HK1257587A1 - Medicine for preventing and treating liver fibrosis and use of the medicine - Google Patents

Abstract

The invention relates to a method for preventing and treating liver fibrosis. The method comprises a step of administrating an effective dosage of plasminogen to a medicine subject.

Description

Medicine for preventing and treating hepatic fibrosis and application thereof

Technical Field

The present invention relates to a method for preventing and treating liver fibrosis comprising administering to a subject an effective amount of plasminogen.

Background

Fibrosis is a pathological change, which is manifested by activated proliferation of fibroblasts, fibrous connective tissue increase in tissues and organs, parenchymal cell reduction, and continuous progress can lead to structural damage and functional loss of tissues and organs. The fibrosis of the vital organs seriously affects the life quality of the patient and even endangers life. Worldwide, tissue fibrosis is the leading cause of disability and death in many diseases, and as shown by the statistics in the united states, about 45% of patients who are fatal to various diseases can be attributed to tissue fibrosis diseases.

Fibrotic diseases include diseases involving multiple systems, such as systemic sclerosis, multifocal fibrosis, scleroderma, renal-derived multiple system fibrosis, and organ-tissue specific diseases, such as skin, heart, lung, liver, renal fibrosis, etc. The etiology of different fibrotic diseases varies, e.g., tissue organ injury, infection, immune response, chronic inflammation, etc., but they are commonly characterized by excessive deposition of extracellular matrix (ECM) in the tissue and organ tissue remodeling^[1-3]。

Cardiac fibrosis occurs in the development and progression of a variety of cardiac diseases, such as viral myocarditis, myocardial infarction, hypertensive heart disease, and the like. Excessive cardiac fibrosis causes impaired cardiac function, which is the leading cause of heart failure in a variety of diseases, where inhibition and reversal of myocardial fibrosis has become an important link in the treatment of cardiovascular diseases.

Hepatic fibrosis is the pathological process of abnormal proliferation of connective tissue in liver and excessive deposition of diffuse extracellular matrix in liver caused by various pathogenic factors. Hepatic fibrosis is caused by various factors, such as viral infection, inflammatory response, oxidative stress, alcohol abuse, and the like. The pathological features of hepatic fibrosis are that there is a lot of fibrous tissue hyperplasia and deposition in the region of the junction and the lobules, but the lobular space is not formed yet, the cirrhosis has the formation of false lobules, the central venous region and the region of the junction have the space, the normal structure of the liver is destroyed, and the further development of hepatic fibrosis is cirrhosis. The chronic liver diseases in China mainly comprise viral hepatitis, and liver tissue fibrosis of the chronic viral hepatitis is related to intrahepatic inflammation, necrosis, virus replication and the like and is reversible in early stage. Therefore, the combination of anti-virus treatment, therapy for adjusting the immune function of the body, protecting liver cells and the like and anti-fibrosis is an active measure for preventing hepatic fibrosis.

The pulmonary fibrosis diseases comprise idiopathic pulmonary fibrosis, sarcoidosis, hypersensitivity pneumonitis, pneumoconiosis, fibrosis caused by drugs and radiation, fibrosis-induced pulmonary alveolitis related to collagen vascular disease and the like with different causes and wide disease spectrum. The main pathological features of the lung tissue include the proliferation of mesenchymal cells in lung tissue, the proliferation and deposition of extracellular matrix, the reconstruction of lung parenchyma and the like. At present, measures such as anti-inflammation, antioxidation, fibroblast proliferation resistance, collagen deposition resistance, lung transplantation and the like are mainly adopted to treat pulmonary fibrosis.

Renal fibrosis, a pathological process in which extracellular matrix and inappropriate connective tissue accumulate in the kidney, leading to structural changes and impaired function of the kidney, is also a common pathway for almost all renal diseases to progress to end-stage renal failure. The renal fibrosis process involves inflammatory reaction, apoptosis of innate cells and immune cells, imbalance of various regulatory fibrosis factors and the like, so the renal fibrosis can be resisted through anti-inflammation, anti-apoptosis, fibrosis factor treatment and other ways.

Chronic lesions of the tissue and organs are often accompanied by fibrosis, for example chronic inflammation of the lung, chronic lesions with pulmonary fibrosis. This is also true of liver fibrosis, such as hepatitis B, hepatitis C, alcoholic liver, fatty liver, schistosomiasis, etc., all with early stage liver fibrosis. The compensatory function of the liver is strong, and fibrotic lesions remain latent in various chronic liver diseases and are generally found when the liver cirrhosis is developed, and actually the liver cirrhosis is a serious stage of the hepatic fibrosis. The chronic nephritis, glomerulonephritis, tubular nephritis and other diseases have renal fibrosis; cardiovascular, cerebrovascular, and lower limb vascular sclerosis, or narrowing, or obstruction, all have vascular fibrosis.

Fibrosis of the skin forms scar tissue. Scar tissue is fibrous connective tissue that has been transformed into an aging stage of mature granulation tissue. In the case of a wound or the like, fibroblasts divide and proliferate, migrate to the damaged site, produce extracellular matrix, form scar tissue, and repair the wound.

Scar formation is a process of progressive fibrosis of granulation tissue. At the moment, more and more reticular fibers and collagen fibers are generated, the reticular fibers are collagenated, the collagen fibers become thick, meanwhile, fibroblasts are fewer and fewer, and a small amount of the rest are converted into fibroblasts; the liquid in the interstitium is gradually absorbed, and the neutrophils, the macrophages, the lymphocytes and the plasma cells disappear in sequence; capillaries close, degenerate, disappear, leaving few arterioles and venules. Thus, the granulation tissue is transformed into a scar tissue consisting mainly of collagen fibers with few blood vessels, which is white to the naked eye and tough.

The scar is tough and lacks elasticity, and the scar contraction can cause organ deformation and dysfunction, so the scar is generated near joints and in important organs, joint spasm or limited movement is often caused, for example, lumen stenosis is caused in cavity organs such as digestive tract, urinary tract and the like, and dyskinesia is caused near the joints. Cicatricial adhesions occurring between organs or between organs and walls of body cavities often affect their function to varying degrees. If extensive fibrosis and vitrescence occur after extensive injury within the organ, hardening of the organ results.

Systemic sclerosis (SSc), also known as scleroderma, is a systemic autoimmune disease characterized by localized or diffuse skin thickening and fibrosis. The pathological changes are characterized by skin fiber hyperplasia and skin-like changes of blood vessel onions, which finally cause skin sclerosis and blood vessel ischemia. This disease is clinically characterized by localized or diffuse skin thickening and fibrosis, which can affect the internal organs (heart, lung and digestive tract, etc.) in addition to skin involvement.

Atherosclerosis often results in ischemic injury to tissues and organs, which in turn causes fibrotic lesions in tissues and organs. Atherosclerosis is a chronic, progressive disease of the arteries, in which fat deposits in the arteries partially or completely block the blood flow. Atherosclerosis occurs when an otherwise smooth, firm intima of an artery becomes rough, thickened, and clogged with fat, fibrin, calcium, and cellular debris. Atherosclerosis is a chronic inflammatory proliferative lesion of the intima of arteries, resulting in stenosis or occlusion of large and medium-sized arterial lumens, causing ischemic injury, fibrosis and even necrosis of corresponding organ tissues.

Atherosclerosis is closely related to diabetes, and is manifested by early onset, high severity and poor prognosis of atherosclerosis, which is the leading cause of death in diabetic patients. Clinical findings show that pathological changes of coronary artery blood vessels of diabetic patients are mainly characterized by more blood vessels with affected lesions, severe coronary artery stenosis, more diffuse and severe lesions, and the mechanism of the pathological changes is mostly considered to be atherosclerosis caused by abnormal blood sugar metabolism, and with further intensive research, more results show that the atherosclerosis caused by diabetes is not caused by a single factor, but is induced and promoted by multiple pathways and more complex mechanisms, such as macrophage polarization, macrophage migration inhibitory factor pathway, glycosylation end product pathway, scavenger receptor up-regulation, insulin resistance, ubiquitin-proteasome system activation, platelet-derived growth factor activation pathway and the like^[4]。

At present, the patients with the fibrotic diseases caused by various reasons are numerous, a plurality of organ tissues of the patients are often affected, an effective treatment method is still lacked, and the social and economic burdens are heavier. The research of the invention finds that the plasminogen can improve the fibrosis of tissues and organs and improve the functions of the tissues and organs, thereby opening up a brand new field for the prevention and treatment of the fibrosis of the tissues and organs and related diseases.

Summary of The Invention

The invention includes the following:

1. a method of preventing or treating hepatic collagen deposition or fibrosis resulting from liver tissue damage in a subject, comprising administering to the subject an effective amount of plasminogen.

2. The method of item 1, wherein the injury comprises ischemia reperfusion injury, drug injury, immune injury, chemical injury, inflammatory injury, injury due to excessive alcohol consumption, injury due to cancer.

3. The method of item 2, wherein the injury comprises an injury caused by fat deposition.

4. A method of preventing or treating hepatic collagen deposition or fibrosis in a subject, comprising administering to the subject an effective amount of plasminogen.

5. A method of preventing or treating hepatic collagen deposition or fibrosis caused or complicated by diabetes in a subject, comprising administering to the subject an effective amount of plasminogen.

6. A method of preventing or treating hepatic collagen deposition or fibrosis caused or complicated by atherosclerosis in a subject, comprising administering to the subject an effective amount of plasminogen.

7. A method of preventing or treating liver collagen deposition or fibrosis caused by or complicated with hyperlipidemia in a subject, comprising administering to the subject an effective amount of plasminogen.

8. The method of item 7, wherein the hyperlipidemia comprises one or more selected from the group consisting of: elevated blood triglyceride levels, elevated blood total cholesterol levels, elevated blood low density lipoproteins, and elevated blood very low density lipoproteins.

9. A method of preventing or treating hepatic collagen deposition or fibrosis caused by a pharmaceutical liver injury in a subject, comprising administering to the subject an effective amount of plasminogen.

10. The method of item 9, wherein the drug is a hepatotoxic drug.

11. The method of item 10, wherein the drug comprises a chemotherapeutic drug, an antibiotic drug, a hypolipidemic drug, an antihypertensive drug, a hypoglycemic drug.

12. A method of preventing or treating alcoholic liver collagen deposition or fibrosis or non-alcoholic liver collagen deposition or fibrosis in a subject comprising administering to the subject an effective amount of plasminogen.

13. A method of preventing or treating hepatic collagen deposition or fibrosis resulting from chronic liver disease in a subject, comprising administering to the subject an effective amount of plasminogen.

14. A method of preventing or treating liver collagen deposition or fibrosis caused by viral hepatitis in a subject, comprising administering to the subject an effective amount of plasminogen.

15. A method of preventing or treating a liver fibrosis related disorder in a subject, comprising administering to the subject an effective amount of plasminogen.

16. The method of clause 15, wherein the liver fibrosis-associated disorder comprises a disorder arising from diminished, impaired, or lost liver function resulting from liver fibrosis.

17. The method according to any of items 1-16, wherein said plasminogen can be administered in combination with one or more other drugs or therapeutic means.

18. The method of item 17, wherein the other medications comprise: blood fat reducing medicine, antiplatelet medicine, blood pressure lowering medicine, vasodilating medicine, blood sugar lowering medicine, anticoagulant medicine, thrombolytic medicine, liver protecting medicine, anti-fibrosis medicine, arrhythmia medicine, heart strengthening medicine, diuretic medicine, antitumor medicine, radiotherapy and chemotherapy medicine, inflammation regulating medicine, immunity regulating medicine, antiviral medicine and antibiotic.

19. The method of any one of items 1-18, wherein the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2, 6, 8, 10 or 12 and still possesses plasminogen activity.

20. The method of any one of items 1 to 19, wherein the plasminogen is a protein which has 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 1 amino acid and still has plasminogen activity, and is added, deleted and/or substituted on the basis of the sequence 2, 6, 8, 10 or 12.

21. The method of any one of items 1-20, wherein the plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.

22. The method of any one of items 1 to 21, said plasminogen being selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen or a plasminogen activity retaining variant thereof.

23. The method of any one of items 1 to 22, wherein the plasminogen is native or synthetic human plasminogen, or a variant or fragment thereof which still retains plasminogen activity.

24. The method of any one of items 1-22, wherein the plasminogen is a human plasminogen ortholog from a primate or rodent, or a variant or fragment thereof that still retains plasminogen activity.

25. The method of any one of items 1 to 24, wherein the amino acid sequence of plasminogen is as shown in sequence 2, 6, 8, 10 or 12.

26. The method of any one of items 1-25, wherein the plasminogen is human native plasminogen.

27. The method of any one of items 1-26, wherein the subject is a human.

28. The method of any one of items 1-27, wherein the subject lacks or lacks plasminogen.

29. The method of any one of items 1-28, wherein the deficiency or deletion is congenital, secondary, and/or local.

30. A plasminogen for use in a method according to any of claims 1-29.

31. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and plasminogen for use in the method of any one of items 1-29.

32. A prophylactic or therapeutic kit comprising: (i) plasminogen for use in a method according to any of claims 1-29 and (ii) means for delivering said plasminogen to said subject.

33. The kit of item 32, wherein the component is a syringe or a vial.

34. The kit of item 32 or 33, further comprising a label or instructions for administering the plasminogen to the subject for performing the method of any one of items 1-29.

35. An article of manufacture, comprising:

a container containing a label; and

comprising (i) plasminogen or a pharmaceutical composition comprising plasminogen for use in a method according to any of claims 1 to 29, wherein the label indicates that the plasminogen or composition is to be administered to the subject for carrying out a method according to any of claims 1 to 29.

36. The kit of any one of claims 32-34 or the article of manufacture of claim 35, further comprising one or more additional components or containers comprising an additional pharmaceutical.

37. The kit or article of manufacture of item 36, wherein the additional agent is selected from the group consisting of: blood fat reducing medicine, antiplatelet medicine, blood pressure lowering medicine, vasodilating medicine, blood sugar lowering medicine, anticoagulant medicine, thrombolytic medicine, liver protecting medicine, anti-fibrosis medicine, arrhythmia medicine, heart strengthening medicine, diuretic medicine, antitumor medicine, radiotherapy and chemotherapy medicine, inflammation regulating medicine, immunity regulating medicine, antiviral medicine and antibiotic.

In one aspect, the invention relates to a method of preventing and/or treating tissue organ collagen deposition or fibrosis and disorders related thereto in a subject, comprising administering to the subject an effective amount of plasminogen, wherein the subject is susceptible to, predisposed to, or suffering from other diseases associated with tissue organ fibrosis. The present invention also relates to the use of plasminogen for the prevention and/or treatment of tissue organ collagen deposition or fibrosis and disorders related thereto in a subject. The invention also relates to the use of plasminogen for the preparation of a medicament for the prevention and/or treatment of tissue organ collagen deposition or fibrosis and disorders related thereto in a subject. In addition, the present invention relates to plasminogen for use in the prevention and/or treatment of tissue organ collagen deposition or fibrosis and disorders related thereto in a subject. In some embodiments, the tissue organ collagen deposition or fibrosis comprises skin fibrosis, vascular fibrosis, cardiac fibrosis, pulmonary fibrosis, liver fibrosis, kidney fibrosis. In other embodiments, the tissue organ collagen deposition or fibrosis is caused by or accompanied by infection, inflammation, hypersensitivity, tumor, tissue ischemia, tissue organ congestion, chemical substances, radiation, or damage caused by environmental contamination. Specifically, the tissue and organ collagen deposition or fibrosis comprises tissue and organ collagen deposition or fibrosis caused by tissue and organ lesion caused by bacterial, viral or parasitic infection, wherein the tissue and organ collagen deposition or fibrosis comprises pulmonary fibrosis caused by mycobacterium tuberculosis infection, liver fibrosis caused by hepatitis b virus, hepatitis c virus or hepatitis e virus infection, and liver fibrosis caused by schistosome infection. In some embodiments, the tissue organ collagen deposition or fibrosis is caused by a sterile inflammatory or autoimmune reaction. Specifically, the tissue organ collagen deposition or fibrosis is chronic glomerulonephritis, pyelonephritis, nephrotic syndrome, renal insufficiency, and kidney fibrosis caused by uremia. In other embodiments, the tissue organ collagen deposition or fibrosis is caused by cancer resulting in tissue organ damage. Specifically, the collagen deposition or fibrosis of the tissue and organ is pulmonary fibrosis caused by lung cancer, hepatic fibrosis caused by liver cancer or renal fibrosis caused by renal cancer. In other embodiments, the tissue organ collagen deposition or fibrosis is caused by chronic ischemic tissue injury. Specifically, the tissue organ collagen deposition or fibrosis is coronary atherosclerosis, cardiac ischemic fibrosis caused by coronary heart disease and/or renal fibrosis caused by chronic ischemic renal injury. In other embodiments, the tissue organ collagen deposition or fibrosis is caused by cardiovascular disease-induced tissue organ congestion. Specifically, the tissue organ collagen deposition or fibrosis is liver congestion or lung congestion. In some embodiments, the tissue organ collagen deposition or fibrosis is drug-induced. Specifically, the tissue organ collagen deposition or fibrosis is drug-induced liver fibrosis or kidney fibrosis. In some embodiments, the tissue organ collagen deposition or fibrosis is pulmonary fibrosis caused by inhaled chemicals or environmental pollutants. In the above embodiments, the tissue organ collagen deposition or fibrosis is caused by systemic lupus erythematosus, systemic sclerosis, ankylosing spondylitis, systemic immune diseases. In some embodiments, the tissue organ fibrosis is idiopathic pulmonary fibrosis.

In the above embodiments, the condition associated with fibrosis in a tissue or organ comprises a condition resulting from a reduction, disorder or loss of function of the tissue or organ due to fibrotic lesions. Specifically, the diseases related to tissue organ fibrosis include atherosclerosis, coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, cerebral ischemia, cerebral infarction, renal insufficiency, uremia, liver dysfunction, liver cirrhosis, hepatic coma, dyspnea, emphysema, pulmonary heart disease, pulmonary fibrosis, and ankylosing spondylitis.

In the above embodiments, the plasminogen can be used in combination with one or more other drugs or therapies. In particular, the plasminogen may be used in combination with one or more drugs selected from the group consisting of: blood fat reducing medicine, antiplatelet medicine, blood pressure lowering medicine, vasodilating medicine, blood sugar lowering medicine, anticoagulant medicine, thrombolytic medicine, liver protecting medicine, anti-fibrosis medicine, arrhythmia medicine, heart strengthening medicine, diuretic medicine, antitumor medicine, radiotherapy and chemotherapy medicine, inflammation regulating medicine, immunity regulating medicine, antiviral medicine and antibiotic.

In the above embodiments, the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2, 6, 8, 10 or 12 and still possesses plasminogen activity.

In the above embodiments, the amino acid of said plasminogen is as shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is a protein that adds, deletes and/or replaces 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 on the basis of sequence 2, 6, 8, 10 or 12 and still has plasminogen activity.

In the above embodiments, the plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity. In particular, the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen or variants thereof retaining plasminogen activity.

In the above embodiments, the plasminogen is native or synthetic human plasminogen, or a variant or fragment thereof which still retains plasminogen activity. In some embodiments, the plasminogen is a human plasminogen ortholog from a primate or rodent, or a variant or fragment thereof that still retains plasminogen activity. For example, plasminogen orthologs from primates or rodents, such as plasminogen orthologs from gorilla, rhesus, mouse, cow, horse, dog. Most preferably, the amino acid sequence of the plasminogen of the present invention is as shown in seq id No. 2, 6, 8, 10 or 12.

In the above embodiments, the subject is a human. In some embodiments, wherein the subject lacks or lacks plasminogen. In particular, the deficiency or deletion is congenital, secondary and/or local.

In one embodiment, the plasminogen is administered systemically or locally, preferably by the following route: topically, intravenously, intramuscularly, subcutaneously, inhalationally, intraspinally, topically, intraarticularly, or rectally. In one embodiment, the topical administration is directly to the area of osteoporosis, for example by means of a dressing, catheter or the like.

In one embodiment, the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer. In one embodiment, the plasminogen is present in an amount of 0.0001-2000mg/kg, 0.001-800 mg/kg, 0.01-600mg/kg, 0.1-400mg/kg, 1-200mg/kg, 1-100mg/kg, 10-100 mg/kg (calculated per kg body weight) or 0.0001-2000mg/cm per day²、0.001-800 mg/cm²、0.01-600mg/cm²、0.1-400mg/cm²、1-200mg/cm²、1-100 mg/cm²、10-100mg/cm²The dose (calculated per square centimeter of body surface area) is preferably administered at least once, preferably at least daily. In the case of topical administration, the above dosages may be further adjusted as appropriate. In one aspect, the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and plasminogen for use in the methods of the invention.

In another aspect, the invention relates to a prophylactic or therapeutic kit comprising: (i) plasminogen for use in the method of the invention and (ii) means (means) for delivering said plasminogen to said subject, in particular said means is a syringe or vial. In some embodiments, the kit further comprises a label or instructions for administering the plasminogen to the subject for performing the methods of the invention.

In another aspect, the present invention is also directed to an article comprising: a container containing a label; and (i) plasminogen or a pharmaceutical composition comprising plasminogen for use in the method of the invention, wherein the label indicates that the plasminogen or composition is to be administered to the subject in order to carry out the method of the invention.

In the above embodiments, the kit or article of manufacture further comprises one or more additional components or containers containing other drugs. In some embodiments, the additional agent is selected from the group consisting of: blood fat reducing medicine, antiplatelet medicine, blood pressure lowering medicine, vasodilating medicine, blood sugar lowering medicine, anticoagulant medicine, thrombolytic medicine, liver protecting medicine, anti-fibrosis medicine, arrhythmia medicine, heart strengthening medicine, diuretic medicine, antitumor medicine, radiotherapy and chemotherapy medicine, inflammation regulating medicine, immunity regulating medicine, antiviral medicine and antibiotic.

Definition of

The "fibrosis" is a lesion of tissues and organs such as lung, liver, kidney, blood vessel, peritoneum, pancreas and skin, after continuous injury caused by inflammation, infection, immune reaction, ischemia, chemical substances, radiation and other reasons, the fibroblast is activated and proliferated, fibrous connective tissues in tissues and organs are increased, parenchymal cells are reduced, and the structures and functions of the tissues and organs are damaged and lost. The term is used interchangeably with "fibrotic lesions". The term "fibrotic lesion" encompasses fibrotic lesions of tissues and organs such as cardiac fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, vascular fibrosis and dermal fibrosis due to various causes, and also encompasses fibrotic lesions of tissues and organs such as cardiac fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, vascular fibrosis and dermal fibrosis which are accompanied in the development and progression of various diseases.

After fibrosis of tissues and organs, the normal structure of the tissues and organs is changed, and the corresponding functions are weakened or lost, so that the related diseases are called as 'tissue and organ fibrosis related diseases'.

"cardiac fibrosis" refers to fibrotic lesions that occur during the development and progression of heart disease caused by or accompanied by damage to cardiac tissue due to various causes (e.g., inflammation, infection, immune response, ischemia, chemicals, radiation). The related diseases caused by the impairment of cardiac function caused by the cardiac fibrosis are called as 'cardiac fibrosis related diseases', and include but are not limited to symptoms and diseases of ischemia of each organ and tissue caused by the impairment of cardiac function, such as coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, cerebral ischemia, dyspnea, renal insufficiency and the like.

"hepatic fibrosis" refers to pathological changes (lesions) resulting from or accompanied by abnormal proliferation of connective tissue in the liver, excessive deposition of diffuse extracellular matrix in the liver, and destruction of normal structures of the liver, caused by various causes such as inflammation, infection (e.g., viral infection), immune response, ischemia, chemicals, radiation, oxidative stress, and alcohol abuse. Further progression of liver fibrosis to cirrhosis is also encompassed by the term "liver fibrosis" of the present invention. Liver fibrosis lesions result in impaired liver function, and the resulting related disorders are referred to as "liver fibrosis-related disorders".

"pulmonary fibrosis" refers to a pathological process resulting from or accompanied by proliferation of mesenchymal cells in lung tissue, proliferative deposition of extracellular matrix, and remodeling of lung parenchyma due to various causes (e.g., inflammation, infection, immune response, ischemia, chemicals, radiation). Pulmonary fibrosis lesions result in impaired lung function, and the associated disorder resulting therefrom is referred to as a "pulmonary fibrosis-associated disorder".

"renal fibrosis" refers to a pathological process in which abnormal accumulation of connective tissue in the kidney, resulting from or accompanied by various causes (e.g., inflammation, infection, immune response, ischemia, chemicals, radiation), leads to structural changes and impaired function of the kidney. Renal fibrotic lesions are a common pathway for the progression of almost all renal diseases to a later stage.

Renal fibrotic lesions lead to impaired renal function, and the resulting related disorders are referred to as "renal fibrosis-related disorders", for example, renal insufficiency, renal failure, uremia, and the like.

Chronic lesions of the tissue and organs are often accompanied by fibrosis, for example chronic inflammation of the lung, chronic lesions with pulmonary fibrosis. The same is true of liver fibrosis, such as hepatitis B, hepatitis C, alcoholic liver, fatty liver, schistosomiasis, etc., all with early stage liver fibrosis. The chronic nephritis, glomerulonephritis, tubular nephritis and other diseases have renal fibrosis; cardiovascular, cerebrovascular, and lower limb vascular sclerosis, or narrowing, or obstruction, all have vascular fibrosis. The term "fibrosis" or "fibrotic lesion" of the present invention encompasses fibrotic lesions that are often accompanied by chronic lesions of various tissues and organs of the body.

Systemic sclerosis, or scleroderma, is a systemic autoimmune disease characterized by localized or diffuse skin thickening and fibrosis. The pathological changes are characterized by skin fiber hyperplasia and skin-like changes of blood vessel onions, which finally cause skin sclerosis and blood vessel ischemia. This disease is clinically characterized by localized or diffuse skin thickening and fibrosis, which can affect the internal organs (heart, lung and digestive tract, etc.) in addition to skin involvement.

"atherosclerosis" is a chronic, progressive disease of the arteries, in which deposited fat partially or completely blocks blood flow. Atherosclerosis is a progressive process. When the concentration of lipids in the blood increases greatly, fatty streaks form along the arterial wall. These streaks can lead to fat and cholesterol deposits that adhere to the otherwise smooth intima of the artery, forming nodules. Below these nodules, fibrotic scar tissue ensues, leading to calcium deposits. The deposited calcium gradually evolved into a chalky hard film (called atheroma) that could not be removed. When an artery connected to a certain tissue or organ in the body is blocked, the ischemic damage of the tissue or organ due to the blockage of the artery in the tissue or organ may cause fibrotic lesions of the tissue or organ, such as fibrosis of the heart, lung, liver, kidney, blood vessel, peritoneum, pancreas, and skin.

With further intensive research, more results show that the atherosclerosis caused by diabetes is not caused by a single factor, but is induced and promoted by multiple ways and more complex mechanisms^[4]. Diabetes and its associated atherosclerosis can lead to tissue, organ damage and fibrosis, such as fibrosis of tissues and organs of the heart, lung, liver, kidney, blood vessels, peritoneum, pancreas, skin, etc.

Detailed Description

Plasmin is a key component of the plasminogen activation system (PA system). It is a broad spectrum protease capable of hydrolyzing several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin and proteoglycans^[5]. In addition, plasmin can activate some metalloprotease precursors (pro-MMPs) to form active metalloproteases (MMPs). Plasmin is therefore considered an important upstream regulator of extracellular proteolysis^[6,7]Plasmin is formed from plasminogen by proteolysis of two physiological PAs, tissue plasminogen activator (tPA) or urokinase plasminogen activator (uPA). since plasminogen is present at relatively high levels in plasma and other body fluids, regulation of the PA system has traditionally been thought to be primarily achieved by the synthesis and activity levels of PAs.the synthesis of components of the PA system is tightly regulated by different factors, such as hormones, growth factors and cytokines.furthermore, specific physiological inhibitors of plasmin and PAs also exist.the primary inhibitor of plasmin is α -antiplasmin (α -antiplasmin). the activity of PAs is simultaneously inhibited by plasminogen activator inhibitor-1 of uPA and tPA(PAI-1) inhibition and regulation of plasminogen activator inhibitor-2 (PAI-2) which primarily inhibits uPA. Certain uPA-specific cell surface receptors (uPAR) with direct hydrolytic activity on the cell surface^[8,9]。

Plasminogen is a single-chain glycoprotein consisting of 791 amino acids and having a molecular weight of about 92 kDa^[10,11]. Plasminogen is synthesized primarily in the liver and is present in large amounts in the extracellular fluid. The plasminogen content in plasma is about 2 μ M. Plasminogen is therefore a large potential source of proteolytic activity in tissues and fluids^[12,^13]. Plasminogen exists in two molecular forms: glutamic acid-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys-plasminogen). Native secreted and uncleaved forms of plasminogen have one amino-terminal (N-terminal) glutamate and are therefore referred to as glutamate-plasminogen. However, in the presence of plasmin, glutamate-plasminogen is hydrolyzed to lysine-plasminogen at Lys76-Lys 77. Lysine-plasminogen has a higher affinity for fibrin and can be activated by PAs at a higher rate than glutamate-plasminogen. The Arg560-Val561 peptide bond of these two forms of plasminogen can be cleaved by uPA or tPA, resulting in the formation of the disulfide-linked, two-chain protease plasmin^[14]Some kringles contain lysine binding sites that mediate the specific interaction of plasminogen with fibrin and its inhibitor α 2-AP a 38kDa fragment of plasminogen, including kringles1-4, has recently been found to be a potent inhibitor of angiogenesis.

The main substrate of plasmin is fibrin, and the dissolution of fibrin is key to prevent pathological thrombosis^[15]. Plasmin also has substrate specificity for several components of the ECM, including laminin, fibronectin, proteoglycans, and gelatin, suggesting that plasmin also plays an important role in ECM remodeling^[11,16,17]. Indirectly, plasmin can also degrade other components of the ECM, including MMP-1, MMP-2, MMP-3, and MMP-9, by converting certain protease precursors to active proteases. Thus, it has been suggested that plasmin may be an important upstream regulator of extracellular proteolysis^[18]. In addition, plasmin has the ability to activate certain potential forms of growth factors^[19-21]. In vitro, plasmin also hydrolyzes components of the complement system and releases chemotactic complement fragments.

"plasmin" is a very important enzyme present in the blood that hydrolyzes fibrin clots into fibrin degradation products and D-dimers.

"plasminogen" is a zymogen form of plasmin, consisting of 810 amino acids, having a molecular weight of about 90kD, calculated from the sequence in swiss prot as the amino acid sequence of native human plasminogen containing a signal peptide (SEQ ID NO: 4), a glycoprotein synthesized predominantly in the liver and capable of circulating in the blood, and the cDNA sequence encoding this amino acid sequence is shown in SEQ ID NO: 3. Full-length plasminogen contains seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAP) domain at the N-terminus, and 5 Kringle domains (Kringle 1-5). Referring to the sequence in swiss prot, the signal peptide includes residues Met1-Gly19, PAP includes residues Glu20-Val98, Kringle1 includes residues Cys103-Cys181, Kringle2 includes residues Glu184-Cys262, Kringle3 includes residues Cys275-Cys352, Kringle4 includes residues Cys377-Cys454, and Kringle5 includes residues Cys481-Cys 560. According to NCBI data, the serine protease domain includes residues Val 581-Arg 804.

Glu-plasminogen is native full-length plasminogen, and consists of 791 amino acids (does not contain a signal peptide of 19 amino acids), and the cDNA sequence encoding this sequence is shown as sequence 1, and the amino acid sequence is shown as sequence 2. In vivo, there is also Lys-plasminogen which is formed by hydrolysis from amino acids 76-77 of Glu-plasminogen as shown in SEQ ID No. 6, and cDNA sequence encoding the amino acid sequence as shown in SEQ ID No. 5. Delta-plasminogen (Delta-plasminogen) is a full-length plasminogen-depleted Kringle2-Kringle5 fragment containing only Kringle1 and serine protease domain^[22,23]The amino acid sequence of delta-plasminogen (SEQ ID NO: 8) has been reported^[23]The cDNA sequence encoding the amino acid sequence is shown as sequence 7. Small plasminogen (Mini-plasminogen) is composed of Kringle5 and a serine protease domain, and it has been reported that it includes residues Val443-Asn791 (starting with the Glu residue of the Glu-plasminogen sequence which does not contain a signal peptide)^[24]The amino acid sequence is shown as sequence 10, and the cDNA sequence for coding the amino acid sequence is shown as sequence 9. On the other hand, microplasminogen (Micro-plasminogen) contains only serine protease domain, and its amino acid sequence has been reported to include residues Ala543-Asn791 (starting with Glu residue of Glu-plasminogen sequence not containing signal peptide)^[25]Patent document CN102154253A also reports that the sequence includes residues Lys531-Asn791 (the Glu residue of the Glu-plasminogen sequence not containing a signal peptide is used as the starting amino acid), and the patent sequence refers to patent document CN102154253A, the amino acid sequence of which is shown as sequence 12, and the cDNA sequence encoding the amino acid sequence is shown as sequence 11.

The plasmin and the plasmin can be used interchangeably and have the same meaning; "plasminogen" is used interchangeably with "plasminogen" and is synonymous.

In the present application, the plasminogen "deficient" means that the plasminogen content or activity in a subject is lower than in a normal human, low enough to affect the normal physiological function of the subject; the plasminogen is "deficient" in the sense that the content or activity of plasminogen in a subject is significantly lower than that of a normal human, even the activity or expression is minimal, and normal physiological function can be maintained only by external supply.

It will be appreciated by those skilled in the art that all of the solutions for plasminogen of the present invention are applicable to plasmin, and thus the solutions described herein encompass both plasminogen and plasmin.

During circulation, plasminogen adopts a closed inactive conformation, but when bound to a thrombus or cell surface, it is converted to active plasmin in an open conformation, mediated by Plasminogen Activator (PA). Active plasmin can further hydrolyze fibrin clots into fibrin degradation products and D-dimers, thereby dissolving thrombus. Wherein the PAp domain of plasminogen contains important determinants for maintaining plasminogen in an inactive closed conformation, and the KR domain is capable of binding to lysine residues present on receptors and substrates. A variety of enzymes are known that are capable of acting as plasminogen activators, including: tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hageman factor), and the like.

"plasminogen active fragment" refers to an active fragment of a plasminogen protein that is capable of binding to a target sequence in a substrate and performing proteolytic functions. The present invention relates to the technical scheme of plasminogen, and the technical scheme of replacing plasminogen by plasminogen active fragments is covered. The plasminogen active fragment is a protein containing serine protease domain of plasminogen, preferably, the plasminogen active fragment contains sequence 14 and a protein with an amino acid sequence which has at least 80%, 90%, 95%, 96%, 97%, 98% and 99% homology with the sequence 14. Thus, the plasminogen of the present invention includes proteins that contain the plasminogen active fragment and still retain the plasminogen activity.

Currently, methods for assaying plasminogen and its activity in blood include: the assay for tissue plasminogen activator activity (t-PAA), the assay for plasma tissue plasminogen activator antigen (t-PAAg), the assay for plasma tissue plasminogen activity (plgA), the assay for plasma tissue plasminogen antigen (plgAg), the assay for plasma tissue plasminogen activator inhibitor activity, the assay for plasma tissue plasminogen activator inhibitor antigen (pPAP), and the assay for plasma plasmin-antiplasmin complex (PAP). Among the most commonly used detection methods are chromogenic substrate methods: streptokinase (SK) and chromogenic substrate are added to the tested plasma, the PLG in the tested plasma is converted into PLM under the action of SK, the latter acts on the chromogenic substrate, and then the absorbance is increased in direct proportion to the plasminogen activity measured by a spectrophotometer. In addition, the plasminogen activity in blood can also be measured by immunochemistry, gel electrophoresis, immunoturbidimetry, radioimmunodiffusion, and the like.

"orthologues or orthologs" refers to homologues between different species, including both protein homologues and DNA homologues, also referred to as orthologs, orthologs. It specifically refers to proteins or genes evolved from the same ancestral gene in different species. The plasminogen of the invention includes native human plasminogen, as well as plasminogen orthologs or orthologs derived from different species having plasminogen activity.

"conservative substitution variants" refer to variants in which a given amino acid residue is changed without changing the overall conformation or function of the protein or enzyme, and include, but are not limited to, substitution of amino acids in the amino acid sequence of a parent protein with amino acids of similar characteristics (e.g., acidic, basic, hydrophobic, etc.). Amino acids with similar properties are well known. For example, arginine, histidine and lysine are hydrophilic basic amino acids and may be interchanged. Likewise, isoleucine is a hydrophobic amino acid and may be replaced by leucine, methionine or valine. Thus, the similarity of two proteins or amino acid sequences of similar function may differ. For example, 70% to 99% similarity (identity) based on the MEGALIGN algorithm. "conservatively substituted variants" also includes polypeptides or enzymes having greater than 60% amino acid identity, preferably greater than 75%, more preferably greater than 85%, and even greater than 90% as determined by the BLAST or FASTA algorithms, and having the same or substantially similar properties or functions as the native or parent protein or enzyme.

By "isolated" plasminogen is meant plasminogen protein that is isolated and/or recovered from its natural environment. In some embodiments, the plasminogen is purified (1) to a purity (by weight) of greater than 90%, greater than 95%, or greater than 98%, as determined by the Lowry method, e.g., greater than 99% (by weight), (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer, or (3) to homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using coomassie blue or silver stain under reducing or non-reducing conditions. Isolated plasminogen also includes plasminogen that has been prepared from recombinant cells by bioengineering techniques and isolated by at least one purification step.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences, fusions with heterologous and homologous leader sequences (with or without an N-terminal methionine residue); and so on.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence, after introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill in 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 necessary to achieve maximum alignment over the full length of the sequences being compared. However, for the purposes of the present invention, percent amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.

In the case where ALIGN-2 is used to compare amino acid sequences, the% amino acid sequence identity of a given amino acid sequence a relative to a given amino acid sequence B (or a given amino acid sequence a that can be expressed as having or comprising some% amino acid sequence identity relative to, with, or for a given amino acid sequence B) is calculated as follows:

fractional X/Y times 100

Wherein X is the number of amino acid residues scored as identical matches in the A and B alignments of the sequence alignment program by the program ALIGN-2, 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. Unless otherwise specifically indicated, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

As used herein, the terms "treat" and "treating" refer to obtaining a desired pharmacological and/or physiological effect. The effect may be a complete or partial prevention of the disease or symptoms thereof, and/or a partial or complete cure of the disease and/or symptoms thereof, and includes: (a) preventing the occurrence of a disease in a subject, which may have a predisposition to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e. blocking its formation; and (c) alleviating the disease and/or symptoms thereof, i.e., causing regression of the disease and/or symptoms thereof.

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, an ungulate (e.g., horse, cow, sheep, pig, goat), and the like.

"therapeutically effective amount" or "effective amount" refers to an amount of plasminogen that is 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 in the subject to be treated, as well as the age, weight, etc.

Preparation of plasminogen in accordance with the invention

Plasminogen can be isolated from nature and purified for further therapeutic use, and can also be synthesized by standard chemical peptide synthesis techniques. When the polypeptide is synthesized chemically, the synthesis may be carried out via a liquid phase or a solid phase. Solid Phase Polypeptide Synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence, is a suitable method for plasminogen chemical synthesis. Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize plasminogen. Techniques for Solid Phase Synthesis are described in Barany and Solid-Phase Peptide Synthesis; pages 3-284 from The Peptides: Analysis, Synthesis, biology, Vol.2: special Methods in peptide Synthesis, Part A., Merrifield, et al J.am.chem.Soc.,85: 2149-; stewart et al, Solid Phase Peptide Synthesis,2nd ed.Pierce chem.Co., Rockford, Ill. (1984); and Ganesan A.2006Mini Rev. Med chem.6:3-10 and Camarero JA et al 2005Protein PeptLett.12: 723-8. Briefly, small insoluble porous beads are treated with a functional unit on which peptide chains are constructed. After repeated cycles of coupling/deprotection, the attached solid phase free N-terminal amine is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine that can be attached to another amino acid. The peptide remains immobilized on the solid phase, after which it is cleaved off.

The plasminogen of the present invention can be produced using standard recombinant methods. For example, a nucleic acid encoding plasminogen is inserted into an expression vector, operably linked to regulatory sequences in the expression vector. Expression control sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Expression control may be a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell (e.g., a COS or CHO cell). 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 in the host organism as episomes or as an integral part of the host chromosomal DNA. Typically, expression vectors contain selectable markers (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate detection of those cells that have been exogenously transformed with the desired DNA sequence.

Coli (Escherichia coli) is an example of a prokaryotic host cell that may be used to clone the subject antibody-encoding polynucleotides. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other Enterobacteriaceae (Enterobacteriaceae), such as Salmonella (Salmonella), Serratia (Serratia), and various Pseudomonas species. In these prokaryotic hosts, expression vectors may also be produced, which will typically contain expression control sequences (e.g., origins of replication) that are compatible with the host cell. In addition, there will be many well known promoters such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or a promoter system from bacteriophage lambda. Promoters will generally control expression, optionally in the case of operator sequences, and have ribosome binding site sequences and the like to initiate and complete transcription and translation.

Other microorganisms, such as yeast, may also be used for expression. Yeast (e.g., saccharomyces cerevisiae (s. cerevisiae)) and Pichia (Pichia) are examples of suitable yeast host cells, wherein suitable vectors have expression control sequences (e.g., promoters), origins of replication, termination sequences, and the like, as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeasts start from promoters which include, inter alia, those from alcohol dehydrogenases, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

In addition to microorganisms, mammalian cells (e.g., mammalian cells cultured in an in vitro cell culture) can also be used to express and produce an anti-Tau antibody of the invention (e.g., a polynucleotide encoding a subject anti-Tau antibody). See Winnacker, From Genes to Clones, VCH Publishers, n.y., n.y. (1987). 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.89:49(1986)), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from the immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al, J.Immunol.148:1149 (1992).

Once synthesized (chemically or recombinantly), the plasminogen described herein can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, High Performance Liquid Chromatography (HPLC), gel electrophoresis, and the like. The plasminogen is substantially pure, e.g., 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, e.g., free of contaminants, such as cellular debris, macromolecules other than the subject antibody, and the like.

Pharmaceutical formulations

Therapeutic formulations can be prepared by mixing plasminogen having the desired purity with an optional Pharmaceutical carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences,16 th edition, Osol, a. ed. (1980)) to form a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients, stabilizers, are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, fucose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and/or a non-ionic surfactant, such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). A preferred lyophilized anti-VEGF antibody formulation is described in WO 97/04801, which is incorporated herein by reference.

The formulations of the invention may also contain more than one active compound as required for the particular condition to be treated, preferably those with complementary activities and without side effects on each other. For example, antihypertensive drugs, antiarrhythmic drugs, drugs for treating diabetes, etc.

The plasminogen of the present invention can be encapsulated in microcapsules prepared by techniques such as coacervation or interfacial polymerization, for example, hydroxymethylcellulose or gel-microcapsules and poly- (methylmethacylate) microcapsules that can be placed in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980).

The plasminogen of the present invention for in vivo administration must be sterile. This can be readily achieved by filtration through sterile filtration membranes before or after lyophilization and reconstitution.

The plasminogen of the invention can be prepared into sustained release preparation. Suitable examples of sustained release formulations include shaped, glycoprotein-containing, solid hydrophobic polymer semi-permeable matrices, such as membranes or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (such as 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. Pat. No. 3773919, EP 58,481), copolymers of L-glutamic acid with gamma ethyl-L-glutamic acid (Sidman, et al, Biopolymers 22:547(1983)), non-degradable ethylene-vinyl acetate (Langer, et al, supra), or degradable lactic acid-glycolic acid copolymers such as Lupron DepotTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly D- (-) -3-hydroxybutyric acid polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid release molecules over 100 days, while some hydrogels release proteins for a shorter period of time. Rational strategies for protein stabilization can be designed based on the relevant mechanisms. For example, if the mechanism of aggregation is found to be intermolecular S — S bond formation through thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling humidity, employing appropriate additives, and developing specific polymer matrix compositions.

Administration and dosage

Administration of the pharmaceutical compositions of the present invention can be accomplished in different ways, e.g., intravenously, intraperitoneally, subcutaneously, intracranially, intrathecally, intraarterially (e.g., via carotid artery), intramuscularly.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of 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 buffered media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include 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.

Medical personnel will determine dosage regimens based on various clinical factors. As is well known in the medical arts, the dosage for any one patient depends on a variety of factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, number and route of administration, general health, and other drugs being administered concurrently. The dosage range of the plasminogen-containing pharmaceutical composition of the invention may be, for example, about 0.0001 to 2000mg/kg, or about 0.001 to 500mg/kg (e.g., 0.02mg/kg, 0.25 mg/kg, 0.5mg/kg, 0.75mg/kg, 10mg/kg, 50mg/kg, etc.) of the subject's body weight per day. For example, the dose may be 1mg/kg body weight or 50mg/kg body weight or in the range 1-50mg/kg, or at least 1 mg/kg. Doses above or below this exemplary range are also contemplated, particularly in view of the above factors. Intermediate doses within the above ranges are also included within the scope of the present invention. The subject may administer such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis. An exemplary dosage schedule includes 1-10mg/kg for several consecutive days. Real-time assessment of treatment efficacy and safety is required during administration of the drug of the present invention.

Articles of manufacture or kits

One embodiment of the present invention relates to a product or kit comprising plasminogen or plasmin of the invention useful in the treatment of cardiovascular diseases and conditions associated therewith resulting from diabetes. The article preferably comprises a container, label or package insert. Suitable containers are bottles, vials, syringes, etc. The container may be made of various materials such as glass or plastic. The container contains a composition that is effective in treating the disease or condition of the invention and has a sterile access port (e.g., the container may be an intravenous solution bag or vial containing a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is plasminogen/plasmin. The label on or attached to the container indicates that the composition is useful for treating cardiovascular disease and conditions related thereto caused by diabetes as described herein. 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 desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes. In addition, the article of manufacture comprises a package insert with instructions for use, including, for example, instructing a user of the composition to administer the plasminogen composition to a patient, as well as other drugs to treat the accompanying disease.

Brief Description of Drawings

FIG. 1 results of liver sirius red staining observation of mouse liver in 28-day carbon tetrachloride-administered plasminogen model. A is blank control group, B is vehicle-giving PBS control group, C is plasminogen-giving group, and D is quantitative analysis result. The results showed significantly less collagen deposition (arrow) to the plasminogen group than to the vehicle PBS control group, and statistically significant differences (P < 0.05); the level of collagen deposition was closer to that of the placebo mice when the vehicle-administered PBS control group was administered. Thus indicating that the plasminogen can reduce the deposition of liver collagen of a liver fibrosis model mouse and improve liver fibrosis.

Fig. 2 ApoE atherosclerosis model mice are representative pictures of sirius red staining of the aortic sinus 30 days after administration of plasminogen. A. C is the vehicle-administered PBS control group, B, D is the plasminogen-administered group. The results show that the area of collagen deposition (arrow) given to the plasminogen group is significantly smaller than that given to the vehicle PBS control group, indicating that plasminogen can reduce aortic sinus fibrosis levels in the atherosclerosis model mice.

FIG. 3 is a representative graph of carbon tetrachloride-induced liver fibrosis in mice model after 14 days of administration of plasminogen. A is blank control group, B is vehicle-giving PBS control group, and C is plasminogen-giving group. The results show that collagen deposition to the plasminogen group is significantly less than to the vehicle PBS control group, and is close to the collagen deposition level of the placebo mouse. Thus indicating that the plasminogen can reduce the deposition of liver collagen and improve the liver fibrosis of the liver fibrosis model mouse.

Figure 416 week hyperlipemia model mice given plasminogen 30 days later aortic sinus sirius red staining representative picture. A. C is the vehicle-administered PBS control group, B, D is the plasminogen-administered group. The results showed that the area of collagen deposition (indicated by arrows) on the intimal wall of aortic sinus in the plasminogen group was significantly smaller than that in the vehicle-administered PBS control group, indicating that plasminogen was able to reduce the level of intimal fibrosis in the aortic sinus wall in the hyperlipidemia model mice.

FIG. 5A representative picture of sirius red staining of skin after 21 days of administration of plasminogen to bleomycin-induced systemic sclerosis model mice. A is a blank control group, B is a vehicle-giving PBS control group, C is a plasminogen-giving group, and D is a PLG activity-impaired group. The results show that in the bleomycin-induced systemic sclerosis mouse model, the collagen fiber bundles on the upper part of the dermis are obviously increased for the solvent PBS group and the PLG activity-damaged group, the collagen fibers are thick and thick, the arrangement is compact, and the dermis layer is thickened; fibroblasts in the dermis of the plasminogen group are obviously less than those in the PBS group, and the thickness of the dermis of the skin is close to the normal level.

Fig. 6a representative picture of sirius red staining of lungs after 21 days of administration of plasminogen to bleomycin-induced systemic sclerosis model mice. A is given vehicle PBS control group, B is given plasminogen group, C is quantitative analysis result. The results show that in the bleomycin-induced systemic sclerosis mouse model, mice were given pulmonary fibrosis (indicated by arrows) to a greater extent in the vehicle PBS group than in the plasminogen group; the lung alveolar wall morphology of mice in the plasminogen group is close to normal, cells at the level of inflammation are obviously reduced, the fibrosis degree is obviously lower than that in the vehicle-fed PBS group, and the statistical difference is obvious (P is expressed as < 0.05).

Figure 7 bleomycin-induced systemic sclerosis model mice are given representative pictures of sirius red staining of the heart after 21 days of plasminogen administration. A is given vehicle PBS control group, B is given plasminogen group. It was found that in the bleomycin-induced systemic sclerosis mouse model, the degree of cardiac collagen deposition (indicated by the arrow) was higher for the vehicle PBS control group than for the plasminogen group. Thus showing that the plasminogen can effectively reduce cardiac fibrosis induced by bleomycin.

Figure 8 bleomycin-induced systemic sclerosis model mice are given representative pictures of sirius red staining of the kidney 21 days after plasminogen administration. A is given vehicle PBS control group, B is given plasminogen group. The results showed that the degree of renal collagen fibrosis (indicated by the arrow) was higher in the bleomycin-induced systemic sclerosis mouse model in the vehicle PBS control group than in the plasminogen group. Thus showing that the plasminogen can effectively reduce the renal fibrosis induced by bleomycin.

FIG. 924-25 weeks old diabetic mice kidney type IV collagen immunostaining observation results after 31 days of plasminogen administration. A is given vehicle PBS control group, B is given plasminogen group. The results show that positive collagen staining (arrow) was significantly greater for plasminogen group IV than for vehicle PBS control, indicating that plasminogen can improve fibrosis of the kidney in diabetic mice.

FIG. 1026 week-old diabetic mice were observed by staining with kidney massson 35 days after administration of plasminogen. A is given vehicle PBS control group, B is given plasminogen group. The results showed that, given vehicle PBS control, mesangial hyperplasia, increased mesangial matrix, mild fibrosis of the renal interstitium (arrow), and blue fibrosis of the hyperplasia. The number of glomerular mesangial cells and stroma in the plasminogen group is obviously less than that in the control group, and the renal interstitial fibrosis is obviously reduced. Indicating that plasminogen can improve fibrotic lesions of the kidney of diabetic mice.

FIG. 1124-25 weeks old diabetic mice were visualized by cardiac massson staining 31 days after administration of plasminogen. A is given vehicle PBS control group, B is given plasminogen group. The results showed that blue-colored hyperplastic collagen fibers (arrows) were visible between the myocardial fibers in the vehicle PBS control group, with mild myocardial fibrosis; a small amount of bluish hyperplastic collagen fibers were observed between the myofibers in the plasminogen group, and the myofibers were significantly reduced compared to the control group. Indicating that plasminogen can improve the fibrosis of the heart in diabetic mice.

FIG. 1217-18 weeks old diabetic mice are representative pictures of sirius red staining of the heart 35 days after administration of plasminogen. A is given vehicle PBS control group, B is given plasminogen group. The results showed that the deposition of collagen fibers (indicated by arrows) was significantly less in mice given plasminogen than in the vehicle-given PBS control group. Indicating that plasminogen is capable of reducing cardiac fibrosis in relatively young (17-18 weeks old) diabetic mice.

FIG. 1326-27 week old diabetic mice are representative pictures of sirius red staining of the heart 35 days after administration of plasminogen. A is given vehicle PBS control group, B is given plasminogen group. The results showed that the collagen deposition (arrow) was significantly less in mice given plasminogen than in the vehicle-given PBS control group. Indicating that plasminogen can attenuate cardiac fibrosis in relatively old (26-27 weeks old) diabetic mice.

FIG. 14 Immunochromatization of Kidney type IV collagen for observation after 7 days of administration of plasminogen to cisplatin renal fibrosis model mice. A is given vehicle PBS control group, B is given plasminogen group. The results showed that the kidney collagen type IV positive expression (arrow) was significantly higher in the vehicle-administered PBS control group than in the plasminogen-administered group. The plasminogen can improve the kidney fibrosis of the cisplatin kidney fibrosis model mouse.

Fig. 15 representative pictures of sirius red staining of the heart 30 days after administration of plasminogen to ApoE atherosclerotic model mice. A is given vehicle PBS control group, B is given plasminogen group. The results show that collagen deposition (arrow) was significantly less for the plasminogen group than for the vehicle-PBS control group, indicating that plasminogen can reduce ApoE atherosclerotic model mouse cardiac fibrosis.

Fig. 16C 57 representative pictures of sirius red staining of the heart 30 days after administration of plasminogen to the hyperlipidemic model mice. A is given vehicle PBS control group, B is given plasminogen group. The results show that collagen deposition (arrow) was significantly less for the plasminogen group than for the vehicle PBS control group, indicating that plasminogen can reduce cardiac fibrosis in the hyperlipidemic model mouse.

FIG. 17 purine-induced chronic kidney injury model mice were observed for 10 days post-administration of plasminogen with sirius red staining of the kidney. A is a vehicle-given PBS control group, B is a plasminogen-given group, C is a PLG activity-impaired group, and D is a quantitative analysis result. Collagen deposition (arrow) was significantly less for the plasminogen group than for the vehicle PBS control group and the PLG activity impaired group, and statistical differences were statistically significant for the plasminogen group versus the PLG activity impaired group (P < 0.05). Indicating that the plasminogen can relieve the kidney fibrosis caused by chronic kidney injury and promote the repair of the kidney injury.

FIG. 1824-25 weeks old diabetic mice were observed with Langerhans' Langerhans red staining after 31 days administration of plasminogen. A is given vehicle PBS control group, B is given plasminogen group, C is quantitative analysis result. The results showed that islet collagen deposition (arrow) was significantly less in mice given plasminogen than in the vehicle-given PBS control group, and the statistical difference was significant (. + -. denotes P < 0.05). It is indicated that plasminogen can improve islet damage and fibrosis caused by diabetes.

FIG. 19 shows the results of the observation of sirius red staining of kidney of mice model of 3% cholesterol hyperlipidemia 30 days after administration of plasminogen. A is blank control group, B is vehicle-giving PBS control group, C is plasminogen-giving group, and D is quantitative analysis result. The results show that the collagen deposition (marked by an arrow) of the kidney is obviously less for the plasminogen group than for the vehicle PBS control group, and the statistical difference is obvious; the plasminogen group fibrosis was restored to substantially normal levels. Thus showing that the plasminogen can effectively reduce the kidney fibrosis of the mouse model of hyperlipemia by 3 percent of cholesterol.

Examples

Example 1 plasminogen reduction of carbon tetrachloride-induced liver fibrosis liver collagen deposition

20C 57 female mice 7-8 weeks old were randomly divided into three groups, 5 mice in the blank control group, 7 mice in the vehicle-added PBS control group and plasmin8 of the original group. Injecting carbon tetrachloride into abdominal cavity of mice in the solvent PBS control group and plasminogen group according to the weight of 1mL/kg, three times per week, and continuously injecting for four weeks to establish hepatic fibrosis model^[36,37]Blank control mice were injected intraperitoneally with corn oil at the corresponding volume. The carbon tetrachloride needs to be diluted by corn oil, and the dilution ratio of the carbon tetrachloride to the corn oil is 1: 3. The injection is started on the day of modeling, which is recorded as day 1, 1mg/0.1 mL/day of human plasminogen is injected into the tail vein of a plasminogen group mouse, PBS with the same volume is injected into the tail vein of a vehicle PBS control group, and the blank control group is not injected and is continuously administered for 28 days. Mice were sacrificed on day 29 and livers fixed in 4% paraformaldehyde for 24 hours. The fixed liver is subjected to alcohol gradient dehydration and xylene transparence and then paraffin embedding. The thickness of the tissue section is 3 μm, the section is washed with water 1 time after being dewaxed to water, and after being stained with 0.1% sirius red for 60 minutes, the section is washed with running water, stained with hematoxylin for 1 minute, washed with running water, differentiated by 1% hydrochloric acid alcohol and ammonia water and turned to blue, washed with running water, dried and then sealed, and the section is observed under an optical microscope of 200 times.

The results showed that collagen deposition was significantly less for the plasminogen group (fig. 1C) than for the vehicle PBS control group (fig. 1B) and the statistical difference was significant (fig. 1D); levels of collagen deposition (indicated by arrows) were more similar to those of the vehicle-administered PBS control group than those of the vehicle-administered PBS control group. Thus indicating that the plasminogen can reduce the deposition of liver collagen of a liver fibrosis model mouse and improve liver fibrosis.

Example 2 plasminogen amelioration of ApoE atherosclerotic mouse aortic sinus fibrosis

13 male ApoE mice at 6 weeks of age were fed high-fat high-cholesterol diet (southeast troffet, TP2031) for 16 weeks to induce an atherosclerosis model^[31,32]. Mice after molding were bled 50 μ l each three days before the administration to detect total cholesterol (T-CHO) content, and were randomly divided into two groups according to T-CHO content, 7 mice were given to vehicle PBS control group, and 6 mice were given to plasminogen group. Initial dosing was defined as day 1, plasminogen activator was administeredIntravenous injection of human plasminogen at 1mg/0.1 mL/day, and tail vein injection of PBS in the same volume in the vehicle PBS control group. The drug is administered for 30 days, mice are sacrificed on the 31 st day, hearts are taken and fixed in 4% paraformaldehyde for 24-48 hours, the hearts are respectively settled in 15% sucrose and 30% sucrose overnight at 4 ℃, OCT embedding is carried out, the thickness of frozen sections is 8 mu m, the hearts are stained with 0.1% sirius red saturated picric acid for 30 minutes, then the sections are washed for 2 minutes by running water, hematoxylin is stained for 1 minute, the sections are washed by running water, 1% hydrochloric acid and alcohol are differentiated, ammonia water is returned to blue, the sections are washed by running water, neutral gum is sealed after being dried, and the sections are observed under 40(2A, 2B) and 200 times (2C, 2D) optical microscopes.

The results showed that the area of intimal collagen deposition (arrow) on the aortic sinus wall was significantly smaller for the plasminogen group (fig. 2B, D) than for the vehicle-administered PBS control group (fig. 2A, C), indicating that plasminogen was able to reduce aortic sinus fibrosis levels in the atherosclerosis model mice.

Example 3 plasminogen improves carbon tetrachloride-induced liver fibrosis

15C 57 female mice, 9 weeks old, were randomly divided into three groups, a blank control group, a vehicle-administered PBS control group and a plasminogen-administered group, each of which was 5 mice. Injecting carbon tetrachloride into abdominal cavity of mice in the solvent PBS control group and plasminogen group according to the weight of 1mL/kg, three times per week, and two weeks continuously to establish hepatic fibrosis model^[36,37]Blank control mice were injected with corresponding volumes of corn oil following the model mouse injection protocol. The carbon tetrachloride needs to be diluted by corn oil, and the dilution ratio of the carbon tetrachloride to the corn oil is 1: 3. The administration is started after the model is established, the day of the administration is recorded as day 1, the mice in the plasminogen group are injected with human plasminogen according to the ratio of 1mg/0.1 mL/tail vein/day, the tail vein of the vehicle PBS control group is injected with PBS with the same volume, the blank control group is not injected and is continuously administered for 14 days. Mice were sacrificed on day 15 and livers were removed and fixed in 4% paraformaldehyde for 24 hours. The fixed liver is subjected to alcohol gradient dehydration and xylene transparence and then paraffin embedding. The tissue slice thickness is 3 μm, the slice is dewaxed to water and washed with water for 1 time, and the amount of the solvent is 0.1% TianlangAfter staining with red star for 60 minutes, washing with running water, staining with hematoxylin for 1 minute, washing with running water, differentiating with 1% hydrochloric acid alcohol and ammonia water to turn blue, washing with running water, drying, sealing, and observing the section under an optical microscope of 200 times.

The results showed that collagen deposition was significantly less for the plasminogen group (fig. 3C) than for the vehicle PBS control group (fig. 3B), and that collagen deposition levels were closer to the placebo control mouse (fig. 3A) for the plasminogen group than for the PBS group. Thus indicating that the plasminogen can reduce the deposition of liver collagen and improve the liver fibrosis of the liver fibrosis model mouse.

Example 4 plasminogen reduction in aortic sinus fibrosis in a 16 week hyperlipidemic model mouse

11 male C57 mice of 6 weeks old were fed high-fat high-cholesterol diet (nantong telofen, cat # TP2031) for 16 weeks to induce hyperlipidemia model^[30,31]This model was designated as a 16-week hyperlipidemia model. And (5) continuously feeding the mice after the molding to high-cholesterol feed. Three days prior to administration, 50. mu.l of each blood was taken to detect total cholesterol (T-CHO) content, and was randomly divided into two groups based on T-CHO content, 6 for vehicle PBS control group, and 5 for plasminogen group. The initial administration is recorded as day 1, the tail vein of plasminogen group is injected with 1mg/0.1 ml/l/day of human plasminogen, and the tail vein of vehicle PBS control group is injected with PBS in the same volume. The administration was performed for 30 days, mice were sacrificed on day 31, and hearts were harvested and fixed in 4% paraformaldehyde for 24-48 hours. The fixed tissue is embedded in paraffin after gradient dehydration with alcohol and xylene transparency. The aortic sinus section thickness is 3 μm, the section is washed with water 1 time after dewaxing and rehydration, after being stained with 0.1% sirius red saturated picric acid for 30 minutes, washed with running water for 2min, stained with hematoxylin for 1 minute, washed with running water, differentiated with 1% hydrochloric acid alcohol, ammonia water turned to blue, washed with running water, dried and then sealed with neutral gum, and observed under 40 (4A, 4B) and 200 times (4C, 4D) optical microscope.

The results showed that the area of intimal collagen deposition (arrow) on the wall of aortic sinus vessels was significantly smaller in the plasminogen-administered group (fig. 4B, 4D) than in the vehicle-administered PBS control group (fig. 4A, 4C), indicating that plasminogen was able to reduce aortic sinus fibrosis levels in mice with hyperlipidemia model.

Example 5 plasminogen reduction of systemic sclerosis skin fibrosis in mice

15 male mice, 12 weeks old, C57, were taken, randomized into three groups, a blank control group, a vehicle-administered PBS (PBS is Phosphate buffered Saline (phospate Buffer Saline), herein vehicle for plasminogen) control group, and a vehicle-administered plasminogen group, each group consisting of 5 mice, and 5 mice with 13 weeks old, impaired PLG activity were taken. The day of experiment initiation is recorded as the 0 th day of weighing and grouping, the next day of molding administration is initiated, the vehicle PBS control group, the plasminogen group and the PLG activity-impaired mice are injected subcutaneously with bleomycin at the rate of 0.1mg/0.1 ml/mouse/day to induce systemic sclerosis^[26]. The blank control group was injected subcutaneously with 0.1 ml/day of saline, and at the same time, plasminogen or PBS was administered and recorded as day 1, and the administration was continued for 21 days. Plasminogen was injected into the plasminogen-group mice at 1mg/0.1 ml/only/day tail vein, the vehicle PBS control group was given PBS of the same volume, and the normal mice and PLG activity-impaired mice were not treated with the administration. Mice were sacrificed on day 22 and dorsal skin tissue was fixed in 4% paraformaldehyde fixative for 24 hours. Fixed skin tissues were paraffin embedded after gradient dehydration with alcohol and xylene clarification. The tissue slice thickness is 3 μm, the slice is dewaxed to water and washed with water for 1 time, stained with 0.1% sirius red saturated picric acid for 30 minutes, washed with running water for 2min, stained with hematoxylin for 1 minute, washed with running water, differentiated with 1% hydrochloric acid alcohol, ammonia water turned to blue, washed with running water, dried, and then sealed with neutral gum, and observed under 100 times optical microscope.

The sirius red staining can lead the collagen to be stained durably, and as a special staining method for pathological sections, the sirius red staining can specifically display the collagen tissues.

The results show that in the bleomycin-induced systemic sclerosis mouse model, the mice (figure 5D) with the vehicle-fed PBS group (figure 5B) and the PLG activity-impaired group have the microscopic observation that the collagen fiber bundles on the upper part of the dermis are obviously increased, the collagen fibers are thick and dense, and the dermis layer is thickened; in the plasminogen group (fig. 5C), fibroblasts in the dermis were significantly less than in the vehicle-PBS control group, and the thickness of the dermal layer was substantially close to normal (fig. 5A). Thus showing that the plasminogen can effectively reduce bleomycin-induced skin fibrosis.

Example 6 plasminogen reduction in pulmonary fibrosis in systemic sclerosis mice

17 male mice, 12 weeks old, C57, were randomly divided into two groups, 11 vehicle PBS control groups and 6 plasminogen groups. The day of experiment was recorded as day 0 and weighed and divided into groups, the day 1 of the experiment was started for model administration, and two groups of mice were injected subcutaneously with bleomycin at 0.1mg/0.1 ml/day to induce systemic sclerosis^[26]And plasminogen or PBS administration was started for 21 days. Plasminogen was injected into mice in the plasminogen group at 1mg/0.1 ml/only/day tail vein, and the vehicle PBS control group was given the same volume of PBS in the same manner. Mice were sacrificed on day 22 and lung tissue was fixed in 4% paraformaldehyde fixative for 24 hours. The fixed lung tissue is subjected to alcohol gradient dehydration and xylene transparence and then paraffin embedding. The tissue slice thickness is 3 μm, the slice is dewaxed to water and washed with water for 1 time, stained with 0.1% sirius red saturated picric acid for 30 minutes, washed with running water for 2min, stained with hematoxylin for 1 minute, washed with running water, differentiated with 1% hydrochloric acid alcohol, ammonia water turned to blue, washed with running water, dried, and then sealed with neutral gum, and observed under 200 times optical microscope.

The study found that in the bleomycin-induced systemic sclerosis mouse model, the collagen fibrosis (indicated by arrows) was observed to a higher degree in the vehicle-administered PBS group (fig. 6A) than in the plasminogen group (fig. 6B) under the mirror; the lung alveolar wall morphology of mice in the plasminogen group is close to the normal level, inflammatory cells are obviously reduced, the fibrosis degree is obviously lower than that in the vehicle-fed PBS group, and the statistical difference is obvious (FIG. 6C). Indicating that the plasminogen can effectively reduce bleomycin-induced systemic sclerosis mouse lung tissue fibrosis.

Example 7 plasminogen reduction of systemic sclerosis mice Heart fibrosis

10 male mice, 12 weeks old C57, were randomly divided into two groups, 5 each for vehicle PBS control and plasminogen. The day of experiment initiation is recorded as day 0 and weighed and grouped, the day 1 is started to make model and dose, and bleomycin is injected subcutaneously according to 0.1mg/0.1 ml/day to induce systemic sclerosis^[26]And administration of plasminogen or PBS was started for 21 consecutive days. Plasminogen was injected into the tail vein of 1mg/0.1 ml/mouse/day in the plasminogen group, the same volume of PBS was injected into the tail vein of the vehicle PBS control group, and the mice were sacrificed on day 22 and the hearts were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed heart is subjected to alcohol gradient dehydration and xylene transparence and then paraffin embedding. The tissue slice thickness is 3 μm, the slice is dewaxed to water and washed with water for 1 time, stained with 0.1% sirius red saturated picric acid for 30 minutes, washed with running water for 2min, stained with hematoxylin for 1 minute, washed with running water, differentiated with 1% hydrochloric acid alcohol, ammonia water turned to blue, washed with running water, dried, and then sealed with neutral gum, and observed under 200 times optical microscope.

It was found that in the bleomycin-induced systemic sclerosis mouse model, higher cardiac collagen deposition was observed under the mirror in the vehicle PBS control group (fig. 7A) than in the plasminogen group (fig. 7B). Thus showing that the plasminogen can effectively reduce cardiac fibrosis induced by bleomycin.

Example 8 plasminogen reduction of Kidney fibrosis in systemic sclerosis mice

10 male mice, 12 weeks old, C57, were randomly divided into two groups, 5 each for vehicle PBS control and plasminogen. The day of experiment start was noted as day 0 and weighed for groups, day 1 the molding administration was started, all mice were subcutaneously injected with bleomycin at 0.1mg/0.1 ml/day to induce systemic sclerosis, and plasminogen or PBS administration was started for 21 consecutive molding administrations. Plasminogen was injected into the tail vein of mice in the plasminogen group at 1mg/0.1 ml/day, and the same volume of PBS was injected into the tail vein of the vehicle PBS control group. Mice were sacrificed on day 22 and kidneys were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidney is subjected to alcohol gradient dehydration and xylene transparence and then paraffin embedding. The tissue slice thickness is 3 μm, the slice is dewaxed to water and washed with water for 1 time, stained with 0.1% sirius red saturated picric acid for 30 minutes, washed with running water for 2min, stained with hematoxylin for 1 minute, washed with running water, differentiated with 1% hydrochloric acid alcohol, ammonia water turned to blue, washed with running water, dried, and then sealed with neutral gum, and observed under 200 times optical microscope.

The results showed that the degree of renal collagen fibrosis (indicated by arrows) was higher in the bleomycin-induced systemic sclerosis mouse model in the vehicle PBS control group (fig. 8A) than in the plasminogen group (fig. 8B). Thus showing that the plasminogen can effectively reduce the renal fibrosis induced by bleomycin.

Example 9 plasminogen reduction of Kidney collagen deposition in diabetic mice

10 db/db male mice 24-25 weeks old are randomly divided into two groups, and a vehicle PBS control group and a plasminogen group are given, and each group is provided with 5 mice. The day of experiment start was recorded as day 0 and the groups were weighed, day 1 on plasminogen or PBS for 31 consecutive days. Plasminogen was injected at 2mg/0.2 ml/only/day tail vein into mice in the plasminogen group, and the same volume of PBS was administered to the vehicle PBS control group. Mice were sacrificed 31 days after plasminogen and kidney tissue was fixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidney tissue is subjected to alcohol gradient dehydration and xylene transparence and then paraffin embedding. The thickness of the tissue slice is 4 μm, and the slice is washed with water 1 time after deparaffinization and rehydration. Incubate with 3% hydrogen peroxide for 15 min, wash with 0.01MPBS for 2 times, each for 5 min. 10% normal sheep serum (Vector laboratories, Inc., USA) was blocked for 1 hour, and after time the serum was spun off and the tissue was circled with a PAP pen. Rabbit anti-mouse polyclonal antibodies against collagen IV (Abcam) were incubated overnight at 4 ℃ and washed 2 times in TBS for 5 minutes each. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times in TBS. Color development was performed according to DAB kit (vectorlaboratoriases, inc., USA), 3 washes followed by hematoxylin counterstaining for 30 seconds, 5 minutes in running water, gradient alcohol dehydration, xylene transparency and transparency, neutral gum mounting, and sections were observed under a 200-fold optical microscope.

Diabetic nephropathy is a chronic complication of diabetes, and glomerulosclerosis and renal interstitial fibrosis are typical pathological changes thereof^[27]。

The results showed that the positive staining of collagen in group IV given plasminogen (fig. 9B) was significantly greater than in the vehicle PBS control group (fig. 9A), indicating that plasminogen can reduce collagen deposition in kidney tissue (arrows), suggesting that plasminogen is expected to prevent diabetes-induced fibrosis in kidney tissue by reducing collagen deposition in kidney tissue.

Example 10 plasminogen improves renal fibrosis in diabetic mice

10 db/db male mice of 26 weeks old were randomly divided into two groups, 5 mice each were given vehicle PBS control group and plasminogen group. The day of experiment start was recorded as day 0 and the groups were weighed, day 1 on plasminogen or PBS for 35 consecutive days. Plasminogen was injected at 2mg/0.2 ml/only/day tail vein into mice in the plasminogen group, and the same volume of PBS was administered to the vehicle PBS control group. Mice were sacrificed on day 36 and kidney tissue was fixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidney tissue is subjected to alcohol gradient dehydration and xylene transparence and then paraffin embedding. The thickness of the tissue slice is 4 μm, and the slice is dewaxed and rehydrated and then placed in potassium dichromate solution overnight. The iron hematoxylin is stained for 3 to 5 minutes and washed slightly with running water. Alcohol differentiation with 1% hydrochloric acid, ammonia water treatment for 1 second, and water washing. Ponceau acid fuchsin solution was stained for 8 minutes and rinsed quickly in water. The 1% phosphomolybdic acid aqueous solution was treated for about 2 minutes and the aniline blue solution was counterstained for 6 minutes. Rinsing with 1% glacial acetic acid for about 1 minute. After dehydrated xylene by absolute ethyl alcohol and transparent, the slices are sealed and observed under an optical microscope with the power of 200 times.

Masson staining can show fibrosis of the tissue. The results showed that the vehicle PBS control group (fig. 10A) was given mesangial hyperplasia, increased mesangial stroma, mild fibrosis of the renal stroma (arrow mark), and hyperplastic fibrosis was blue. The plasminogen group (fig. 10B) showed significantly less mesangial cells and stroma than the control group, and significantly reduced renal interstitial fibrosis. Indicating that the plasminogen can improve the fibrosis of the kidney of the diabetic mouse.

Example 11 plasminogen improves cardiac fibrosis in 24-25 week old diabetic mice

10 db/db male mice 24-25 weeks old are randomly divided into two groups, and a vehicle PBS control group and a plasminogen group are given, and each group is provided with 5 mice. The day of experiment start was recorded as day 0 and the groups were weighed, day 1 on plasminogen or PBS for 31 consecutive days. Plasminogen was injected at 2mg/0.2 ml/only/day tail vein into mice in the plasminogen group, and the same volume of PBS was administered to the vehicle PBS control group. Mice were sacrificed 31 days after plasminogen and heart tissue fixed in 4% paraformaldehyde fixative for 24 hours. The fixed heart tissue is subjected to alcohol gradient dehydration and xylene transparence and then paraffin embedding. The thickness of the tissue slice is 4 μm, and the slice is dewaxed and rehydrated and then placed in potassium dichromate solution overnight. The iron hematoxylin is stained for 3 to 5 minutes and washed slightly with running water. Alcohol differentiation with 1% hydrochloric acid, ammonia water treatment for 1 second, and water washing. Ponceau acid fuchsin solution was stained for 8 minutes and rinsed quickly in water. The 1% phosphomolybdic acid aqueous solution was treated for about 2 minutes and the aniline blue solution was counterstained for 6 minutes. Rinsing with 1% glacial acetic acid is about 1 minute. After dehydrated xylene by absolute ethyl alcohol and transparent, the slices are sealed and observed under an optical microscope with the power of 200 times.

The most common complication of diabetes is excessive accumulation of connective tissue (pathological fibrosis), and interstitial fibrosis of myocardium may be the characteristic pathological change of diabetic cardiomyopathy^[28-29]。

Masson staining can show fibrosis of the tissue. The results showed that blue-colored hyperplastic collagen fibers (arrows) were visible between the myocardial fibers in the vehicle PBS control group (fig. 11A), with mild myocardial fibrosis; a small amount of bluish hyperplastic collagen fibers were seen between the myocardial fibers in the plasmin-treated group (fig. 11B), and myocardial fibrosis was significantly reduced compared to the control group. Indicating that the plasminogen can improve the fibrosis of the heart of the diabetic mouse.

Example 12 plasminogen reduction of Heart collagen deposition in 17-18 week old diabetic mice

8 db/db male mice 17-18 weeks old are randomly divided into two groups, and a vehicle PBS control group and a plasminogen group are given, wherein each group is provided with 4 mice. The day of experiment start was recorded as day 0 and the groups were weighed, day 1 on plasminogen or PBS for 35 consecutive days. Plasminogen was injected at 2mg/0.2 ml/only/day tail vein into mice in the plasminogen group, and the same volume of PBS was administered to the vehicle PBS control group. Mice were sacrificed 35 days after plasminogen and heart tissue fixed in 4% paraformaldehyde fixative for 24 hours. The fixed heart is subjected to alcohol gradient dehydration and xylene transparence and then paraffin embedding. The tissue slice thickness is 3 μm, the slice is dewaxed to water and washed with water for 1 time, stained with 0.1% sirius red saturated picric acid for 30 minutes, washed with running water for 2min, stained with hematoxylin for 1 minute, washed with running water, differentiated with 1% hydrochloric acid alcohol, ammonia water turned to blue, washed with running water, dried, and then sealed with neutral gum, and observed under 200 times optical microscope.

The results show that the deposition of collagen fibers (arrows) was significantly less for the mice given the plasminogen group (fig. 12B) than for the vehicle PBS control group (fig. 12A). Indicating that plasminogen can reduce the deposition of collagen in heart tissue, indicating that plasminogen is expected to reduce the fibrosis of heart tissue in relatively young (17-18 weeks old) diabetic mice by reducing the deposition of collagen in heart tissue.

Example 13 plasminogen reduction of cardiac collagen deposition in diabetic mice of 26-27 weeks old

9 db/db male mice of 26-27 weeks old are randomly divided into two groups, 5 mice are given to a vehicle PBS control group and 4 mice are given to a plasminogen group. The day of experiment start was recorded as day 0 and the groups were weighed, day 1 on plasminogen or PBS for 35 consecutive days. Plasminogen was injected at 2mg/0.2 ml/only/day tail vein into mice in the plasminogen group, and the same volume of PBS was administered to the vehicle PBS control group. Mice were sacrificed 35 days after plasminogen and heart tissue fixed in 4% paraformaldehyde fixative for 24 hours. The fixed heart is subjected to alcohol gradient dehydration and xylene transparence and then paraffin embedding. The tissue slice thickness is 3 μm, the slice is washed with water 1 time after dewaxing to water, after staining with 0.1% sirius red for 60 minutes, washed with running water, stained with hematoxylin for 1 minute, washed with running water, differentiated with 1% hydrochloric acid alcohol and ammonia water to turn blue, washed with running water, dried, and then sealed, and observed under an optical microscope of 200 times.

The results show that the deposition of collagen fibers (arrows) was significantly less for the mice given the plasminogen group (fig. 13B) than for the vehicle PBS control group (fig. 13A). Indicating that the plasminogen can reduce the deposition of the collagen of the heart tissue, the plasminogen is expected to reduce the fibrosis of the heart tissue of the relatively old (26-27 weeks old) diabetic mice by reducing the deposition of the collagen of the heart tissue.

Example 14 plasminogen reduction of fibrosis in the kidney of cisplatin Kidney fibrosis model mice

10 healthy male C57 mice, 8-9 weeks old, were randomly divided into two groups, 5 each, vehicle PBS control and plasminogen. After grouping is finished, cisplatin is injected into abdominal cavity once according to 10mg/Kg of body weight to establish a kidney fibrosis model^[30]. After modeling, plasminogen was administered to the plasminogen group at 1mg/0.1 ml/l/day by tail vein injection, and the vehicle PBS control group was administered with the same volume of PBS. The day of experiment start was recorded as day 0, the body weight was weighed and divided, day 1, an intraperitoneal cisplatin molding was started, and plasminogen or vehicle PBS was administered within 3 hours after molding for a period of 7 days. Mice were sacrificed on day 8 and kidneys were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidney tissue is dehydrated and dimethyl by alcohol gradientAfter the benzene was transparent, paraffin embedding was performed. The thickness of the tissue slice is 5 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. The citric acid is restored for 30 minutes, and the water is gently rinsed after the mixture is cooled for 10 minutes at room temperature. Incubate with 3% hydrogen peroxide for 15 minutes and pen the tissue with PAP. Blocking 10% sheep serum (vectorlaboratoria, inc., USA) for 1 hour; after the time is up, the sheep blood clear liquid is discarded. Rabbit anti-mouse IV collagen antibody (Abcam) was incubated overnight at 4 ℃ and washed 2 times in TBS for 5 minutes each. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times in TBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes followed by hematoxylin counterstaining for 30 seconds, flowing water rewet for 5 minutes, and then 1 wash with TBS. The sections were observed under a 200-fold optical microscope.

Cisplatin is a broad-spectrum anti-tumor drug with wide clinical application and reliable curative effect, but has serious renal toxicity, mainly causes renal tubular and renal interstitial injury, and finally develops renal fibrosis^[30]. The results of this experiment show that the positive expression of renal collagen type IV (arrow) is significantly higher in the vehicle-administered PBS control group (fig. 14A) than in the plasmin-administered group (fig. 14B). Indicating that plasminogen can improve kidney fibrosis of cisplatin kidney fibrosis model mice.

Example 15 plasminogen improves ApoE atherosclerotic mouse Heart fibrosis levels

13 ApoE male mice of 6 weeks old are fed with high-fat high-cholesterol feed (Nantong terioflofine, TP2031) for 16 weeks to induce atherosclerosis^[31,32]. Three days before administration, 50. mu.L of blood was taken from each mouse, total cholesterol concentration was measured, and the mice were randomly divided into two groups according to the measurement results, 7 mice were given to the vehicle PBS control group and 6 mice were given to the plasminogen group. The administration was started on day 1, mice in the plasminogen group were injected intravenously with 1mg/0.1 ml/l/day of human plasminogen, and the vehicle PBS control group was injected intravenously with the same volume of PBS. The administration is carried out for 30 days, and high-fat and high-cholesterol feed is fed all the time. Mice were sacrificed on day 31 and hearts were fixed in 4% paraformaldehyde for 24-48 hours. After being fixedThe tissues were paraffin embedded after gradient dehydration with alcohol and xylene clarification. The thickness of the tissue section is 3 μm, the section is washed with water 1 time after dewaxing and rehydration, after being stained with 0.1% sirius red saturated picric acid for 30 minutes, washed with running water for 2 minutes, stained with hematoxylin for 1 minute, washed with running water, differentiated with 1% hydrochloric acid alcohol, ammonia water rewet, washed with running water, dried, and then the neutral gum sealing piece is observed under an optical microscope of 200 times.

The results show that the collagen deposition (arrows) was significantly less for the plasminogen group (fig. 15B) than for the vehicle PBS control group (fig. 15A), suggesting that plasminogen can prevent and reduce atherosclerosis-induced cardiac fibrosis by reducing collagen deposition in heart tissue of ApoE atherosclerotic model mice.

Example 16 plasminogen reduction of cardiac fibrosis in a hyperlipidemic model mouse

6 weeks old C57 male mice 11 were fed with high-fat high-cholesterol diet (Nantong Telofei, TP2031) for 16 weeks to induce hyperlipidemia^[33,34]. Three days prior to dosing, 50 μ L of blood was taken from each mouse, total cholesterol concentrations were measured, and the mice were randomized into two groups according to them, 6 for vehicle PBS control group and 5 for plasminogen group. The initial dose was recorded as day 1, mice in the plasminogen group were injected intravenously with 1mg/0.1 ml/l/day of human plasminogen and the vehicle PBS control group was injected intravenously with the same volume of PBS in the tail vein. The administration is carried out for 30 days, and high-fat and high-cholesterol feed is fed all the time. Mice were sacrificed on day 31 and heart tissue was fixed in 4% paraformaldehyde for 24-48 hours. The fixed tissue is embedded in paraffin after gradient dehydration with alcohol and xylene transparency. The thickness of the tissue section is 3 μm, the section is washed with water 1 time after dewaxing and rehydration, after being stained with 0.1% sirius red saturated picric acid for 30 minutes, washed with running water for 2 minutes, stained with hematoxylin for 1 minute, washed with running water, differentiated with 1% hydrochloric acid alcohol, ammonia water rewet, washed with running water, dried, and then the neutral gum sealing piece is observed under an optical microscope of 200 times.

The results show that the collagen deposition (arrow) was significantly less for the plasminogen group (fig. 16B) than for the vehicle PBS control group (fig. 16A), suggesting that plasminogen could prevent and reduce hyperlipidemia-induced cardiac fibrosis by reducing the collagen deposition in heart tissue of the mouse model with hyperlipidemia.

Example 17 plasminogen repair fibrosis in the Kidney of model Chronic renal failure

12 PLG-active normal male mice 8-9 weeks old and 6 PLG-active impaired male mice, the PLG-active normal mice were randomly divided into two groups, 6 each for the plasminogen group and the vehicle-PBS control group. Three groups of mice were fed with 0.25% purine feed (Nantong Telofei) daily to establish a model of chronic renal failure^[35]. The day of molding was recorded as day 1, and administration was started. Plasminogen was administered at 1mg/0.1 ml/day to the plasminogen group, PBS was administered at the same volume to the vehicle PBS control group in the same manner for 10 consecutive days of molding, and the PLG activity-impaired mice were not treated. Mice were sacrificed on day 11 and kidneys were fixed in 4% paraformaldehyde for 24 hours. The fixed kidney is subjected to alcohol gradient dehydration and xylene transparence and then paraffin embedding. The thickness of the tissue section is 3 μm, the section is washed with water 1 time after being dewaxed to water, and after being stained with 0.1% sirius red for 60 minutes, the section is washed with running water, stained with hematoxylin for 1 minute, washed with running water, differentiated by 1% hydrochloric acid alcohol and ammonia water and turned to blue, washed with running water, dried and then sealed, and the section is observed under an optical microscope of 200 times.

The results show that collagen deposition (arrow) was significantly less for the plasminogen group (fig. 17B) than for the vehicle PBS control group (fig. 17A) and the PLG activity impaired group (fig. 17C), and that the plasminogen group was statistically significantly different from the PLG activity impaired group (P ═ 0.018) (fig. 17D). The plasminogen can obviously reduce the deposition of collagen in the kidney tissue of animals with chronic kidney injury, and prevent and reduce the kidney fibrosis caused by the chronic kidney injury.

Example 18 plasminogen reduces islet collagen deposition in diabetic mice

16 db/db male mice 24-25 weeks old were randomly divided into two groups, 10 plasminogen groups and 6 vehicle PBS control groups. 2mg/0.2 ml/day of human plasminogen is injected into tail vein of plasminogen group, and PBS with the same volume is injected into tail vein of vehicle PBS control group. The day of experiment start was recorded as day 0 weight group and day 1 started plasminogen or PBS for 31 consecutive days. Mice were sacrificed on day 32 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue section is 3 μm, the section is washed with water 1 time after being dewaxed to water, and after being stained with 0.1% sirius red for 60 minutes, the section is washed with running water, stained with hematoxylin for 1 minute, washed with running water, differentiated by 1% hydrochloric acid alcohol and ammonia water and turned to blue, washed with running water, dried and then sealed, and the section is observed under an optical microscope of 200 times.

The results showed that islet collagen deposition (arrow) was significantly lower in the plasminogen mice (fig. 18B) than in the vehicle PBS control group (fig. 18A) and the statistical difference was significant (fig. 18C). The plasminogen can obviously reduce the deposition of collagen in pancreatic tissues of diabetic mice, and prevent and reduce the damage and fibrosis of the pancreas.

Example 19 plasminogen reduction 3% Cholesterol hyperlipidemia Kidney fibrosis in model mice

9-week-old male C57 mice were fed 3% cholesterol high-fat diet (Nantong terioflofine) for 4 weeks to induce hyperlipidemia^[30,31]The model was designated as a 3% cholesterol hyperlipidemia model, and mice after molding were further fed with 3% cholesterol hyperlipidemia diet. Another 5 male C57 mice of the same week age were used as a blank control group and were fed with normal maintenance diet during the experiment. Three days before administration, 50 μ L of blood was taken from each mouse, total cholesterol was detected, model mice were randomly divided into two groups according to total cholesterol concentration and body weight, and plasminogen group and vehicle-added PBS control group were given, 8 each. The initial administration is recorded as day 1, the mice in the plasminogen group are injected intravenously with 1mg/0.1 ml/day of human plasminogen, and the vehicle is given PBS controlGroup tail vein was injected with the same volume of PBS. Mice were dosed 30 days after day 30 dosing, sacrificed at day 31, and kidneys were harvested and fixed in 4% paraformaldehyde for 24-48 hours. The fixed tissue is embedded in paraffin after gradient dehydration with alcohol and xylene transparency. The slice thickness is 3 μm, the slice is washed with water 1 time after dewaxing and rehydration, after being stained with 0.1% sirius red saturated picric acid for 30 minutes, washed with running water for 2 minutes, stained with hematoxylin for 1 minute, washed with running water, differentiated with 1% hydrochloric acid alcohol, ammonia water is rewet, washed with running water, dried, and then the gel is sealed with neutral gum, and observed under an optical microscope of 200 times.

The results showed that kidney collagen deposition (arrow) was significantly less for the plasminogen group (fig. 19C) than for the vehicle PBS control group (fig. 19B) and the statistical difference was significant (fig. 19D); the plasminogen group fibrosis was returned to essentially normal levels (fig. 19A). Thus showing that the plasminogen can effectively reduce the kidney fibrosis of the mouse model of hyperlipemia by 3 percent of cholesterol.

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Sequence listing

<110> Shenzhen Rizhen Life sciences research institute Limited

<120> a method for preventing and treating hepatic fibrosis

<130>PDK03581

<160>14

<170>PatentIn version 3.5

<210>1

<211>2376

<212>DNA

<213> native plasminogen (Glu-PLG, Glu-plasminogen) nucleic acid sequence without signal peptide

<400>1

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggaatgtat gcattgcagt ggagaaaact atgacggcaa aatttccaag 540

accatgtctg gactggaatg ccaggcctgg gactctcaga gcccacacgc tcatggatac 600

attccttcca aatttccaaa caagaacctg aagaagaatt actgtcgtaa ccccgatagg 660

gagctgcggc cttggtgttt caccaccgac cccaacaagc gctgggaact ttgtgacatc 720

ccccgctgca caacacctcc accatcttct ggtcccacct accagtgtct gaagggaaca 780

ggtgaaaact atcgcgggaa tgtggctgtt accgtgtccg ggcacacctg tcagcactgg 840

agtgcacaga cccctcacac acataacagg acaccagaaa acttcccctg caaaaatttg 900

gatgaaaact actgccgcaa tcctgacgga aaaagggccc catggtgcca tacaaccaac 960

agccaagtgc ggtgggagta ctgtaagata ccgtcctgtg actcctcccc agtatccacg 1020

gaacaattgg ctcccacagc accacctgag ctaacccctg tggtccagga ctgctaccat 1080

ggtgatggac agagctaccg aggcacatcc tccaccacca ccacaggaaa gaagtgtcag 1140

tcttggtcat ctatgacacc acaccggcac cagaagaccc cagaaaacta cccaaatgct 1200

ggcctgacaa tgaactactg caggaatcca gatgccgata aaggcccctg gtgttttacc 1260

acagacccca gcgtcaggtg ggagtactgc aacctgaaaa aatgctcagg aacagaagcg 1320

agtgttgtag cacctccgcc tgttgtcctg cttccagatg tagagactcc ttccgaagaa 1380

gactgtatgt ttgggaatgg gaaaggatac cgaggcaaga gggcgaccac tgttactggg 1440

acgccatgcc aggactgggc tgcccaggag ccccatagac acagcatttt cactccagag 1500

acaaatccac gggcgggtct ggaaaaaaat tactgccgta accctgatgg tgatgtaggt 1560

ggtccctggt gctacacgac aaatccaaga aaactttacg actactgtga tgtccctcag 1620

tgtgcggccc cttcatttga ttgtgggaag cctcaagtgg agccgaagaa atgtcctgga 1680

agggttgtag gggggtgtgt ggcccaccca cattcctggc cctggcaagt cagtcttaga 1740

acaaggtttg gaatgcactt ctgtggaggc accttgatat ccccagagtg ggtgttgact 1800

gctgcccact gcttggagaa gtccccaagg ccttcatcct acaaggtcat cctgggtgca 1860

caccaagaag tgaatctcga accgcatgtt caggaaatag aagtgtctag gctgttcttg 1920

gagcccacac gaaaagatat tgccttgcta aagctaagca gtcctgccgt catcactgac 1980

aaagtaatcc cagcttgtct gccatcccca aattatgtgg tcgctgaccg gaccgaatgt 2040

ttcatcactg gctggggaga aacccaaggt acttttggag ctggccttct caaggaagcc 2100

cagctccctg tgattgagaa taaagtgtgc aatcgctatg agtttctgaa tggaagagtc 2160

caatccaccg aactctgtgc tgggcatttg gccggaggca ctgacagttg ccagggtgac 2220

agtggaggtc ctctggtttg cttcgagaag gacaaataca ttttacaagg agtcacttct 2280

tggggtcttg gctgtgcacg ccccaataag cctggtgtct atgttcgtgt ttcaaggttt 2340

gttacttgga ttgagggagt gatgagaaat aattaa 2376

<210>2

<211>791

<212>PRT

<213> native plasminogen (Glu-PLG, Glu-plasminogen) amino acid sequence without signal peptide

<400>2

Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser

1 5 10 15

Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala

20 25 30

Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His

35 40 45

Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser

50 55 60

Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr

65 70 75 80

Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met

85 90 95

Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser

100 105 110

Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu

115 120 125

Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp

130 135 140

Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu

145 150 155 160

Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly

165 170 175

Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser

180 185 190

Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys

195 200 205

Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro

210 215 220

Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile

225 230 235 240

Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys

245 250 255

Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val

260 265 270

Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His

275 280285

Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr

290 295 300

Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn

305 310 315 320

Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser

325 330 335

Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr

340 345 350

Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly

355 360 365

Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser

370 375 380

Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala

385 390 395 400

Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro

405 410 415

Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu

420 425 430

Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val

435 440 445

Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe

450 455 460

Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly

465 470 475 480

Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile

485 490 495

Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys

500 505 510

Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn

515 520 525

Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro

530 535 540

Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly

545 550 555 560

Arg Val Val Gly Gly Cys Val Ala His Pro His Ser TrpPro Trp Gln

565 570 575

Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu

580 585 590

Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser

595 600 605

Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val

610 615 620

Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu

625 630 635 640

Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala

645 650 655

Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr

660 665 670

Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr

675 680 685

Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val

690 695 700

Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val

705 710 715 720

Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser

725 730 735

Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys

740 745 750

Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro

755 760 765

Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile

770 775 780

Glu Gly Val Met Arg Asn Asn

785 790

<210>3

<211>2433

<212>DNA

<213> nucleic acid sequence of native plasminogen (derived from swiss prot) containing signal peptide

<400>3

atggaacata aggaagtggt tcttctactt cttttatttc tgaaatcagg tcaaggagag 60

cctctggatg actatgtgaa tacccagggg gcttcactgt tcagtgtcac taagaagcag 120

ctgggagcag gaagtataga agaatgtgca gcaaaatgtg aggaggacga agaattcacc 180

tgcagggcat tccaatatca cagtaaagag caacaatgtg tgataatggc tgaaaacagg 240

aagtcctcca taatcattag gatgagagat gtagttttat ttgaaaagaa agtgtatctc 300

tcagagtgca agactgggaa tggaaagaac tacagaggga cgatgtccaa aacaaaaaat 360

ggcatcacct gtcaaaaatg gagttccact tctccccaca gacctagatt ctcacctgct 420

acacacccct cagagggact ggaggagaac tactgcagga atccagacaa cgatccgcag 480

gggccctggt gctatactac tgatccagaa aagagatatg actactgcga cattcttgag 540

tgtgaagagg aatgtatgca ttgcagtgga gaaaactatg acggcaaaat ttccaagacc 600

atgtctggac tggaatgcca ggcctgggac tctcagagcc cacacgctca tggatacatt 660

ccttccaaat ttccaaacaa gaacctgaag aagaattact gtcgtaaccc cgatagggag 720

ctgcggcctt ggtgtttcac caccgacccc aacaagcgct gggaactttg tgacatcccc 780

cgctgcacaa cacctccacc atcttctggt cccacctacc agtgtctgaa gggaacaggt 840

gaaaactatc gcgggaatgt ggctgttacc gtgtccgggc acacctgtca gcactggagt 900

gcacagaccc ctcacacaca taacaggaca ccagaaaact tcccctgcaa aaatttggat 960

gaaaactact gccgcaatcc tgacggaaaa agggccccat ggtgccatac aaccaacagc 1020

caagtgcggt gggagtactg taagataccg tcctgtgact cctccccagt atccacggaa 1080

caattggctc ccacagcacc acctgagcta acccctgtgg tccaggactg ctaccatggt 1140

gatggacaga gctaccgagg cacatcctcc accaccacca caggaaagaa gtgtcagtct 1200

tggtcatcta tgacaccaca ccggcaccag aagaccccag aaaactaccc aaatgctggc 1260

ctgacaatga actactgcag gaatccagat gccgataaag gcccctggtg ttttaccaca 1320

gaccccagcg tcaggtggga gtactgcaac ctgaaaaaat gctcaggaac agaagcgagt 1380

gttgtagcac ctccgcctgt tgtcctgctt ccagatgtag agactccttc cgaagaagac 1440

tgtatgtttg ggaatgggaa aggataccga ggcaagaggg cgaccactgt tactgggacg 1500

ccatgccagg actgggctgc ccaggagccc catagacaca gcattttcac tccagagaca 1560

aatccacggg cgggtctgga aaaaaattac tgccgtaacc ctgatggtga tgtaggtggt 1620

ccctggtgct acacgacaaa tccaagaaaa ctttacgact actgtgatgt ccctcagtgt 1680

gcggcccctt catttgattg tgggaagcct caagtggagc cgaagaaatg tcctggaagg 1740

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 1800

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 1860

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 1920

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 1980

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 2040

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 2100

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 2160

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 2220

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 2280

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 2340

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 2400

acttggattg agggagtgat gagaaataat taa 2433

<210>4

<211>810

<212>PRT

<213> amino acid sequence of native plasminogen (derived from swiss prot) containing signal peptide

<400>4

Met Glu His Lys Glu Val Val Leu Leu Leu Leu Leu Phe Leu Lys Ser

1 5 10 15

Gly Gln Gly Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser

20 25 30

Leu Phe Ser Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu

35 40 45

Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe

50 55 60

Gln Tyr His Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg

65 70 75 80

Lys Ser Ser Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys

85 90 95

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg

100 105 110

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser

115 120 125

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser

130 135 140

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln

145 150 155 160

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys

165 170 175

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn

180 185 190

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala

195 200 205

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe

210 215 220

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu

225 230 235 240

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu

245 250 255

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr

260 265 270

Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala

275 280 285

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro

290 295 300

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp

305 310 315 320

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His

325 330 335

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys

340 345 350

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro

355 360 365

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser

370 375 380

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser

385 390 395 400

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr

405 410 415

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp

420 425 430

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr

435 440 445

Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro

450 455 460

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp

465 470475 480

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr

485 490 495

Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg

500 505 510

His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys

515 520 525

Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr

530 535 540

Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

545 550 555 560

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

565 570 575

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

580 585 590

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

595 600 605

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

610 615 620

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

625 630 635 640

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

645 650 655

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

660 665 670

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

675 680 685

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

690 695 700

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

705 710 715 720

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

725 730 735

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

740 745 750

Thr Asp Ser Cys Gln GlyAsp Ser Gly Gly Pro Leu Val Cys Phe Glu

755 760 765

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

770 775 780

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

785 790 795 800

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

805 810

<210>5

<211>2145

<212>DNA

<213> LYS77-PLG (Lys-plasminogen) nucleic acid sequences

<400>5

aaagtgtatc tctcagagtg caagactggg aatggaaaga actacagagg gacgatgtcc 60

aaaacaaaaa atggcatcac ctgtcaaaaa tggagttcca cttctcccca cagacctaga 120

ttctcacctg ctacacaccc ctcagaggga ctggaggaga actactgcag gaatccagac 180

aacgatccgc aggggccctg gtgctatact actgatccag aaaagagata tgactactgc 240

gacattcttg agtgtgaaga ggaatgtatg cattgcagtg gagaaaacta tgacggcaaa 300

atttccaaga ccatgtctgg actggaatgc caggcctggg actctcagag cccacacgct 360

catggataca ttccttccaa atttccaaac aagaacctga agaagaatta ctgtcgtaac 420

cccgataggg agctgcggcc ttggtgtttc accaccgacc ccaacaagcg ctgggaactt 480

tgtgacatcc cccgctgcac aacacctcca ccatcttctg gtcccaccta ccagtgtctg 540

aagggaacag gtgaaaacta tcgcgggaat gtggctgtta ccgtgtccgg gcacacctgt 600

cagcactgga gtgcacagac ccctcacaca cataacagga caccagaaaa cttcccctgc 660

aaaaatttgg atgaaaacta ctgccgcaat cctgacggaa aaagggcccc atggtgccat 720

acaaccaaca gccaagtgcg gtgggagtac tgtaagatac cgtcctgtga ctcctcccca 780

gtatccacgg aacaattggc tcccacagca ccacctgagc taacccctgt ggtccaggac 840

tgctaccatg gtgatggaca gagctaccga ggcacatcct ccaccaccac cacaggaaag 900

aagtgtcagt cttggtcatc tatgacacca caccggcacc agaagacccc agaaaactac 960

ccaaatgctg gcctgacaat gaactactgc aggaatccag atgccgataa aggcccctgg 1020

tgttttacca cagaccccag cgtcaggtgg gagtactgca acctgaaaaa atgctcagga 1080

acagaagcga gtgttgtagc acctccgcct gttgtcctgc ttccagatgt agagactcct 1140

tccgaagaag actgtatgtt tgggaatggg aaaggatacc gaggcaagag ggcgaccact 1200

gttactggga cgccatgcca ggactgggct gcccaggagc cccatagaca cagcattttc 1260

actccagaga caaatccacg ggcgggtctg gaaaaaaatt actgccgtaa ccctgatggt 1320

gatgtaggtg gtccctggtg ctacacgaca aatccaagaa aactttacga ctactgtgat 1380

gtccctcagt gtgcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 1440

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 1500

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 1560

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 1620

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 1680

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 1740

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 1800

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 1860

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1920

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1980

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 2040

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 2100

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 2145

<210>6

<211>714

<212>PRT

<213> LYS77-PLG (Lys-plasminogen) amino acid sequence

<400>6

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg

1 5 10 15

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser

20 25 30

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser

35 40 45

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln

50 55 60

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys

65 70 75 80

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn

85 90 95

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala

100 105 110

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe

115 120 125

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu

130 135 140

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu

145 150 155 160

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr

165 170 175

Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala

180 185 190

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro

195 200 205

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp

210 215 220

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His

225 230 235 240

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys

245 250 255

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro

260 265 270

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser

275 280 285

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser

290 295 300

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr

305 310 315 320

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp

325 330 335

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr

340 345 350

Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro

355 360 365

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp

370 375 380

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr

385 390 395 400

Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg

405 410 415

His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys

420 425 430

Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr

435 440 445

Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

450 455 460

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

465 470 475 480

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

485 490 495

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

500 505 510

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

515 520 525

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

530 535 540

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

545 550 555 560

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

565 570 575

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

580 585 590

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

595 600 605

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

610 615 620

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

625 630 635 640

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

645 650 655

Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu

660 665 670

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

675 680 685

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

690 695 700

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

705 710

<210>7

<211>1245

<212>DNA

<213> Delta-plg (Delta-plasminogen) nucleic acid sequence

<400>7

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 540

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 600

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 660

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 720

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 780

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 840

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 900

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 960

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1020

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1080

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 1140

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 1200

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 1245

<210>8

<211>414

<212>PRT

<213> Delta-plg (Delta-plasminogen) amino acid sequence

<400>8

Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser

1 5 10 15

Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala

20 25 30

Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His

35 40 45

Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser

50 55 60

Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr

65 70 75 80

Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met

85 90 95

Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser

100 105 110

Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu

115 120 125

Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp

130 135 140

Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu

145 150 155 160

Glu Cys Glu GluAla Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val

165 170 175

Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His

180 185 190

Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met

195 200 205

His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala

210 215 220

Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile

225 230 235 240

Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile

245 250 255

Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu

260 265 270

Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala

275 280 285

Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe

290 295 300

Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu

305 310 315 320

Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr

325 330 335

Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His

340 345 350

Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu

355 360 365

Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp

370 375 380

Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val

385 390 395 400

Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

405 410

<210>9

<211>1104

<212>DNA

<213> Mini-plg (Small plasminogen) nucleic acid sequence

<400>9

gtcaggtggg agtactgcaa cctgaaaaaa tgctcaggaa cagaagcgag tgttgtagca 60

cctccgcctg ttgtcctgct tccagatgta gagactcctt ccgaagaaga ctgtatgttt 120

gggaatggga aaggataccg aggcaagagg gcgaccactg ttactgggac gccatgccag 180

gactgggctg cccaggagcc ccatagacac agcattttca ctccagagac aaatccacgg 240

gcgggtctgg aaaaaaatta ctgccgtaac cctgatggtg atgtaggtgg tccctggtgc 300

tacacgacaa atccaagaaa actttacgac tactgtgatg tccctcagtg tgcggcccct 360

tcatttgatt gtgggaagcc tcaagtggag ccgaagaaat gtcctggaag ggttgtaggg 420

gggtgtgtgg cccacccaca ttcctggccc tggcaagtca gtcttagaac aaggtttgga 480

atgcacttct gtggaggcac cttgatatcc ccagagtggg tgttgactgc tgcccactgc 540

ttggagaagt ccccaaggcc ttcatcctac aaggtcatcc tgggtgcaca ccaagaagtg 600

aatctcgaac cgcatgttca ggaaatagaa gtgtctaggc tgttcttgga gcccacacga 660

aaagatattg ccttgctaaa gctaagcagt cctgccgtca tcactgacaa agtaatccca 720

gcttgtctgc catccccaaa ttatgtggtc gctgaccgga ccgaatgttt catcactggc 780

tggggagaaa cccaaggtac ttttggagct ggccttctca aggaagccca gctccctgtg 840

attgagaata aagtgtgcaa tcgctatgag tttctgaatg gaagagtcca atccaccgaa 900

ctctgtgctg ggcatttggc cggaggcact gacagttgcc agggtgacag tggaggtcct 960

ctggtttgct tcgagaagga caaatacatt ttacaaggag tcacttcttg gggtcttggc 1020

tgtgcacgcc ccaataagcc tggtgtctat gttcgtgttt caaggtttgt tacttggatt 1080

gagggagtga tgagaaataa ttaa 1104

<210>10

<211>367

<212>PRT

<213> Mini-plg (plasminogen) amino acid sequence

<400>10

Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala

1 5 10 15

Ser Val Val Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr

20 25 30

Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly

35 40 45

Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala

50 55 60

Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg

65 70 75 80

Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly

85 90 95

Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys

100 105 110

Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln

115 120 125

Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala

130 135 140

His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly

145 150 155 160

Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr

165 170 175

Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val

180 185 190

Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu

195 200 205

Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala

210 215 220

Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro

225 230 235 240

Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys

245 250 255

Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu

260 265 270

Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg

275 280 285

Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly

290 295 300

His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro

305 310 315 320

Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser

325 330 335

Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg

340 345 350

Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

355 360 365

<210>11

<211>750

<212>DNA

<213> Micro-plg (microplasminogen) nucleic acid sequence

<400>11

gccccttcat ttgattgtgg gaagcctcaa gtggagccga agaaatgtcc tggaagggtt 60

gtaggggggt gtgtggccca cccacattcc tggccctggc aagtcagtct tagaacaagg 120

tttggaatgc acttctgtgg aggcaccttg atatccccag agtgggtgtt gactgctgcc 180

cactgcttgg agaagtcccc aaggccttca tcctacaagg tcatcctggg tgcacaccaa 240

gaagtgaatc tcgaaccgca tgttcaggaa atagaagtgt ctaggctgtt cttggagccc 300

acacgaaaag atattgcctt gctaaagcta agcagtcctg ccgtcatcac tgacaaagta 360

atcccagctt gtctgccatc cccaaattat gtggtcgctg accggaccga atgtttcatc 420

actggctggg gagaaaccca aggtactttt ggagctggcc ttctcaagga agcccagctc 480

cctgtgattg agaataaagt gtgcaatcgc tatgagtttc tgaatggaag agtccaatcc 540

accgaactct gtgctgggca tttggccgga ggcactgaca gttgccaggg tgacagtgga 600

ggtcctctgg tttgcttcga gaaggacaaa tacattttac aaggagtcac ttcttggggt 660

cttggctgtg cacgccccaa taagcctggt gtctatgttc gtgtttcaag gtttgttact 720

tggattgagg gagtgatgag aaataattaa 750

<210>12

<211>249

<212>PRT

<213> Micro-plg (microplasminogen) amino acid sequence

<400>12

Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys

1 5 10 15

Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro

20 25 30

Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly

35 40 45

Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu

50 55 60

Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln

65 70 75 80

Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu

85 90 95

Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser

100 105 110

Pro Ala Val Ile Thr Asp Lys ValIle Pro Ala Cys Leu Pro Ser Pro

115 120 125

Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly

130 135 140

Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu

145 150 155 160

Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly

165 170 175

Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr

180 185 190

Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys

195 200 205

Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala

210 215 220

Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr

225 230 235 240

Trp Ile Glu Gly Val Met Arg Asn Asn

245

<210>13

<211>684

<212>DNA

<213> nucleic acid sequence of serine protease (Domain)

<400>13

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 60

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 120

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 180

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 240

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 300

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 360

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 420

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 480

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 540

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 600

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 660

acttggattg agggagtgat gaga 684

<210>14

<211>228

<212>PRT

<213> amino acid sequence of serine protease (Domain)

<400>14

Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val

1 5 10 15

Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile

20 25 30

Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro

35 40 45

Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn

50 55 60

Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu

65 70 75 80

Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val

85 90 95

Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val

100 105 110

Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln

115 120 125

Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile

130 135 140

Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln

145 150 155 160

Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys

165 170 175

Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr

180 185 190

Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn

195 200 205

Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu

210 215 220

Gly Val Met Arg

225

Claims (10)

1. A method of preventing or treating hepatic collagen deposition or fibrosis resulting from liver tissue damage in a subject, comprising administering to the subject an effective amount of plasminogen.

2. The method of claim 1, wherein said injury comprises ischemia reperfusion injury, drug injury, immunological injury, chemical injury, inflammatory injury, injury due to excessive alcohol consumption, injury due to cancer.

3. The method of claim 2, wherein the damage comprises damage caused by fat deposition.

4. A method of preventing or treating hepatic collagen deposition or fibrosis in a subject, comprising administering to the subject an effective amount of plasminogen.

5. A method of preventing or treating hepatic collagen deposition or fibrosis caused or complicated by diabetes in a subject, comprising administering to the subject an effective amount of plasminogen.

6. A method of preventing or treating hepatic collagen deposition or fibrosis caused or complicated by atherosclerosis in a subject, comprising administering to the subject an effective amount of plasminogen.

7. A method of preventing or treating liver collagen deposition or fibrosis caused by or complicated with hyperlipidemia in a subject, comprising administering to the subject an effective amount of plasminogen.

8. The method of claim 7, wherein the hyperlipidemia comprises one or more selected from the group consisting of: elevated blood triglyceride levels, elevated blood total cholesterol levels, elevated blood low density lipoproteins, and elevated blood very low density lipoproteins.

9. A method of preventing or treating hepatic collagen deposition or fibrosis caused by a pharmaceutical liver injury in a subject, comprising administering to the subject an effective amount of plasminogen.

10. The method of claim 9, wherein the drug is a hepatotoxic drug.