Anticoagulant peptide as well as preparation method and application thereof

Abstract

The invention discloses an anticoagulant peptide as well as a preparation method and application thereof. According to the method, the ultramicro anticoagulant peptide RF7 is detected from a complex crude peptide extract by optimizing an extraction and separation mode of ground beetle anticoagulant activity, and the screened active anticoagulant peptide is subjected to high-throughput analysis and identification of a peptide fragment sequence by adopting a NanoLC-Q-Orbitrap-MS / MS method. Through searching and comparison of an intelligent data processing system and a polypeptide database, biological information and pharmaceutical components of the anticoagulant peptide are comprehensively analyzed. And finally, by constructing molecular level, cell and animal models, pharmacokinetic and pharmacodynamic researches and the like are carried out on the anticoagulant peptide with obvious activity, the safety and effectiveness of the medicine are evaluated, and a research foundation is laid for development and druggability evaluation of a novel anticoagulant medicine.

Description

Technical Field

[0001] The present invention relates to a peptide and a preparation method and application thereof, in particular to an anticoagulant peptide and a preparation method and application thereof, belonging to the field of bioactive peptides. Background Art

[0002] Thrombotic diseases are a type of cardiovascular disease with a high incidence, mortality and clinical disability rate, so antithrombotic therapy is crucial for the prevention and treatment of cardiovascular diseases. Currently, commonly used anticoagulants such as heparin and warfarin have good clinical efficacy, but they interfere with the delicate balance of procoagulant and anticoagulant functions in the body and have potential bleeding risks. Therefore, the search for new anticoagulant drugs with good anticoagulant effects and low bleeding risks has become a research hotspot for cardiovascular diseases.

[0003] Bioactive peptides are compounds between proteins and amino acids, with a molecular weight of less than 6000 Da and composed of two or more amino acid residues. Due to their unique structure and sequence, they have good biocompatibility, can specifically interact with target proteins, and have a low incidence of adverse reactions, and have gradually become popular candidate drugs in current drug development. Animal-based traditional Chinese medicines are rich in proteins and are an important source of bioactive peptides. Hirudin, a polypeptide extracted from leeches, is the strongest natural thrombin inhibitor discovered so far. The analgesic ziconotide is extracted from the venom of sea snails and exerts analgesic effects by effectively and selectively blocking neuronal N-type voltage-gated calcium channels (N-VSCCs). As a traditional blood-activating and stasis-removing medicine, the traditional Chinese medicine earthworm is widely used in clinical practice for its powerful effect of breaking blood and removing blood stasis. Modern pharmacological studies have shown that earthworms have pharmacological effects such as dissolving thrombus, anticoagulation, and regulating blood lipids. Earthworms contain a variety of active proteins (enzymes), amino acids, unsaturated fatty acids, trace elements, alkaloids, and fat-soluble vitamins, among which protein and polypeptide components account for more than 60%. The literature currently reports that the main component of its anticoagulant activity is protein peptides. However, few anticoagulant active components have been obtained so far, and most are still in the in vitro and animal experimental stage, and the active components and mechanisms of action are still unclear. Therefore, further research is needed to extract and isolate new anticoagulant peptides with low bleeding risk from Eupolyphaga sinensis. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide an anticoagulant peptide with good anticoagulant effect; another purpose of the present invention is to provide a method for preparing an anticoagulant peptide; another purpose of the present invention is to provide an application of an anticoagulant peptide.

[0005] Technical solution: An anticoagulant peptide of the present invention has the following structural formula:

[0006] In another aspect, the present invention provides a method for preparing the above-mentioned anticoagulant peptide, comprising the following steps:

[0007] Fmoc-Phe-Wang-Resin resin was activated, the protective group Fmoc was removed, and then Fmoc-Arg-OH was connected to block the activation site to obtain compound 1; Remove the protecting group Fmoc in compound 1 and connect Fmoc-Ile-OH to obtain compound 2; Remove the protecting group Fmoc in compound 2 and connect Fmoc-Asp-OH to obtain compound 3; The protecting group Fmoc in compound 3 was removed, and Fmoc-Ser-OH was connected to obtain compound 4; The protecting group Fmoc in compound 4 was removed, and Fmoc-Ser-OH was connected to obtain compound 5; Remove the protecting group Fmoc in compound 5 and connect Fmoc-Arg-OH to obtain compound 6; The protecting group Fmoc in compound 6 was removed and the resin was cleaved to obtain the target product RF7.

[0008] Furthermore, the reagent used for activating Fmoc-Phe-Wang-Resin resin is dichloromethane.

[0009] Furthermore, the reagent used for cleaving the resin is trifluoroacetic acid.

[0010] Furthermore, the reagent used to block the activation sites was a mixture of dichloromethane, methanol, and diisopropylethylamine (80:15:5).

[0011] Furthermore, the reagent used to remove the protecting group Fmoc in each compound is diisobutyl ketone.

[0012] Furthermore, after removing the protecting group Fmoc in compound 6, the obtained product is washed, the resin is cut, filtered, and precipitated to obtain purified RF7.

[0013] Furthermore, after removing the protecting group Fmoc in compound 6, the product was washed with N,N-dimethylformamide (DMF), dichloromethane (DCM) and methanol, respectively.

[0014] Furthermore, after removing the protecting group Fmoc in compound 6, the reagent used for cutting the resin is trifluoroacetic acid.

[0015] Furthermore, after removing the protecting group Fmoc in compound 6, the reagent used for precipitation is glacial ether.

[0016] In another aspect, the present invention provides a use of the above anticoagulant peptide in the preparation of anticoagulant drugs.

[0017] In another aspect, the present invention provides a method for extracting a crude peptide extract from Eupolyphaga sinensis, comprising the following steps: Dissolve the Eupolyphaga sinensis powder in hydrochloric acid solution, shake at 37°C, add 2% pepsin, shake at 37°C. Adjust the pH to 8.0, add 4% trypsin, shake at 37°C. Take out the crude extract and boil it in water to inactivate trypsin. Cool the solution, centrifuge it, take the supernatant and freeze-thaw it three times at -20°C, remove the precipitate during the freeze-thaw process, and take the supernatant and freeze-dry it.

[0018] On the other hand, the present invention provides a method for separating and screening the anticoagulant peptide RF7 from the crude peptide extract of Eupolyphaga sinensis, comprising the following steps: Identification: After the crude peptide extract of Eupolyphaga sinensis was dialyzed and desalted, mass spectrometry was performed to obtain the original mass spectrometry data of several peptides. The original mass spectrometry data was matched with the data in the Eupolyphaga sinensis peptide database. The matching protein and peptide sequences were found in the database through the mass of the primary and secondary fragments of the peptide segment to achieve the identification of the peptide sequence in the sample; Computer activity screening: analyze the biological activity of the identified peptides, predict the peptide toxicity, and screen out peptides with good biological activity, good water solubility, non-toxicity and not included in the Eupolyphaga database for molecular docking; Molecular docking: The Auto Dock Vina program was used for docking studies to obtain predictions of the binding free energy and binding constant of the docked ligand. The affinity of the optimal docking position of the ligand and protein target complex was determined by the E value (kcal / mol). Based on the above results, the anticoagulant peptide RF7 was screened.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: combining the multi-dimensional three-in-one mass spectrometry peptide group technology, through the intelligent data processing system and the peptide database search and comparison, the biological information and pharmaceutical components of the anticoagulant peptides are comprehensively analyzed, and a new anticoagulant peptide RF7 with good anticoagulant effect and low bleeding risk is screened from the complex earthworm crude peptide extract. Finally, by constructing molecular level, cell and animal models, the pharmacokinetics and pharmacodynamics of the anticoagulant peptides with obvious activity are studied, the safety and effectiveness of the drugs are evaluated, and the research foundation is laid for the development of new anticoagulant drugs and the evaluation of drugability. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The results of the comparison of the anticoagulant effects of different crude peptide extracts are shown in Figure 1. (A) shows the effect on the activated partial thromboplastin time (APTT); (B) shows the effect on the prothrombin time (PT); (C) shows the effect on the thrombin time (TT); (D) shows the effect on the fibrinogen coagulation time (FCT); IM in the figure is the immersion extraction method, EHM is the enzymatic extraction method, FC is the freeze-thaw process, and DT is the degreasing process; a in the figure is P <0.05, b is P <0.05, c is P <0.01, d is P <0.001.

[0021] Figure 2 This is the result of investigating the anticoagulant activity of crude peptides extracted by the enzymatic freeze-thaw method. Among them, (A) is the effect of crude peptide on activated partial thromboplastin time (APTT); (B) is the effect of crude peptide on prothrombin time (PT); (C) is the effect of crude peptide on thrombin time (TT); (D) is the effect of crude peptide on fibrinogen clotting time (FCT); (E) is the effect of crude peptide on thrombin clotting time (TCT); (F) is the volume of thrombin consumed by crude peptide in thrombin titration method; (G) is the effect of crude peptide on plasma recalcification time (PRT); (H) is the effect of crude peptide on fibrinogen in fibrinogen plate method; (I) is the effect of crude peptide on fibrin in fibrin plate method; Note: The numbers in fibrinogen plate and fibrin plate, 1-3 are blank controls: PBS buffer, 4-6 are urokinase (0.1 mg / mL), 7-9 are crude peptides (30 mg / mL), 10-12 are crude peptides (60 mg / mL), 13-15 are crude peptides (90 mg / mL), 16 is the control without any sample added; ns in the figure is P >0.05, for P <0.05, for P <0.01, for P <0.001, for P <0.0001.

[0022] Figure 3 The results of the investigation of the anticoagulant activity and bleeding risk of crude peptides in rats. Among them, (A) shows the effect of crude peptide administration on rat activated partial thromboplastin time (APTT); (B) shows the effect of crude peptide administration on rat prothrombin time (PT); (C) shows the effect of crude peptide administration on rat thrombin time (TT); (D) shows the effect of crude peptide administration on the normal coagulation system of mice; Note: ns in the figure is P >0.05, forP <0.05, for P <0.01, for P <0.0001.

[0023] Figure 4 The molecular docking results of the anticoagulant peptide RF7 with thrombin and fibrinogen are shown in Figure 1. (A) is a 3D view of the interaction between the anticoagulant peptide RF7 and thrombin and the hydrogen bonds formed by its amino acid residues; (B) is a 3D view of the interaction between the anticoagulant peptide RF7 and fibrinogen and the hydrogen bonds formed by its amino acid residues.

[0024] Figure 5 The molecular docking results of the anticoagulant peptide RF7 and FXIa, FXIIa and FXIIIa. Among them, (A) is a 3D view of the interaction between the anticoagulant peptide RF7 and FXIa and the formation of hydrogen bonds between its amino acid residues; (B) is a 3D view of the interaction between the anticoagulant peptide RF7 and the original FXIIa and the formation of hydrogen bonds between its amino acid residues; (C) is a 3D view of the interaction between the anticoagulant peptide RF7 and the original FXIIIa and the formation of hydrogen bonds between its amino acid residues.

[0025] Figure 6 The following are the relevant characterization diagrams of peptide RF7. Among them, (A) is the UV spectrum of peptide RF7 (0.02 mg / mL); (B) is the circular dichroism spectrum of peptide RF7 (0.5 mg / mL); (C) is the infrared spectrum of peptide RF7; (D) is the potential characterization of peptide RF7 (5 mg / mL).

[0026] Figure 7 The results of the in vitro anticoagulant activity of RF7 are shown in Figure 2. (A) shows the effect of RF7 on activated partial thromboplastin time (APTT); (B) shows the effect of RF7 on prothrombin time (PT); (C) shows the effect of RF7 on thrombin time (TT); (D) shows the effect of RF7 on plasma recalcification time (PRT); (E) shows the effect of RF7 on fibrinogen clotting time (FCT); (F) shows the effect of RF7 on thrombin clotting time (TCT); Note: ns in the figure is P >0.05, for P <0.05, for P <0.01, for P <0.001, for P <0.0001.

[0027] Figure 8The results of RF7's in vitro antithrombotic activity are shown in the figure. Among them, (A) is the morphology of the blood clot after incubation of RF7 with the blood clot; (B) is the weight of the blood clot after incubation of RF7 with the blood clot; (C) is the morphology of the blood clot formed after the sample is incubated with whole blood for 10 minutes; (D) is the dry weight of the blood clot formed after the sample is incubated with whole blood for 10 minutes. Note: In Figure A, ① is normal saline, ② is earthworm enzyme, ③ is 5 mg / mL RF7, ④ is 10 mg / mL RF7, ⑤ is 15 mg / mL RF7, ⑥ is 20 mg / mL RF7, ⑦ is 25 mg / mL RF7, ⑧ is 30 mg / mL RF7; in the figure for P <0.05, for P <0.01, for P <0.001, for P <0.0001.

[0028] Fig. 9 The results of the anticoagulant effect of RF7 in animals. Among them, (A) shows the effect of different concentrations of RF7 (50, 75, 100 mg / kg) on ​​the morphology of rat neck vessels induced by ferric chloride; (B) shows the HE staining results of different concentrations of RF7 (50, 75, 100 mg / kg) on ​​ferric chloride-induced rat neck vessels; (C) shows the morphology of the tail tissue of each group of mice in the carrageenan-induced tail thrombosis model; (D) shows the staining results of the mouse tail tissue; (E) shows the wet weight of rat neck vessels after ferric chloride induction; (F) shows the length of the mouse black tail; (G) shows the rate of the mouse black tail; Note: in the figure for P <0.05, for P <0.01, for P <0.001, for P <0.0001.

[0029] Fig.10 The results of the safety study of peptide RF7 are shown in the figure. Among them, (A) shows the effect of peptide RF7 on the survival rate of HUVEC cells; (B) shows the effect of RF7 on the morphology of red blood cells; (C) shows the hemolysis rate of RF7 at different concentrations; (D) shows the schematic diagram of the mouse tail bleeding experiment; (E) shows the bleeding time of the mouse tail; (F) shows the amount of bleeding in the mouse tail; (G) shows the HE staining results of RF7 on common organs of rats (the scale is 50 μm); Note: ns in the figure is P >0.05, for P <0.001.

[0030] Fig.11 The blood drug concentration and time curve of RF7 in rats. (A) is the blood drug concentration and time curve at different time points in rats after oral administration of 200 mg / kg of RF7 (n = 4); (B) is the blood drug concentration and time curve at different time points in rats after tail vein injection of 100 mg / kg of RF7 (n = 4). DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0032] The embodiment of the present invention optimizes the extraction and separation method of the anticoagulant activity of the earthworm, thereby detecting ultra-trace anticoagulant peptides from complex crude peptide extracts, and adopts the NanoLC-Q-Orbitrap-MS / MS method to perform high-throughput analysis and identification of peptide sequences for the screened active anticoagulant peptides. Through the search and comparison of the intelligent data processing system and the peptide database, the biological information and pharmaceutical components of the anticoagulant peptides are comprehensively analyzed. Finally, by constructing molecular, cell and animal models, the pharmacokinetics and pharmacodynamics of anticoagulant peptides with obvious activity are studied, the safety and effectiveness of the drugs are evaluated, and the research foundation is laid for the development of new anticoagulant drugs and the evaluation of drugability.

[0033] Example 1

[0034] In this example, four methods, namely, immersion method, enzymatic hydrolysis method, repeated freezing and thawing of enzymatic hydrolysis solution, and repeated freezing and thawing of defatted enzymatic hydrolysis, were used to extract crude peptides.

[0035] (1) Immersion method. Take the fine powder of Eupolyphaga sinensis, add eight times the amount of water to soak for 1 hour, ultrasonicate for 30 minutes, and extract at 4℃ for 10 hours. Centrifuge at 2560g for 10 minutes, take the supernatant, add seven times the amount of water to the precipitate, ultrasonicate for 30 minutes, and extract at 4℃ for 8 hours. Centrifuge at 2560g for 10 minutes, take the supernatant. Combine the supernatants, freeze-dry, and store at 4℃ for later use.

[0036] (2) Enzymatic hydrolysis. Dissolve 2.0 g of powder in 40 mL of hydrochloric acid solution (1:20, w / v) with a pH of 1.5, shake at 37°C for half an hour, add 2% pepsin, and shake at 37°C for 3 hours. Adjust the pH to 8.0 with 1.0 M NaOH, add 4% trypsin, and shake at 37°C for 3 hours. Take out the crude extract and boil it in a water bath for 15 minutes to inactivate trypsin. When the solution cools to about 40°C, centrifuge at 2560 g for 10 minutes, and freeze-dry the supernatant.

[0037] (3) Repeated freeze-thaw of the enzymatic solution. Take 2 g of powder and dissolve it in 40 mL of hydrochloric acid solution with a pH of 1.5 (1:20, w / v), shake at 37°C for half an hour, add 2% pepsin, and shake at 37°C for 3 hours. Adjust the pH to 8.0 with 1.0 M NaOH, add 4% trypsin, and shake at 37°C for 3 hours. Take out the crude extract and boil it in a water bath for 15 minutes to inactivate trypsin. When the solution cools to about 40°C, centrifuge it at 2560 g for 10 minutes, take the supernatant and freeze-thaw it three times in a -20°C refrigerator to remove the precipitate precipitated during the freeze-thaw process, and take the supernatant for freeze-drying.

[0038] (4) Delipidation and enzymatic solution, repeated freeze-thaw. Take 2 g of sieved earthworm powder, add 10 times the amount of petroleum ether, shake at 37°C overnight, centrifuge at 2560 g for 10 minutes, remove the supernatant, and defatted powder in a fume hood overnight. After overnight, the defatted powder was dissolved in 40 mL of hydrochloric acid solution (1:20, w / v) with a pH of 1.5, shake at 37°C for half an hour, add 2% pepsin, and shake at 37°C for 3 hours. Adjust the pH to 8.0 with 1.0 M NaOH, add 4% trypsin, and shake at 37°C for 3 hours. Take out the crude extract and boil it in a water bath for 15 minutes to inactivate trypsin. When the solution cools to about 40°C, centrifuge at 2560 g for 10 minutes, take the supernatant and freeze-thaw it three times in a -20°C refrigerator, remove the precipitate precipitated during the freeze-thaw process, and take the supernatant for freeze-drying.

[0039] More than 60% of the components of Eupolyphaga sinensis are proteins, and the crude peptides obtained by different extraction methods have different activities. The appearance properties, water solubility, extraction rate and protein content of the crude peptides obtained by four extraction methods, namely, immersion method, enzymatic hydrolysis method, enzyme thawing freeze-thaw method and delipidase thawing freeze-thaw method, were compared. In terms of color properties, the extract of the immersion method is yellow-brown, the extract of the enzymatic hydrolysis method is a mixture of yellow-brown fragments and yellow-white powder, the extract of the enzyme thawing freeze-thaw method is dark brown, and the extract of the delipidase thawing freeze-thaw method is brown-yellow. The water solubility of the extract to a certain extent reflects the content of water-soluble amino acids, and some of the water-soluble amino acids play an important role in cardiovascular diseases. For example, lysine has excellent hydration, can form a hydration layer, can resist the adhesion of blood components, and specifically binds to plasminogen (Plg) and its activator tissue plasminogen activator (t-PA), lysing early thrombus (fibrin clot). Arginine can affect the conformation of thrombin, thereby affecting the anticoagulant effect; in addition, L-arginine can prolong APTT and PT, inhibit the speed of blood clot formation, and has anticoagulant activity. Therefore, water-soluble indicators have a certain reference for anticoagulant activity. The extract obtained by the enzymatic thawing freeze-thaw method has the best water solubility. At a concentration of 20 mg / mL, it is completely dissolved and no precipitation is produced in the solution. The Chinese medicine earthworm is often administered orally, and enters the blood circulation after digestion and absorption in the gastrointestinal tract, and is degraded by pepsin and trypsin to exert its efficacy. As shown in Table 1, the enzymatic hydrolysis method uses pepsin and trypsin to simulate the in vivo environment, the extraction conditions are mild, the extraction efficiency is high, reaching 52.94%, and the degree of damage to the protein is small, and the protein content reaches 93.38%. The enzymatic thawing freeze-thaw method undergoes three freeze-thaw cycles on the basis of the enzymatic hydrolysis method. During the freeze-thaw process, some endogenous animal proteins will be removed, and the active ingredients will be further purified, so the extraction rate and protein content are reduced. The enzymatic thawing freeze-thaw defatting method uses petroleum ether to defatting to remove some fat-soluble components, and at the same time, a part of the protein peptides will be lost, and the extraction rate and protein content will be further reduced. As one of the most traditional extraction methods, the immersion method is simple and gentle to operate, but it has problems such as low extraction rate and long time consumption. The immersion method takes more than 12 hours, and the protein content obtained is not high (45.53%).

[0040] Table 1 Extraction rate and protein content of different extraction methods of Eupolyphaga sinensis (Note: IM: immersion; EHM: enzymatic hydrolysis; FC: freeze-thaw; DT: defatting)

[0041] The above four extraction methods were tested for activity respectively to select the best extraction method. The activity test of the four extraction methods was anticoagulant activity test, and the best extraction method was selected through anticoagulant activity test. The crude peptide extract obtained by the best extraction method was subjected to fibrinolytic activity test, in vivo anticoagulant activity test and bleeding risk investigation.

[0042] 1. Anticoagulant activity assay (1) APTT, PT, TT and FCT determination. Blood was collected from the abdominal aorta of SD rats, anticoagulated with 3.8% sodium citrate (blood: sodium citrate = (9:1), V / V), and centrifuged at 1306 g for 8 min. The supernatant was collected to obtain platelet-poor plasma (PPP). Coagulation time was determined using an SC40 semi-automatic coagulation analyzer and a diagnostic kit. In brief, PPP (40 μL) and APTT reagent (50 μL) were mixed with various concentrations of sample solution (10 μL) or saline (10 μL), pre-incubated at 37°C for 3 min, and CaCl was added. 2 (50 μL), start the coagulation pathway, and record APTT. PPP (40 μL) was mixed with sample solutions of various concentrations (10 μL) or saline (10 μL), pre-incubated at 37°C for 3 minutes, PT reagent (100 μL) was added, the coagulation pathway was started, and PT was recorded. PPP (80 μL) was mixed with sample solutions of various concentrations (20 μL) or saline (20 μL), pre-incubated at 37°C for 3 minutes, TT reagent (100 μL) was added, the coagulation pathway was started, and TT was recorded. Extracts of Eupolyphaga sinensis, thrombin and fibrinogen were dissolved in Tris-HCl buffer (35 mM Tris, 25 mM NaCl, pH 7.4). After 100 μL of extracts of different concentrations and 50 μL of fibrinogen (1 mg / mL) were pre-incubated at 37°C for 3 min, 50 μL of thrombin (10 U / mL) was added, and the time for fibrinogen to convert to fibrin was measured on a semi-automatic coagulation analyzer. The measurements were performed three times in parallel, and Tris-HCl buffer was used as a blank control.

[0043] (2) Determination of plasma recalcification time. PPP was prepared as described above. 50 μL PPP and 50 μL of different concentrations (30, 60, and 90 mg / mL) of crude peptide extracts from Eupolyphaga sinensis or saline were added to the test cup, pipetted evenly, incubated at 37°C for 3 min, and 50 μL of 25 mM CaCl was added. 2 Solution, immediately start the semi-automatic coagulometer to measure the coagulation time.

[0044] (3) Determination of antithrombin activity. Take the crude peptide and prepare sample solutions with concentrations of 30, 60 and 90 mg / mL using Tris-HCl buffer. Take 50 μL of the sample, add 50 μL of (human) fibrinogen (1 mg / mL), shake well, and incubate at 37°C for 5 minutes. Add thrombin (10 U / mL) dropwise until coagulation occurs. When adding thrombin, add 5 μL dropwise every 1 minute, and shake gently while adding; record the volume of thrombin solution consumed.

[0045] (4) Determination of thrombin clotting time. Take 100 μL of crude peptide extracts of different concentrations, add 50 μL of thrombin (10 U / mL), pre-incubate at 37°C for 30 min, add 50 μL of fibrinogen (1 mg / mL), and measure the thrombin clotting time on a semi-automatic coagulometer. The measurement was performed three times in parallel, and Tris-HCl buffer was used as a blank control.

[0046] 2. Fibrinolytic activity assay (1) Fibrinogen plate. Use PBS (50 mM NaH 2 PO4, 0.15 M NaCl, pH 7.2) to prepare 20 mL of 1% agarose solution, heat it to completely melt, cool it to about 40℃, add 1 mL of fibrinogen (5 mg / mL), stir well and immediately pour it into a glass culture dish with a diameter of 9 cm. After the agar is completely solidified, place the culture dish on a piece of white paper printed with a 1.6 cm × 1.6 cm small square matrix, use a 3 mm diameter puncher to punch small holes on the matrix points, and after punching the holes, use a 200 μL pipette to carefully absorb the liquid in the holes. After adding 10 μL of the sample to be tested, urokinase and PBS buffer to the small holes, the fiber plate is placed in a 37℃ incubator for 15 h, stained with Coomassie Brilliant Blue staining solution (Coomassie Brilliant Blue R-250 0.25%, glacial acetic acid 5%, methanol 4.5%) for 15 minutes, and then decolorized with elution solution until a clear solvent circle can be seen.

[0047] (2) Fibrin plate. Prepare 20 mL of 1% agarose solution with PBS, heat it to completely melt, cool it to about 40°C, add 1 mL of fibrinogen (5 mg / mL), immediately add 20 μL of 100 U / mL thrombin, stir evenly, pour into the culture dish, and start punching after incubation at room temperature for half an hour, add the sample, incubate for 15 hours, stain with Coomassie Brilliant Blue staining solution, and then decolorize with elution solution until a clear solvent circle can be seen.

[0048] (3) SDS-PAGE. Incubate the sample with fibrinogen (1 mg / mL) at 37°C for 30 min, add protein loading buffer, boil in water for 5 min, centrifuge at 9000 g for 2 min, load 10 μL of supernatant, and run the gel at 80 V. Stain with 0.25% Coomassie Brilliant Blue, decolorize until obvious bands are visible, and process the gel block photos using Image J 2.0 software.

[0049] 3. In vivo anticoagulant activity assay Twenty-four SD rats were randomly divided into a blank control group and low, medium and high dose groups of Eupolyphaga sinensis, with 6 rats in each group. The control group was gavaged with an equal amount of normal saline, and the low, medium and high dose groups of Eupolyphaga sinensis were gavaged with 200, 400 and 800 mg / kg, respectively. 0.5 mL of blood was collected from the orbit at 0.5 h, 1.0 h, 1.5 h, 2.0 h and 3.0 h after administration, anticoagulated with 3.8% sodium citrate (1:9), centrifuged at 1306 g for 8 min, and the supernatant was collected to determine its APTT, PT and TT.

[0050] 4. Bleeding risk assessment Twenty-four ICR mice were randomly divided into a control group and low, medium, and high-dose groups of Eupolyphaga sinensis, with 6 mice in each group. The control group was gavaged with corresponding saline, and the low, medium, and high-dose groups of Eupolyphaga sinensis were gavaged with 200, 400, and 800 mg / kg, respectively. Half an hour after administration, the mice were anesthetized, the tails were cut off 3 mm from the tip of the tails, and the tails were placed in 37°C saline, and the time when the bleeding stopped was recorded. At the end of the experiment, the wounds of the mice were cleaned with iodine to avoid infection.

[0051] The results of crude peptide extraction and activity determination are as follows: 1. Activity screening results of crude peptides Thrombosis is a complex pathological process, which is usually caused by multiple links or factors. APTT, PT, TT and FCT are usually used as anticoagulant detection indicators to compare the anticoagulant activity of crude peptides obtained by different extraction methods (immersion method, enzymatic hydrolysis method, enzyme thawing freeze-thaw method and enzyme thawing freeze-thaw defatting method). Figure 1 A and Figure 1 As shown in Figure B, except for the crude peptides obtained by enzymatic hydrolysis (20 and 30 mg / mL), which had a significant effect on APTT, the crude peptides extracted by other methods had no significant difference in their effects on APTT and PT at different concentrations (10, 20, 30 mg / mL). At higher concentrations (20 and 30 mg / mL), the crude peptides obtained by the four methods slightly prolonged TT ( Figure 1 C in Figure 1 The crude peptides obtained by the above four extraction methods all had a significant effect on FCT, especially the enzymatic thawing freeze-thaw method, which could significantly prolong FCT, which was significantly different from other methods and was concentration-dependent. Considering the anticoagulant effect, yield and protein content, the best extraction method was the enzymatic thawing and repeated freeze-thaw method.

[0052] 2. Anticoagulant effect of crude peptide The anticoagulant effect of crude peptides after repeated freeze-thaw was further investigated. Compared with the blank group, crude peptides with a concentration of 160 mg / mL or more prolonged APTT by 3 seconds, and high concentrations (90 mg / mL) prolonged APTT by more than 4 seconds ( Figure 2A in the figure). There was also a tendency to prolong PT, with the highest concentration (90 mg / mL) extending it by 2 seconds ( Figure 2 The crude peptide had a more significant effect on TT. The effect of 20 mg / mL crude peptide on TT began to show significant differences. 90 mg / mL prolonged TT by 33.7 seconds ( Figure 2 C in the figure). The APTT, PT, and TT data indicate that the crude peptide mainly affects the common pathway of the coagulation cascade and has a significant inhibitory effect on the conversion of fibrinogen to fibrin, possibly by inhibiting thrombin or fibrinogen. When only fibrinogen, thrombin, and crude peptide are present in the system, the crude peptide prolongs FCT by 11.67 seconds at the lowest dose (10 mg / mL) and by 521.77 seconds at the high dose (90 mg / mL). Figure 2 D), the thrombin clotting time was prolonged by 436 seconds ( Figure 2 E in the figure), fibrinogen is hardly converted into fibrin by thrombin. The anticoagulant activity of crude peptide was determined by the classic thrombin titration method. There was an obvious threading phenomenon at the end point of the titration, and the liquid did not drip due to gravity after inversion. As the concentration of crude peptide increased, the amount of thrombin consumed gradually increased. In the end, compared with the blank group, 30 μL more thrombin was consumed ( Figure 2 F in the sample). Citric acid can chelate calcium ions in plasma, interrupting the coagulation cascade and thus blocking the formation of thrombus. The addition of calcium ions can restore the coagulation cascade and allow the blood to coagulate normally. When anticoagulants are present in the blood, the plasma recalcification time will be prolonged. When the crude peptide is pre-incubated with plasma and calcium ions are added to initiate the coagulation cascade, the fibrin formation time is significantly prolonged compared to the blank, and at a high dose (90 mg / mL) it is prolonged by 164.7 seconds ( Figure 2 G in the figure). Both the fibrinogen plate and the fibrin plate are semi-quantitative methods. Under appropriate conditions, the size of the dissolution zone is proportional to the concentration of the added sample. It can detect both plasmin and plasminogen activators. When the added sample has fibrinolytic activity, it can hydrolyze fibrin into fragments. The staining ability of Coomassie Brilliant Blue for fragments is not as high as that for intact fibrin. Therefore, under the dark blue background, the area where the plasmin is located appears light blue. Urokinase is a first-generation thrombolytic drug that hydrolyzes fibrin and fibrinogen by activating plasminogen. Under the blue background, it can be observed that the positive drug urokinase (labeled 4-6) has an obvious dissolution zone, while the crude peptides of different concentrations (labeled 7-15) cannot see the dissolution zone, even if the concentration has reached 90 mg / mL (labeled 13-15) ( Figure 2 H and I in the figure). This indicates that the crude peptide may not have a fibrinolytic effect and cannot hydrolyze fibrinogen or cut fibrin and fibrinogen by activating plasminogen.

[0053] 3. Anticoagulant activity of crude peptide in vivo The crude peptide entered the rats through the gastrointestinal tract, and the efficacy of the crude peptide in rats was investigated by measuring APTT, PT and TT at different time points. The crude peptide entered the rats and was first absorbed by the gastrointestinal tract and entered the blood. Due to the different absorption of the anticoagulant active ingredients, there was no obvious prolongation trend for APTT at 0.5 h. At 1 h, the low-dose group (200 mg / kg) prolonged by 4.25s, which was a significant difference; after the crude peptide passed through the enterohepatic circulation, the blood drug concentration was further increased. At 2 h, the APTT extension time showed a dose-dependent manner. The three low, medium and high dose groups (200, 400, and 800 mg / kg) prolonged by 1.87, 4.3, and 8.73 seconds, respectively ( Figure 3 The crude peptide had almost no significant effect on PT, except for the low-dose group (200 mg / kg) which prolonged PT by 2.43 seconds at 1 h. Figure 3 The crude peptide mainly exerts anticoagulant activity by prolonging TT. During the period of 0.5-3 h, TT was prolonged, and the prolongation time at 0.5, 2, and 3 h was significantly different. At 0.5 h, the prolongation time was 4.37, 8.45, and 14.4 seconds, respectively. At 2 h, the high-dose group (800 mg / kg) was prolonged by 5.6 seconds. At 3 h, the three dose groups (200, 400, and 800 mg / kg) were prolonged by 4.62, 5, and 6.42 seconds, respectively. Figure 3 C in the figure). The anticoagulant activity of crude peptide in rats is consistent with the in vitro efficacy, mainly prolonging TT time, and also partially affecting APTT and PT. This indicates that crude peptide mainly exerts anticoagulant activity by inhibiting coagulation factors (thrombin, fibrinogen, fibrin, etc.) on the common pathway.

[0054] 4. Investigation of bleeding risk of crude peptide The effect of the crude extract of Eupolyphaga sinensis on the normal coagulation function of mice was investigated by a mouse bleeding experiment. The average bleeding time of the blank control group was 321.5 ± 116.4 s, and the average bleeding time of the low (200 mg / kg), medium (400 mg / kg), and high dose (800 mg / kg) groups of the crude peptide extract of Eupolyphaga sinensis were 289.0 ± 62.1 s, 318.5 ± 90.9 s, and 293.8 ± 61.0 s, respectively, which showed no significant difference compared with the blank control group, indicating that the crude peptide extract had no effect on the normal hemostasis of mice after tail amputation ( Figure 3 D in the figure). Currently, anticoagulants are mainly associated with a high risk of bleeding. The mouse tail bleeding experiment showed that the bleeding time of mice given crude peptide extracts remained almost the same as that of mice given saline, indicating that the crude peptide extract has a low risk of bleeding. This further indicates that the crude extract of Eupolyphaga sinensis has great research value. Future research needs to further explore the specific components in the crude extract that exert their efficacy and examine their mechanism of action.

[0055] Example 2 In this example, the crude peptides extracted in Example 1 were used for anticoagulant peptide identification and screening, so as to screen out the best active peptides in the crude peptide extract of Eupolyphaga sinensis.

[0056] 1. Identification After the crude peptides of Eupolyphaga sinensis were dialyzed and desalted, the samples were analyzed using a Thermo Nano LC system coupled with an Orbitrap Fusion Lumos. 3 μL of sample was taken and separated by the chromatographic column. The elution was performed for 120 min at a flow rate of 600 nL / min. The mobile phase A was 0.1% formic acid in water, and the mobile phase B was pure acetonitrile. The gradient elution was 3% B-8% B in 5 min, 5-85 min: 5% B-28% B, 85-102 min: 28%-38% B, 102 min-110 min: 38% B-100% B, and 110-120 min: 100% B. The mass spectrometry conditions were: positive ion mode, the primary mass spectrometer detector was Orbitrap, the resolution was 60K, the mass scan range was m / z 350-2000, the maximum injection time was 50 ms; MIPS filtration was used, the filtration mode was peptide, the discharge time was 40 s, and the mass tolerance was ppm. The secondary scan was isolated with a quadrupole, using HCD mode collision fragmentation, collision energy 30%, and Orbitrap detection of secondary fragments, with a resolution of 15K, a maximum injection time of 22 ms, and an AGC control of 5.0×10 4 The raw data obtained by mass spectrometry were imported into Xcalibur4.1.31 software to obtain the total ion current of the sample. The Eupolyphaga database provided by the Key Laboratory of Natural Medicinal Chemistry of China Pharmaceutical University was imported into Proteome Discoverer 2.1 software. The raw data of mass spectrometry were matched with the data in the database according to the set parameters. The matching protein and peptide sequences were found in the database through the mass of the primary and secondary fragments of the peptide segment to achieve the identification of the peptide sequence in the sample.

[0057] 2. Computer activity screening PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to analyze the biological activity of the identified peptides. The score ranged from 0 to 1. The higher the score, the higher the possibility that the peptide had biological activity. The water solubility of the peptide is an important indicator for exploring functional peptides. Therefore, Innovagen (http: / / www.innovagen.com / proteomics-tools) was used to evaluate its water solubility. Peptides with a score greater than 0.5 were selected as potential active peptides, and further toxicity prediction and sequence comparison were performed. The toxicity of peptides was predicted by the online tool ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / design.php), and the online database BIOPEP-UWM (https: / / biochemia.uwm.edu.pl / ) was used to investigate whether it had been reported and included in the database. Molecular docking was further studied for peptides with good biological activity, good water solubility, non-toxicity, and not included in the database.

[0058] 3. Molecular docking The Auto Dock Vina program was used for docking studies, which provides predictions of the binding free energy and binding constant of the docked ligand. The affinity of the optimal docking position of the ligand and protein target complex was determined by the E value (kcal / mol). The specific operations are as follows: First, prepare the ligand: the 2D and 3D structures of the peptide were drawn by ChemDraw20.0, and the 3D structure was optimized using MM 2The force field was used for energy minimization and saved as a PDB file. Secondly, the receptor was prepared: the X-ray crystal structure of the coagulation factor (thrombin, PDB code: 2BVR; fibrinogen, PDB code: 2HPC; FXIIIa, PDB code: 5MHO; FXIIa, PDB code: 6L63; FXIa, PDB code: 6HHC) was downloaded from the RCSB Protein Database (www.rcsb.org). The downloaded PDB file was imported into the PyMOL software, and the commands "remove solvent" and "remove organic" were entered in sequence to remove the water molecules and ligands present in the receptor protein. The receptor protein was imported into the AutoDockTools (version 1.5.6) software, hydrogenated, selected as a receptor, and saved as a PDBQT file. After that, the ligand was imported, hydrogenated, selected as a ligand, and the PDBQT file was exported. The docking parameters were set, the entire receptor was selected as the docking box, the number of dockings was changed to 10, and AutoDock Vina was run. The docking results were analyzed, the one with the lowest binding energy was selected, the binding affinity was calculated, and it was displayed in kcal / mol. Finally, the interaction between the peptide and the coagulation factor was displayed using PyMOL software.

[0059] The results of identification and screening of new anticoagulant peptides are as follows: 1. Mass spectrometry identification The crude peptides extracted by enzyme thawing and freezing were detected by mass spectrometry, and the total ion current diagram obtained showed that there were multiple proteins and peptides in the crude peptide extract of Eupolyphaga sinensis. After searching the database, 221 peptides were identified from the Eupolyphaga sinensis extract. Since the human body surface contains many proteins, such as keratin present in the outer layer of skin, hair, and nails, which may contaminate the experimental samples, it is necessary to import the contamination protein database and remove the peptides from the contamination protein database when searching the database. Therefore, 213 peptides were finally obtained, with a molecular weight of 745-2984Da and a number of amino acids in the peptides between 6 and 28.

[0060] 2. Computer virtual activity screening Table 2 Screened active peptides

[0061] In order to screen the active peptides in the extract of Eupolyphaga sinensis, PeptideRank was used to evaluate the biological activity of each peptide. Since the PeptideRank score represents the probability that the peptide has biological activity, peptides with a PeptideRank score of more than 0.5 were selected for further analysis. As shown in Table 2, 4 peptides have a PeptideRank score greater than 0.5, indicating that these peptides may have higher biological activity. Safety affects the application of bioactive peptides and is an important issue. The potential allergic and toxicity of the peptides are expected to exclude peptides with potential risks. The results of the ToxinPred tool showed that all four peptides were non-toxic and could be used as potential bioactive peptides. Water solubility is also a key factor restricting the bioavailability of peptides. The Innovagen results showed that peptides 3 and 2 have good water solubility. According to the prediction results of the ToxinPred software, peptide 2 has a higher hydrophilicity, reaching 0.76. It was imported into the BIOPEP-UWM online database and was not included. The sequence of peptide 2 is RSSDIRF, and the number of its amino acids is less than 10. Studies have shown that peptide activity decreases with increasing peptide chain length. Therefore, the peptide RSSDIRF, i.e. RF7, was selected for subsequent research.

[0062] 3. Molecular docking The docking score is the approximate potential energy of the ligand binding to the macromolecule. The lower the score, the stronger the affinity between the ligand and the target protein. A docking score < –5.0 kcal / mol usually indicates that the anticoagulant peptide and the enzyme (target) have good binding affinity, thereby inhibiting the enzymatic activity of the target protein. Thrombin is the core of the blood coagulation process. It cuts soluble fibrinogen into fibrin chains and forms insoluble clots with platelets, blood cells and other substances. Therefore, thrombin is an important target in antithrombotic drugs. Thrombin generally has four binding sites, namely the active site, two positively charged anion binding sites (exosite 1 and exosite 2) and the sodium ion binding site. In addition, it also contains a self-catalytic hydrolysis ring and a W60d ring. The docking score of the anticoagulant peptide RF7 with thrombin reached -7.6 kcal / mol, and it formed 10 hydrogen bonds with Trp-60A, Ser-214, Trp-215, Gly-216, Gly-219, Asp-189, Ala-190, and Ser-195 residues of thrombin, with an average hydrogen bond distance (Å) of 2.69 ( Figure 4 A in the figure). Mainly related to Na + Binding site interactions. + The binding site mainly regulates the activity of thrombin by fixing sodium ions through coordination bonds. According to the sodium ion binding situation, it can be divided into fast thrombin and slow thrombin. The sodium-free form (called the "slow form") has anticoagulant activity. Asp-189 is negatively charged. +Ser-195 and Asp-189 together affect the optimal conformation of binding substrate. Under physiological conditions, the anticoagulant peptide FR7 is positively charged, which is conducive to binding with Na + Binding site interactions. + Binding is thought to allosterically regulate thrombin activation, and peptide RF7 binds to Na + The main amino acids in the binding site form hydrogen bonds to inhibit Na + Binding to thrombin causes thrombin to be in a sodium-free mode, thereby exerting anticoagulant activity.

[0063] Fibrin, formed after fibrinogen is hydrolyzed by thrombin, is an important component of thrombus. Inhibiting fibrinogen plays an important role in inhibiting thrombosis. Fibrinogen is composed of three chains, α, β, and γ, through 29 disulfide bonds. Fibrinogen is negatively charged at a pH of 7.40, which is also conducive to the interaction between RF7 and fibrinogen through the difference in charge. The affinity of the anticoagulant peptide RF7 to fibrinogen is -7.9 kcal / mol, and it forms 14 hydrogen bonds with Asp-318, Asp-320, Asn-319, Arg-275, Tyr-278, Asn-308, Gly-309, Asp-291, Ser-306, Lys-302 and Lys-31 residues of fibrinogen, with an average hydrogen bond distance (Å) of 2.87 ( Figure 4 The above results indicate that there is an interaction between RF7 and fibrinogen.

[0064] FXIa is located in the endogenous pathway of the coagulation pathway and can effectively inhibit the progression of coagulation with a lower risk of bleeding. Studies have shown that compared with wild-type mice, mice with FXI gene deficiency are much less susceptible to arterial and venous thrombosis. More importantly, mice with FXI gene deficiency grow healthily and do not show bleeding symptoms. In addition, patients with hemophilia C rarely experience thrombotic events due to the lack of FXI in their blood. Therefore, FXIa is an attractive target for antithrombotic therapy. Each subunit of FXIa contains four apple domains (N-terminus) and a protease domain (C-terminus). The catalytic triad of FXIa is present in the protease domain, which is composed of His-57, Asp-102 and Ser-195. Therefore, the current research on FXIa inhibitors is mainly focused on the protease domain. The FXIa apple domain is associated with binding to platelets, heparin, high molecular weight kininogen and many proteins (such as thrombin, factor XIIa and glycoprotein Ibα). The affinity of anticoagulant peptide RF7 to fibrinogen (FXIa) was -5.3 kcal / mol, and it formed nine hydrogen bonds with amino acids Ser-500, Leu-501, Ser-503, Leu-589, Tyr-511, Val-612, and Asn-610 of FXIa, with an average hydrogen bond distance (Å) of 2.39 ( Figure 5 The docking results with FXIa showed that RF7 did not act on the protease domain of FXIa, but interacted with the apple domain to change the three-dimensional structure of FXIa, thereby inhibiting the activity of FXIa.

[0065] As the first step of the intrinsic coagulation pathway, FXIIa is activated by negatively charged substances in the blood, which further activates factor FXIa. The docking score of the anticoagulant peptide RF7 with FXIIa is 7.45 kcal / mol, and it forms 8 hydrogen bonds with His-384, Gly-428, Glu-430, Tyr-605, Asp-602, and Gln-581 of FXIIa, with an average hydrogen bond distance (Å) of 2.175 ( Figure 5B in the figure). Gly-193 and Ser-19 are conserved residues of FXIIa, which form an oxygen anion hole, which is a characteristic of serine proteases and is required for their proteolytic cleavage of substrates. RF7 did not interact with Gly-193 and Ser-19 and had a low inhibitory effect on FXIIa. FXIIIa is a transglutaminase that cross-links glutamine and lysine residues of proteins through isopeptide bonds, which affects the size of the formed thrombus. In addition, FXIIIa-mediated fibrin cross-linking increases the rigidity and stability of the clot, making the clot less soluble. The docking score of the anticoagulant peptide RF7 with FXIIIa is -4.06 kcal / mol, and it forms eight hydrogen bonds with Glu-415, Lys-418, Pro-564, Lys-565, and Arg-333 of FXIIa, with an average hydrogen bond distance (Å) of 2.4 ( Figure 5 C in the figure). The active center of FXIIIa consists of a catalytic triad consisting of Cys-314, His-373 and Asp-396, and a catalytic dyad (His-342, Glu-401) located on the active side of FXIIIa. The docking results showed that RF7 did not act on the active center of FXIIIa, but exerted its effect by changing its three-dimensional conformation.

[0066] Example 3

[0067] The present invention provides a method for preparing the anticoagulant peptide RF7 as follows:

[0068] The anticoagulant peptide RF7 was prepared by solid phase synthesis. After Fmoc-Phe-Wang-Resin resin was activated, Fmoc-Arg-OH was connected; the activation site was blocked to obtain compound 1; the protecting group Fmoc in compound 1 was removed, and Fmoc-Ile-OH was connected to obtain compound 2; the protecting group Fmoc in compound 2 was removed, and Fmoc-Asp-OH was connected to obtain compound 3; the protecting group Fmoc in compound 3 was removed, and Fmoc-Ser-OH was connected to obtain compound 4; the protecting group Fmoc in compound 4 was removed, and Fmoc-Ser-OH was connected to obtain compound 5; the protecting group Fmoc in compound 5 was removed, and Fmoc-Arg-OH was connected to obtain compound 6; the protecting group Fmoc in compound 6 was removed, and the resin was cut to obtain the target product RF7.

[0069] The details are as follows: First, swell the Fmoc-Phe-Wang-Resin resin with dichloromethane (DCM) for half an hour (solid-liquid ratio of 1:20), wash it with N,N-dimethylformamide (DMF) three times, block the active sites on the resin with a mixture of dichloromethane, methanol and diisopropylethylamine (80:15:5), and wash it with DMF five times. Deprotect it with diisobutyl ketone (DBLK) for 15 minutes, wash it with DMF six times, and start coupling the first amino acid Fmoc-Arg-OH for half an hour. If the test is qualified (the solution is bright yellow and the resin is transparent), repeat the DMF washing, DBLK deprotection, and connect the second amino acid until all the amino acids are connected (the molar ratio of the amino acid added to the resin is twice). After the amino acid connection is completed, DBLK deprotects for 15 minutes to remove the Fmoc protecting group of the last amino acid to be coupled, and then wash it with DMF, DCM and methanol three times each, cut the resin with trifluoroacetic acid, filter it, precipitate the peptide with ice ether, and freeze-dry it. The dried polypeptide powder was purified using a protein purifier, and the purified components were collected to finally obtain polypeptide RF7.

[0070] The characterization method of the prepared polypeptide RF7 is as follows: The AB4000 mass spectrometer (AB Sciex, United States) and AVANCE NEO 600 NMR (Bruke, Germany) were used to verify whether the peptides were successfully synthesized, and the purity of the synthesized peptides was investigated using an Agilent 1260 high-performance liquid chromatograph (Agilent Technologies, United States). The detection wavelength was 220 nm, buffer B was 0.1% trifluoroacetic acid in water, and buffer A was 0.1% trifluoroacetic acid in acetonitrile. Gradient separation was performed on a chromatographic column (250 × 4.6 mm, Boston Green ODS-AQ) at a flow rate of 1 mL / min. The gradient of the liquid phase separation was: from 0 to 25 min, the linear gradient of buffer B was from 85% to 60%; from 25 to 25.1 min, the linear gradient of buffer B was from 60% to 0%; from 25.1 to 30 min, buffer B was kept at 0%. The secondary structure of the peptide was characterized using a JASCO-810 circular dichroism spectropolarimeter (Japan SCO Corporation, Japan), the charge was investigated using a Malven Nano ZS90 particle size potentiometer (Malvern Instruments Ltd., England), and the functional group structure was characterized using a TENSOR 27 infrared spectrometer (Bruke, Germany).

[0071] The characterization results of peptide RF7 are as follows: The anticoagulant peptide RF7 (C37 H 61 N 13 O 12 , MW = 879.97), electrospray ionization mass spectrometry (ESI-MS) scanning gave m / z = 440.8 [M+2H] 2+ and m / z = 880.5 [M+H] + Two ion peaks. The NMR data of RF7 are as follows: 1H NMR (600 MHz, D2O) δ 7.42 (dd, J = 8.2, 6.8 Hz, 2H), 7.39 – 7.34(m, 1H), 7.34 – 7.31 (m, 2H), 4.72 (dd, J = 8.9, 5.3 Hz, 1H), 4.63 (t, J =5.9 Hz, 1H), 4.55 (t, J = 5.4 Hz, 1H), 4.20 – 4.14 (m, 2H), 4.01 – 3.89 (m,4H), 3.33 – 3.26 (m, 3H), 3.21 (t, J = 7.0 Hz, 2H), 3.09 (dd, J = 14.1, 8.9Hz, 1H), 2.97 (dd, J = 16.9, 6.0 Hz, 1H), 2.87 (dd, J = 16.9, 7.3 Hz, 1H),2.07 – 1.98 (m, 2H), 1.89 – 1.67 (m, 5H), 1.59 (dddd, J = 23.8, 16.9, 13.7,6.7 Hz, 2H), 1.47 (ddd, J = 13.6, 7.4, 3.5 Hz, 1H), 1.26 – 1.14 (m, 2H), 0.90(t, J = 7.4 Hz, 3H), 0.84 (d, J = 6.8 Hz, 3H). The MS and NMR data showed that RF7 had been successfully synthesized. The purity of RF7 was 96.18% by HPLC. RF7 has UV-Vis absorption at 200-300 nm, with a maximum absorption at 210 nm ( Figure 6 The circular dichroism spectrum showed that RF7 had double negative peaks at 222 nm and 208 nm, and a positive peak at 190 nm ( Figure 6 B in the figure indicates that the structure of RF7 is mainly α-helical. The infrared chromatographic peak of RF7 is at 1669.53 cm -1 and 1532.45 cm -1 It shows the characteristic absorption of amide, 3287.74 cm -1Shows the characteristic absorption of carboxyl groups ( Figure 6 The website predicts that the isoelectric point of RF7 is 10.35. In physiological environment, it is positively charged in theory. The potential results show that RF7 is positively charged, which is consistent with the theory ( Figure 6 D in.

[0072] Pharmaceutical research on peptide RF7, including in vitro anticoagulant activity of RF7, in vitro inhibition of thrombus formation, in vivo anticoagulant effect investigation, safety investigation of RF7 and pharmacokinetic study: 1. Anticoagulant activity (1) In vitro anticoagulant activity of RF7 Referring to the APTT, PT, TT kits and Example 1, the effects of polypeptide RF7 on APTT, PT, TT, FCT, TCT and PRT were determined.

[0073] (2) Inhibition of thrombus formation in vitro 2 mL of blood was collected from the orbital venous plexus of SD rats, incubated at room temperature for 2 h, and allowed to coagulate naturally. The blood clot was washed with saline, divided, and 55 mg of blood clot and 100 microliters of sample were added to each EP tube, incubated at 37°C for 12 h, and the blood clot was washed with saline and weighed. 200 μL of whole blood was added to the EP tube containing 50 μL of samples of different concentrations, and left naturally at room temperature for 10 min. The formed thrombus was taken out of the EP tube, the liquid was absorbed with filter paper, and the wet weight of the thrombus was weighed; to reduce the error, the thrombus was naturally air-dried, and the dry weight of the thrombus was weighed. Among them, the negative blank control was added with saline, and the positive control was selected with earthworm enzyme.

[0074] 2. Investigation of in vivo anticoagulant effect (1) Ferric chloride-induced rat carotid artery thrombosis model SD rats were randomly divided into six groups, namely, blank group (normal saline), model group (normal saline), positive drug (5 mg / kg earthworm enzyme), low, medium and high dose groups (50, 75, 100 mg / kg RF7), with 6 rats in each group. 3 The rats were anesthetized by inducing common carotid artery thrombosis. After administration through the tail vein, ketamine (100 mg / mL) was injected intraperitoneally at a dose of 1 mL / kg. The left common carotid artery was isolated and 10% FeCl 3The filter paper strip (0.5 cm x 0.5 cm) soaked in the solution was applied around the common carotid artery. After 10 minutes, the filter paper was removed and the blood vessel was rinsed with saline three times. After 15 minutes, the 0.5 cm segment of the common carotid artery thrombosis site wrapped by the filter paper strip was precisely cut off and placed on the filter paper to absorb the floating blood. The wet weight of the blood vessel containing the thrombus was measured. The heart, liver, spleen, lung, kidney and blood vessels of the rat were fixed with 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin-eosin (HE) to observe the degree of tissue damage.

[0075] (2) Carrageenan-induced mouse tail thrombosis model ICR mice weighing 26-30 g were randomly divided into six groups, namely, blank group, model group, positive drug group, and three RF7 low, medium, and high dose groups, marked as groups A, B, C, D, E, and H. All six groups were administered via tail vein, of which groups A and B were given normal saline, group C was given earthworm enzyme (5 mg / kg), and groups D, E, and H were given 50, 75, and 100 mg / kg of RF7 solution, respectively). The six groups were given 1% carrageenan immediately after the first administration, and 50 mg / kg was intraperitoneally administered to model. The mice were weighed and observed on the second and third days, and the mice were killed on the third day to observe the length of the tail thrombus, and the tail was cut 4 cm away from the tip of the mouse tail, decalcified and embedded, and HE staining was used to observe pathological damage.

[0076] 3. Safety assessment of RF7 (1) Biocompatibility 100 μL HUVECs cells were added to a 96-well plate and incubated at 37°C with 5% CO 2 Culture overnight (cell density is 4 × 10 3 cells / well). To explore the cytotoxicity of peptide RF7 to HUVECs cells, the concentration range of RF7 was set to 0.0005, 0.005, 0.05, 0.1, 0.5, 1, 5, and 10 mg / mL, and the control group was added with an equal volume of PBS solution. After HUVECs cells were incubated for 24 h after administration, 10 μL of cell counting reagent-8 (CCK-8) solution was added to each well. Then the cells were incubated for 2 h under the same conditions, and the absorbance value of each well at 450 nm was measured using a SpectraMaxM2e multi-function microplate reader (Molecular Devices, United States). The cell viability percentage was calculated by the following formula, and each concentration point was operated in parallel 5 times.

[0077] Cell viability (%) = OD sample / OD control × 100% Among them, OD sample and OD control represent the absorbance values ​​of the sample group and the control group at 450 nm, respectively.

[0078] (2) Hemolysis rate investigation 2 mL of blood was collected from the orbital venous plexus of rats, anticoagulated with sodium heparin, centrifuged at 200 g, the supernatant was removed, the lower layer solution was fixed to 10 mL, centrifuged at 400 g for 5 min, the supernatant was removed, and repeated 3 times until the supernatant had no obvious red color. 500 μL of red blood cells in the lower layer were taken and fixed to 25 mL to obtain a 2% red blood cell suspension. All samples were prepared with normal saline. 70 μL of red blood cell suspension was incubated with the same volume of sample at 37°C for 4 h, centrifuged at 800 g for 5 min, 100 μL of supernatant was taken, and the absorbance at 540 nm was measured. Among them, pure water was used as a positive control and normal saline was used as a negative control. The hemolysis rate was determined according to the following formula.

[0079] (A 1 -A 0 ) / (A 2 -A 0 ) × 100% Among them A 1 : Absorbance value of the experimental group, A 0 : Absorbance value of positive control, A 2 : Absorbance value of negative control (3) Bleeding risk experiment Thirty ICR mice were randomly divided into a blank group, a heparin group, and a low-, medium-, and high-dose group of peptide RF7, with 6 mice in each group. The control group was given the corresponding saline through the tail vein, the heparin group was injected with 5 mg / kg sodium heparin through the tail vein, and the low-, medium-, and high-dose groups of peptide RF7 were given 50, 75, and 100 mg / kg RF7 solution through the tail vein, respectively. Five minutes after administration, the mice were anesthetized, cut off 3 mm from the tip of the tail, and placed in 37°C saline. The time when bleeding stopped was recorded. At the end of the experiment, the wound of the mouse was cleaned with iodine to avoid infection. The blood flowing out of the tail of the mouse was collected, centrifuged at 4000 g, the supernatant was removed, the red blood cells were resuspended with 1 mL of 1% Triton X-100 solution, and 200 μL of the above red blood cell solution was taken to measure its absorbance at 562 nm. Among them, the higher the absorbance value, the more red blood cells and the higher the amount of bleeding.

[0080] 4. Pharmacokinetic studies SD rats were given 100 mg / kg RF7 solution through the tail vein and 200 mg / kg RF7 solution orally. After administration, blood was collected from the orbit at 2, 5, 10, 20, 30, 45, 60, 80, 100, and 120 min, respectively, and plasma was collected by centrifugation. After biological sample pretreatment, the RF7 concentration in rat plasma at different time points was determined by LC-MS / MS, and the peak area was substituted into the accompanying standard curve to calculate the blood drug concentration. The blood drug concentration-time curve of RF7 was plotted, and the blood drug concentration-time data were processed using DAS 2.2.7 software to calculate the pharmacokinetic parameters.

[0081] The results of pharmaceutical studies on peptide RF7 are as follows: 1. Anticoagulant activity (1) In vitro anticoagulant activity The anticoagulant activity of RF7 was evaluated by APTT, PT, TT, FCT, TCT and PRT. RF7 had no significant effect on APTT at concentrations below 8 mg / mL. When it reached 10 mg / mL, it prolonged APTT by 3.73 seconds, and at 20 mg / mL, it prolonged APTT by 10.1 seconds ( Figure 7 A in the figure). Molecular docking results showed that RF7 had certain interactions with FXIa and FIXa, and it may prolong APTT by inhibiting these two coagulation factors. RF7 may prolong PT time by inhibiting FVIIa. At 20 mg / mL, RF7 prolonged PT by 5.7 seconds ( Figure 7 B in the figure). In the molecular docking results, RF7 has the lowest docking score for thrombin and fibrinogen, which indicates that RF7 has the strongest inhibitory effect on them. The TT results show that RF7 has a significant difference in the extension time of TT at 2 mg / mL, reaching 2.93 seconds, and can extend 32.43 seconds at the highest concentration (20 mg / mL). Figure 7 C in the coagulation cascade). Calcium ions are essential ions in the coagulation cascade and are also coagulation factor IV. Without calcium ions, the coagulation cascade will be inhibited. PRT examines the effect on the endogenous pathway of the coagulation cascade. RF7 can inhibit coagulation factors in the endogenous coagulation pathway, such as FXIa, FIXa, etc. to prolong PRT. The experimental results show that when the RF7 concentration reaches 20mg / mL, the PRT can be extended by 109.2 seconds ( Figure 7 D in the figure). APTT, PT, and TT are commonly used clinical indicators for detecting the coagulation system. The results show that RF7 has a significant effect on TT, mainly affecting the coagulation factors on the common pathway, namely, fibrinogen and thrombin. RF7 was incubated with thrombin and fibrinogen, respectively, and the fibrinogen clotting time (FCT) and thrombin clotting time (TCT) were measured. When the incubation concentration of RF7 and thrombin reached 10 mg / mL, FCT reached the maximum measurement time, and fibrin could not be formed ( Figure 7 When the concentration of RF7 and fibrinogen incubated reached 8 mg / mL, TCT reached the maximum measurement time and fibrin could not be formed ( Figure 7 The results showed that RF7 has an inhibitory effect on both fibrinogen and thrombin, and its effect on fibrinogen may be stronger than that on thrombin. In summary, RF7 has obvious anticoagulant activity, which mainly inhibits the activity of thrombin and fibrinogen, thereby inhibiting the common pathway.

[0082] (2) Antithrombotic effect in vitro The rat blood was naturally coagulated without anticoagulants to obtain blood clots. The same weight of blood clots were taken and incubated with the samples to investigate whether RF7 could dissolve the blood clots. Figure 8 As shown in A, after RF7 was incubated with blood clots, the volume of the blood clots gradually decreased with the increase of RF7 concentration. The weight of the blood clots was measured. The blood clots in the blank group did not dissolve and the weight still reached 55.4 mg. As the concentration of RF7 increased to 30 mg / mL, only 14.23 mg of blood clots remained ( Figure 8 B in the figure shows that RF7 dissolves blood clots by hydrolyzing fibrin. In the in vitro anticoagulation mechanism study, RF7 not only hydrolyzes fibrinogen, but also has an inhibitory effect on thrombin and FXIa. RF7 was incubated with whole blood for a certain period of time, and the weight of the clot after the blood naturally coagulated was measured to investigate the in vitro antithrombotic effect of RF7. The results are shown in Figure 8 As shown in C, the blood clot formed in the blank group was the largest, and as the concentration of RF7 increased, the blood clot formed gradually decreased. The wet weight of the blood clot was measured. The wet weight of the blood clot in the normal saline group was 96.83 ± 2.59 mg, and the blood clot weight in the positive drug control group earthworm enzyme group (5 mg / mL) was 69.93 ± 25.61 mg. Compared with the normal saline group, the blood clot formation was reduced after RF7 was incubated with whole blood, and as the concentration increased, the blood clot and its weight gradually became smaller. The blood clot weight in the highest concentration group (30 mg / mL) was only 30.2 ± 6.7 mg. Because the blood clot contains a high amount of water, in order to reduce errors, the dry weight of the blood clot in each group was measured. The results are shown in Figure 8 As shown in D, the blood clot weight in the saline group was 17.6 ± 2.59 mg, the blood clot weight in the earthworm enzyme group was 69.93 ± 25.61 mg, and the blood clot weight in the highest RF7 concentration group (30 mg / mL) was 3.87 ± 1.33 mg. The above results show that RF7 can inhibit the formation of blood clots. In summary, RF7 can dissolve the already formed blood clots by hydrolyzing fibrin, and can also inhibit the formation of blood clots by inhibiting coagulation factors such as FXIa and thrombin, and has significant anti-thrombotic activity in vitro.

[0083] 2. Investigation of in vivo anticoagulant effect (1) Ferric chloride model To evaluate the antithrombotic effect of RF7 in animals, an acute carotid artery thrombosis model was established in rats. Ferric chloride can damage endothelial cells, activate platelets and the coagulation cascade system, and form local arterial thrombosis. Fig. 9 As shown in A, RF7 can significantly reduce the 3 The induced carotid thrombosis wet weight inhibited thrombosis. After the carotid artery was fixed with paraformaldehyde, the blood vessels in the blank group appeared white as a whole, and no thrombus formation occurred in the blood vessels. The model group was dark brown, and the blood vessels were completely blocked by thrombi, indicating that the model was successful; there was a small amount of thrombus formation in the blood vessels of the positive drug group, and the blood vessel walls appeared yellow; the low, medium and high dose groups showed obvious dose dependence, and the thrombi in the blood vessels gradually decreased. The HE staining results showed that the vascular wall of the blank group was a three-layer structure of the inner membrane, the middle membrane and the outer membrane from the inside to the outside, with a complete tissue structure. A small amount of endothelial cells were visible on the inner membrane, the elastic membrane of the middle membrane was wavy, the structure was clear, and blood vessels and nerve fibers were visible in the outer membrane. There was no obvious thinning or damage to the vascular wall, and no thrombus formation occurred in the lumen. The wall of the model group became thinner, the structure was unclear, and the wavy structure of the elastic membrane disappeared and became flat. Thrombus formation was visible in the lumen, suspected to be a mixed thrombus, accounting for about 80% of the entire lumen. After administration, the thrombus content in the lumen gradually decreased ( Fig. 9 B in the figure). The rat neck blood vessels were weighed. The blood vessel weight of the blank group was 2.98 ± 0.45 mg. The blood vessel weight of the model group was 6.38 ± 1.28 mg due to the formation of thrombus in the blood vessel. The drug administration can significantly reduce the formation of thrombus in the blood vessel. The high-dose group (100 mg / kg) has a higher efficacy than the positive drug group and can significantly reduce the weight of the blood vessel ring ( Fig. 9 E in.

[0084] (2) Mouse carrageenan Carrageenan is a sulfated polysaccharide extracted from seaweed. Once it enters the animal body, it will cause tissue inflammation, damage vascular endothelial cells, activate the coagulation system, and cause thrombosis. There was no thrombosis in the tail of the mice in the blank control group. The tail tissue was normal and the blood vessels of the mice could be clearly seen. The mice in the model group and the drug-treated group gradually formed thrombi from the tip of the tail, causing the tail tissue of the mice to appear black ( Fig. 9 C). The antithrombotic effect of RF7 in the mouse tail thrombosis model was further demonstrated by sectioning 4 cm from the tip of the mouse tail and performing hematoxylin-eosin staining. Fig. 9As shown in D, no thrombosis was observed in the blood vessels of the blank group mice, while thrombosis was observed in the blood vessels of the tail tissue of the model group mice, which tended to be mixed thrombosis, accounting for about 75% of the lumen. After administration, the thrombosis in the tail vessels of the mice in the earthworm enzyme group (5 mg / kg) accounted for about 30%-50% of the lumen, and a small amount of inflammatory cell infiltration was observed around some blood vessels. After the administration of the peptide RF7, no thrombosis was observed in the blood vessels at a medium dose (75 mg / kg). The antithrombotic activity of RF7 in vivo was further evaluated by measuring the length of black thrombus in the tail of mice. The normal group mice had no black tail length, while the model group had a black tail length of 53.12 ± 13.10 mm. After administration of positive drug earthworm enzyme and polypeptide RF7, the thrombosis formation in the tail of mice was inhibited to varying degrees, and the length of thrombus in the tail of mice decreased significantly. The black tail length of the low-dose RF7 group (50 mg / kg) was 32.92 ± 14.03 mm, and the degree of thrombosis inhibition was greater than that of the earthworm enzyme group (42.65 ± 8.94 mm). When the dose of polypeptide RF7 reached 100 mg / kg, the length of the black tail of mice was only 13.93 ± 11.32 mm ( Fig. 9 F in the figure). Since the tail length of each mouse is different, in order to further reduce the error, the black tail rate of mice was calculated. The black tail rate of mice in the model group was 57.52 ± 13.79%, which decreased to 43.47 ± 8.40% after administration of earthworm enzyme. After administration of different concentrations of RF7 (50, 75, 100 mg / kg), the black tail rate gradually decreased to 35.21 ± 14.82%, 33.38 ± 12.09%, and 17.31 ± 12.02%, respectively ( Fig. 9 In conclusion, RF7 can alleviate carrageenan-induced tail thrombosis in mice and has good antithrombotic activity.

[0085] 3. RF7 safety evaluation HUVEC cells were used to investigate the cellular biotoxicity of RF7. The experimental results showed that the cell survival rate was close to 100% within the concentration range of 10 mg / mL ( Fig.10 A in the figure shows that RF7 has little cytotoxicity. After RF7 was co-cultured with 2% erythrocytes for 4 h, there was no rupture of erythrocytes, which was consistent with the results of the saline group ( Fig.10 When the RF7 concentration reached 20 mg / mL, the hemolysis rate was less than 1% ( Fig.10C in the figure indicates that RF7 does not cause hemolysis and has good biosafety. Bleeding risk is the biggest side effect of current anticoagulants. The bleeding risk of RF7 was investigated by a mouse tail bleeding experiment. Low, medium and high concentrations (50, 75, 100 mg / kg) of RF7 were administered through the tail vein. The mice were anesthetized 5 minutes later, the blood vessels in the tail of the mice were damaged, and the mice were placed in normal saline to allow natural coagulation. The bleeding time was recorded, the collected blood was centrifuged, the red blood cells were broken, the amount of bleeding was investigated by absorbance value, and compared with the positive control heparin (5 mg / kg) ( Fig.10 D in the figure). The bleeding time of the group injected with normal saline as the blank control was 160.13 seconds, while the bleeding risk of the heparin group (5 mg / kg) was serious, with a bleeding time of 559.33 seconds, which was significantly different from the blank group. The bleeding times of the low, medium and high dose groups of RF7 (50, 75, and 100 mg / kg) were 161.83, 180.5, and 197.97 seconds, respectively, which were not significantly different from the blank group ( Fig.10 E in the figure). The amount of bleeding in different groups can be further shown by the absorbance value. The higher the absorbance value, the greater the amount of bleeding and the higher the risk of bleeding. The absorbance value of the blank group was 0.09, the heparin group (5 mg / kg) was 1.13, and the absorbance values ​​of the RF7 low, medium and high dose groups (50, 75, 100 mg / kg) were 0.21, 0.20, and 0.46, respectively, which were not significantly different from the blank group ( Fig.10 F in the figure). The results of bleeding time and bleeding volume showed that the bleeding risk of RF7 was much lower than that of heparin, and there was no significant difference with the blank group. The bleeding risk was low and it could be used as a candidate drug for anticoagulant drugs. After SD rats were intravenously administered low, medium and high concentrations (50, 75, 100 mg / kg) of RF7, the anesthetized rats were killed by dislocating the neck and the important organs (heart, liver, spleen, lung and kidney) were taken for HE staining. The results are shown in Fig.10 As shown in G, the staining results of the three low, medium and high dose groups (50, 75 and 100 mg / kg) were consistent with those of the blank group, and there was no obvious acute or chronic pathological toxicity or inflammatory cells in the main organs. In summary, RF7 has no cytotoxicity, no hemolytic risk, low bleeding risk, no damage to the main organs of rats, and high safety.

[0086] 4. Pharmacokinetic study of RF7 Table 3 Pharmacokinetic parameters of RF7

[0087] After SD rats were given RF7 solution by tail vein and oral gavage, 0.3 mL of blood was collected from the rats' orbits at 2, 5, 10, 20, 30, 45, 60, 80, 100, and 120 min, respectively. After biological sample pretreatment, LC-MS / MS was used to determine the RF7 concentration in rat plasma at different time points, and the peak area was substituted into the accompanying standard curve to calculate the blood drug concentration, and the blood drug concentration-time curve of RF7 was drawn ( Fig.11 The blood drug concentration-time data were processed by DAS 2.2.7 software, and the pharmacokinetic parameters of RF7 were analyzed by non-compartmental model fitting. The results are shown in Table 3. After oral absorption, RF7 reached C max (299.50 ± 114.18 ng / mL), AUC 0-∞ was (7564.65 ± 1497.56) ng·h / mL; after tail vein administration, C max (1171.00 ± 664.34 ng / mL), AUC 0-∞ The absolute bioavailability of RF7 was calculated to be 13.90%, which is not suitable for oral administration. The HPLC method was established to investigate the plasma stability and gastrointestinal stability of RF7. RF7 would break in plasma and then be gradually hydrolyzed into amino acids. The plasma stability of RF7 was low, and only 46.53% remained after incubation with plasma for 30 minutes.

[0088] Table 4 Stability of RF7 in artificial gastric juice and artificial intestinal juice

[0089] Artificial gastric juice and artificial intestinal juice were used to simulate the human gastrointestinal tract to investigate the stability of RF7 in the gastrointestinal tract. The results are shown in Table 4. RF7 is relatively stable in gastric juice, still containing 78.42% after 3 hours of incubation, but it is unstable in artificial gastric juice, with only 1.3% left after 3 hours. RF7 is an acidic polypeptide, which is more stable in an acidic environment than in an alkaline environment. Secondly, RF7 is a linear polypeptide, which is much less stable than a cyclic peptide and is easily hydrolyzed into amino acids by various enzymes in plasma. Therefore, RF7 has a low half-life and low absolute bioavailability.

Claims

1. An anticoagulant peptide, characterized in that It has the following structural formula: 。 2. A method for preparing the anticoagulant peptide according to claim 1, characterized in that: The following steps are involved: After activation of Fmoc-Phe-Wang-Resin resin, Fmoc-Arg-OH was connected to block the activation site to obtain compound 1; Remove the protecting group Fmoc in compound 1 and connect Fmoc-Ile-OH to obtain compound 2; Remove the protecting group Fmoc in compound 2 and connect Fmoc-Asp-OH to obtain compound 3; The protecting group Fmoc in compound 3 was removed, and Fmoc-Ser-OH was connected to obtain compound 4; The protecting group Fmoc in compound 4 was removed, and Fmoc-Ser-OH was connected to obtain compound 5; Remove the protecting group Fmoc in compound 5 and connect Fmoc-Arg-OH to obtain compound 6; The protecting group Fmoc in compound 6 was removed and the resin was cleaved to obtain the target product RF7.

3. The method for preparing the anticoagulant peptide according to claim 2, characterized in that: The reagent used for activation of Fmoc-Phe-Wang-Resin resin is dichloromethane.

4. The method for preparing the anticoagulant peptide according to claim 2, characterized in that: The reagent used to block the activated sites was a mixture of dichloromethane, methanol and diisopropylethylamine.

5. The method for preparing the anticoagulant peptide according to claim 2, characterized in that: The reagent used to remove the Fmoc protecting group in each compound is diisobutyl ketone.

6. The method for preparing the anticoagulant peptide according to claim 2, characterized in that: After removing the protecting group Fmoc in compound 6, the obtained product is washed, the resin is cut, filtered, and precipitated to obtain purified RF7.

7. The method for preparing the anticoagulant peptide according to claim 6, characterized in that: After removing the protecting group Fmoc in compound 6, the product was washed with N,N-dimethylformamide, dichloromethane and methanol respectively.

8. The method for preparing the anticoagulant peptide according to claim 6, characterized in that: After removing the protecting group Fmoc in compound 6, the reagent used to cut the resin is trifluoroacetic acid.

9. The method for preparing the anticoagulant peptide according to claim 6, characterized in that: After removing the protecting group Fmoc in compound 6, the reagent used for precipitation was glacial ether.

10. Use of the anticoagulant peptide according to claim 1 in the preparation of anticoagulant drugs.

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