Method for prevention or treatment of acute and chronic thrombosis

Abstract

The present invention relates to the use of plasminogen in the dissolution of fresh and old thrombus. Compared with other existing thrombolytic drugs, the plasminogen of the present invention can specifically dissolve thrombus without causing side effects such as bleeding. The drug of the present invention also has the advantages of dissolving both fresh and old thrombus, with a long half-life and controllable thrombolytic strength. Therefore, plasminogen may become a brand-new strategy for dissolving thrombus in vivo.

Claims

1. A method of preventing and/or eliminating an arterial and venous thrombosis in a subject, comprising administering to the subject an effective amount of plasminogen alone.

2. The method according to claim 1, wherein said thrombus comprises fresh thrombus and old thrombus.

3. The method of claim 1, wherein the thrombosis is a thrombosis caused by a disease selected from the group consisting of a blood system disease, a circulatory system disease, an autoimmune disease, a metabolic disorder disease and an infectious disease.

4. The method of claim 1, wherein the thrombosis is a large vascular thrombosis, small vascular thrombosis or microvascular thrombosis, secondary to diabetes.

5. The method of claim 1, wherein the thrombosis is a thrombosis caused by large and/or small vascular lesions.

6. A method of preventing and/or treating thrombosis-related diseases in a subject, comprising administering to the subject an effective amount of plasminogen alone, wherein the plasminogen prevents and/or treats the thrombosis-related disease in the subject by eliminating the thrombus.

7. The method according to claim 6, wherein the thrombosis-related disease comprises a disease selected from the group consisting of pancreatitis and cirrhosis caused by portal vein thrombosis; renal embolism caused by renal vein thrombosis; systemic sepsis, pulmonary embolism, cerebral thrombosis and deep vein thrombosis caused by internal jugular vein thrombosis; organ infarction caused by arterial or venous thrombosis.

8. The method according to claim 6, wherein the thrombosis-related disease comprises a disease selected from the group consisting of diabetic nephropathy, diabetic retinopathy, diabetic liver disease, diabetic heart disease, diabetic enteropathy and diabetic neuropathy.

9. The method according to claim 1, wherein the plasminogen is administered in combination with a therapeutic drug for other diseases accompanying the thrombosis.

10. The method according to claim 1, wherein the plasminogen is a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the SEQ ID No.2, 6, 8, 10 or 12 and retaining a plasminogen activity.

11. The method according to claim 7, wherein the organ infarction caused by arterial or venous thrombosis comprises a disease selected from the group consisting of cerebral infarction, myocardial infarction, thrombotic stroke, atrial fibrillation, unstable angina pectoris, intractable angina pectoris, transient ischemic attack and pulmonary embolism.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the thrombolytic effect of different doses of plasminogen on 20-hour old thrombus in the presence of 125 ng tPA, incubation at 37 C. for 1 hour.

(2) FIG. 2 shows the thrombolytic effect of different doses of plasminogen on 20-hour old thrombus in the presence of 125 ng tPA, incubation at 37 C. for 2 hours.

(3) FIG. 3 shows the thrombolytic effect of different doses of plasminogen on 20-hour old thrombus when incubated at 37 C. for 2 hours at 10 ng tPA.

(4) FIG. 4 shows the thrombolytic effect of different doses of plasminogen on 72-hour old thrombus when incubated at 37 C. for 2 hours at 125 ng tPA.

(5) FIG. 5 shows the thrombolytic effect of different doses of plasminogen on 72-hour old thrombus when incubated at 37 C. for 2 hours at 10 ng tPA.

(6) FIG. 6 shows changes in thrombolysis rates over time with the addition of 10 ng of tPA and 1 mg of plg or 5 g of tPA alone to 20-hour old thrombus.

(7) FIG. 7 shows the thrombolytic effect of different doses of plasminogen on 20-hour old thrombus when incubated at 37 C. for 1 hour at 100 ng uPA.

(8) FIG. 8 shows the thrombolytic effect of different doses of plasminogen on 20-hour old thrombus when incubated at 37 C. for 2 hours at 1 ng uPA.

(9) FIG. 9 shows the results of a specific adsorption experiment of plasminogen for thrombus in vivo.

(10) FIG. 10 shows the thrombolytic effect of different doses of plasminogen on 30-minute fresh thrombus when incubated at 37 C. for 2 hours at 125 ng tPA.

(11) FIG. 11 shows the results of detection of the concentration of D-dimer in serum after 15 days of plasminogen administration in 24-25 weeks old diabetic mice.

(12) FIG. 12 shows cardiac fibrin immunohistochemical staining results after 31 days of PBS (A) or plasminogen (B) administration in 24-25 weeks old diabetic mice.

(13) FIG. 13 shows renal fibrin immunohistochemical staining results after 31 days of PBS (A) or plasminogen (B) administration in 24-25 weeks old diabetic mice.

(14) FIG. 14 shows liver fibrin immunohistochemical staining results after 31 days of PBS (A) or plasminogen (B) administration in 24-25 weeks old diabetic mice.

(15) FIG. 15 shows sciatic nerve fibrin immunohistochemical staining after 15 days of PBS (A) or plasminogen (B) administration in 24-25 weeks old mice with late diabetic nerve damage.

EXAMPLE

(16) Materials and Methods:

(17) In Vivo Experiments:

Experimental Animals

(18) C57 mice (6-8 weeks old) are purchased from Experimental Animal Center of Southern Medical University. Purchased mice are kept in barrier environment animal rooms. Db/db mice are purchased from Nanjing Institute of Biomedical Research.

Experimental Design and Administration

(19) After the dissection of carotid arteries from all the animals in the control group and the experimental group, unilateral carotid artery thrombosis is modeled with a filter paper containing 10% FeCl.sub.3 for 5 minutes. Intravenous injection of plasminogen is started within 1 hour after the model is established, and the control group is intravenously injected with an equal volume of PBS. After 3 hours, the corresponding jugular vein thrombi and the muscles near the contralateral vein are removed. The thrombi and the muscles near the contralateral vein are homogenized using a grinder, and the supernatant is removed after centrifugation. The supernatant is assayed for its total protein by BCA method, and the plasminogen content in the homogenate is measured by enzyme-linked immunosorbent assay, to calculate the plasminogen content in the certain amount of total protein. Study the specificity of thrombolysis in vivo by plasminogen.

(20) In addition, 24-25-week-old db/db mice are administered solvent PBS or plasminogen through tail veins respectively, as control and experimental animals. After 31 days, eyeballs are taken for D-dimer detection and immunohistochemical staining of fibrin is performed on nerve, liver, kidney and heart, to study the thrombolytic effect of plasminogen in vivo.

(21) Blood D-Dimer Analysis

(22) Eyeballs are taken from the mice to draw blood and obtain plasma. Experiments are performed according to the D-dimer kit (Wuhan USCN, China). After the test is completed, a reading is performed at 450 nm using a microplate reader (Biotek, USA) for data analysis.

(23) Immunohistochemical Analysis

(24) Nerve, liver, kidney and heart are collected, and fixed in 10% neutral formalin for more than 24 hours. The fixed tissues are dehydrated by gradient ethanol and embedded in paraffin. The paraffin is sectioned to a thickness of 5 m and the sections are washed once after deparaffinization to water. Then circle the tissues with a PAP pen. Incubate with hydrogen peroxide diluted with 0.3% methanol for 15 minutes and wash three times. Block with 10% normal serum homologous to the secondary antibody for 10 minutes and absorb excess serum. Incubate with primary antibody for 30 minutes at room temperature or overnight at 4 and wash three times with TBS. Incubate with HRP-labeled secondary antibody for 30 minutes at room temperature and wash three times with TBS. Stain according to DAB kit (vector laboratories, Inc., USA), counterstain with hematoxylin for 30 seconds, flush with water for 5 minutes and then wash once with TBS. Gradient dehydration, clearing and mounting are followed. The antibodies used are: the marker antibody is anti-Fibrinogen antibody (Abcam). Sections are observed under an optical microscope (Olympus, BX43).

(25) In Vitro Thrombolytic Experimental Design:

(26) Healthy human plasma is collected in an ELISA 96-well plate. Add a fixed amount of thrombin (Sigma, USA) to form a thrombus, and then perform the following different experiments. Add fixed amounts of tPA, uPA (sigma, USA) and different amounts of plasminogen, fixed amounts of plasminogen and different amounts of tPA, uPA, streptokinase (sigma, USA), and add PBS in the control group. Incubate for different lengths of time until thrombolysis occurs. The absorbance readings and the time of each measurement are observed and recorded on a microplate reader (Biotek, USA) at the wavelength of OD405. The data is analyzed.

Example 1 Thrombolytic Effect of Different Doses of Plasminogen on 20-Hour Old Thrombus when Incubated at 37 C. for 1 Hour at 125 ng tPA

(27) Whole blood of two SD rats is individually collected into Eppendorf (EP) tubes and the supernatant is discarded after incubation at 37 C. for 20 h to form old thrombus.sup.[33, 34]. Add PBS and wash repeatedly for 5-10 times until the added PBS solution becomes clear. Dry the thrombus with absorbent paper as much as possible. Then place the thrombus evenly in each EP tube and weigh the thrombus. Try to make the weight of each thrombus consistent. The thrombi are divided into PBS blank control group, 125 ng tPA control group, 20 g tPA control group, 0.2 mg plasminogen group, 1 mg plasminogen group and 2 mg plasminogen group. 3 tubes per group. 1 mL PBS is added in PBS blank control group; 1 mL PBS and 125 ng tPA are added in 125 ng tPA control group; 1 mL PBS and 20 g tPA are added in 20 g tPA control group; 1 mL PBS, 125 ng tPA and 0.2 mg plasminogen are added in 0.2 mg plasminogen group; 1 mL PBS, 125 ng tPA and 1 mg plasminogen are added in 1 mg plasminogen group; 1 mL PBS, 125 ng tPA and 2 mg plasminogen are added in 2 mg plasminogen group. All reactions are performed in an incubator at 37. After incubation for 1 hour, the supernatant is aspirated. Dry the thrombus with absorbent paper as much as possible and weigh the thrombus. Calculate the thrombolysis rate.

(28) According to the literature, the content of tPA is 5-10 ng/mL under normal physiological conditions.sup.[35], while in the case of strenuous exercise or venous congestion, the content of tPA in the body increases from 20 times to 100 times, that is, over 100 ng/mL.sup.[36]. Therefore, the dose of tPA used in this experiment is 125 ng/mL to mimic the naturally occurring tPA content in the case of in vivo thrombosis.

(29) The results show that for old thrombi formed in vitro for 20 hours, the thrombolysis rates when adding 0.2 mg, 1 mg, 2 mg of plasminogen under the condition of 125 ng tPA are significantly higher than those when adding 125 ng of tPA alone and the statistical differences are extremely significant, indicating that in the case of naturally occurring tPA levels in the presence of thrombosis in the body, the addition of 0.2 mg or more of plasminogen for 1 hour can significantly promote thrombolysis. Under the condition of 125 ng tPA, adding 1 mg plasminogen can achieve the same thrombolytic effect of in vivo injection of 20 g tPA (according to instructions for alteplase for injection produced by Boehringer Ingelheim, the dose required for thrombolysis in the case of thrombosis in vivo is converted into the required injection dose in rats). That is to achieve the same thrombolysis rate, if there is 1 mg plasminogen in vivo, the required tPA amount can be reduced to the original 1/160. In addition, under the condition of 125 ng tPA, the addition of plasminogen 1 mg reaches the peak of plasminogen thrombolysis, and the addition of 1 times more plasminogen has a decreasing trend in the thrombolysis rate, indicating there is saturation for the addition of plasminogen and the saturation is about 1 to 2 mg (FIG. 1).

Example 2 Thrombolytic Effect of Different Doses of Plasminogen on 20-Hour Old Thrombus when Incubated at 37 C. for 2 Hours at 125 ng tPA

(30) Whole blood of two SD rats is individually collected into EP tubes and the supernatant is discarded after incubation at 37 C. for 20 h to form old thrombus.sup.[33, 34]. Add PBS and wash repeatedly for 5-10 times until the added PBS solution becomes clear. Dry the thrombus with absorbent paper as much as possible. Then place the thrombus evenly in each EP tube and weigh the thrombus. Try to make the weight of each thrombus consistent. The thrombi are divided into PBS blank control group, 125 ng tPA control group, 20 g tPA control group, 0.2 mg plasminogen group, 1 mg plasminogen group and 2 mg plasminogen group. 3 tubes per group. 1 mL PBS is added in PBS blank control group; 1 mL PBS and 125 ng tPA are added in 125 ng tPA control group; 1 mL PBS and 20 g tPA are added in 20 g tPA control group; 1 mL PBS, 125 ng tPA and 0.2 mg plasminogen are added in 0.2 mg plasminogen group; 1 mL PBS, 125 ng tPA and 1 mg plasminogen are added in 1 mg plasminogen group; 1 mL PBS, 125 ng tPA and 2 mg plasminogen are added in 2 mg plasminogen group. All reactions are performed in an incubator at 37. After incubation for 2 hours, the supernatant is aspirated. Dry the thrombus with absorbent paper as much as possible and weigh the thrombus. Calculate the thrombolysis rate.

(31) According to the literature, the content of tPA is 5-10 ng/mL under normal physiological conditions.sup.[35], while in the case of strenuous exercise or venous congestion, the content of tPA in the body increases from 20 times to 100 times, that is, over 100 ng/mL.sup.[36]. Therefore, the dose of tPA used in this experiment is 125 ng/mL to mimic the naturally occurring tPA content in the case of in vivo thrombosis.

(32) The results show that for old thrombi formed in vitro for 20 hours, compared with Example 1, the thrombolysis rate increases as the reaction time prolonged in each group. The thrombolysis rates when adding 0.2 mg, 1 mg, 2 mg of plasminogen under the condition of 125 ng tPA are significantly higher than those when adding 125 ng of tPA alone and the statistical differences are extremely significant, indicating that in the case of naturally occurring tPA doses in the presence of thrombosis in the body, the addition of 0.2 mg or more of plasminogen for 2 hours can significantly promote thrombolysis. After 2 hours of reaction, the thrombolytic effects of the 1 mg and 2 mg plasminogen group are superior to the normal injection dose in vivo of 20 g tPA control group (according to instructions for alteplase for injection produced by Boehringer Ingelheim, the dose required for thrombolysis in the case of thrombosis in vivo is converted into the required injection dose in rats). That is to achieve the same thrombolysis rate, if there is 1 mg of plasminogen in the system, the required tPA amount can be reduced to less than 1/160 of the amount of tPA required (20 g) without 1 mg of plasminogen in the system (FIG. 2).

Example 3 Thrombolysis Rate on 20-Hour Old Thrombus at 10 ng tPA Increases with Increasing Plasminogen Dose

(33) Whole blood of two SD rats is individually collected into EP tubes and the supernatant is discarded after incubation at 37 C. for 20 h to form old thrombus.sup.[33, 34]. Add PBS and wash repeatedly for 5-10 times until the added PBS solution becomes clear. Dry the thrombus with absorbent paper as much as possible. Then place the thrombus evenly in each EP tube and weigh the thrombus. Try to make the weight of each thrombus consistent. The thrombi are divided into PBS blank control group, 10 ng tPA control group, 0.2 mg plasminogen control group, 0.2 mg plasminogen group, 1 mg plasminogen group and 2 mg plasminogen group. 3 tubes per group. 1 mL PBS is added in PBS blank control group; 1 mL PBS and 10 ng tPA are added in 10 ng tPA control group; 1 mL PBS and 0.2 mg plasminogen are added in 0.2 mg plasminogen control group; 1 mL PBS, 10 ng tPA and 0.2 mg plasminogen are added in 0.2 mg plasminogen group; 1 mL PBS, 10 ng tPA and 1 mg plasminogen are added in 1 mg plasminogen group; 1 mL PBS, 10 ng tPA and 2 mg plasminogen are added in 2 mg plasminogen group. All reactions are performed in an incubator at 37. After incubation for 2 hours, the supernatant is aspirated. Dry the thrombus with absorbent paper as much as possible and weigh the thrombus. Calculate the thrombolysis rate.

(34) According to the literature, the content of tPA is 5-10 ng/mL under normal physiological conditions.sup.[35]. Therefore, the dose of tPA used in this experiment is 10 ng/mL to mimic the naturally occurring tPA content in normal physiological conditions in vivo.

(35) The results show that for old thrombi formed in vitro for 20 hours, the thrombolysis rate of each groups added with plasminogen is higher than that of the control group in which the physiological dose of tPA alone is added, under the condition of naturally occurring tPA content (10 ng) under normal physiological conditions in the body and the statistical differences are extremely significant. Moreover, with the increase of the amount of plasminogen additive, the corresponding thrombolysis rate also shows a gradient increase trend, indicating that the rate of dissolution of 20-hour old thrombus can be adjusted by adjusting the dose of plasminogen. In addition, in the presence of 0.2 mg plasminogen, the thrombolysis efficiency is significantly higher in the group with the in vivo physiological level of tPA (10 ng) than in the group without the addition of tPA, and the thrombolytic effect of adding 0.2 mg plasminogen alone is similar to that of adding control PBS, indicating that physiological levels of tPA play a key role in thrombolysis by plasminogen (FIG. 3).

Example 4 Thrombolysis Rate on 72-Hour Old Thrombus at 125 ng tPA Increases with Increasing Plasminogen Dose

(36) Whole blood of two SD rats is individually collected into EP tubes and the supernatant is discarded after incubation at 37 C. for 72 hours to form old thrombus.sup.[36]. Add PBS and wash repeatedly for 5-10 times until the added PBS solution becomes clear. Dry the thrombus with absorbent paper as much as possible. Then place the thrombus evenly in each EP tube and weigh the thrombus. Try to make the weight of each thrombus consistent. The thrombi are divided into PBS blank control group, 125 ng tPA control group, 0.2 mg plasminogen control group, 0.2 mg plasminogen group, 1 mg plasminogen group and 2 mg plasminogen group. 3 tubes per group. 1 mL PBS is added in PBS blank control group; 1 mL PBS and 125 ng tPA are added in 125 ng tPA control group; 1 mL PBS and 0.2 mg plasminogen are added in 0.2 mg plasminogen control group; 1 mL PBS, 125 ng tPA and 0.2 mg plasminogen are added in 0.2 mg plasminogen group; 1 mL PBS, 125 ng tPA and 1 mg plasminogen are added in 1 mg plasminogen group; 1 mL PBS, 125 ng tPA and 2 mg plasminogen are added in 2 mg plasminogen group. All reactions are performed in an incubator at 37. After incubation for 2 hours, the supernatant is aspirated. Dry the thrombus with absorbent paper as much as possible and weigh the thrombus. Calculate the thrombolysis rate.

(37) According to the literature, the content of tPA is 5-10 ng/mL under normal physiological conditions.sup.[35], while in the case of strenuous exercise or venous congestion, the content of tPA in the body increases from 20 times to 100 times, that is, over 100 ng/mL.sup.[36]. Therefore, the dose of tPA used in this experiment is 125 ng/mL to mimic the naturally occurring tPA content in the case of in vivo thrombosis.

(38) The results show that for old thrombi formed in vitro for 72 hours, the thrombolysis rates with addition of plasminogen under the condition of 125 ng tPA are higher than those when adding 125 ng of tPA alone and the statistical differences are extremely significant, indicating that in the case of naturally occurring tPA dose (125 ng) in the presence of thrombosis in the body, the addition of 0.2 mg or more of plasminogen for 2 hours can significantly promote thrombolysis on 72-hour old thrombus. Moreover, with the gradient increase of the dose of plasminogen additive, its thrombolysis rate also shows a gradient increase trend, indicating that the rate of dissolution of old thrombus can be adjusted by adjusting the dose of plasminogen. In addition, the thrombolysis rate of adding 4 mg of plasminogen exceeds the thrombolysis rate of 20 g tPA of normal injection dose (according to instructions for alteplase for injection produced by Boehringer Ingelheim, the dose required for thrombolysis in the case of thrombosis in vivo is converted into the required injection dose in rats) in vivo in this experiment, indicating that under the condition of naturally occurring tPA dose (125 ng) in the presence of thrombosis in the body, the effect of adding plasminogen alone to dissolve old thrombus is superior to that of existing thrombolytic drugs (FIG. 4), which shows that plasminogen can be a thrombolytic material with better thrombolytic effect.

(39) In addition, in Example 2, the addition of 125 ng of tPA alone significantly increases the ability to dissolve the 20-hour thrombus compared to the control PBS group. However, in the present example, for the 72-hour old thrombus, similar to the in vivo situation, the thrombolytic effect is almost the same for the group adding 125 ng tPA alone and the control PBS group, indicating that as the thrombus is getting older, the thrombolytic capacity of tPA naturally produced under physiological conditions gradually decreases, which in one aspect indicates that the model used in the examples can mimic the situation in vivo to some extent.

Example 5 Thrombolysis Rate on 72-Hour Old Thrombus at 10 ng tPA Increases with Increasing Plasminogen Dose

(40) Whole blood of two SD rats is individually collected into EP tubes and the supernatant is discarded after incubation at 37 C. for 72 h to form old thrombus.sup.[36]. Add PBS and wash repeatedly for 5-10 times until the added PBS solution becomes clear. Dry the thrombus with absorbent paper as much as possible. Then place the thrombus evenly in each EP tube and weigh the thrombus. Try to make the weight of each thrombus consistent. The thrombi are divided into PBS blank control group, 10 ng tPA control group, 20 g tPA control group, 0.2 mg plasminogen control group, 0.2 mg plasminogen group, 1 mg plasminogen group, 2 mg plasminogen group and 4 mg plasminogen group. 3 tubes per group. 1 mL PBS is added in PBS blank control group; 1 mL PBS and 10 ng tPA are added in 10 ng tPA control group; 1 mL PBS and 20 g tPA are added in 20 g tPA control group; 1 mL PBS and 0.2 mg plasminogen are added in 0.2 mg plasminogen control group; 1 mL PBS, 10 ng tPA and 0.2 mg plasminogen are added in 0.2 mg plasminogen group; 1 mL PBS, 10 ng tPA and 1 mg plasminogen are added in 1 mg plasminogen group; 1 mL PBS, 10 ng tPA and 2 mg plasminogen are added in 2 mg plasminogen group; 1 mL PBS, 10 ng tPA and 4 mg plasminogen are added in 4 mg plasminogen group. All reactions are performed in an incubator at 37. After incubation for 2 hours, the supernatant is aspirated. Dry the thrombus with absorbent paper as much as possible and weigh the thrombus. Calculate the thrombolysis rate.

(41) According to the literature, the content of tPA is 5-10 ng/mL under normal physiological conditions.sup.[35]. Therefore, the dose of tPA used in this experiment is 10 ng/mL to mimic the naturally occurring tPA content in normal physiological conditions in vivo.

(42) The experimental result shows that for old thrombi formed in vitro for 72 hours, the thrombolysis rate of adding plasminogen is higher than that of adding 10 ng tPA alone when the normal physiologic tPA content is 10 ng/mL in the body and the difference is extremely significant. It is demonstrated that under the condition of naturally occurring tPA dose (10 ng) in the presence of thrombosis in the body, the addition of 0.2 mg or more of plasminogen for 2 hours can significantly promote the dissolution of 72-hour old thrombus. As the dose of plasminogen added increases, the thrombolysis rate also shows a gradient increase, indicating that the rate of dissolving old thrombus can be adjusted by adjusting the dose of plasminogen. Furthermore, the thrombolysis rate of the group with 4 mg of plasminogen added is similar to that of the normal tPA injection dose (according to instructions for alteplase for injection produced by Boehringer Ingelheim, the dose required for thrombolysis in the case of thrombosis in vivo is converted into the required injection dose in rats) (FIG. 5), indicating that under the condition of physiological level of tPA dose (10 ng), the effect of adding plasminogen alone to dissolve old thrombus can reach the effect of existing thrombolytic drugs. In this sense, plasminogen is expected to become a new thrombolytic drug for old thrombus.

Example 6 Plasminogen Moderately Dissolves 20-Hour Old Thrombus

(43) Whole blood of two SD rats is individually collected into EP tubes and the supernatant is discarded after incubation at 37 C. for 20 hours to form old thrombus.sup.[33, 34]. Add PBS and wash repeatedly for 5-10 times until the added PBS solution becomes clear. Dry the thrombus with absorbent paper as much as possible. Then place the thrombus evenly in each EP tube and weigh the thrombus. Try to make the weight of each thrombus consistent. The thrombi are divided into two groups and 12 samples per group. The first group is tPA control group, in which 1 mL PBS and 5 g tPA are added; the second group is plasminogen group, in which 1 mL PBS, 10 ng tPA and 1 mg plasminogen are added. Pre-experiments prove that the thrombolysis rates of these two groups for 20-hour old thrombi are similar within 2 hours (data not shown). All reactions are performed in an incubator at 37. Samples are collected at 0.5 h, 1 h, 1.5 h and 2 h respectively and three samples are collected from the two groups respectively at each time point. Aspirate the supernatant. Dry the thrombus with absorbent paper as much as possible and weigh the thrombus. Calculate the thrombolysis rate.

(44) According to the literature, the content of tPA is 5-10 ng/mL under normal physiological conditions.sup.[35]. Therefore, the dose of tPA used in this experiment is 10 ng/mL to mimic the naturally occurring tPA content in normal physiological conditions in vivo.

(45) Experiments show that for a 20-hour old thrombus, the total thrombolysis rate increases in both groups over time, but between 0 and 0.5 hours and 0.5 to 1 hour, the thrombolytic curve slope of plasminogen group is lower than that of tPA group (FIG. 6). Table 1 shows the comparison of the thrombolytic efficiency of 1 mg of plasminogen and the thrombolytic efficiency of 5 g of tPA alone over time in the presence of 10 ng of tPA. As shown in Table 1, 75% of the total thrombolysis rate for 2 hours in the plasminogen group is concentrated within about 1 hour, and 75% of the total thrombolysis rate for 2 hours in the tPA control group is concentrated in about 0.5 hours. These data clearly show that, relative to tPA, the thrombolysis rate of plasminogen is more moderate than that of tPA (FIG. 6, Table 1).

(46) TABLE-US-00001 TABLE 1 Changes of the thrombolytic efficiency of 1 mg plasminogen and the thrombolytic efficiency of 5 g tPA alone over time in the presence of 10 ng tPA Total Total Total Total 50% of the 75% of the thrombolysis thrombolysis thrombolysis thrombolysis total total rate after rate after rate after rate after thrombolysis thrombolysis incubation for incubation for incubation for incubation for rate for rate for 0.5 hours 1 hour 1.5 hours 2 hours 2 hours 2 hours 10 ng 22.80% 33.55% 36.91% 45.42% 22.71% 34.07% tPA + 1 mg Plg Group 5 g tPA 41.28% 46.88% 49.88% 57.77% 28.88% 43.33% Group

Example 7 Plasminogen Promotes the Dissolution of 20-Hour Old Thrombus Under the Condition of 100 ng uPA

(47) Whole blood of two SD rats is individually collected into EP tubes and the supernatant is discarded after incubation at 37 C. for 20 hours to form old thrombus.sup.[33, 34]. Add PBS and wash repeatedly for 5-10 times until the added PBS solution becomes clear. Dry the thrombus with absorbent paper as much as possible. Then place the thrombus evenly in each EP tube and weigh the thrombus. Try to make the weight of each thrombus consistent. The thrombi are divided into PBS blank control group, 100 ng uPA control group, 0.2 mg plasminogen control group, 0.2 mg plasminogen group, 1 mg plasminogen group and 2 mg plasminogen group. 3 tubes per group. 1 mL PBS is added at the beginning in PBS blank control group; 1 mL PBS and 100 ng uPA are added in 100 ng uPA control group; 1 mL PBS and 0.2 mg plasminogen are added in 0.2 mg plasminogen control group; 1 mL PBS, 100 ng uPA and 0.2 mg plasminogen are added in 0.2 mg plasminogen group; 1 mL PBS, 100 ng uPA and 1 mg plasminogen are added in 1 mg plasminogen group; 1 mL PBS, 100 ng uPA and 2 mg plasminogen are added in 2 mg plasminogen group. All reactions are performed in an incubator at 37. After incubation for 1 hour, the supernatant is aspirated. Dry the thrombus with absorbent paper as much as possible and weigh the thrombus. Calculate the thrombolysis rate.

(48) According to the literature, the tPA Michaelis constant is 0.1810.sup.7 mol/L during the enzymatic reaction with plasminogen as substrate.sup.[37], while the Michaelis constant of uPA is 2.4310.sup.7 mol/L.sup.[38]. In other words, under the same reaction conditions, the affinity of tPA is about 10 times that of uPA within the same reaction time. Therefore, in this experiment, the dose of uPA is estimated to be 100 ng/ml according to the 10 ng tPA/ml used in Example 3.

(49) The results show that for old thrombi formed in vitro for 20 hours, after changing the plasminogen activator 10 ng tPA to 100 ng uPA, the thrombolysis rates of the groups adding 0.2 mg, 1 mg, 2 mg of plasminogen are significantly higher than those when adding 100 ng of uPA alone and the statistical differences are extremely significant (**P<0.01; ***P<0.001).

(50) It is demonstrated that in the case of 100 ng of uPA dose, the addition of 0.2 mg or more of plasminogen for 1 hour can significantly promote thrombolysis and with the increase of the plasminogen additive gradient, the thrombolysis rate also increases significantly (FIG. 7). It indicates that under the condition of 100 ng uPA, plasminogen can promote the dissolution of old thrombus.

Example 8 Plasminogen Promotes the Dissolution of 20-Hour Old Thrombus Under the Condition of 1 ng uPA

(51) Whole blood of two SD rats is individually collected into EP tubes and the supernatant is discarded after incubation at 37 C. for 20 hours to form old thrombus.sup.[33, 34]. Add PBS and wash repeatedly for 5-10 times until the added PBS solution becomes clear. Dry the thrombus with absorbent paper as much as possible. Then place the thrombus evenly in each EP tube and weigh the thrombus. Try to make the weight of each thrombus consistent. The thrombi are divided into PBS blank control group, 1 ng uPA control group, 0.2 mg plasminogen control group, 0.2 mg plasminogen group, 1 mg plasminogen group and 2 mg plasminogen group. 3 tubes per group. 1 mL PBS is added in PBS blank control group; 1 mL PBS and 1 ng uPA are added in 1 ng uPA control group; 1 mL PBS and 0.2 mg plasminogen are added in 0.2 mg plasminogen control group; 1 mL PBS, 1 ng uPA and 0.2 mg plasminogen are added in 0.2 mg plasminogen group; 1 mL PBS, 1 ng uPA and 1 mg plasminogen are added in 1 mg plasminogen group; 1 mL PBS, 1 ng uPA and 2 mg plasminogen are added in 2 mg plasminogen group. All reactions are performed in an incubator at 37. After incubation for 2 hours, the supernatant is aspirated. Dry the thrombus with absorbent paper as much as possible and weigh the thrombus. Calculate the thrombolysis rate.

(52) According to the literature, the content of uPA is 1 ng/mL under normal physiological conditions.sup.[35]. Therefore, the dose of uPA used in this experiment is 1 ng/mL to mimic the naturally occurring uPA content in normal physiological conditions in vivo. The results show that for old thrombi formed in vitro for 20 hours, when the usage of uPA is reduced to 1 ng of normal body content, the thrombolysis rate of old thrombus is generally slow. However, the thrombolysis rates in the 1 mg and 2 mg plasminogen groups are significantly higher than those in the 1 ng uPA control group, with statistical differences. It indicates that under the condition of 1 ng uPA, the addition of plasminogen significantly promotes the dissolution of old thrombus (FIG. 8).

Example 9 Rebleeding Experiment after Intravenous Injection of tPA and Plasminogen in Mice

(53) Fifty-five 11-week-old C57 wild-type male mice are selected and general anesthesia is performed with 3% pentobarbital. Cut 3 mm of tails respectively, place tails in 37 warm water and observe the condition of tail bleeding.sup.[39]. After hemostasis, the mice are randomly divided into two groups, 5 in the tPA group and 50 in the plasminogen group. In the tPA group, 400 g/0.05 mL/body of tPA is injected through the orbital vein; in the plasminogen group, 1 mg/0.05 mL/body of plasminogen is injected through the orbital vein. During the experiment, the mouse tail vein is always placed in warm water at 37 C. The condition of experimental bleeding is observed for 20 minutes and recorded.

(54) The experimental results show that intravenous injection of 400 g tPA can cause rebleeding in tail wounds of wounded mice that have already been coagulated, which is a common side effect of tPA drugs. However, mice injected intravenously with 1 mg of plasminogen do not have such side effects (Table 2), suggesting that plasminogen is safer than tPA.

(55) TABLE-US-00002 TABLE 2 In vivo hemorrhage experimental results after intravenous injection of tPA or plasminogen in mice Rebleeding condition (20 minutes after injection) Drugs Total number of injected Yes No mice tPA 400 g 2 3 5 Plg 1 mg 0 50 50

Example 10 Specific Adsorption Experiment of Plasminogen on Thrombus In Vivo

(56) Nine wild-type mice are selected and are randomly divided into three groups, solvent PBS control group, 0.2 mg plasminogen group and 1 mg plasminogen group. 3 mice per group. General anesthesia is performed by using 3% pentobarbital and the jugular veins of the mice are isolated. Venous thrombus is formed by applying absorbent paper (3 mm5 mm) impregnated with 10% FeCl.sub.3 solution to the jugular vein for 5 minutes. Immediately after thrombus formation, plasminogen or solvent PBS is administered. In solvent PBS control group, 100 l PBS is injected through tail vein and in 1 mg plasminogen group and 0.2 mg plasminogen group, 1 mg and 0.2 mg plasminogen are administered by tail vein injection, respectively. After 3 hours, the corresponding jugular vein thrombi and the muscles near the contralateral vein are removed. The thrombi and the muscles near the contralateral vein are homogenized using a grinder, and the supernatant is removed after centrifugation. The supernatant is assayed for its total protein by BCA method, and the plasminogen content in the homogenate is measured by enzyme-linked immunosorbent assay, to calculate the plasminogen content in the certain amount of total protein.

(57) The results show that the content of plasminogen in thrombus after thrombus formation is significantly higher than that in muscle. In addition, the plasminogen content in the thrombus is further increased after intravenous injection of plasminogen. These results indicate that in the presence of in vivo thrombi plasminogen can specifically bind to thrombi (FIG. 9) and further exerts thrombolytic effects, whereas once tPA is injected into blood vessels, it will non-specifically catalyze thrombolysis in blood vessels. The results of this experiment indicate that plasminogen has a significant advantage of specific thrombolysis over tPA.

Example 11 Thrombolysis Rate of 30-Minute Fresh Thrombus after Adding Plasminogen is Significantly Increased

(58) Whole blood of two SD rats is individually collected into EP tubes and the supernatant is discarded after incubation at 37 C. for 30 minutes to form fresh thrombus.sup.[33]. Add PBS and wash repeatedly for 5-10 times until the added PBS solution becomes clear. Dry the thrombus with absorbent paper as much as possible. Then place the thrombus evenly in each EP tube and weigh the thrombus. Try to make the weight of each thrombus consistent. The thrombi are divided into PBS blank control group, 125 ng tPA control group, 0.2 mg plasminogen group, 1 mg plasminogen group and 2 mg plasminogen group. 2 tubes per group. 1 mL PBS is added in PBS blank control group; 1 mL PBS and 125 ng tPA are added in tPA control group; 1 mL PBS, 125 ng tPA and 0.2 mg plasminogen are added in 0.2 mg plasminogen group; 1 mL PBS, 125 ng tPA and 1 mg plasminogen are added in 1 mg plasminogen group; 1 mL PBS, 125 ng tPA and 2 mg plasminogen are added in 2 mg plasminogen group. All reactions are performed in an incubator at 37. After incubation for 2 hours, the supernatant is aspirated. Dry the thrombus with absorbent paper as much as possible and weigh the thrombus. Calculate the thrombolysis rate.

(59) According to the literature, the content of tPA is 5-10 ng/mL under normal physiological conditions.sup.[35], while in the case of strenuous exercise or venous congestion, the content of tPA in the body increases from 20 times to 100 times, that is, over 100 ng/mL.sup.[36]. Therefore, the dose of tPA used in this experiment is 125 ng/mL to mimic the naturally occurring tPA content in the case of in vivo thrombosis.

(60) This experiment shows that for fresh thrombi formed in vitro for 30 minutes, the thrombolysis rate shows a gradient increase trend under the condition of gradient increase of plasminogen dose. In addition, the thrombolysis rates in each plasminogen group are higher than those in the control group where tPA alone is added, and the statistical differences are extremely significant. These results indicate that in the case of naturally occurring tPA levels in the presence of thrombosis in the body, the addition of 0.2 mg or more of plasminogen for 1 hour can significantly promote thrombolysis (FIG. 10). It is demonstrated that plasminogen can not only promote the dissolution of old thrombus, but also promote the dissolution of fresh thrombus.

Example 12 Plasminogen Promotes Dissolution of Microthrombus Caused by Diabetes

(61) Ten 24-25-week-old db/db male mice are randomly divided into two groups, solvent PBS-treated control group and plasminogen-treated group. 5 mice per group. The day starting the experiment is recorded as day 0 when mice are weighed and grouped. The second day of the experiment when starting administration of plasminogen or PBS is recorded as day 1. The continuous administration is performed for 15 days. The mice in the plasminogen-treated group are injected with plasminogen at a dose of 2 mg/0.2 mL/body/day through tail vein, and those in the solvent PBS-treated control group are administered the same volume of PBS. On the 16th day, eyeballs are taken to draw blood and after the whole blood is left standing, serum is used to detect D-dimer content in blood.

(62) The results show that after 15 days of administration, the content of D-dimer in the plasminogen-treated group is significantly increased (FIG. 11), suggesting that the microthrombus caused by diabetes is significantly dissolved after the administration of plasminogen.

Example 13 Plasminogen Promotes Thrombolysis in Cardiac Tissue in Late-Stage Diabetic Mice

(63) Ten 24-25-week-old db/db male mice are randomly divided into two groups, solvent PBS-treated control group and plasminogen-treated group. 5 mice per group. The day starting the experiment is recorded as day 0 when mice are weighed and grouped. The second day of the experiment when starting administration of plasminogen or PBS is recorded as day 1. The continuous administration is performed for 31 days. The mice in the plasminogen-treated group are injected with plasminogen at a dose of 2 mg/0.2 mL/body/day through tail vein, and those in the solvent PBS-treated control group are administered the same volume of PBS. Mice are sacrificed on day 32 and hearts are collected and fixed in 10% neutral formalin for 24 hours. The fixed cardiac tissue is dehydrated in gradient ethanol and cleared in xylene, followed by being paraffin-embedded. The thickness of the tissue section is 5 After dewaxing and rehydration, the sections are washed with water once, incubated with 3% hydrogen peroxide for 15 minutes, and washed with water twice for 5 minutes each time. Block with 10% normal sheep serum (Vector laboratories, Inc., USA) for 1 hour; then discard the sheep serum and circle the tissue with a PAP pen. Incubate with rabbit anti-mouse fibrinogen antibody (Abcam) at 4 overnight and wash with TBS twice for 5 minutes each time. Incubate with the secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam) for 1 hour at room temperature and wash with TBS twice for 5 minutes each time. Stain according to DAB kit (Vector laboratories, Inc., USA), counterstain with hematoxylin for 30 seconds after washing with water 3 times and flush with water for 5 minutes. Gradient dehydration, clearing and mounting are followed. Sections are observed under a microscope at 400 times.

(64) Fibrinogen is a precursor of fibrin. In the presence of tissue damage, as a stress response to the body's damage, fibrinogen is hydrolyzed into fibrin.sup.[40-42], so fibrin level can be used as a sign of the degree of damage. Fibrin is also a major component of thrombus formed after tissue damage. Therefore, fibrin level can also be used as a marker of thrombus.

(65) The results show that compared with the solvent PBS-treated control group (FIG. 12A), the plasminogen-treated group (FIG. 12B) has a lighter positive staining of fibrin in cardiac tissue of mice, indicating the reduction of fibrin deposition in the cardiac tissue of the plasminogen-treated group, which reflects that plasminogen can promote the repair of cardiac tissue damage caused by diabetes, and it also demonstrates that plasminogen can promote the dissolution of cardiac tissue thrombus.

Example 14 Plasminogen Promotes Thrombolysis in Renal Tissue in Late-Stage Diabetic Mice

(66) Twenty 24-25-week-old db/db male mice are randomly divided into two groups, solvent PBS-treated control group and plasminogen-treated group. 10 mice per group. The day starting the experiment is recorded as day 0 when mice are weighed and grouped. The second day of the experiment when starting administration of plasminogen or PBS is recorded as day 1. The continuous administration is performed for 31 days. The mice in the plasminogen-treated group are injected with plasminogen at a dose of 2 mg/0.2 mL/body/day through tail vein, and those in the solvent PBS-treated control group are administered the same volume of PBS. Mice are sacrificed on day 32 and kidneys are collected and fixed in 10% neutral formalin for 24 hours. The fixed renal tissue is dehydrated in gradient ethanol and cleared in xylene, followed by being paraffin-embedded. The thickness of the tissue section is 5 m. After dewaxing and rehydration, the sections are washed with water once, incubated with 3% hydrogen peroxide for 15 minutes, and washed with water twice for 5 minutes each time. Block with 10% normal sheep serum (Vector laboratories, Inc., USA) for 1 hour; when time is up, discard the sheep serum and circle the tissue with a PAP pen. Incubate with rabbit anti-mouse fibrinogen antibody (Abcam) at 4 overnight and wash with TBS twice for 5 minutes each time. Incubate with the secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam) for 1 hour at room temperature and wash with TBS twice for 5 minutes each time. Stain according to DAB kit (Vector laboratories, Inc., USA), counterstain with hematoxylin for 30 seconds after washing with water 3 times and flush with water for 5 minutes. Gradient dehydration, clearing and mounting are followed. Sections are observed under a microscope at 200 times.

(67) Fibrinogen is a precursor of fibrin. In the presence of tissue damage, as a stress response to the body's damage, fibrinogen is hydrolyzed into fibrin.sup.[40-42], so fibrin level can be used as a sign of the degree of damage. Fibrin is also a major component of thrombus formed after tissue damage. Therefore, fibrin level can also be used as a marker of thrombus.

(68) The results show that the plasminogen-treated group (FIG. 13B) has a lighter positive staining of fibrinogen than the solvent PBS-treated control group (FIG. 13A). It indicates that injection of plasminogen can significantly reduce renal fibrin deposition in diabetic mice, which shows that plasminogen has significant repair effect on kidney damage in diabetic mice, and it also demonstrates that plasminogen can promote the dissolution of renal tissue thrombus.

Example 15 Plasminogen Promotes Thrombolysis in Liver Tissue in Late-Stage Diabetes

(69) Ten 24-25-week-old db/db male mice are randomly divided into two groups, solvent PBS-treated control group and plasminogen-treated group. 5 mice per group. The day starting the experiment is recorded as day 0 when mice are weighed and grouped. The second day of the experiment when starting administration of plasminogen or PBS is recorded as day 1. The continuous administration is performed for 31 days. The mice in the plasminogen-treated group are injected with plasminogen at a dose of 2 mg/0.2 mL/body/day through tail vein, and those in the solvent PBS-treated control group are administered the same volume of PBS. Mice are sacrificed on day 32 and livers are collected and fixed in 10% neutral formalin for 24 hours. The fixed liver tissue is dehydrated in gradient ethanol and cleared in xylene, followed by being paraffin-embedded. The thickness of the tissue section is 5 After dewaxing and rehydration, the sections are washed with water once, incubated with 3% hydrogen peroxide for 15 minutes, and washed with water twice for 5 minutes each time. Block with 10% normal sheep serum (Vector laboratories, Inc., USA) for 1 hour; when time is up, discard the sheep serum and circle the tissue with a PAP pen. Incubate with rabbit anti-mouse fibrinogen antibody (Abcam) at 4 overnight and wash with TBS twice for 5 minutes each time. Incubate with the secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam) for 1 hour at room temperature and wash with TBS twice for 5 minutes each time. Stain according to DAB kit (Vector laboratories, Inc., USA), counterstain with hematoxylin for 30 seconds after washing with water 3 times and flush with water for 5 minutes. Gradient dehydration, clearing and mounting are followed. Sections are observed under a microscope at 200 times.

(70) Fibrinogen is a precursor of fibrin. In the presence of tissue damage, as a stress response to the body's damage, fibrinogen is hydrolyzed into fibrin.sup.[40-42], so fibrin level can be used as a sign of the degree of damage. Fibrin is also a major component of thrombus formed after tissue damage. Therefore, fibrin level can also be used as a marker of thrombus.

(71) The study finds that compared with the solvent PBS-treated control group (FIG. 14A), the plasminogen-treated group (FIG. 14B) has a lighter positive staining of fibrin in liver tissue of mice, indicating that injection of plasminogen can significantly reduce liver fibrin deposition in diabetic mice, which reflects that plasminogen has significant repair effect on liver damage in diabetic mice, and it also demonstrates that plasminogen can promote the dissolution of liver tissue thrombus.

Example 16 Plasminogen Promotes Thrombolysis in Nerve Tissue in Mice with Late-Stage Diabetic Nerve Damage

(72) Ten 24-25-week-old db/db male mice are randomly divided into two groups, solvent PBS-treated control group and plasminogen-treated group. 5 mice per group. The day starting the experiment is recorded as day 0 when mice are weighed and grouped. The second day of the experiment when starting administration of plasminogen or PBS is recorded as day 1. The continuous administration is performed for 15 days. The mice in the plasminogen-treated group are injected with plasminogen at a dose of 2 mg/0.2 mL/body/day through tail vein, and those in the solvent PBS-treated control group are administered the same volume of PBS. Mice are sacrificed on day 16 and sciatic nerves are collected and fixed in 10% neutral formalin for 24 hours. The fixed sciatic nerve is dehydrated in gradient ethanol and cleared in xylene, followed by being paraffin-embedded. The thickness of the tissue section is 5 After dewaxing and rehydration, the sections are washed with water once and then the tissues are circled with a PAP pen. Incubate with 3% TBS diluted hydrogen peroxide for 15 minutes and wash with water 3 times. Block with 10% normal sheep serum (Vector laboratories, Inc., USA) for 1 hour and absorb excess serum. Incubate with rabbit anti-mouse fibrinogen antibody (Abcam) at room temperature for 1 hour or at 4 overnight and wash with TBS 3 times. Incubate with the secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam) for 1 hour at room temperature and wash with TBS 3 times. Stain according to DAB kit (Vector laboratories, Inc., USA), counterstain with hematoxylin for 30 seconds after washing with water 3 times and flush with water for 5 minutes. Gradient dehydration, clearing and mounting are followed. Sections are observed under a microscope at 400 times.

(73) Fibrinogen is a precursor of fibrin. In the presence of tissue damage, as a stress response to the body's damage, fibrinogen is hydrolyzed into fibrin.sup.[40-42], so fibrin level can be used as a sign of the degree of damage. Fibrin is also a major component of thrombus formed after tissue damage. Therefore, fibrin level can also be used as a marker of thrombus.

(74) The study finds that compared with the solvent PBS-treated control group (FIG. 15A), the sciatic nerve fibrin levels are reduced in mice in the plasminogen-treated group (FIG. 15B), indicating that plasminogen has the function of degrading fibrin levels and the damage is repaired to a certain extent, which also demonstrates that plasminogen can promote the dissolution of thrombi around nerve tissue.

Summary of Experimental Results

(75) Experiments in the embodiments of the present invention include two parts of in vitro thrombolysis and in vivo thrombolysis of plasminogen.

(76) In vitro thrombolysis mimics the conditions of in vivo thrombolysis. 10 ng/mL tPA is selected to mimic the naturally occurring tPA levels in the body under normal physiological conditions, and 125 ng/mL tPA is selected to mimic the naturally occurring tPA levels in the case of thrombosis in the body, to study plasminogen thrombolytic capacity.

(77) The experimental study of the present invention shows that under the condition of 10 ng/mL tPA or 125 ng/mL tPA, plasminogen has very good thrombolytic effect whether it is a 20-hour old thrombus or a 72-hour old thrombus. And with the increase of plasminogen dose, the thrombolytic efficiency increases.

(78) Plasminogen has a strong ability to dissolve fresh thrombus, and the thrombolysis rate can reach over 80% after two hours of incubation.

(79) We also study the thrombolytic effect of plasminogen in the presence of uPA. Under the conditions of 1 ng/mL uPA or 100 ng/mL uPA, plasminogen also has very good thrombolytic effect. And with the increase of plasminogen dose, the thrombolytic efficiency increases.

(80) In the experiments in vivo, late-stage diabetic mice are administered 2 mg of plasminogen daily for 15 consecutive days and the D-dimer content in the serum is significantly increased. At the same time, the fibrin levels of heart, liver, kidney and nerve tissues significantly decrease, indicating that plasminogen can obviously promote the dissolution of thrombi caused by diabetes-induced damage in these tissues and fibrin degradation, which proves that the administration of plasminogen to experimental animals can also achieve significant thrombolytic effect.

(81) The mouse jugular vein thrombosis model experiment shows that plasminogen can bind to thrombus in vivo very specifically.

(82) In addition, our study showed that thrombolysis of plasminogen is more moderate than that of tPA, and tail-bleeding experiments in mice showed no bleeding side effects of plasminogen.

(83) In summary, plasminogen has a very good thrombolytic capacity, especially for the old thrombus, and has the characteristics of high specificity, moderate strength, quick effect and no bleeding side effects.

REFERENCES

(84) [1] Yuan guiqing, Thrombotic disease has become an important disease that threatens human health and life. Chinese Journal of Laboratory Medicine, 2004, 8(27)487. [2] Alexander C M and Werb, Z. (1991). Extracellular matrix degradation. In Cell Biology of Extracellular Matrix, Hay E D, ed. (New York: Plenum Press), pp. 255-302. [3] Werb, Z., Mainardi, C. L., Vater, C. A., and Harris, E. D., Jr. (1977). Endogenous activiation of latent collagenase by rheumatoid synovial cells. Evidence for a role of plasminogen activator. N. Engl. J. Med. 296, 1017-1023. [4] He, C. S., Wilhelm, S. M., Pentland, A. P., Marmer, B. L., Grant, G. A., Eisen, A. Z., and Goldberg, G. I. (1989). Tissue cooperation in a proteolytic cascade activating human interstitial collagenase. Proc. Natl. Acad. Sci. U. S. A 86, 2632-2636. [5] Stoppelli, M. P., Corti, A., Soffientini, A., Cassani, G., Blasi, F., and Assoian, R. K. (1985). Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes. Proc. Natl. Acad. Sci. U. S. A 82, 4939-4943. [6] Vassalli, J. D., Baccino, D., and Belin, D. (1985). A cellular binding site for the Mr 55,000 form of the human plasminogen activetor, urokinase. J. Cell Biol. 100, 86-92. [7] Wiman, B. and Wallen, P. (1975). Structural relationship between glutamic acid and lysine forms of human plasminogen and their interaction with the NH2-terminal activation peptide as studied by affinity chromatography. Eur. J. Biochem. 50, 489-494. [8] Saksela, O. and Rifkin, D. B. (1988). Cell-associated plasminogen activation: regulation and physiological functions. Annu. Rev. Cell Biol. 4, 93-126 [9] Raum, D., Marcus, D., Alper, C. A., Levey, R., Taylor, P. D., and Starzl, T. E. (1980). Synthesis of human plasminogen by the liver. Science 208, 1036-1037 [10] Wallen P (1980). Biochemistry of plasminogen. In Fibrinolysis, Kline D L and Reddy K K N, eds. [11] Sottrup-Jensen, L., Zaj del, M., Claeys, H., Petersen, T. E., and Magnusson, S. (1975). Amino-acid sequence of activation cleavage site in plasminogen: homology with pro part of prothrombin. Proc. Natl. Acad. Sci. U. S. A 72, 2577-2581. [12] Collen, D. and Lijnen, H. R. (1991). Basic and clinical aspects of fibrinolysis and thrombolysis. Blood 78, 3114-3124. [13] Alexander, C. M. and Werb, Z. (1989). Proteinases and extracellular matrix remodeling. Curr. Opin. Cell Biol. 1, 974-982. [14] Mignatti, P. and Rifkin, D. B. (1993). Biology and biochemistry of proteinases in tumor invasion. Physiol Rev. 73, 161-195. [15] Collen, D. (2001). Ham-Wasserman lecture: role of the plasminogen system in fibrin-homeostasis and tissue remodeling. Hematology. (Am. Soc. Hematol. Educ. Program.) 1-9. [16] Rifkin, D. B., Moscatelli, D., Bizik, J., Quarto, N., Blei, F., Dennis, P., Flaumenhaft, R., and Mignatti, P. (1990). Growth factor control of extracellular proteolysis. Cell Differ. Dev. 32, 313-318. [17] Andreasen, P. A., Kjoller, L., Christensen, L., and Duffy, M. J. (1997). The urokinase-type plasminogen activator system in cancer metastasis: a review. Int. J. Cancer 72, 1-22. [18] Rifkin, D. B., Mazzieri, R., Munger, J. S., Noguera, I., and Sung, J. (1999). Proteolytic control of growth factor availability. APMIS 107, 80-85. [19] Hillis L D, et. al. High dose intravenous streptokinase for acute myocardial infarction: preliminary results of a multicenter trial. J Am Coll Cardiol. 1985; 6:957-962. [20] Smalling R W. A fresh look at the molecular pharmacology of plasminogen activators: from theory to test tube to clinical outcomes. Am J Health-Syst Pharm 1997; 54(suppl 1):S17-S22. [21] Nobel S, McTavish D. Reteplase: a review of it pharmacological properties and clinical efficiency in the management of acute myocardial infarction. [J]. Drug, 1996, 52(4):589-605. [22] Abdoli-Nasab Ml, Jalali-Javaran M, Expression of the truncated tissue plasminogen activator (K2S) gene in tobacco chloroplast, Mol Biol Rep (2013) 40:5749-5758 [23] Gottlob R. (1975) Plasminogen and plasma inhibitors inarterial and venous thrombi of various ages. In: Progress inchemical fibrinolysis and thrombolysis. vol. 1. Raven Press, New York, pp. 23-36. [24] Sabovic M, Lijnen H R, Keber D, Collen D. (1989) Effect ofretraction on the lysis of human clots with fibrin specific andnon-fibrin specific plasminogen activators. Thromb Haemost, 62, 1083-1087. [25] Potter van Loon B J, Rijken D C, Brommer E J, van der MaasAP. (1992) The amount of plasminogen, tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in human thrombi and the relation to ex-vivo lysibility. Thromb Haemost, 67, 101-105. [26] Hacke W, Kaste M, Bluhmki E, Brozman M, Dvalos A et al. (2008) Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke. N Engl J Med 359: 1317-1329. [27] Lees K R, Bluhmki E, von Kummer R, Brott T G, Toni D et al. (2010) Time to treatment with intravenous alteplase and outcome in stroke: anupdated pooled analysis of Ecass, Atlantis, Ninds, and Epithet trials. Lancet 375. [28] Marder V J, Novokhatny V. Direct fibrinolytic agents: biochemical attributes, preclinical foundation and clinical potential [J]. Journal of Thrombosis and Haemostasis, 2010, 8(3): 433-444. [29] Hunt J A, Petteway Jr S R, Scuderi P, et al. Simplified recombinant plasmin: production and fu-nctional comparison of a novel thrombolytic molecule with plasma-derived plasmin [J]. Thromb Haemost, 2008, 100(3): 413-419. [30] Sottrup-Jensen L, Claeys H, Zajdel M, et al. The primary structure of human plasminogen: Isolation of two lysine-binding fragments and one mini-plasminogen (MW, 38,000) by elastase-catalyzed-specific limited proteolysis [J]. Progress in chemical fibrinolysis and thrombolysis, 1978, 3: 191-209. [31] Nagai N, Demarsin E, Van Hoef B, et al. Recombinant human microplasmin: production and potential therapeutic properties [J]. Journal of Thrombosis and Haemostasis, 2003, 1(2): 307-313. [32] Valery V. Novokhatny, Gary J. Jesmok, Locally delivered plasmin: why should it be superior to plasminogen activators for direct thrombolysis, Trends in Pharmacological Sciences Vol. 25 No. 2 Feb. 2004 [33]V. Novokhatny, K. Talylor and T. P. Zimmerman, Thrombolytic potency of acid-stabilized plasmin: superiority over tissue-type plasminogen activator in an in vitro model of catheter-assisted thrombolysis, Journal of Thrombosis and Haemostasis, 1: 1034-1041 [34] F Bachmann, Springer, Fibrinolytics and antifibrinolytics, 2001, 146(4):670 [35] R. B. Aisinal and L. I. Mukhametova, Structure and Function of Plasminogen/Plasmin System, Russian Journal of Bioorganic Chemistry, 2014, Vol. 40, No. 6, pp. 590-605. [36] Kyle Landskroner, M S, Neil Olson, D V M, PhD, and Gary Jesmok, PhD, Cross-Species Pharmacologic Evaluation of Plasmin as a Direct-Acting Thrombolytic Agent: Ex Vivo Evaluation for Large Animal Model Development, J Vasc Intery Radiol 2005; 16:369-377. [37] Edvin L. Madison, Gary S. Coombs, and Dacid R. Corey, Substrate Specificity of Tissue Type Plasminogen Activator, The Journal of biological, Chemistry, 1995, Vol. 270, No. 13, pp. 7558-7562. [38] Kei Takahashi, Hau C. Kwaan, Enki Koh, ardMasatakaTanabe, Enzymatic Properties Of The Phosphorylated Urokinase-Type Plasminogen Activator Isolated From A Human Carcinomatous Cell Line, Biochemical and Biophysical Research Communications, 1992 Pages 1473-1481 [39] Hui Y H, Huang N H, Ebbert L et al. Pharmacokinetic comparisons of tail-bleeding with cannula- or retro-orbital bleeding techniques in rats using six marketed drugs. J Pharmacol Toxicol Methods. 2007 September-October; 56(2):256-64. [40] Jae Kyu Ryu, Mark A. Petersen, Sara G. Murray et al. Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation. Nature Communications, 2015, 6:8164. [41] Dimitrios Davalos, Katerina Akassoglou. Fibrinogen as a key regulator of inflammation in disease. Seminars in Immunopathology, 2012. 34(1):43-62. [42] Valvi D, Mannino D M, Mullerova H, et al. Fibrinogen, chronic obstructive pulmonary disease (COPD) and outcomes in two United States cohorts. Int J Chron Obstruct Pulmon Dis 2012; 7:173-82.