上述血栓相关疾病是继发的和/或局部的血栓相关疾病。
在一个实施方案中，纤溶酶原与序列2、6、8、10或12具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。在一个实施方案中，纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、δ-纤溶酶原或其任意组合。在一个实施方案中，纤溶酶原是选自如下的保守取代变体：Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、δ-纤溶酶原或微纤溶酶原。在一个实施方案中，纤溶酶原为人天然纤溶酶原，例如序列2所示的纤溶酶原的直向同系物，例如，来自灵长类动物或啮齿类动物的纤溶酶原直向同系物，例如来自大猩猩、恒河猴、鼠、牛、马、狗的纤溶酶原直向同系物。最优选，本发明的纤溶酶原的氨基酸序列如序列2、6、8、10或12所示。
在一个实施方案中，所述纤溶酶原通过全身或局部给药，优选通过以下途径施用：表面、静脉内、肌内、皮下、吸入、椎管内、局部注射、关节内注射或通过直肠。在一个实施方案中，所述局部给药通过在血栓区域应用含有纤溶酶原的敷料和/或导管来进行。
在一个实施方案中，所述纤溶酶原与适当的多肽载体或稳定剂组合施用。在一个实施方案中，所述纤溶酶原以每天0.0001-2000mg/kg、0.001-800mg/kg、0.01-600mg/kg、0.1-400mg/kg、1-200mg/kg、1-100mg/kg、10-100mg/kg(以每公斤体重计算)或0.0001-2000mg/cm2、0.001-800mg/cm2、0.01-600mg/cm2、0.1-400mg/cm2、1-200mg/cm2、1-100mg/cm2、10-100 mg/cm2(以每平方厘米体表面积计算)的剂量施用，优选至少重复一次，优选至少每天施用。在局部施用的情况下，上述剂量还可以根据情况进一步调整。
上述纤溶酶原可以单独施用，也可以与其它药物联合使用以预防和/或治疗与病理性血栓发生相伴的其它疾病，所述其它药物包括，例如，心血管疾病治疗药物、心律失常治疗药物、糖尿病治疗药物等，。
另一方面，本发明涉及纤溶酶原在制备预防和/或消除受试者动、静脉血栓的药物、制品、药盒中的用途。本发明还涉及一种制药方法，包括将纤溶酶原与药学可接受载体共同制备成预防和/或消除受试者动、静脉血栓的药物、制品、药盒。在一个实施方案中，其中所述血栓包括新鲜血栓(急性血栓)和陈旧血栓(慢性血栓)。在一个实施方案中，所述血栓为血液系统疾病、循环系统疾病、自身免疫疾病、代谢紊乱疾病或感染性疾病导致的血栓。在一个实施方案中，所述血栓为继发于糖尿病的大、小血管、微血管血栓。在一个实施方案中，所述血栓为大小血管病变导致的血栓。
同时，本发明涉及纤溶酶原在制备预防和/或消除受试者病理性血栓的药物、制品、药盒中的用途，以及纤溶酶原在制备预防和/或治疗受试者血栓相关疾病的药物、制品、药盒中的用途。本发明还涉及一种制备药物的方法，包括将纤溶酶原与药学可接受载体一起制备成预防和/或消除受试者病理性血栓的药物、制品、药盒，或预防和/或治疗受试者血栓相关疾病的药物、制品、药盒。所述血栓为新鲜血栓和/或陈旧血栓，所述血栓相关疾病为新鲜血栓和/或陈旧血栓诱导的疾病。所述受试者为哺乳动物，优选为人。
在一个实施方案中，所述受试者纤溶酶或者纤溶酶原低下。具体地，所述低下是先天的、继发的和/或局部的。
在一个实施方案中，上述血栓为静脉血栓和/或动脉血栓。所述血栓相关疾病包括门静脉血栓导致的胰腺炎、肝硬化；肾静脉血栓导致的肾栓塞；颈内静脉血栓引起的全身性败血症、肺栓塞、脑血栓；动脉血栓导致的器官的梗死，包括但不限于：脑梗塞、心肌梗死、血栓性中风、房颤、不稳定性心绞痛、顽固性心绞痛、短暂性脑缺血发作、肺栓塞、糖尿病大小血管栓塞等。
在一个实施方案中，所述血栓相关疾病为糖尿病性肾病、糖尿病性视网膜病、糖尿病性肝病、糖尿病性心脏病、糖尿病性肠病、包括糖尿病性神经痛等的糖尿病性神经病变。
在一个实施方案中，上述血栓是继发的和/或局部的血栓；上述血栓相关疾病是继发的和/或局部的血栓相关疾病。
在一个实施方案中，纤溶酶原与序列2、6、8、10或12具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。在一个实施方案中，纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、δ-纤溶酶原或其任意组合。在一个实施方案中，纤溶酶原是选自如下的保守取代变体：Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、δ-纤溶酶原或微纤溶酶原。在一个实施方案中，纤溶酶原为人天然纤溶酶原，例如序列2所示的纤溶酶原的直向同系物，例如，来自灵长类动物或啮齿类动物的纤溶酶原直向同系物，例如来自大猩猩，恒河猴、鼠、牛、马，狗的纤溶酶原直向同系物。最优选，本发明的纤溶酶原的氨基酸序列如序列2、6、8、10或12所示。
在一个实施方案中，所述纤溶酶原通过全身或局部给药，优选通过以下途径施用：表面、静脉内、肌内、皮下、吸入、椎管内、局部注射、关节内注射或通过直肠。在一个实施方案中，所述局部给药通过在血栓区域应用含有纤溶酶原的敷料和/或导管来进行。
上述纤溶酶原可以单独施用，也可以与其它药物联合使用以治疗与病理性血栓发生相伴的其它疾病，所述其它药物包括，例如，心血管疾病治疗药物，心律失常治疗药物，糖尿病治疗药物等。
在一个实施方案中，所述纤溶酶原与适当的多肽载体或稳定剂组合施用。在一个实施方案中，所述纤溶酶原以每天0.0001-2000mg/kg、0.001-800mg/kg、0.01-600mg/kg、0.1-400mg/kg、1-200mg/kg、1-100mg/kg、10-100mg/kg(以每公斤体重计算)或0.0001-2000mg/cm2、0.001-800mg/cm2、 0.01-600mg/cm2、0.1-400mg/cm2、1-200mg/cm2、1-100mg/cm2、10-100mg/cm2(以每平方厘米体表面积计算)的剂量施用，优选至少重复一次，优选至少每天施用。在局部施用的情况下，上述剂量还可以根据情况进一步调整。
另一方面，本发明涉及用于预防和/或消除受试者动、静脉血栓的纤溶酶原，以及用于预防和/或消除受试者动、静脉血栓的、包含纤溶酶原的药物组合物。在一个实施方案中，其中所述血栓包括新鲜血栓和陈旧血栓。在一个实施方案中，所述血栓为血液系统疾病、循环系统疾病、自身免疫疾病、代谢紊乱疾病或感染性疾病导致的血栓。在一个实施方案中，所述血栓为继发于糖尿病的大、小血管、微血管血栓。在一个实施方案中，所述血栓为大小血管病变导致的血栓。同时，本发明涉及用于预防和/或治疗血栓相关疾病的纤溶酶原，以及用于预防和/或治疗血栓相关疾病的、包含纤溶酶原的药物组合物。上述血栓为新鲜血栓和/或陈旧血栓，所述血栓相关疾病为新鲜血栓和/或陈旧血栓诱导的疾病。在一个实施方案中，上述血栓为静脉血栓和/或动脉血栓。所述血栓相关疾病包括门静脉血栓导致的胰腺炎、肝硬化；肾静脉血栓导致的肾栓塞；颈内静脉血栓引起的全身性败血症、肺栓塞、脑血栓；动脉血栓导致的器官的梗死，包括但不限于：脑梗塞、心肌梗死、血栓性中风、房颤、不稳定性心绞痛、顽固性心绞痛、短暂性脑缺血发作、肺栓塞、糖尿病大小血管栓塞等。
在一个实施方案中，所述血栓相关疾病为糖尿病性肾病、糖尿病性视网膜病、糖尿病性肝病、糖尿病性心脏病、糖尿病性肠病、包括糖尿病性神经痛等的糖尿病性神经病变。
在一个实施方案中，上述血栓是先天的、继发的和/或局部的血栓；上述血栓相关疾病是先天的、继发的和/或局部的血栓相关疾病。
在一个实施方案中，纤溶酶原与序列2、6、8、10或12具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。在一个实施方案中，纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。 在一个实施方案中，纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、δ-纤溶酶原或其任意组合。在一个实施方案中，纤溶酶原是选自如下的保守取代变体：Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、δ-纤溶酶原或微纤溶酶原。在一个实施方案中，纤溶酶原为人天然纤溶酶原，例如序列2所示的纤溶酶原的直向同系物，例如，来自灵长类动物或啮齿类动物的纤溶酶原直向同系物，例如来自大猩猩、恒河猴、鼠、牛、马、狗的纤溶酶原直向同系物。最优选，本发明的纤溶酶原的氨基酸序列如序列2、6、8、10或12所示。
在一个实施方案中，所述纤溶酶原通过全身或局部给药，优选通过以下途径施用：表面、静脉内、肌内、皮下、吸入、椎管内、局部注射、关节内注射或通过直肠。在一个实施方案中，所述局部给药通过在血栓区域应用含有纤溶酶原的敷料和/或导管来进行。
在一个实施方案中，所述纤溶酶原与适当的多肽载体或稳定剂组合施用。在一个实施方案中，所述纤溶酶原以每天0.0001-2000mg/kg、0.001-800mg/kg、0.01-600mg/kg、0.1-400mg/kg、1-200mg/kg、1-100mg/kg、10-100mg/kg(以每公斤体重计算)或0.0001-2000mg/cm2、0.001-800mg/cm2、0.01-600mg/cm2、0.1-400mg/cm2、1-200mg/cm2、1-100mg/cm2、10-100mg/cm2(以每平方厘米体表面积计算)的剂量施用，优选至少重复一次，优选至少每天施用。在局部施用的情况下，上述剂量还可以根据情况进一步调整。
另一个方面，本发明涉及用于预防和/或消除受试者动、静脉血栓的、包含纤溶酶原的制品或药盒。在一个实施方案中，其中所述血栓包括新鲜血栓和陈旧血栓。在一个实施方案中，所述血栓为血液系统疾病、循环系统疾病、自身免疫疾病、代谢紊乱疾病或感染性疾病导致的血栓。在一个实施方案中，所述血栓为继发于糖尿病的大、小血管、微血管血栓。在一个实施方案中，所述血栓为大小血管病变导致的血栓。在一个实施方案中，所述制品或药盒包含含有有效剂量的纤溶酶原的容器。进一步的，所述制品或药盒还包含包含含有一种或多种其它药物的容器，其中所述其它药物为伴随血栓形成的其它疾病的治疗药物。该药盒还可包含使用说明书，说明所述纤溶酶原可以用于预防和/或治疗所述动、静脉血栓，或血栓相关疾病，并且可以进一步说明，所述纤溶酶原可以在其它药物施用之前，同时， 和/或之后施用。在一个实施方案中，所述其它药物可以是心血管疾病治疗药物，心律失常治疗药物，糖尿病治疗药物等，以治疗与病理性血栓发生相伴的其它疾病。在具体的上述方案中，上述血栓为新鲜血栓和/或陈旧血栓，所述血栓相关疾病为新鲜血栓和/或陈旧血栓诱导的疾病。在一个实施方案中，上述血栓为静脉血栓和/或动脉血栓。所述血栓相关疾病包括门静脉血栓导致的胰腺炎、肝硬化；肾静脉血栓导致的肾栓塞；颈内静脉血栓引起的全身性败血症、肺栓塞、脑血栓；动脉血栓导致的器官的梗死，包括但不限于：脑梗塞、心肌梗死、血栓性中风、房颤、不稳定性心绞痛、顽固性心绞痛、短暂性脑缺血发作、肺栓塞、糖尿病大小血管栓塞等。
在一个实施方案中，所述血栓相关疾病为糖尿病性肾病、糖尿病性视网膜病、糖尿病性肝病、糖尿病性心脏病、糖尿病性肠病、包括糖尿病性神经痛等的糖尿病性神经病变。
在一个实施方案中，上述血栓是先天的、继发的和/或局部的血栓；上述血栓相关疾病是先天的、继发的和/或局部的血栓相关疾病。
在一个实施方案中，纤溶酶原与序列2、6、8、10或12具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。在一个实施方案中，纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、δ-纤溶酶原或其任意组合。在一个实施方案中，纤溶酶原是选自如下的保守取代变体：Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、δ-纤溶酶原或微纤溶酶原。在一个实施方案中，纤溶酶原为人天然纤溶酶原，例如序列2所示的纤溶酶原的直向同系物，例如，来自灵长类动物或啮齿类动物的纤溶酶原直向同系物，例如来自大猩猩，恒河猴、鼠、牛、马，狗的纤溶酶原直向同系物。最优选，本发明的纤溶酶原的氨基酸序列如序列2、6、8、10或12所示。
在一个实施方案中，所述受试者纤溶酶或者纤溶酶原低下。具体地，所述低下是先天的、继发的和/或局部的。
本发明明确涵盖了属于本发明实施方案之间的技术特征的所有组合，并且这些组合后的技术方案在本申请中已经明确公开，就像上述技术方案已经单独且明确公开一样。另外，本发明还明确涵盖各个实施方案及其要素的所有亚组合，并且在本文中公开，就像每一个此类亚组合单独且明确在本文中公开一样。
发明详述
1.定义
“血栓”是凝血过程中形成的产物。凝血过程是机体保持封闭高压循环系统完整性的防御机制。在正常情况下，该过程应保持非活化状态，但当组织受到损伤时，需要立即启动该机制以减少血液外渗。当血管受损时，在凝血酶的作用下溶于血浆中的纤维蛋白原(fibrinogen)将最终转变为不溶于水的纤维蛋白(fibrin)多聚体，并彼此交织成网，将血细胞网罗在内，形成血凝块，完成凝血过程。在该过程中，血凝块与伤处的大小比例至关重要。因此初始血凝块形成的分子(纤维蛋白，凝血酶)和溶解血凝块的分子(纤溶酶、纤溶酶原激活剂等)间应存在平衡。但在病理过程中，该平衡的破坏将导致过量的血凝块形成分子，进而形成血栓(thrombus)，该血栓为“病理性血栓”。
在人体中，血栓可在具有血流的任何位置发生，目前主要将其分为两大类：静脉血栓和动脉血栓。静脉血栓由在静脉中产生的血凝块导致。最常见的静脉血栓类型为：深静脉血栓(DVT)，其通常影响肢体静脉如股静脉，导致受影响部位的疼痛和红肿；门静脉血栓，其可影响肝门静脉，进而导致胰腺炎、肝硬化、憩室炎或胆管癌；肾静脉血栓，导致肾栓塞；颈内静脉血栓，其可引起全身性败血症、肺栓塞等多种并发症；脑静脉血栓，导致患者呈现头痛、视觉异常中风等症状。动脉血栓则可能导致在体内几乎任何器官的梗死，其引发的病症包括但不限于：脑梗塞、心肌梗死、血栓性中风、动脉粥样硬化疾病、不稳定性心绞痛、顽固性心绞痛、短暂性脑缺血发作、肺栓塞等。
“血栓相关疾病”为血栓形成和血栓栓塞两种病理过程所引起的疾病。本发明血栓相关疾病的术语明确涵盖由血栓形成和血栓栓塞引起的所有疾病。
血栓形成(thrombosis)是指在一定条件下，血液有形成分在血管内(多数为小血管)形成栓子，造成血管部分或完全堵塞，相应部位血液供应障碍的病理过程。依血栓组成成分可分为血小板血栓、红细胞血栓、纤维蛋白血栓、混合血栓等。按血管种类可分为动脉性血栓、静脉性血栓及毛细血管性血栓。
血栓栓塞(thromboembolism)是血栓由形成部位脱落，在随血流移动的过程中部分或全部堵塞某些血管，引起相应组织和(或)器官缺血、缺氧、坏死(动脉血栓)及淤血、水肿(静脉血栓)的病理过程。
静脉血栓形成以下肢深静脉血栓形成最为多见，常见于深静脉如腘静脉、股静脉、肠系膜静脉及门静脉等。多为红细胞血栓或纤维蛋白血栓。主要表现有：(1)血栓形成的局部肿胀、疼痛；(2)血栓远端血液回流障碍：如远端水肿、胀痛、皮肤颜色改变、腹水等；(3)血栓脱落后栓塞血管引起相关脏器功能障碍，如肺梗死的症状、体征等。
动脉血栓形成多见于冠状动脉、脑动脉、肠系膜动脉及肢体动脉等，血栓类型早期多为血小板血栓，随后为纤维蛋白血栓。临床表现有：(1)发病多较突然，可有局部剧烈疼痛，如心绞痛、腹痛、肢体剧烈疼痛等；(2)相关供血部位组织缺血、缺氧所致的器官、组织结构及功能异常，如心肌梗死、心力衰竭、心源、性休克、心律失常、意识障碍及偏瘫等；(3)血栓脱落引起脑栓塞、肾栓塞、脾栓塞等相关症状及体征；(4)供血组织缺血性坏死引发的临床表现，如发热等。毛细血管血栓形成常见于DIC、TTP及溶血尿毒症综合征(HUS)等。临床表现往往缺乏特异性，主要为皮肤粘膜栓塞性坏死、微循环衰竭及器官功能障碍。
“糖尿病”是由遗传因素、免疫功能紊乱、微生物感染及其毒素、自由基毒素、精神因素等等各种致病因子作用于机体导致胰岛功能减退、胰岛素抵抗等而引发的糖、蛋白质、脂肪、水和电解质等一系列代谢紊乱综合征，临床上以高血糖为主要特点。
“糖尿病并发症”是由糖尿病过程中血糖控制不良导致的身体其他器官或组织的损害或功能障碍，其中包括肝脏、肾脏、心脏、视网膜、神经系统的损害或功能障碍等。据世界卫生组织统计，糖尿病并发症高达100多种，是目前已知并发症最多的一种疾病。而这些糖尿病的并发症主要是由于患者各器官的大血管、小血管和微血管受损所致。
“糖尿病性大血管病变”主要指主动脉及各器官动脉等发生的动脉粥样硬化。其发病机理包括以下方面：(1)持续性高血糖使血液粘滞度和凝固性升高，进而引起动脉血管弹性减弱乃至丧失；(2)脂类代谢异常，其促使胆固醇和胆固醇脂在细胞内堆积，导致动脉粥样硬化发生与发展；(3)动脉壁内皮细胞损伤，血流动力学改变使血液机械性地长期冲击血管内皮，引起内皮损伤，进而导致血小板、纤维蛋白等在损伤部位粘附聚集形成血栓，并可进一步导致炎症；(4)参与凝血机制的糖蛋白因子增多，促进血小板和纤维蛋白聚集粘附于损伤的内皮下层而溶解能力下降，进而形成血栓。
“糖尿病性微血管病变”指糖尿病患者机体各器官或组织微循环不同程度的异常导致的微血管病变。微血管病变形成的过程大致为：微循环功能性改变，内皮损伤，基膜增厚，血粘度增高，红细胞聚集，血小板粘附和聚集，最后导致微血栓形成和/或微血管闭塞。
上述的两种“糖尿病性血管病变”导致组织或器官局部血管损伤、血流不畅、细胞缺氧、形成血凝块、血栓和炎症，并进一步影响周边的组织及器官功能，进而导致糖尿病并发症，例如，糖尿病性心脏病、糖尿病性肠病、糖尿病性肾病、糖尿病性视网膜病、糖尿病性肝病、糖尿病性神经病变。
“糖尿病性肾病”是糖尿病微血管并发症，主要指糖尿病性肾小球硬化症，一种以血管损害为主的肾小球病变，其特征包括蛋白尿、高血压、水肿、肾小球硬化症、血管结构改变和小管间质性疾病(tubulointerstitial disease)。糖尿病肾病的第一临床证据通常是尿液中存在白蛋白尿，例如微白蛋白尿(microalbuminuria)或大量白蛋白尿(macroalbuminuria)。
“糖尿病性神经病变”或称为“糖尿病性神经病”是由糖尿病引起的神经系统损伤造成，包括感觉神经受损、运动神经受损和自主神经受损。其中感觉神经受损通常较为严重，常见症状包括但不限于：肢体疼痛、感觉减退、麻木、灼热、冰凉，以及糖尿病神经性疼痛，包括但不限于糖尿病并发症引发的自发性疼痛、痛觉减退(hypoalgesia)、痛觉超敏(hyperalgesia)等等。
“糖尿病性神经痛”是糖尿病神经病变的最常见形式，通常由糖尿病感觉神经受损所致。主要的疼痛通常伴有温度和触觉丧失，疼痛发生以下肢感觉居多，同时也发生在上肢和躯干。一般可分为周围和中枢神经疼痛。 周围神经疼痛通过周围神经的损伤引起，而中枢神经疼痛通过中枢神经系统和/或脊髓损伤引起。
“糖尿病肝损伤”是指由糖尿病引起的肝脏组织学和功能变化的病变。其主要由糖尿病引起的大血管、微血管病变导致。已知糖尿病可引起的肝损伤包括：肝酶学异常，其可引起肝细胞内二氧化碳蓄积、酸中毒、氧供减少、氧消耗增加，使肝脏转氨酶活性增加，胆红素代谢紊乱，重者可引起肝细胞坏死；脂肪肝，在引起脂肪肝的所有病因中，糖尿病占第三位，其中21％～78％糖尿病患者伴有脂肪肝；肝炎、硬化和肝癌，其中糖尿病患者中病毒性肝炎的患病率约为正常人的2-4倍，原发性肝癌的发生率约为正常人的4倍。
在临床上，由糖尿病引起的肝病及其相关症状包括但不限于：肝酶学异常、肝区不适和触痛、肝肿大、脾肿大、肝脾肿大、肝炎、脂肪肝、胆管炎、肝硬化、肝坏死和肝癌等。
“糖尿病性心血管病”指由糖尿病引起的心血管系统的组织学和功能变化的病变，其为最常见的糖尿病并发症之一，主要由糖尿病引起的大血管、微血管病变导致。其中，患者临床可表现为心电图异常、心脏扩大、心率失常、心绞痛、无痛性心肌梗死、心力衰竭。据统计，大约70％～80％的糖尿病患者最后死于心血管并发症。
“糖尿病视网膜病变”也称“糖尿病视网膜病”，指由糖尿病引起的视网膜组织学和功能变化的病变，主要由糖尿病引起的大血管、微血管病变导致。糖尿病性视网膜病变是最常见的糖尿病眼病，常造成视力减退或失明。据统计，50％的糖尿病患者在病程的10年左右将出现该病变，15年以上则达80％。糖尿病病情越重，年龄越大，发病的几率越高。
通过CT或MRI等技术检查患者血栓组织时，可见病灶呈新鲜或陈旧血栓。首发病灶在急性发作期为新鲜灶，病灶组织缺血中心部分坏死，部分有恢复可能，周边区域未受影响。此时的治疗目的应主要是防止“中心梗死区”扩大。而陈旧血栓则为组织缺血中心的完全坏死，治疗目的应致力于使梗死区周边组织功能继续得到改善。陈旧性血栓存在较高的复发风险，因此对陈旧性血栓的患者，治疗和预防同样重要，在减少症状程度的同时也应降低高复发率。目前的多种溶栓药物对于急性期的新鲜血栓治疗效果尚可，但治疗陈旧性血栓则效果较差。
“纤溶酶”是存在于血液中的一种非常重要的酶，能将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体。
“纤溶酶原”是纤溶酶的酶原形式，根据swiss prot中的序列，按含有信号肽的天然人源plasminogen氨基酸序列(序列4)计算由810个氨基酸组成，分子量约为92kD，主要在肝脏中合成并能够在血液中循环的糖蛋白，编码该氨基酸序列的cDNA序列如序列3所示。全长的PLG包含七个结构域：位于C末端的丝氨酸蛋白酶结构域、N末端的Pan Apple(PAp)结构域以及5个Kringle结构域(Kringle1-5)。参照swiss prot中的序列，其信号肽包括残基Met1-Gly19，PAp包括残基Glu20-Val98，Kringle1包括残基Cys103-Cys181，Kring1e2包括残基Glu184-Cys262，Kring1e3包括残基Cys275-Cys352，Kring1e4包括残基Cys377-Cys454，Kring1e5包括残基Cys481-Cys560。根据NCBI数据，丝氨酸蛋白酶域包括残基Val581-Arg804。
Glu-纤溶酶原是天然全长的纤溶酶原，由791个氨基酸组成(不含有19个氨基酸的信号肽)，编码该序列的cDNA序列如序列1所示，其氨基酸序列如序列2所示。在体内，还存在一种是从Glu-纤溶酶原的第76-77位氨基酸处水解从而形成的Lys-纤溶酶原，如序列6所示，编码该氨基酸序列的cDNA序列如序列5所示。δ-plasminogen(δ-plasminogen)是全长纤溶酶原缺失了Kringle2-Kringle5结构的片段，仅含有Kringle1和丝氨酸蛋白酶域[28， 29]，有文献报道了δ-plasminogen的氨基酸序列(序列8)[30]，编码该氨基酸序列的cDNA序列如序列7。Mini-plasminogen由Kringle5和丝氨酸蛋白酶域组成，有文献报道其包括残基Val443-Asn791(以不含有信号肽的Glu-plg序列的Glu残基为起始氨基酸)[31]，其氨基酸序列如序列10所示，编码该氨基酸序列的cDNA序列如序列9所示。而Micro-plasminogen仅含有丝氨酸蛋白酶结构域，有文献报道其氨基酸序列包括残基Ala543-Asn791(以不含有信号肽的Glu-plg序列的Glu残基为起始氨基酸)[32]，也有专利CN102154253A报道其序列包括残基Lys531-Asn791(以不含有信号肽的Glu-plg序列的Glu残基为起始氨基酸)，本专利序列参考专利CN102154253A，其氨基酸序列如序列12所示，编码该氨基酸序列的cDNA序列如序列11所示。
本发明的“纤溶酶”与“纤维蛋白溶酶”、“纤维蛋白溶解酶”可互换使用，含义相同；“纤溶酶原”与“纤维蛋白溶酶原”、“纤维蛋白溶解酶原”可互换使用，含义相同。
本发明的“新鲜血栓”和“急性血栓”可以互换使用；“陈旧血栓”和“慢性血栓”可以互换使用。
在循环过程中，纤溶酶原采用封闭的非活性构象，但当结合至血栓或细胞表面时，在纤溶酶原激活剂(plasminogen activator，PA)的介导下，其转变为呈开放性构象的活性纤溶酶。具有活性的纤溶酶可进一步将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体，进而溶解血栓。其中纤溶酶原的PAp结构域包含维持纤溶酶原处于非活性封闭构象的重要决定簇，而KR结构域则能够与存在于受体和底物上的赖氨酸残基结合。已知多种能够作为纤溶酶原激活剂的酶，包括：组织纤溶酶原激活剂(tPA)、尿激酶纤溶酶原激活剂(uPA)、激肽释放酶和凝血因子XII(哈格曼因子)等。
“纤溶酶原活性片段”指在纤溶酶原蛋白中，能够与底物中的靶序列结合并发挥蛋白水解功能的活性片段。本发明涉及纤溶酶原的技术方案涵盖了用纤溶酶原活性片段代替纤溶酶原的技术方案。本发明所述的纤溶酶原活性片段为包含纤溶酶原的丝氨酸蛋白酶域的蛋白质，优选，本发明所述的纤溶酶原活性片段包含序列14、与序列14具有至少80％、90％、95％、96％、97％、98％、99％同源性的氨基酸序列的蛋白质。因此，本发明所述的纤溶酶原包括含有该纤溶酶原活性片段、并且仍然保持该纤溶酶原活性的蛋白。
目前，对于血液中纤溶酶原及其活性测定的方法包括：对组织纤溶酶原激活剂活性的检测(t-PAA)、血浆组织纤溶酶原激活剂抗原的检测(t-PAAg)、对血浆组织纤溶酶原活性的检测(plgA)、血浆组织纤溶酶原抗原的检测(plgAg)、血浆组织纤溶酶原激活剂抑制物活性的检测、血浆组织纤溶酶原激活剂抑制物抗原的检测、血浆纤溶酶-抗纤溶酶复合物检测(PAP)。其中最常用的检测方法为发色底物法：向受检血浆中加链激酶(SK)和发色底物，受检血浆中的PLG在SK的作用下，转变成PLM，后者作用于发色底物，随后用分光光度计测定，吸光度增加与纤溶酶原活性成正比。此外也可采用免疫化学法、凝胶电泳、免疫比浊法、放射免疫扩散法等对血液中的纤溶酶原活性进行测定。
“直系同源物或直系同系物(ortholog)”指不同物种之间的同源物，既包括蛋白同源物也包括DNA同源物，也称为直向同源物、垂直同源物。其具体指不同物种中由同一祖先基因进化而来的蛋白或基因。本发明的纤溶酶原包括人的天然纤溶酶原，还包括来源于不同物种的、具有纤溶酶原活性的纤溶酶原直系同源物或直系同系物。
“保守取代变体”是指其中一个给定的氨基酸残基改变但不改变蛋白质或酶的整体构象和功能，这包括但不限于以相似特性(如酸性、碱性、疏水性，等)的氨基酸取代亲本蛋白质中氨基酸序列中的氨基酸。具有类似性质的氨基酸是众所周知的。例如，精氨酸、组氨酸和赖氨酸是亲水性的碱性氨基酸并可以互换。同样，异亮氨酸是疏水氨基酸，则可被亮氨酸、蛋氨酸或缬氨酸替换。因此，相似功能的两个蛋白或氨基酸序列的相似性可能会不同。例如，基于MEGALIGN算法的70％至99％的相似度(同一性)。“保守取代变体”还包括通过BLAST或FASTA算法确定具有60％以上的氨基酸同一性的多肽或酶，若能达75％以上更好，最好能达85％以上，甚至达90％以上为最佳，并且与天然或亲本蛋白质或酶相比具有相同或基本相似的性质或功能。
“分离的”纤溶酶原是指从其天然环境分离和/或回收的纤溶酶原蛋白。在一些实施方案中，所述纤溶酶原会纯化(1)至大于90％、大于95％、或大于98％的纯度(按重量计)，如通过Lowry法所确定的，例如超过99％(按重量计)，(2)至足以通过使用旋转杯序列分析仪获得N端或内部氨基酸序列的至少15个残基的程度，或(3)至同质性，该同质性是通过使用考马斯蓝或银染在还原性或非还原性条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确定的。分离的纤溶酶原也包括通过生物工程技术从重组细胞制备，并通过至少一个纯化步骤分离的纤溶酶原。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用，指任何长度的氨基酸的聚合形式，其可以包括遗传编码的和非遗传编码的氨基酸，化学或生物化学修饰的或衍生化的氨基酸，和具有经修饰的肽主链的多肽。该术语包括融合蛋白，包括但不限于具有异源氨基酸序列的融合蛋白，具有异源和同源前导序列(具有或没有N端甲硫氨酸残基)的融合物；等等。
关于参照多肽序列的“氨基酸序列同一性百分数(％)”定义为在必要时引入缺口以实现最大百分比序列同一性后，且不将任何保守替代视为序列同 一性的一部分时，候选序列中与参照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式实现，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员能决定用于比对序列的适宜参数，包括对所比较序列全长实现最大对比需要的任何算法。然而，为了本发明的目的，氨基酸序列同一性百分数值是使用序列比较计算机程序ALIGN-2产生的。
在采用ALIGN-2来比较氨基酸序列的情况中，给定氨基酸序列A相对于给定氨基酸序列B的％氨基酸序列同一性(或者可表述为具有或包含相对于、与、或针对给定氨基酸序列B的某一％氨基酸序列同一性的给定氨基酸序列A)如下计算：
分数X/Y乘100
其中X是由序列比对程序ALIGN-2在该程序的A和B比对中评分为相同匹配的氨基酸残基的数目，且其中Y是B中的氨基酸残基的总数。应当领会，在氨基酸序列A的长度与氨基酸序列B的长度不相等的情况下，A相对于B的％氨基酸序列同一性会不等于B相对于A的％氨基酸序列同一性。除非另有明确说明，本文中使用的所有％氨基酸序列同一性值都是依照上一段所述，使用ALIGN-2计算机程序获得的。
如本文中使用的，术语“治疗”、“处理”和“消除”指获得期望的药理和/或生理效果。所述效果可以是完全或部分预防疾病或其症状，和/或部分或完全治愈疾病和/或其症状，并且包括：(1)预防疾病在受试者体内发生，所述受试者可以具有疾病的素因，但是尚未诊断为具有疾病；(2)抑制疾病，即阻滞其形成；和(3)减轻疾病和/或其症状，即引起疾病和/或其症状消退。
术语“个体”、“受试者”和“患者”在本文中可互换使用，指哺乳动物，包括但不限于鼠(大鼠，小鼠)、非人灵长类、人、犬、猫、有蹄动物(例如马、牛、绵羊、猪、山羊)等。
“治疗有效量”或“有效量”指在对哺乳动物或其它受试者施用以治疗疾病时足以实现对疾病的所述预防和/或治疗的纤溶酶原的量。“治疗有效量”会根据所使用的纤溶酶原、要治疗的受试者的疾病和/或其症状的严重程度以及年龄、体重等而变化。
2.本发明纤溶酶原的制备
纤溶酶原可以从自然界分离并纯化用于进一步的治疗用途，也可以通过标准的化学肽合成技术来合成。当通过化学合成多肽时，可以经液相或固相进行合成。固相多肽合成(SPPS)(其中将序列的C末端氨基酸附接于不溶性支持物，接着序贯添加序列中剩余的氨基酸)是适合纤溶酶原化学合成的方法。各种形式的SPPS，诸如Fmoc和Boc可用于合成纤溶酶原。用于固相合成的技术描述于Barany和Solid-Phase Peptide Synthesis；第3-284页于The Peptides：Analysis，Synthesis，Biology.第2卷：Special Methods in Peptide Synthesis，Part A.，Merrifield，等J.Am.Chem.Soc.，85：2149-2156(1963)；Stewart等，Solid Phase Peptide Synthesis，2nd ed.Pierce Chem.Co.，Rockford，Ill.(1984)；和Ganesan A.2006Mini Rev.Med Chem.6：3-10和Camarero JA等2005Protein Pept Lett.12：723-8中。简言之，用其上构建有肽链的功能性单元处理小的不溶性多孔珠。在偶联/去保护的重复循环后，将附接的固相游离N末端胺与单个受N保护的氨基酸单元偶联。然后，将此单元去保护，露出可以与别的氨基酸附接的新的N末端胺。肽保持固定在固相上，之后将其切掉。
可以使用标准重组方法来生产本发明的纤溶酶原。例如，将编码纤溶酶原的核酸插入表达载体中，使其与表达载体中的调控序列可操作连接。表达调控序列包括但不限于启动子(例如天然关联的或异源的启动子)、信号序列、增强子元件、和转录终止序列。表达调控可以是载体中的真核启动子系统，所述载体能够转化或转染真核宿主细胞(例如COS或CHO细胞)。一旦将载体掺入合适的宿主中，在适合于核苷酸序列的高水平表达及纤溶酶原的收集和纯化的条件下维持宿主。
合适的表达载体通常在宿主生物体中作为附加体或作为宿主染色体DNA的整合部分复制。通常，表达载体含有选择标志物(例如氨苄青霉素抗性、潮霉素抗性、四环素抗性、卡那霉素抗性或新霉素抗性)以有助于对外源用期望的DNA序列转化的那些细胞进行检测。
大肠杆菌(Escherichia coli)是可以用于克隆主题抗体编码多核苷酸的原核宿主细胞的例子。适合于使用的其它微生物宿主包括杆菌，诸如枯草芽孢杆菌(Bacillus subtilis)和其他肠杆菌科(Enterobacteriaceae)，诸如沙门氏菌属(Salmonella)、沙雷氏菌属(Serratia)、和各种假单胞菌属(Pseudomonas)物 种。在这些原核宿主中，也可以生成表达载体，其通常会含有与宿主细胞相容的表达控制序列(例如复制起点)。另外，会存在许多公知的启动子，诸如乳糖启动子系统、色氨酸(trp)启动子系统、β-内酰胺酶启动子系统，或来自噬菌体λ的启动子系统。启动子通常会控制表达，任选在操纵基因序列的情况中，并且具有核糖体结合位点序列等，以启动并完成转录和翻译。
其他微生物，诸如酵母也可用于表达。酵母(例如酿酒酵母(S.cerevisiae))和毕赤酵母(Pichia)是合适的酵母宿主细胞的例子，其中合适的载体根据需要具有表达控制序列(例如启动子)、复制起点、终止序列等。典型的启动子包含3-磷酸甘油酸激酶和其它糖分解酶。诱导型酵母启动于特别包括来自醇脱氢酶、异细胞色素C、和负责麦芽糖和半乳糖利用的酶的启动子。
除微生物外，哺乳动物细胞(例如在体外细胞培养物中培养的哺乳动物细胞)也可以用于表达并生成本发明的纤溶酶原(例如编码主题抗-Tau抗体的多核苷酸)。参见Winnacker，From Genes to Clones，VCH Publishers，N.Y.，N.Y.(1987)。合适的哺乳动物宿主细胞包括CHO细胞系、各种Cos细胞系、HeLa细胞、骨髓瘤细胞系、和经转化的B细胞或杂交瘤。用于这些细胞的表达载体可以包含表达控制序列，如复制起点，启动子和增强子(Queen等，Immunol.Rev.89：49(1986))，以及必需的加工信息位点，诸如核糖体结合位点、RNA剪接位点、多聚腺苷酸化位点，和转录终止子序列。合适的表达控制序列的例子是白免疫球蛋白基因、SV40、腺病毒、牛乳头瘤病毒、巨细胞病毒等衍生的启动子。参见Co等，J.Immunol.148：1149(1992)。
一旦合成(化学或重组方式)，可以依照本领域的标准规程，包括硫酸铵沉淀、亲和柱、柱层析、高效液相层析(HPLC)、凝胶电泳等来纯化本发明所述的纤溶酶原。该纤溶酶原是基本上纯的，例如至少约80％至85％纯的，至少约85％至90％纯的，至少约90％至95％纯的，或98％至99％纯的或更纯的，例如不含污染物，所述污染物如细胞碎片，除主题抗体以外的大分子，等等。
3.药物配制剂
可以通过将具有所需纯度的纤溶酶原与可选的药用载体、赋形剂，或稳定剂(Remington′s Pharmaceutical Sciences，16版，Osol，A.ed.(1980))混合形成冻干制剂或水溶液制备治疗配制剂。可接受的载体、赋形剂、稳定剂 在所用剂量及浓度下对受者无毒性，并包括缓冲剂例如磷酸盐、柠檬酸盐及其它有机酸；抗氧化剂包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；氯化己烷双胺；氯化苄烷铵(benzalkonium chloride)，苯索氯铵；酚、丁醇或苯甲醇；烷基对羟基苯甲酸酯如甲基或丙基对羟基苯甲酸酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；间甲酚)；低分子量多肽(少于约10个残基)；蛋白质如血清白蛋白、明胶或免疫球蛋白；亲水聚合物如聚乙烯吡咯烷酮；氨基酸如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖及其它碳水化合物包括葡萄糖、甘露糖、或糊精；螯合剂如EDTA；糖类如蔗糖、甘露醇、岩藻糖或山梨醇；成盐反离子如钠；金属复合物(例如锌-蛋白复合物)；和/或非离子表面活性剂，例如TWEENTM，PLURONICSTM或聚乙二醇(PEG)。优选冻干的抗-VEGF抗体配制剂在WO 97/04801中描述，其包含在本文中作为参考。
本发明的配制剂也可含有需治疗的具体病症所需的一种以上的活性化合物，优选活性互补并且相互之间没有副作用的那些。例如，抗高血压的药物、抗心律失常的药物、治疗糖尿病的药物等。
本发明的纤溶酶原可包裹在通过诸如凝聚技术或界面聚合而制备的微胶囊中，例如，可置入在胶质药物传送系统(例如，脂质体，白蛋白微球，微乳剂，纳米颗粒和纳米胶囊)中或置入粗滴乳状液中的羟甲基纤维素或凝胶-微胶囊和聚-(甲基丙烯酸甲酯)微胶囊中。这些技术公开于Remington′s Pharmaceutical Sciences 16th edition，Osol，A.Ed.(1980)。
用于体内给药的本发明的纤溶酶原必需是无菌的。这可以通过在冷冻干燥和重新配制之前或之后通过除菌滤膜过滤而轻易实现。
本发明的纤溶酶原可制备缓释制剂。缓释制剂的适当实例包括具有一定形状且含有糖蛋白的固体疏水聚合物半通透基质，例如膜或微胶囊。缓释基质实例包括聚酯、水凝胶(如聚(2-羟基乙基-异丁烯酸酯)(Langer等，J.Biomed.Mater.Res.，15：167-277(1981)；Langer，Chem.Tech.，12：98-105(1982))或聚(乙烯醇)、聚交酯(美国专利3773919，EP 58，481)、L-谷氨酸与γ乙基-L-谷氨酸的共聚物(Sidman，等，Biopolymers 22：547(1983))，不可降解的乙烯-乙烯乙酸酯(ethylene-vinyl acetate)(Langer，等，出处同上)，或可降解的乳酸-羟基乙酸共聚物如Lupron DepotTM(由乳酸-羟基乙酸共聚物和亮氨酰脯氨酸(leuprolide)乙酸酯组成的可注射的微球体)，以及聚D-(-)-3- 羟丁酸。聚合物如乙烯-乙酸乙烯酯和乳酸-羟基乙酸能持续释放分子100天以上，而一些水凝胶释放蛋白的时间却较短。可以根据相关机理来设计使蛋白稳定的合理策略。例如，如果发现凝聚的机理是通过硫代二硫键互换而形成分子间S-S键，则可通过修饰巯基残基、从酸性溶液中冻干、控制湿度、采用合适的添加剂、和开发特定的聚合物基质组合物来实现稳定。
4.给药和剂量
可以通过不同方式，例如通过静脉内、腹膜内、皮下、颅内、鞘内、动脉内(例如经由颈动脉)、肌内、鼻内、表面或皮内施用或脊髓或脑投递来实现本发明药物组合物的施用。气溶胶制剂如鼻喷雾制剂包含活性剂的纯化的水性或其它溶液及防腐剂和等渗剂。将此类制剂调节至与鼻粘膜相容的pH和等渗状态。
在一些情况中，可以以下方式修饰或配制本发明的纤溶酶原药物组合物，从而提供其穿过血脑屏障的能力。可以通过各种肠内和胃肠外施用路径，包括口服、静脉内等对患有血栓和/或血栓相关疾病的个体施用此类纤溶酶原的组合物。
用于胃肠外施用的制备物包括无菌水性或非水性溶液、悬浮液和乳剂。非水性溶剂的例子是丙二醇、聚乙二醇、植物油如橄榄油，和可注射有机酯，如油酸乙酯。水性载体包括水、醇性/水性溶液、乳剂或悬浮液，包括盐水和缓冲介质。胃肠外媒介物包含氯化钠溶液、林格氏右旋糖、右旋糖和氯化钠、或固定油。静脉内媒介物包含液体和营养补充物、电解质补充物，等等。也可以存在防腐剂和其他添加剂，诸如例如，抗微生物剂、抗氧化剂、螯合剂、和惰性气体，等等。
在一些实施方案中，本发明的纤溶酶原与促进穿过血脑屏障的药剂配制在一起。在一些情况中，本发明的纤溶酶原直接或经接头与促进穿过血脑屏障的载体分子、肽或蛋白质融合。在一些实施方案中，本发明的纤溶酶原与结合内源血脑屏障(BBB)受体的多肽融合。连接纤溶酶原与结合内源BBB受体的多肽，促进穿过BBB。结合内源BBB受体的合适的多肽包括抗体，例如单克隆抗体，或其抗原结合片段，其特异性结合内源BBB受体。合适的内源BBB受体包括但不限于胰岛素受在一些情况中，抗体是囊封于脂质体中的。体、转铁蛋白受体、脂蛋白受体、和胰岛素样生长因子受体。参见例如美国专利公开文本No.2009/0156498。
医务人员会基于各种临床因素确定剂量方案。如医学领域中公知的，任一患者的剂量取决于多种因素，包括患者的体型、体表面积、年龄、要施用的具体化合物、性别、施用次数和路径、总体健康、和同时施用的其它药物。本发明包含纤溶酶原的药物组合物的剂量范围可以为例如每天约0.0001至2000mg/kg，或约0.001至500mg/kg(例如0.02mg/kg，0.25mg/kg，0.5mg/kg，0.75mg/kg，10mg/kg，50mg/kg等等)受试者体重。例如，剂量可以是1mg/kg体重或50mg/kg体重或在1-50mg/kg的范围，或至少1mg/kg。高于或低于此例示性范围的剂量也涵盖在内，特别是考虑到上述的因素。上述范围中的中间剂量也包含在本发明的范围内。受试者可以每天、隔天、每周或根据通过经验分析确定的任何其它日程表施用此类剂量。例示性的剂量日程表包括连续几天1～10mg/kg。在本发明的药物施用过程中需要实时评估、定期评估血栓和血栓相关疾病的治疗效果和安全性。
5.治疗效力和安全性评价
治疗效力
对纤溶酶原的治疗效力评价主要通过监测以下指标的来进行：
(1)治疗1周后血栓溶解率。例如，可以通过导管注入造影剂，每日评估溶栓情况，对每一个血管区域进行评分，完全开放者为0分，部分闭塞者为1分，完全闭塞为2分。按照溶栓前总分减去溶栓后总分，除以溶栓前总分所得的比率划分不同的溶栓等级，一级＜50％，二级为50％～90％，三级为血栓完全溶解。
(2)6个月后血管通畅率，例如可通过内窥镜、CT血管造影分析、彩色多普勒超声等方法评估血管通畅率。通过治疗后血管通畅率百分比与治疗前是否有统计学显著的提高来判断治疗有效性。
(3)6个月后血管闭塞和/或静脉返流率。通过统计治疗后血管闭塞和/或静脉返流率的降低来判断药剂对于溶栓率的改善。
(4)其他评估指标；例如，血管腔内回声改变、血管壁厚度对比、和2年后血栓后遗症发生率等。例如，血管壁厚度和腔内回声可以通过灰阶超声进行评估，而髂、股静脉血流和股静脉瓣膜功能膜不全可以使病人采取站立姿势用多普勒超声进行评估。
安全性评估
对纤溶酶原药物治疗血栓后的安全性进行评估，所述评估主要包括监测治疗后不良事件的发生率。一般将严重出血、栓塞、中风和死亡定为严重不良事件，而次要出血和其他轻微症状的并发症定为次要不良事件。
对于安全性评估而言，最为常见的不良事件是出血，如颅内出血(又称出血性中风，包括蛛膜下出血、硬膜下出血等)。本发明所述严重出血一般指颅内出血或出血严重程度足以导致死亡、手术、停止治疗或需要输血的出血事件，包括“大出血(major hemorrhage)事件”和“威胁生命的出血事件”。而次要出血是指导管鞘边的出血、和/或改变溶血栓药剂、抗凝剂或抗血小板药物的剂量、或通过压迫可以止住的出血。所述“大出血”、“大出血事件”具体是指血色素含量降低至少2.0g/L或输血至少2单位的血液，或关键部位或器官中的症状性出血。而比“大出血”严重程度更高的出血事件，即大出血事件的子类，称为“威胁生命的出血事件”，包括致命性出血、症状性颅内出血、血色素降低至少5.0g/L或需要输血超过4单位的血液或需要心肌收缩剂或必需手术的出血。
此外，对于评估具有大出血事件风险因子的患者，视情况严重程度对其施用剂量进行微调并对其用药后不良事件进行随访监测至少3个月，优选为6个月及以上。所述大出血风险包括但不限于(1)年龄75岁及以上，(2)具有先前出血事件的病史，(3)具有降低的肌酸酐清除率，其小于80mL/分钟或小于50mL/分钟。
6.制品或药盒
本发明的一个实施方案涉及一种制品或药盒，其包含可用于治疗血栓的本发明纤溶酶原。所述制品优选包括一个容器，标签或包装插页。适当的容器有瓶子、小瓶、注射器等。容器可由各种材料如玻璃或塑料制成。所述容器含有组合物，所述组合物可有效治疗本发明的疾病或病症并具有无菌入口(例如所述容器可为静脉内溶液包或小瓶，其含有可被皮下注射针穿透的塞子的)。所述组合物中至少一种活性剂为纤溶酶原。所述容器上或所附的标签说明所述组合物用于治疗本发明所述血栓和血栓相关疾病。所述制品可进一步包含含有可药用缓冲液的第二容器，诸如磷酸盐缓冲的盐水、林格氏溶液以及葡萄糖溶液。其可进一步包含从商业和使用者角度来看所需的其它物质，包括其它缓冲液、稀释剂、过滤物、针和注射器。此 外，所述制品包含带有使用说明的包装插页，包括例如指示所述组合物的使用者将纤溶酶原组合物以及治疗伴随的疾病的其它药物给药患者。
附图简述：
图1显示在125ng tPA存在的条件下，37℃温育1小时，不同剂量纤溶酶原对20小时陈旧血栓溶解效果。
图2显示125ng tPA存在时，37℃温育2小时，不同剂量纤溶酶原对20小时陈旧血栓的溶解效果。
图3显示在10ng tPA条件下，37℃温育2小时，不同剂量的纤溶酶原对20小时陈旧血栓溶栓效果。
图4显示在125ng tPA条件下，37℃温育2小时，不同剂量的纤溶酶原对72小时陈旧血栓的溶栓效果。
图5显示在10ng tPA条件下，37℃温育2小时，不同剂量的纤溶酶原对72小时陈旧血栓的溶栓效果。
图6显示20小时陈旧血栓在加入10ng的tPA和1mg的plg或者单独加入5μg的tPA后随着时间的延长血栓溶栓率的变化。
图7显示在100ng uPA条件下，37℃温育1小时，不同剂量的纤溶酶原对20小时陈旧血栓溶效果。
图8显示在1ng uPA条件下，37℃温育2小时，不同剂量的纤溶酶原对20小时陈旧血栓溶效果。
图9显示纤溶酶原对于体内血栓的特异性吸附实验结果。
图10显示在125ng tPA条件下，37℃温育2小时，不同浓度的纤溶酶原对于30分钟新鲜血栓的溶栓效果。
图11显示24-25周龄糖尿病小鼠给予纤溶酶原15天后血清中D-二聚体的浓度检测结果。
图12显示24-25周糖尿病小鼠给予PBS(A)或纤溶酶原(B)31天心脏纤维蛋白免疫组化染色结果。
图13显示24-25周糖尿病小鼠给予PBS(A)或纤溶酶原(B)31天后肾脏纤维蛋白免疫组化染色结果。
图14 24-25显示周糖尿病小鼠给予PBS(A)或纤溶酶原(B)31天后肝脏纤维蛋白免疫组化染色结果。
图15 24-25周糖尿病后期神经损伤小鼠给予PBS(A)或纤溶酶原(B)15天后坐骨神经纤维蛋白免疫组化染色。
实施例
材料与方法：
体内实验：
实验动物
C57小鼠(6-8周龄)购自南方医科大学实验动物中心。购买的小鼠饲养在屏障环境动物房内。db/db小鼠购自南京生物医药研究所。
实验设计及给药方式
对照组和实验组所有动物经手术方式游离颈动脉后，用含10％FeCl3的滤纸外敷5min进行单侧颈动脉血栓造模，造模后1h内开始静脉注射纤溶酶原，对照组静脉注射等体积PBS来进行。3小时后取出相应颈静脉血栓及对侧静脉附近的肌肉。将血栓及对侧静脉附近肌肉使用研磨器进行匀浆，离心后取上清，将上清利用BCA法检测其总蛋白，酶联免疫法检测匀浆液中的纤溶酶原含量，计算出一定蛋白总量中的纤溶酶原含量。研究纤溶酶原体内溶栓的特异性。
此外，24-25周龄db/db小鼠作为对照组和实验组动物，分别尾静脉给予溶媒PBS或纤溶酶原。31天后摘眼球取血行D-二聚体检测，取神经、肝、肾、心脏行纤维蛋白免疫组化染色，研究纤溶酶原体内溶栓效果。
血液D-二聚体分析
小鼠摘眼球取血，获得血浆，按照D-二聚体试剂盒(武汉优尔生，中国)进行实验操作，试验完成后使用酶标仪(Biotek美国)在450nm处进行读数，进行数据分析。
免疫组化分析
取神经、肝、肾、心脏，在10％中性福尔马林固定24小时以上。固定后的组织经过乙醇梯度脱水，包埋在石蜡中，并进行石蜡切片，切片厚度5μm，切片脱蜡至水后水洗1次，然后用PAP笔圈出组织。以0.3％甲醇稀释的双氧水孵育15分钟，水洗3次。10％与二抗同源的正常血清封闭10分钟，吸走多余血清。一抗室温孵育30分钟或4度过夜，TBS洗3次。HRP标记的二抗室温孵育30分钟，TBS洗3次。按DAB试剂盒(vector  laboratories，Inc.，USA)显色，苏木素复染30秒，流水返蓝5分钟，然后TBS洗1次。梯度脱水透明并封片。用到的抗体有：标志物抗体有Fibrin(ogen)(Abcam)。切片在光学显微镜(Olympus，BX43)下观察。
体外溶血栓实验设计：
取健康人血浆于ELISA 96孔板中，加入固定量的凝血酶(Sigma，美国)形成血栓，然后进行下面的不同实验。加入固定量tPA、uPA(sigma，美国)和不同量的纤溶酶原，固定量的纤溶酶原和不同量的tPA、uPA、链激酶(sigma，美国)，对照组加入PBS。孵育不同时间至有血栓溶解，在酶标仪(Biotek，美国)上OD405的波长观察记录吸光值读数和每次测量的时间。进行分析数据。
实施例1 20小时陈旧血栓在125ng tPA条件下，37℃温育1小时，不同剂量纤溶酶原的溶栓效果
采集2只SD大鼠全血至Eppendorf(EP)管内，37℃温育20h后弃上清，形成陈旧血栓[33，34]。加入PBS反复清洗5-10次，直至所加PBS溶液变澄清。用吸水纸尽量吸干血栓水分，然后将血栓平均分布各个EP管内，称量血栓重量，尽量使每管血栓重量一致。将血栓分为PBS空白对照组、125ng tPA对照组、20μg tPA对照组，0.2mg纤溶酶原组，1mg纤溶酶原组以及2mg纤溶酶原组，每组3管。PBS空白对照组加入1mL PBS；125ng tPA对照组加入1mL PBS和125ng tPA；20μg tPA对照组加入1mL PBS和20μg tPA；0.2mg纤溶酶原组加入1mL PBS和125ng tPA以及0.2mg纤溶酶原；1mg纤溶酶原组加入1mL PBS和125ng tPA以及1mg纤溶酶原；2mg纤溶酶原组加入1mL PBS和125ng tPA以及2mg纤溶酶原。所有反应在37℃培养箱中进行，温育1小时后吸去上清，吸水纸尽量吸干血栓，并称血栓的重量，计算溶栓率。
根据文献报道，在正常生理情况下tPA的含量为5-10ng/mL[35]，而当剧烈运动或静脉淤血情况下，体内tPA的含量会增至20倍至100倍，即达到100ng/mL以上[36]。因此，本实验中使用的tPA剂量为125ng/mL以模仿体内血栓情况下自然产生的tPA含量。
结果显示，对于体外20个小时形成的陈旧血栓，在125ng的tPA条件下，添加纤溶酶原0.2mg、1mg、2mg的溶栓率比单独添加125ng tPA的溶 栓率有明显的升高，统计差异均极显著，说明在体内出现血栓时自然产生的tPA水平的情况下，添加0.2mg或更多的纤溶酶原1小时可以显著促进溶栓。在125ng tPA条件下，添加纤溶酶原1mg就可以达到体内注射剂量20μg tPA(根据Boehringer Ingelheim公司生产的注射用阿普替酶说明书在体内血栓情况下溶栓需使用的剂量换算成的大鼠所需注射剂量)相同的溶栓效果。即达到同样的溶栓率，如果体内存在1mg纤溶酶原，所需tPA量可以减少到原有的1/160。此外，在125ng tPA条件下，添加纤溶酶原1mg达到了纤溶酶原溶栓的峰值，再添加多1倍的纤溶酶原其溶栓率有下降趋势，说明纤溶酶原的添加存在饱和度，饱和度在1到2mg左右(图1)。
实施例2 20小时陈旧血栓在125ng tPA条件下，37℃温育2小时，不同剂量纤溶酶原的溶栓效果
采集2只SD大鼠全血至EP管内，37℃温育20h后弃上清，形成陈旧血栓[33，34]。加入PBS反复清洗5-10次，直至所加PBS溶液变澄清。用吸水纸尽量吸干血栓水分，然后将血栓平均分布各个EP管内，称量血栓重量，尽量使每管血栓重量一致。将血栓分为PBS空白对照组、125ng tPA对照组、20μg tPA对照组，0.2mg纤溶酶原组，1mg纤溶酶原组以及2mg纤溶酶原组，每组3管。PBS空白对照组开始时加入1mL PBS；125ng tPA对照组加入1mLPBS和125ng tPA；20μg tPA对照组加入1mL PBS和20μg tPA；0.2mg纤溶酶原组加入1mL PBS和125ng tPA以及0.2mg纤溶酶原；1mg纤溶酶原组加入1mL PBS和125ng tPA以及1mg纤溶酶原；2mg纤溶酶原组加入1mL PBS和125ng tPA以及2mg纤溶酶原。所有反应在37℃培养箱中进行，温育2小时后，吸去上清，吸水纸尽量吸干血栓，并称血栓的重量，计算溶栓率。
根据文献报道，在正常生理情况下tPA的含量为5-10ng/mL[35]，而当剧烈运动，或静脉淤血情况下，体内tPA的含量会增至20倍至100倍，即达到100ng/mL以上[36]。因此，本实验中使用的tPA剂量为125ng/mL以模仿体内血栓情况下自然产生的tPA含量。
结果显示，对于在体外20个小时形成的陈旧血栓，与实施例1相比，每组随着反应时间的延长，溶栓率也相应增加。在125ng的tPA条件下，添加纤溶酶原0.2mg、1mg、2mg的溶栓率比单独添加125ng tPA的溶栓率有明显的升高，统计差异均极显著。说明在体内出现血栓时自然产生的 tPA剂量的情况下，添加0.2mg或更多的纤溶酶原2小时可以显著促进溶栓。在反应2小时后，1mg、2mg纤溶酶原组的溶栓效果要优于体内正常注射剂量20μg tPA对照组(根据Boehringer Ingelheim公司生产的注射用阿普替酶说明书在体内血栓情况下溶栓需使用的剂量换算成的大鼠所需注射剂量)，即达到同样的溶栓率，如果体系内存在1mg纤溶酶原，所需tPA量可以减少到体系内没有1mg纤溶酶原时所需tPA量(20μg)的不到1/160(图2)。
实施例3 20小时陈旧血栓在10ng tPA条件下溶栓率随着纤溶酶原剂量增加而升高
采集2只SD大鼠全血至EP管内，37℃温育20h后弃上清，形成陈旧血栓[33，34]。加入PBS反复清洗5-10次，直至所加PBS溶液变澄清。用吸水纸尽量吸干血栓水分，然后将血栓平均分布各个EP管内，称量血栓重量，尽量使每管血栓重量一致。将血栓分为PBS空白对照组、10ng tPA对照组、0.2mg纤溶酶原对照组，0.2mg纤溶酶原组，1mg纤溶酶原组以及2mg纤溶酶原组，每组3管。PBS空白对照组开始时加入1mL PBS；10ng tPA对照组加入1mL PBS和10ng tPA；0.2mg纤溶酶原对照组加入1mL PBS和0.2mg纤溶酶原；0.2mg纤溶酶原组加入1mL PBS和10ng tPA以及0.2mg纤溶酶原；1mg纤溶酶原组加入1mL PBS和10ng tPA以及1mg纤溶酶原；2mg纤溶酶原组加入1mL PBS和10ng tPA以及2mg纤溶酶原。所有反应在37℃培养箱中进行，温育2小时后，吸去上清，吸水纸尽量吸干血栓，并称血栓的重量，计算溶栓率。
根据文献报道，在正常生理情况下tPA的含量为5-10ng/mL[35]，因此，本实验中使用的tPA剂量为10ng/mL以模仿体内正常生理情况下自然产生的tPA含量。
结果显示，对于在体外20个小时形成的陈旧血栓，在体内正常生理情况下自然产生的tPA含量(10ng)的条件下，添加纤溶酶原的各组溶栓率均高于只添加体内生理剂量tPA的对照组的溶栓率，统计差异均极显著。而且随着纤溶酶原添加剂量的增加，相应的溶栓率也呈梯度升高趋势，说明了可以通过调节纤溶酶原的剂量调节溶解20小时血栓的速度。此外，在0.2mg纤溶酶原存在的情况下，添加体内生理水平的tPA(10ng)的组的溶栓效率要极显著地高于不添加tPA的组，而只添加0.2mg的纤溶酶原的溶栓 效果与添加对照PBS的溶栓效果类似，说明生理水平的tPA对于纤溶酶原发挥溶栓作用起到关键作用(图3)。
实施例4 72小时陈旧血栓在125ng tPA条件下溶栓率随着纤溶酶原剂量增加而升高
采集2只SD大鼠全血至EP管内，37℃温育72小时后弃上清，形成陈旧血栓[36]。加入PBS反复清洗5-10次，直至所加PBS溶液变澄清。用吸水纸尽量吸干血栓水分，然后将血栓平均分布各个EP管内，称量血栓重量，尽量使每管血栓重量一致。将血栓分为PBS空白对照组、125ng tPA对照组、0.2mg纤溶酶原对照组，0.2mg纤溶酶原组，1mg纤溶酶原组以及2mg纤溶酶原组，每组3管。PBS空白对照组开始时加入1mL PBS；125ng tPA对照组加入1mL PBS和125ng tPA；0.2mg Plg对照组加入1mLPBS和0.2mg Plg；0.2mg纤溶酶原组加入1mL PBS和125ng tPA以及0.2mg纤溶酶原；1mg纤溶酶原组加入1mL PBS和125ng tPA以及1mg纤溶酶原；2mg纤溶酶原组加入1mL PBS和125ng tPA以及2mg纤溶酶原。所有反应在37℃培养箱中进行，温育2小时后，吸去上清，吸水纸尽量吸干血栓，并称血栓的重量，计算溶栓率。
根据文献报道，在正常生理情况下tPA的含量为5-10ng/mL[35]，而当剧烈运动，或静脉淤血情况下，体内tPA的含量会增至20倍至100倍，即达到100ng/ml以上[36]。因此，本实验中使用的tPA剂量为125ng/ml以模仿体内血栓情况下自然产生的tPA含量。
结果显示，对于体外72个小时形成的陈旧血栓，在125ng tPA条件下，添加纤溶酶原的溶栓率要高于单独添加125ng tPA的溶栓率，且差异极显著，说明在体内出现血栓时自然产生的tPA剂量(125ng)的情况下，添加0.2mg或更多的纤溶酶原2小时可以显著促进溶解72小时的陈旧血栓。而且随着添加纤溶酶原剂量梯度的增加，其溶栓率也呈梯度递增趋势，说明了可以通过调节纤溶酶原的剂量调节溶解陈旧血栓的速度。此外，添加纤溶酶原4mg的溶栓率在本次实验中超过了体内正常注射剂量20μg tPA(根据Boehringer Ingelheim公司生产的注射用阿普替酶说明书在体内血栓情况下溶栓需使用的剂量换算成的大鼠所需注射剂量)的溶栓率，说明在体内出现血栓时自然产生的tPA剂量(125ng)的情况下，只添加纤溶酶原溶解陈旧血 栓的效果优于现有溶栓药物的效果(图4)，表明纤溶酶原可以成为溶栓效果更优的溶栓物质。
此外，在实施例2中，相较于对照PBS组，单独添加125ng的tPA能显著增加溶解20小时血栓的能力。但是，在本实施例中，对于72小时的陈旧血栓，与体内情况类似，单独添加125ng的tPA组与对照PBS组的溶栓效果几乎相同，说明随着血栓逐渐陈旧，生理情况下自然产生的tPA的溶栓能力逐步下降，从侧面说明了实施例使用的模型能够一定程度模仿体内情况。
实施例5 72小时陈旧血栓在10ng tPA条件下溶栓率随着纤溶酶原剂量增加而升高
采集2只SD大鼠全血至EP管内，37℃温育72h后弃上清，形成陈旧血栓[36]。加入PBS反复清洗5-10次，直至所加PBS溶液变澄清。用吸水纸尽量吸干血栓水分，然后将血栓平均分布各个EP管内，称量血栓重量，尽量使每管血栓重量一致。将血栓分为PBS空白对照组、10ng tPA对照组、20μg tPA对照组，0.2mg纤溶酶原对照组，0.2mg纤溶酶原组，1mg纤溶酶原组，2mg纤溶酶原组以及4mg纤溶酶原组，每组3管。PBS空白对照组加入1mL PBS；10ng tPA对照组加入1mL PBS和10ng tPA；20μg tPA对照组加入1mL PBS和20μg tPA；0.2mg Plg对照组加入1mL PBS和0.2mg Plg；0.2mg纤溶酶原组加入1mL PBS和10ng tPA以及0.2mg纤溶酶原；1mg纤溶酶原组加入1mL PBS和10ng tPA以及1mg纤溶酶原；2mg纤溶酶原组加入1mL PBS和10ng tPA以及2mg纤溶酶原；4mg纤溶酶原组加入1mL PBS和10ng tPA以及4mg纤溶酶原。所有反应在37℃培养箱中进行，反应2小时，吸去上清，吸水纸尽量吸干血栓，并称血栓的重量，计算溶栓率。
根据文献报道，在正常生理情况下tPA的含量为5-10ng/mL[35]，因此，本实验中使用的tPA剂量为10ng/mL以模仿体内正常生理情况下自然产生的tPA含量。
此实验结果表明，对于在体外72个小时形成的陈旧血栓，在体内正常生理tPA含量10ng/mL的条件下，添加纤溶酶原的溶栓率要高于单独添加10ng tPA的溶栓率，且差异极显著。说明在体内出现血栓时自然产生的tPA剂量(10ng)的情况下，添加0.2mg或更多的纤溶酶原2小时可以显著促 进溶解72小时的陈旧血栓。并且随着添加纤溶酶原剂量的增加，其溶栓率也呈梯度增加，说明了可以通过调节纤溶酶原的剂量调节溶解陈旧血栓的速度。而且添加4mg纤溶酶原组的溶栓率接近正常tPA注射剂量(根据Boehringer Ingelheim公司生产的注射用阿普替酶说明书在体内血栓情况下溶栓需使用的剂量换算成的大鼠所需注射剂量)的溶栓率(图5)，说明在生理水平的tPA剂量(10ng)的情况下，只添加纤溶酶原后其溶解陈旧血栓的效果就可以达到现有溶栓药物的效果，在这个意义上，纤溶酶原有希望成为一种新的陈旧血栓溶解药物。
实施例6纤溶酶原温和溶解20小时陈旧血栓
采集2只SD大鼠全血至EP管内，37℃温育20h后弃上清，形成陈旧血栓[33，34]。加入PBS反复清洗5-10次，直至所加PBS溶液变澄清。用吸水纸尽量吸干血栓水分，然后将血栓平均分布各个EP管内，称量血栓重量，尽量使每管血栓重量一致。将血栓分为两组，每组12个样品。第一组为tPA对照组，加入1mL PBS及5μg tPA；第二组为纤溶酶原组，加入1mL PBS、10ng tPA及1mg纤溶酶原。预实验证明，这两组对于20小时陈旧血栓在2小时内的溶解率类似(数据未显示)。所有反应在37℃培养箱中进行，分别在第0.5h、1h、1.5h、2h取样，每个时间点两组分别取三个样品，吸去上清，吸水纸尽量吸干血栓，并称血栓的重量，计算溶栓率。
根据文献报道，在正常生理情况下tPA的含量为5-10ng/mL[35]，因此，本实验中使用的tPA剂量为10ng/mL以模仿体内正常生理情况下自然产生的tPA含量。
实验显示，对于20小时陈旧血栓，两组总溶栓率随着时间的延长而升高，但在0到0.5小时和0.5到1小时两个区间，纤溶酶原组的溶栓曲线斜率均低于tPA组(图6)。表1显示10ng tPA存在条件下，1mg纤溶酶原溶栓效率与单独5ug tPA的溶栓效率随时间变化的对比。如表1所示，纤溶酶原组2小时的总溶栓率的75％集中在约1小时内，而tPA对照组2小时总溶栓率的75％集中在约0.5小时。这些数据清楚地说明，相对于tPA，纤溶酶原的溶栓速度相对于tPA的溶栓速度更为温和(图6，表1)。
表1 10ng tPA存在条件下，1mg纤溶酶原溶栓效率与单独5ug tPA溶栓效率的随着时间的变化。
Figure PCTCN2016110448-appb-000001
实施例7在100ng uPA条件下，纤溶酶原促进20小时陈旧血栓溶解
采集2只SD大鼠全血至EP管内，37℃温育20h后弃上清，形成陈旧血栓[33，34]。加入PBS反复清洗5-10次，直至所加PBS溶液变澄清。用吸水纸尽量吸干血栓水分，然后将血栓平均分布各个EP管内，称量血栓重量，尽量使每管血栓重量一致。将血栓分为PBS空白对照组、100ng uPA对照组、0.2mg纤溶酶原对照组，0.2mg纤溶酶原组，1mg纤溶酶原组以及2mg纤溶酶原组，每组3管。PBS空白对照组开始时加入1mL PBS；100nguPA对照组加入1mL PBS和100ng uPA；0.2mg纤溶酶原对照组加入1mLPBS和0.2mg纤溶酶原；0.2mg纤溶酶原组加入1mL PBS和100ng uPA以及0.2mg纤溶酶原；1mg纤溶酶原组加入1mL PBS和100ng uPA以及1mg纤溶酶原；2mg纤溶酶原组加入1mL PBS和100ng uPA以及2mg纤溶酶原。所有反应在37℃培养箱中进行，温育1小时后，吸去上清，吸水纸尽量吸干血栓，并称血栓的重量，计算溶栓率。
根据文献报道，在以纤溶酶原为底物的酶促反应过程中tPA米氏常数为0.18×10-7mol/L[37]，而uPA的米氏常数为2.43×10-7mol/L[38]，也就是说，在相同的反应条件，相同的反应时间内tPA的亲和力约为uPA的10倍，所以本次实验中，根据实施例3使用的10ng tPA/ml估算出uPA的使用剂量为100ng/ml。
结果显示，对于在体外20个小时形成的陈旧血栓，在改变纤溶酶原激活剂10ng tPA为100ng uPA后，添加纤溶酶原0.2mg、1mg、2mg各组的溶栓率比单独添加100ng uPA的溶栓率有明显的升高，统计差异均极显著(**P＜0.01；***P＜0.001)。说明在100ng的uPA剂量的情况下，添加0.2mg 或更多的纤溶酶原1小时可以显著促进溶栓，并且随着纤溶酶原添加剂量梯度的增加，其溶栓率也显著升高(图7)。说明100ng uPA条件下，纤溶酶原能够促进陈旧血栓溶解。
实施例8在1ng uPA条件下，纤溶酶原促进20小时陈旧血栓溶解
采集2只SD大鼠全血至EP管内，37℃温育20h后弃上清，形成陈旧血栓[33，34]。加入PBS反复清洗5-10次，直至所加PBS溶液变澄清。用吸水纸尽量吸干血栓水分，然后将血栓平均分布各个EP管内，称量血栓重量，尽量使每管血栓重量一致。将血栓分为PBS空白对照组、1ng uPA对照组、0.2mg Plg对照组，0.2mg纤溶酶原组，1mg纤溶酶原组以及2mg纤溶酶原组，每组3管。PBS空白对照组加入1mL PBS；1ng uPA对照组加入1mL PBS和1ng uPA；0.2mg Plg对照组加入1mL PBS和0.2mg Plg；0.2mg纤溶酶原组加入1mL PBS和1ng uPA以及0.2mg纤溶酶原；1mg纤溶酶原组加入1mL PBS和1ng uPA以及1mg纤溶酶原；2mg纤溶酶原组加入1mL PBS和1ng uPA以及2mg纤溶酶原。所有反应在37℃培养箱中进行，温育2小时后，吸去上清，吸水纸尽量吸干血栓，并称血栓的重量，计算溶栓率。
根据文献报道，在正常生理情况下uPA的含量为1ng/mL[35]，因此，本实验中使用的uPA剂量为1ng/mL以模仿体内正常生理情况下自然产生的uPA含量。
结果显示，对于在体外20个小时形成的陈旧血栓，在把uPA的使用量降为体内正常含量1ng时，陈旧血栓的溶栓率的溶栓总体较为缓慢。但1mg和2mg的纤溶酶原组溶栓率显著高于1ng uPA对照组，具有统计差异。说明1ng uPA条件下，添加纤溶酶原对于陈旧血栓的溶解有显著的促进作用(图8)。
实施例9小鼠静脉注射tPA及纤溶酶原后再出血实验
选择11周龄C57野生型雄性小鼠55只，使用3％戊巴比妥进行全身麻醉，分别剪尾3mm，将尾巴置于37℃温水中，观察尾巴出血状况[39]，。止血后随机分成两组，tPA组5只，纤溶酶原组50只。tPA组眼眶静脉注射tPA 400μg/0.05mL/只；纤溶酶原组眼眶静脉注射1mg/0.05mL/只纤溶酶原，实验过程中始终将小鼠尾静脉放置于37℃温水中，观察实验出血状况20分钟，并记录。
实验结果显示，静脉注射400μg tPA能够致使受伤小鼠原已凝结的尾部伤口发生再出血现象，此为tPA药物的一个普遍的副作用。但静脉注射1mg纤溶酶原的小鼠没有此种副作用出现(表2)，说明纤溶酶原相较于tPA的安全性更好。
表2小鼠静脉注射tPA或纤溶酶原后体内出血实验结果
Figure PCTCN2016110448-appb-000002
实施例10纤溶酶原对体内血栓的特异性吸附实验
选择野生型雄性小鼠9只，随机分为3组，溶媒PBS对照组、0.2mg纤溶酶原组和1mg纤溶酶原组，每组3只。使用3％戊巴比妥进行全身麻醉，将小鼠颈静脉分离，用浸有10％的FeCl3溶液的吸水纸(3mm×5mm)敷于颈静脉5分钟形成静脉血栓。形成血栓后立即开始给予纤溶酶原或溶媒PBS。溶媒PBS对照组尾静脉注射100ul的PBS，1mg纤溶酶原组和0.2mg纤溶酶原组分别尾静脉注射给予1mg、0.2mg纤溶酶原。3小时后取出相应颈静脉血栓及对侧静脉附近的肌肉。将血栓及对侧静脉附近肌肉使用研磨器进行匀浆，离心后取上清，将上清利用BCA法检测其总蛋白，酶联免疫法检测匀浆液中的纤溶酶原含量，计算出一定蛋白总量中的纤溶酶原含量。
结果显示，血栓形成后血栓中纤溶酶原的含量均明显高于肌肉中的纤溶酶原含量。而且静脉注射纤溶酶原后血栓中纤溶酶原的含量进一步增加。这些结果说明在体内血栓的存在下，纤溶酶原能够特异性地结合到血栓上(图9)并进一步发挥溶血栓作用，而tPA一旦注射进入血管，就会非特异性地在血管内催化溶栓反应。该实验结果说明纤溶酶原相较于tPA有显著的特异性溶栓优点。
实施例11 30分钟的新鲜血栓在添加纤溶酶原后的溶栓率显著升高
采集2只SD大鼠全血至EP管内，37℃温育30分钟后，弃上清，形成新鲜血栓[33]。加入PBS反复清洗5-10次，直至所加PBS溶液变澄清。用吸水纸尽量吸干血栓水分，然后将血栓平均分布各个EP管内，称量血栓重量，尽量使每管血栓重量一致。将血栓分为PBS空白对照组、125ng tPA 对照组、0.2mg纤溶酶原组，1mg纤溶酶原组以及2mg纤溶酶原组，每组2管。PBS空白对照组加入1mL PBS；tPA对照组加入1mL PBS和125ng tPA；0.2mg纤溶酶原组加入1mL PBS和125ng tPA以及0.2mg纤溶酶原；1mg纤溶酶原组加入1mL PBS和125ng tPA以及1mg纤溶酶原；2mg纤溶酶原组加入1mL PBS和125ng tPA以及2mg纤溶酶原。所有反应在37℃培养箱中进行，温育2小时后，吸去上清，吸水纸尽量吸干血栓，并称血栓的重量，计算溶栓率。
根据文献报道，在正常生理情况下tPA的含量为5-10ng/mL[35]，而当剧烈运动，或静脉淤血情况下，体内tPA的含量会增至20倍至100倍，即达到100ng/mL以上[36]。因此，本实验中使用的tPA剂量为125ng/mL以模仿体内血栓情况下自然产生的tPA含量。
此实验说明对于在体外30分钟形成的新鲜血栓，在梯度增加纤溶酶原使用剂量的条件下，溶栓率呈梯度升高趋势，并且纤溶酶原各组溶栓率均高于单独添加tPA对照组的溶栓率，统计差异极其显著。这些结果说明在体内出现血栓时自然产生的tPA水平的情况下，添加0.2mg或更多的纤溶酶原1小时可以显著促进溶栓(图10)，说明纤溶酶原不仅能够促进溶解陈旧血栓，也能够促进溶解新鲜血栓。
实施例12纤溶酶原促进糖尿病造成的微血栓的溶解
24-25周龄db/db雄鼠10只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组，每组各5只。实验开始当天记为第0天称重分组，实验第二天开始给纤溶酶原或PBS并记为第1天，连续给药15天。给纤溶酶原组小鼠按2mg/0.2mL/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。在第16天摘眼球取血，全血静置后血清用于检测血液中D-二聚体含量。
结果显示，给药15天后，给纤溶酶原组血清中D-二聚体含量显著上升(图11)，说明给纤溶酶原后，由于糖尿病造成的微血栓显著溶解。
实施例13纤溶酶原促进糖尿病晚期小鼠心脏组织血栓溶解
24-25周龄db/db雄鼠10只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组各5只。实验开始当天记为第0天称重分组，实验第二天开始给纤溶酶原或PBS并记为第1天，连续给药31天。给纤溶酶原组小鼠按2mg/0.2mL/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组给予相同体积的 PBS。在第32天处死小鼠并取心脏在10％中性福尔马林固定液中固定24小时。固定后的心脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为5μm，切片脱蜡复水后水洗1次，以3％双氧水孵育15分钟，水洗2次，每次5分钟。10％的正常羊血清液(Vectorlaboratories，Inc.，USA)封闭1小时；之后弃除羊血清液，用PAP笔圈出组织。兔抗小鼠纤维蛋白(原)抗体(Abcam)4℃孵育过夜，TBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，TBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下400倍下观察。
纤维蛋白原是纤维蛋白的前体，在组织存在损伤的情况下，作为机体对损伤的一种应激反应，纤维蛋白原水解成纤维蛋白[40-42]，因此可将纤维蛋白水平作为损伤程度的一个标志。纤维蛋白也是组织损伤后形成血栓的主要成分，因此，也可将纤维蛋白水平作为血栓的一个标志。
结果显示，与给溶媒PBS对照组(图12A)相比，给纤溶酶原组(图12B)的小鼠心脏组织纤维蛋白阳性着色较浅，说明给纤溶酶原组心脏组织沉积的纤维蛋白减少，反映纤溶酶原能够促进糖尿病所致心脏组织损伤修复，也说明纤溶酶原能够促进心脏组织血栓的溶解。
实施例14纤溶酶原促进糖尿病后期小鼠肾脏组织血栓溶解
24-25周龄db/db雄鼠20只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组各10只。实验开始当天记为第0天称重分组，实验第二天开始给纤溶酶原或PBS并记为第1天，连续给药31天。给纤溶酶原组小鼠按2mg/0.2mL/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。在第32天处死小鼠并取肾脏在10％中性福尔马林固定液中固定24小时。固定后的肾脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为5μm，切片脱蜡复水后水洗1次。以3％双氧水孵育15分钟，水洗2次，每次5分钟。10％的正常羊血清液(Vector laboratories，Inc.，USA)封闭1小时；时间到后，弃除羊血清液，用PAP笔圈出组织。兔抗小鼠纤维蛋白(原)抗体(Abcam)4℃孵育过夜，TBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，TBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染 30秒，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下200倍下观察。
纤维蛋白原是纤维蛋白的前体，在组织存在损伤的情况下，作为机体对损伤的一种应激反应，纤维蛋白原水解成纤维蛋白[40-42]，因此可将纤维蛋白水平作为损伤程度的一个标志。纤维蛋白也是组织损伤后形成血栓的主要成分，因此，也可将纤维蛋白水平作为血栓的一个标志。
结果显示，给纤溶酶原组(图13B)比给溶媒PBS对照组(图13A)纤维蛋白原阳性着色浅。说明注射纤溶酶原能够显著降低糖尿病小鼠肾脏纤维蛋白沉积，反映出纤溶酶原对糖尿病小鼠肾脏损伤有显著的修复作用，也说明纤溶酶原能够促进肾脏组织血栓的溶解。
实施例15纤溶酶原促进糖尿病晚期肝组织血栓溶解
24-25周龄db/db雄鼠10只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组各5只。实验开始当天记为第0天称重分组，实验第二天开始给纤溶酶原或PBS并记为第1天，连续给药31天。给纤溶酶原组小鼠按2mg/0.2mL/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。在第32天处死小鼠并取肝脏组织在10％中性福尔马林固定液中固定24小时。固定后的肝脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为5μm，切片脱蜡复水后水洗1次。以3％双氧水孵育15分钟，水洗2次，每次5分钟。10％的正常羊血清液(Vector laboratories，Inc.，USA)封闭1小时；时间到后，弃除羊血清液，用PAP笔圈出组织。兔抗小鼠纤维蛋白(原)抗体(Abcam)4℃孵育过夜，TBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，TBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下200倍下观察。
纤维蛋白原是纤维蛋白的前体，在组织存在损伤的情况下，作为机体对损伤的一种应激反应，纤维蛋白原水解成纤维蛋白[40-42]，因此可将纤维蛋白水平作为损伤程度的一个标志。纤维蛋白也是组织损伤后形成血栓的主要成分，因此，也可将纤维蛋白水平作为血栓的一个标志。
研究发现，与给溶媒PBS对照组(图14A)相比，给纤溶酶原组(图14B)的小鼠其肝脏组织纤维蛋白阳性着色浅，说明注射纤溶酶原能够显著降低 糖尿病小鼠肝脏纤维蛋白沉积，反映出纤溶酶原对糖尿病小鼠肝脏损伤有显著修复作用，也说明纤溶酶原能够促进肝脏组织血栓的溶解。
实施例16纤溶酶原促进糖尿病后期神经损伤小鼠神经组织血栓溶解
24-25周龄db/db雄鼠10只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组，每组各5只。实验开始当天记为第0天称重分组，实验第二天开始给纤溶酶原或PBS并记为第1天，连续给药15天。给纤溶酶原组小鼠按2mg/0.2mL/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。在第16天处死小鼠并取坐骨神经在10％中性福尔马林固定液中固定24小时。固定后的坐骨神经经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为5μm，切片脱蜡复水后水洗1次，然后用PAP笔圈出组织。以3％TBS稀释的双氧水孵育15分钟，水洗3次。10％正常羊血清(Vector laboratories，Inc.，USA)封闭1小时，吸走多余血清。兔抗小鼠纤维蛋白(原)抗体(Abcam)室温孵育1小时或4℃孵育过夜，TBS洗3次。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，TBS洗3次。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下400倍下观察。
纤维蛋白原是纤维蛋白的前体，在组织存在损伤的情况下，作为机体对损伤的一种应激反应，纤维蛋白原水解成纤维蛋白[40-42]，因此可将纤维蛋白水平作为损伤程度的一个标志。纤维蛋白也是组织损伤后形成血栓的主要成分，因此，也可将纤维蛋白水平作为血栓的一个标志。
研究发现，与给溶媒PBS对照组(图15A)相比，给纤溶酶原组(图15B)的小鼠其坐骨神经纤维蛋白的水平降低，说明纤溶酶原具有降解纤维蛋白水平的功能，损伤得到一定程度的修复，也说明纤溶酶原能够促进神经组织周围血栓的溶解。
实验结果总结：
本发明实施例实验包括纤溶酶原体外溶栓和体内溶栓两部分。
体外溶栓模拟体内溶栓条件，选取10ng/mL tPA模仿正常生理情况下体内自然产生的tPA量，125ng/mL tPA模仿体内血栓情况下自然产生的tPA量，来研究纤溶酶原的溶栓能力。
本发明实验研究显示，10ng/mL tPA或125ng/mL tPA条件下，无论是20小时的陈旧血栓还是72小时的陈旧血栓，纤溶酶原都具有非常好的溶栓效果，并且随着纤溶酶原剂量的增大溶栓效率随之增大。
纤溶酶原对新鲜血栓具有很强的溶解能力，温育两小时溶栓率可达到80％以上。
我们也研究了uPA存在条件下纤溶酶原溶栓效果。1ng/mL uPA或100ng/mL uPA条件下，纤溶酶原同样都具有非常好的溶栓效果，并且随着纤溶酶原剂量的增加溶栓效率随之增大。
体内实验中，糖尿病后期小鼠连续15天每天给予2mg纤溶酶原，血清中D-二聚体的含量显著增加。同时，心脏、肝脏、肾脏、神经组织纤维蛋白水平显著下降，说明纤溶酶原能明显促进这些组织中由糖尿病组织损伤引发的血栓的溶解，纤维蛋白降解，证明给药实验动物纤溶酶原同样可以达到显著的溶栓效果。
小鼠颈静脉血栓模型实验显示，纤溶酶原能够非常特异地结合体内血栓。
此外，我们研究显示与tPA相比纤溶酶原对血栓的溶解更加温和，并且小鼠的tail-bleeding实验表明纤溶酶原没有出血副作用。
综上所述，纤溶酶原具有非常好的溶栓能力，特别是针对陈旧血栓，并且具有特异性高、温和、见效快以及无出血副作用的特点。
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Claims (15)

  1. 一种预防和/或消除受试者动、静脉血栓的方法，包括给药受试者有效量的纤溶酶原。
  2. 根据权利要求1的方法，其中所述血栓包括新鲜血栓和陈旧血栓。
  3. 权利要求1或2的方法，其中所述血栓为血液系统疾病、循环系统疾病、自身免疫疾病、代谢紊乱疾病或感染性疾病导致的血栓。
  4. 权利要求1-3任一项的方法，其中所述血栓为继发于糖尿病的大、小血管、微血管血栓。
  5. 权利要求1-4任一项的方法，其中所述血栓为大小血管病变导致的血栓。
  6. 一种预防和/或治疗受试者血栓相关疾病的方法，包括给药受试者有效量的纤溶酶原，其中所述纤溶酶原通过消除血栓预防和/或治疗受试者所述血栓相关疾病。
  7. 根据权利要求6的方法，其中所述血栓相关疾病为门静脉血栓导致的胰腺炎、肝硬化；肾静脉血栓导致的肾栓塞；颈内静脉血栓引起的全身性败血症、肺栓塞、脑血栓、深静脉血栓；动脉和静脉血栓导致的器官的梗死，包括但不限于：脑梗塞、心肌梗死、血栓性中风、房颤、不稳定性心绞痛、顽固性心绞痛、短暂性脑缺血发作、肺栓塞。
  8. 根据权利要求6的方法，其中所述血栓相关疾病为糖尿病性肾病、糖尿病性视网膜病、糖尿病性肝病、糖尿病性心脏病、糖尿病性肠病、糖尿病性神经病变。
  9. 根据权利要求1-8任一项的方法，其中所述纤溶酶原可与伴随血栓形成的其它疾病的治疗药物联合施用。
  10. 根据权利要求1-10任一项的方法，其中所述纤溶酶原为与序列2、6、8、10或12具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的蛋白质。
  11. 一种用于预防和/或消除受试者动、静脉血栓的制品，其包含含有有效剂量的纤溶酶原的容器，和指导施用所述制品预防和/或消除受试者动、静脉血栓的说明书。
  12. 一种用于预防和/或治疗受试者血栓相关疾病的制品，其包含含有有效剂 量的纤溶酶原的容器，和指导施用所述制品预防和/或治疗受试者血栓相关疾病的说明书。
  13. 权利要求11或12的制品，进一步包含含有一种或多种其它药物的容器。
  14. 权利要求13的制品，其中所述其它药物为伴随血栓形成的其它疾病的治疗药物。
  15. 权利要求13或14的制品，其中所述说明书进一步说明所述纤溶酶原可以在所述其它药物施用之前，同时，和/或之后施用。
PCT/CN2016/110448 2015-12-18 2016-12-16 一种用于预防或治疗急性及慢性血栓的方法 WO2017101866A1 (zh)

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DK16874923.2T DK3395359T3 (da) 2015-12-18 2016-12-16 Plasminogen til anvendelse til forebyggelse eller behandling af akut trombose og kronisk trombose
CA3008185A CA3008185C (en) 2015-12-18 2016-12-16 Method for prevention or treatment of acute and chronic thrombosis
JP2018550633A JP2019500422A (ja) 2015-12-18 2016-12-16 急性及び慢性血栓を予防または治療することに用いる方法
CN202211684331.2A CN115845037A (zh) 2015-12-18 2016-12-16 一种用于预防或治疗急性及慢性血栓的方法
EP16874923.2A EP3395359B1 (en) 2015-12-18 2016-12-16 Plasminogen for use in preventing or treating acute thrombosis and chronic thrombosis
CN201680073669.0A CN108472342B (zh) 2015-12-18 2016-12-16 一种用于预防或治疗急性及慢性血栓的方法
US16/063,534 US10864257B2 (en) 2015-12-18 2016-12-16 Method for prevention or treatment of acute and chronic thrombosis
ES16874923T ES2968254T3 (es) 2015-12-18 2016-12-16 Plasminógeno para su uso en la prevención o el tratamiento de la trombosis aguda y la trombosis crónica

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