An antithrombotic polypeptide and an antithrombotic drug

Abstract

The application provides an antithrombotic polypeptide and an antithrombotic drug, and belongs to the technical field of biological medicines. The application provides an antithrombotic polypeptide, and the amino acid sequence of the antithrombotic polypeptide is shown in SEQ ID No. 1. The antithrombotic polypeptide can significantly prolong plasma recalcification time, has excellent anticoagulant activity, and can effectively inhibit the formation of arterial thrombosis induced by ferric chloride and the formation of venous thrombosis induced by inferior vena cava ligation. The antithrombotic polypeptide has simple structure, is easy to be artificially synthesized, and has outstanding anticoagulant activity, and can be used as an effective component for the research and preparation of an antithrombotic drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antithrombotic polypeptide and an antithrombotic drug. Background Technology

[0002] Thrombosis is a pathological process in the living cardiovascular system where blood abnormally coagulates or forms solid masses by aggregation and adhesion of blood components. A thrombus can either adhere to the vessel wall in situ or detach and travel through the bloodstream as an embolus, causing distal vascular embolism, blocking blood supply to tissues and organs, leading to local ischemic and hypoxic damage, and in severe cases, organ failure and endangering life.

[0003] Despite continuous advancements in the prevention and treatment of thrombotic diseases in recent years, thrombosis-related diseases remain among the leading causes of death worldwide. The Virchow triad—hemodynamic abnormalities, alterations in blood composition, and vascular endothelial injury—works synergistically to mediate thrombus formation and remains the core theory explaining the mechanism of thrombosis. Clinically, most existing antithrombotic drugs carry a high risk of bleeding and have significant limitations in therapeutic safety, restricting their clinical applicability to some extent. Therefore, developing novel anticoagulant and antithrombotic candidate molecules with superior safety profiles and exploring new drug design strategies have become important research directions in the field of thrombosis prevention and treatment.

[0004] Previous studies have attempted to discover natural anticoagulant peptides from biological resources. In 2015, Australian scholars SL Ranasinghe et al. (Ranasinghe SL, Fischer K, Gobert GN, et al. A novel coagulation inhibitor from Schistosoma japonicum [J]. Parasitology, 2015, 142(14): 1663-1672.) screened and obtained the anticoagulant peptide SjKI-1 from the Schistosoma japonicum cDNA library. However, its in vitro and in vivo anticoagulant effects are limited and cannot meet the needs of clinical application. Further development of novel anticoagulant peptides with better activity is still needed. Summary of the Invention

[0005] This invention provides an antithrombotic polypeptide and an antithrombotic drug. The polypeptide has excellent anticoagulant effect and can effectively improve the hypercoagulable state of blood in patients with thrombosis. It is suitable for the preparation of antithrombotic drugs.

[0006] The present invention provides an antithrombotic polypeptide, the amino acid sequence of which is shown in SEQ ID No. 1.

[0007] This invention also provides the application of the above-mentioned antithrombotic peptides in the preparation of antithrombotic drugs.

[0008] In one specific embodiment of the present invention, the therapeutic effect of the antithrombotic drug includes at least one of the following: prolonging plasma recalcification time and inhibiting thrombosis.

[0009] In one specific embodiment of the present invention, the antithrombotic drug is used to prevent arterial thrombosis and venous thrombosis.

[0010] In one specific embodiment of the present invention, the arterial thrombosis includes arterial thrombosis induced by FeCl3, and the venous thrombosis includes venous thrombosis induced by inferior vena cava ligation.

[0011] In one specific embodiment of the present invention, when treating arterial and venous thrombosis, the effective dose of the antithrombotic polypeptide in the antithrombotic drug is 0.006~0.1 mg / kg.

[0012] In one specific embodiment of the present invention, when treating arterial thrombosis, the effective dose of the antithrombotic polypeptide in the antithrombotic drug is 0.03 mg / kg; When treating venous thrombosis, the effective dose of the antithrombotic polypeptide in the antithrombotic drug is 0.03 mg / kg.

[0013] The present invention also provides an antithrombotic drug, the active ingredient of which includes the above-mentioned antithrombotic polypeptide, and also includes pharmaceutically acceptable excipients.

[0014] In one specific embodiment of the present invention, the concentration of the antithrombotic polypeptide in the drug is 0.01~0.3 μM.

[0015] In one specific embodiment of the present invention, the concentration of the antithrombotic polypeptide in the drug is 0.1 μM.

[0016] Beneficial Effects: This invention provides an antithrombotic polypeptide with the amino acid sequence shown in SEQ ID No. 1. This antithrombotic polypeptide can significantly prolong plasma recalcification time and possesses excellent anticoagulant activity; simultaneously, it can effectively inhibit ferric chloride-induced arterial thrombosis and inferior vena cava ligation-induced venous thrombosis. The antithrombotic polypeptide of this invention has a simple structure, is easy to synthesize artificially, and exhibits outstanding anticoagulant activity, making it suitable as an active ingredient for the research and preparation of antithrombotic drugs. Attached Figure Description

[0017] Figure 1 To prolong plasma recalcification time with different concentrations of peptide GD8; in the figure, A: absorbance at 650 nm; B: time corresponding to maximum absorbance; n=3. p <0.001; Figure 2This study presents the effect of peptide GD8 on a FeCl3-induced mouse carotid artery thrombosis model. A represents the blood flow monitoring results of the FeCl3-induced mouse carotid artery thrombosis model after administration of the saline group (NC) and different concentrations of GD8; B represents the statistical results of A based on blood flow perfusion units; n=6. p <0.01, p <0.001; Figure 3 This image shows the effect of peptide GD8 in a mouse inferior vena cava (IVC) thrombosis model. A: Photographs of IVC thrombi in the saline group (NC), GD8-0.03 mg / kg group, and GD8-0.1 mg / kg group; B: Statistical results of IVC thrombus weight in the three groups; C: Statistical results of IVC thrombus length and weight in the three groups; n=3. P <0.05, p <0.01. Detailed Implementation

[0018] The present invention provides an antithrombotic polypeptide, the amino acid sequence of which is shown in SEQ ID No. 1.

[0019] The amino acid sequence of the antithrombotic polypeptide of the present invention is shown in SEQ ID No.1: GKMTRRGD, with a molecular weight of 920.05 Daltons, abbreviated as GD8.

[0020] This invention does not specifically limit the preparation method and source of the antithrombotic peptide. It can be prepared by exogenous expression or directly synthesized by chemical synthesis. In one embodiment, the GD8 of this invention is obtained based on a full-sequence synthesis and purification method, such as synthesis using an automated peptide synthesizer. This invention does not specifically limit the method of synthesis using an automated peptide synthesizer; peptide synthesis methods well known to those skilled in the art can be used. The purification method of this invention preferably uses reversed-phase high-performance liquid chromatography (RP-HPLC). This invention does not have strict requirements on the specific steps of the R-HPLC method; methods well known to those skilled in the art can be used to ensure that the purity of the obtained GD8 is above 95%. The GD8 of this invention is relatively simple, easy to synthesize artificially, can strongly prolong plasma recalcification time, has a very significant anticoagulant effect, and strongly inhibits ferric chloride-induced arterial thrombosis.

[0021] This invention also provides the application of the above-mentioned antithrombotic peptides in the preparation of antithrombotic drugs.

[0022] The thrombi described in this invention include arterial thrombi and venous thrombi. In one embodiment, FeCl3 is used to induce the formation of arterial thrombi, and inferior vena cava ligation is used to induce venous thrombi.

[0023] The effective dose of the polypeptide GD8 described in this invention is 0.006~0.1 mg / kg. In one embodiment, by constructing an animal model of ferric chloride-induced thrombosis, the polypeptide GD8 was administered at a dose of 0.006~0.1 mg / kg. Compared with the control group, it could significantly inhibit FeCl3-induced blood flow reduction and inhibit ferric chloride-induced arterial thrombosis.

[0024] In another embodiment, by constructing an animal model of venous thrombosis induced by inferior vena cava ligation, the peptide GD8 was administered at a dose of 0.006~0.1 mg / kg. Compared with the control group, it significantly reduced the weight and length of thrombi in the inferior vena cava, inhibited the formation of venous thrombi, and showed good venous thrombosis prevention and control effects in a dose-dependent manner.

[0025] The present invention also provides an antithrombotic drug, the active ingredient of which includes the above-mentioned antithrombotic polypeptide, and also includes pharmaceutically acceptable excipients.

[0026] The concentration of peptide GD8 in the drug described in this invention is 0.01~0.3 μM, such as 0.01 μM, 0.05 μM, 0.1 μM, 0.2 μM or 0.3 μM, or any concentration within the range formed by any two of the above exemplary concentrations. This invention does not have strict requirements on the specific type of pharmaceutically acceptable excipients; conventional selection based on the drug's dosage form is sufficient.

[0027] To further illustrate the present invention, the following detailed description of an antithrombotic polypeptide and an antithrombotic drug provided by the present invention is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1: Synthesis of peptide GD8 (GKMTRRGD) The following steps were performed using Fmoc-Lys(Boc)-Wang Resin: 1. Resin swelling Weigh 5g of Fmoc-Leu-Wang Resin with a substitution degree of 0.3, add it to the reaction column, add 50 ml of DCM to swell, and soak for 30 min.

[0029] 2. Remove protection Remove DCM from the reaction column and add 20% piperidine / DMF solution to remove Fmoc. Purge with nitrogen for 30 min, then remove the column. Wash with DMF 5 times and remove the column again.

[0030] 3. Condensation reaction Generally, TBTU / DIEA activator is used for condensation. TBTU and linked amino acids are 3 times the amount of feed, dissolved in 50 ml of DMF and added to the reaction column. DIEA (1.55 ml) is added, nitrogen is used for agitation, and the reaction time is 1 hour.

[0031] 4. Testing and washing Take a small amount of resin from the reaction column and place it in a small test tube. Wash it once with DMF, discard the DMF, and then add 3 drops each of solution A (ninhydrin alcohol solution), solution B (a mixed solution of 20% alcohol and 80% phenol), and solution C (re-distilled pyridine). Place the tube in a heater at 120 °C for 3 min. Remove the tube and observe the color of the solution and resin. If the resin is colorless or pale yellow, it indicates that the condensation reaction is complete. Stop the reaction, remove the resin, wash it three times with DMF, and remove the resin again.

[0032] 5. Reinvestment If the resin is found to be a different color, such as blue, green, or purple, it indicates that the reaction is incomplete. Continue to dry and wash it three times, then add the same amount again to react until the reaction is complete.

[0033] 6. Continue to condense Repeat steps 2-5 to link the remaining amino acids, using the same algorithm to calculate and weigh different amounts of amino acids, while keeping the amount of TBTU / DIEA constant.

[0034] 7. Contraction After all amino acids are linked, repeat step 2, then wash three times with DCM and three times with methanol, dry under vacuum, pour out the resin, dry it, and put it into a 500 ml beaker.

[0035] 8. Cutting Pour 100 ml of cutting fluid (volume ratio of TFA: methyl sulfide: phenol: EDT: water = 86:5:4:3:2) into a beaker, place a magnetic stir bar in the beaker, and place the beaker on a magnetic stirrer. Stir for 2 hours. After filtering with a sintered sand funnel, wash the resin in the sintered sand core twice with TFA. Add the cutting fluid to 600 ml of ice-cold diethyl ether to precipitate the peptide, forming a peptide-diethyl ether suspension. Centrifuge the suspension, discard the supernatant, wash three times with diethyl ether, and then bake with a baking lamp to remove residual diethyl ether. The resulting solid is the crude peptide.

[0036] 9. Purification The crude peptide was dissolved in water, filtered, and then separated and purified by high performance liquid chromatography (HPLC). The purity of the final target peptide was over 95%. When using HPLC, a 100DAC column was selected. The sample was added to the column, and the gradient was selected according to the elution time of the crude product for separation. The target peptide was identified by LC-MS.

[0037] 10. Safety Inspection (1) Cytotoxicity test The cellular safety of peptide GD8 was evaluated using human umbilical vein endothelial cells (HUVECs). HUVECs in logarithmic growth phase were seeded in 96-well plates, with a blank control group, a saline control group, and different concentrations of peptide GD8 (0.01 μM, 0.1 μM, and 0.3 μM) in each group, with six replicates per group. Cells were incubated at 37°C in a 5% CO2 incubator for 24 h. Cell viability was assessed using the CCK-8 assay. Results showed that within the effective application concentration range described in this invention, there was no significant difference in cell viability between the peptide GD8 concentration groups and the control group, indicating no significant cytotoxicity to normal vascular endothelial cells and good cell biocompatibility.

[0038] (2) Hemolysis safety test Fresh, healthy Kunming rats were charged with anticoagulated whole blood. Red blood cells were separated by centrifugation, washed with physiological saline, and resuspended to prepare a red blood cell suspension. A physiological saline negative control group and a distilled water positive lysis group were set up. Different concentrations of peptide GD8 (0.01–0.3 μM) were added to each group, and the suspensions were incubated at 37°C for 30 min. The supernatant was centrifuged, and the absorbance was measured to calculate the hemolysis rate. The results showed that the peptide GD8 of this invention has an extremely low hemolysis rate within the effective concentration range, with no significant hemolytic effect, and does not pose a risk of red blood cell rupture and hemolysis, indicating good safety.

[0039] (3) Preliminary observation of acute toxicity in mice Healthy Kunming mice were selected and divided into a blank control group and a high-dose peptide GD8 administration group. The mice were administered a single dose via tail vein at a dose significantly higher than the effective therapeutic dose. The mice were observed for 14 consecutive days, and their mental state, diet, water intake, activity level, and weight changes were recorded. During the observation period, all mice in each group maintained good mental state, normal diet and activity, with no deaths, behavioral abnormalities, or significant abnormal weight fluctuations. This preliminarily indicates that the peptide has no acute toxicity within the administered dose range, providing a good foundation for safe application as an active ingredient in antithrombotic drugs.

[0040] Example 2: Pharmacological experiment of peptide GD8 - plasma recalcification time experiment The polypeptide GD8 obtained in Example 1 was dissolved in water to obtain anticoagulant polypeptide solutions with concentrations of 0.01 μM, 0.1 μM and 0.3 μM. Blood from healthy individuals and sodium citrate were mixed at a volume ratio of 9:1 to obtain normal human plasma. 40 μL of normal human plasma was mixed with 10 μL of different concentrations of peptide GD8 (0 μM, 0.01 μM, 0.1 μM, and 0.3 μM), and incubated at 37°C for 10 min. Then, 50 μL of preheated CaCl2 solution at 37°C was added, and the mixture was immediately mixed. The OD was measured using a microplate reader. 650 The result is as follows Figure 1 As shown, the peptide GD8 provided by this invention exhibits a concentration-dependent inhibitory effect on plasma recalcification time, indicating that the anticoagulant peptide GD8 can inhibit the coagulation reaction.

[0041] Example 3: Pharmacological experiment of peptide GD8 - FeCl3-induced neck thrombosis model Kunming rats were randomly divided into a saline group (NC) and different concentrations of GD8 (0.006 mg / kg, 0.03 mg / kg, 0.1 mg / kg) administration groups, and administered the drug via tail vein injection. Immediately after administration, mice in each treatment group were anesthetized. The carotid artery was then isolated, and a waterproof paper sheet was placed below the common carotid artery. A filter paper sheet pre-soaked in 10% FeCl3 solution was then placed over the carotid artery. After 2 minutes, the filter paper sheet was removed, and residual FeCl3 was washed away with saline. Blood flow changes were detected using laser Doppler ultrasound. The results are as follows: Figure 2 As shown.

[0042] according to Figure 2 It can be seen that no thrombus was formed in either the saline group (NC) or the GD8 administration group after 7 min of FeCl3 induction (the black area above the white paper represents the arterial vessels); however, after 18 min, the saline group (NC) formed obvious arterial thrombus (the black vessels were almost invisible above the white paper), the arterial vessels were completely blocked, and the blood perfusion units dropped to the minimum. Although the blood perfusion units in the administration group decreased, they did not drop to the minimum, and the vessels were not completely blocked (the black vessels were still visible above the white paper). This indicates that the peptide GD8 provided by this invention can significantly inhibit the reduction of blood flow induced by FeCl3 and inhibit the ferric chloride-induced arterial thrombosis.

[0043] As can be seen from the above, the polypeptide GD8 described in this invention can strongly prolong the plasma recalcification time, has a very significant anticoagulant effect, and strongly inhibits ferric chloride-induced arterial thrombosis.

[0044] Example 4: Pharmacological experiment of polypeptide GD8 - inferior vena cava ligation thrombosis model Kunming rats were randomly divided into three groups: a saline group (NC), a GD8-0.03 mg / kg administration group, and a GD8-0.1 mg / kg administration group, administered via tail vein injection. Following administration, the mice were anesthetized with isoflurane inhalation. The inferior vena cava and its collateral branches were exposed and dissected via abdominal incision. The main trunk and all branches of the inferior vena cava were ligated to construct an inferior vena cava thrombosis model. Thrombus formation was induced for 4-6 hours. The thrombus-containing segment of the inferior vena cava was then removed, photographed, and its length measured. After dehydration, the thrombus was weighed. The results are as follows: Figure 3 As shown.

[0045] according to Figure 3 It can be seen that significant thrombi formed in the inferior vena cava of the saline group (NC), with longer and heavier thrombi; while the thrombus length and weight of the GD8-treated group were significantly reduced, indicating that the peptide GD8 provided by the present invention can significantly inhibit the formation of venous thrombosis induced by inferior vena cava ligation.

[0046] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antithrombotic polypeptide, characterized in that, The amino acid sequence of the antithrombotic polypeptide is shown in SEQ ID No.

1.

2. The use of the antithrombotic polypeptide of claim 1 in the preparation of antithrombotic drugs.

3. The application according to claim 2, characterized in that, The therapeutic effects of the antithrombotic drug include at least one of the following: prolonging plasma recalcification time and inhibiting thrombosis.

4. The application according to claim 2, characterized in that, The antithrombotic drugs are effective against arterial thrombosis and venous thrombosis.

5. The application according to claim 4, characterized in that, The arterial thrombosis includes arterial thrombosis induced by FeCl3, and the venous thrombosis includes venous thrombosis induced by inferior vena cava ligation.

6. The application according to claim 5, characterized in that, When treating arterial and venous thrombosis, the effective dose of the antithrombotic polypeptide in the antithrombotic drug is 0.006~0.1 mg / kg.

7. The application according to claim 5 or 6, characterized in that, When treating arterial thrombosis, the effective dose of the antithrombotic polypeptide in the antithrombotic drug is 0.03 mg / kg; When treating venous thrombosis, the effective dose of the antithrombotic polypeptide in the antithrombotic drug is 0.03 mg / kg.

8. An antithrombotic drug, characterized in that, The active ingredient includes the antithrombotic polypeptide of claim 1, and also includes pharmaceutically acceptable excipients.

9. The drug according to claim 8, characterized in that, The concentration of the antithrombotic peptide in the drug is 0.01~0.3 μM.

10. The drug according to claim 8 or 9, characterized in that, The concentration of the antithrombotic peptide in the drug is 0.1 μM.

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