A polypeptide sr14 and its use

Abstract

The application belongs to the technical fields of polypeptide and biological medicine, and particularly relates to a polypeptide SR14 and application thereof. The amino acid sequence of the polypeptide SR14 is SCEPCQTLAVRSYR. The polypeptide SR14 composed of the amino acids can be effectively combined with a platelet receptor, interfere with the aggregation of thrombin-activated platelets, and then achieve the effect of anti-thrombus. Meanwhile, the polypeptide SR14 has a low risk of bleeding, and can be used for the preparation of anti-thrombus drugs.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide and biomedical technology, specifically relating to a polypeptide SR14 and its applications. Background Technology

[0002] Platelets are the smallest blood cells, normally disc-shaped. When blood vessel walls are damaged, platelets are activated, deform, aggregate, and adhere to the vessel wall. Simultaneously, platelets release granulation factors, which feed back into the coagulation system, promoting the coagulation process. Because platelets possess various types of membrane protein receptors, different agonists can activate them through different receptors, making the activation process complex. Since platelets play crucial roles in hemostasis and thrombosis, and possess abundant therapeutic targets, antiplatelet drugs are currently a major focus in antithrombotic drug development. Based on the various protein receptors and enzymes within platelets, a series of antiplatelet antithrombotic drugs have been developed, primarily consisting of membrane protein receptor inhibitors and enzyme activity inhibitors.

[0003] Current antiplatelet antithrombotic drugs such as clopidogrel and hirudin can effectively reduce the incidence of cardiovascular and cerebrovascular diseases. However, due to the significant bleeding side effects of these drugs in clinical use, the use of higher doses to achieve better therapeutic effects is limited.

[0004] Thrombin, a serine protease, is a major platelet agonist. It binds to receptors on platelets, triggering signal transduction and activating platelets. Recently discovered bioactive peptides with antiplatelet aggregation effects can inhibit platelet activation and aggregation induced by various pathways, providing new strategies and a basis for the development of novel antiplatelet drugs. Currently, there are no reports in existing technologies of peptides that possess both antiplatelet aggregation efficacy and low bleeding risk. Summary of the Invention

[0005] The purpose of this invention is to provide a polypeptide SR14 and its application, wherein the polypeptide SR14 has both strong antithrombotic properties and low bleeding risk, and can be used in the preparation of antithrombotic drugs.

[0006] The present invention provides a polypeptide SR14, wherein the amino acid sequence of the polypeptide SR14 is SCEPCQTLAVRSYR.

[0007] This invention also provides the application of the polypeptide SR14 described in the above technical solution in the preparation of antithrombotic drugs.

[0008] Preferably, the antithrombotic drug includes an antiplatelet drug.

[0009] Preferably, the antiplatelet agent comprises a drug having antiplatelet aggregation and / or antithrombin activation effects.

[0010] Preferably, the antiplatelet drug includes drugs that have antiplatelet aggregation and antithrombin activation effects.

[0011] The present invention also provides a drug for preventing and treating thrombotic diseases, the drug comprising an effective dose of the polypeptide SR14 described in the above-described technical solution and a pharmaceutically acceptable carrier.

[0012] Beneficial effects:

[0013] This invention provides a polypeptide SR14, the amino acid sequence of which is SCEPCQTLAVRSYR. The polypeptide SR14 of this invention can effectively bind to platelet receptors, interfering with thrombin-activated platelet aggregation, thereby achieving an antithrombotic effect; simultaneously, the polypeptide SR14 has a low bleeding risk. The above-mentioned effects of the polypeptide have the potential for use in the preparation of antithrombotic drugs. Attached Figure Description

[0014] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0015] Figure 1 This is an example of platelet aggregation activated by thrombin under different concentrations of polypeptide SR14 in Example 2.

[0016] Figure 2 This is a statistical analysis of the maximum aggregation rate of thrombin-activated platelets under different concentrations of peptide SR14 in Example 2.

[0017] Figure 3 This refers to the effect of polypeptide SR14 in Example 3 on thrombin activation of downstream platelet pathways;

[0018] Figure 4 The effect of polypeptide SR14 on the release of downstream calcium ions from platelets activated by thrombin in Example 3;

[0019] Figure 5 This refers to the effect of polypeptide SR14 on changes in blood flow in blood vessels caused by ferric chloride in Example 4.

[0020] Figure 6 The effect of polypeptide SR14 on tail bleeding in mice in Example 4. Detailed Implementation

[0021] This invention provides a polypeptide SR14, the amino acid sequence of which is SCEPCQTLAVRSYR (SEQ ID NO.1). Preferably, the polypeptide SR14 is a synthetically produced polypeptide with a molecular weight of 1612.82 Da.

[0022] This invention synthesizes the polypeptide SR14 using a solid-phase Fmoc synthesis method. This invention does not specifically limit the steps of the Fmoc synthesis method; polypeptides with the same amino acid sequence as the polypeptide SR14 obtained using Fmoc synthesis methods in the art are all within the scope of protection of this invention.

[0023] The present invention has found that the polypeptide SR14 can effectively bind to platelet receptors, interfere with thrombin-activated platelet aggregation, and thus achieve an antithrombotic effect; at the same time, the polypeptide SR14 has a low bleeding risk.

[0024] Based on the advantages of the aforementioned polypeptide SR14, this invention provides the application of the polypeptide SR14 described in the above technical solution in the preparation of antithrombotic drugs. The antithrombotic drugs of this invention preferably include antiplatelet drugs, more preferably drugs with antiplatelet aggregation and / or thrombin activation inhibition effects, and most preferably drugs with both antiplatelet aggregation and thrombin activation inhibition effects.

[0025] This invention also provides a drug for preventing and treating thrombotic diseases, the drug comprising an effective dose of the polypeptide SR14 described in the above-mentioned technical solution and a pharmaceutically acceptable carrier. This invention does not specifically limit the type of carrier; a suitable drug carrier can be selected based on the drug's dosage form.

[0026] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1

[0028] A method for preparing polypeptide SR14, comprising the following steps:

[0029] The polypeptide SR14 was synthesized in an ordered manner on the polymeric resin Fmoc-Arg(PBf)-Wang Resin according to its amino acid sequence characteristics (SCEPCQTLAVRSYR).

[0030] 1. Resin swelling

[0031] Weigh out Fmoc-Arg(PBf)-Wang Resin, pour it into a solid-phase synthesis reaction column, add 50 mL of dichloromethane (DCM) to swell, and soak for 5 min.

[0032] 2. Remove protection

[0033] The DCM in the reaction column was dried using a vacuum pump. A 20% piperidine / DMF (dimethylformamide) solution was added to remove Fmoc. The column was agitated with nitrogen for 30 minutes, then dried. The column was washed five times with DMF and then dried again.

[0034] 3. Condensation reaction

[0035] The condensation reaction was carried out using a combination of TBTU and DIEA activators. TBTU and the linked amino acid were weighed and calculated based on 3 times the feed amount. After being dissolved in 50 mL of LDMF, the mixture was added to the reaction column. DIEA (1.55 mL) was added and the reaction was agitated with nitrogen for 1 h.

[0036] 4. Testing and washing

[0037] Take a small amount of resin from the reaction column using a sampling tube, pour it into a small test tube, wash it once with DMF, then discard the DMF. Add 3 drops each of solutions A, B, and C (solution A: ninhydrin alcohol solution; solution B: a mixture of 20% volume anhydrous ethanol and 80% volume phenol; solution C: redistilled pyridine solution). Heat the test tube at 120°C for 3 minutes. Observe the color of the solution and resin in the small test tube. If the solution is yellow and the resin is colorless or pale yellow, it indicates that the condensation reaction is complete. Stop the reaction in the reaction column, dry it, and wash it three times with DMF.

[0038] 5. Re-investment

[0039] If the resin is detected to be a different color, such as green, blue, or purple, it indicates that the reaction is incomplete. In this case, the resin should be dried and washed three times. Weigh the same amount of TBTU and the linked amino acid from the previous reaction and react them again in the reaction column. Repeat step 4 until the reaction is complete.

[0040] 6. Continue to condense

[0041] Repeat steps 2-5 for subsequent amino acid ligation, keeping the amount of amino acids weighed and the amount of TBTU / DIEA constant.

[0042] 7. Contraction

[0043] After the last amino acid is attached, repeat step 2, then wash three times with DCM and three times with methanol. After drying, pour out the resin and put it into a 500mL beaker to dry.

[0044] 8. Cutting

[0045] Add 100 mL of cutting fluid (volume ratio of trifluoroacetic acid: benzyl sulfide: phenol: 1,2-ethylenedithiol: water = 86:5:4:3:2) to a beaker, place the beaker on a magnetic stirrer and stir for 2 h. Then filter with a sintered sand funnel, wash the resin in the sintered sand core twice with trifluoroacetic acid, add room temperature ether to the filtered cutting fluid, at which point the polypeptide precipitates to form a polypeptide ether suspension, centrifuge at 3100 g for 30 s, discard the supernatant, add ether to wash, repeat the washing 3 times, and the solid obtained after drying is the crude target polypeptide.

[0046] 9. Purification and preparation

[0047] The crude product was dissolved in water, filtered, and then separated and purified by high performance liquid chromatography (HPLC) to finally obtain a high-purity target polypeptide with a purity of over 95%.

[0048] Specific process: The crude target peptide is dissolved in a certain volume of acetonitrile (CAN)∶H2O (1∶4) solution, and a small sample is taken to analyze the peak time of the sample.

[0049] High-performance liquid chromatography (HPLC) analysis: A C18 column is selected, and the dissolved crude peptide is passed through the column. The crude peptide is separated according to its elution time. Impurities are separated from the desired sample based on the different elution peaks in the chromatogram. The accurate molecular weight of the chromatographic peaks is determined on ESI-MS, and finally, the high-purity target product is obtained.

[0050] The collected products that passed the mass spectrometry test were freeze-dried and concentrated, then dispensed into cryovials and stored at -20°C.

[0051] Example 2

[0052] The steps for verifying the antiplatelet aggregation activity of peptide SR14 are as follows:

[0053] The anti-platelet aggregation activity of peptide SR14 was verified by platelet aggregation assay. Platelet aggregation was detected by turbidimetric assay, and the steps are as follows:

[0054] 1. After thoroughly mixing the blood sample with the anticoagulant, balance the mixture and centrifuge at 150g for 10 minutes at room temperature. After centrifugation, take the upper light yellow suspended liquid (platelet-rich plasma (PRP)) into a 1.5mL centrifuge tube, centrifuge at 400g for 5 minutes at room temperature, and discard the supernatant.

[0055] 2. Resuspend the separated platelet pellet using benchtop solution A. Centrifuge the resuspended platelet suspension at 400g for 5 minutes at room temperature. Discard the supernatant after centrifugation, and repeat 2-3 times. After the final wash, discard the supernatant and resuspend the platelets in benchtop solution B. Dilute the platelet concentration to 10 using benchtop solution B.5 / mL;

[0056] 3. Turn on the platelet aggregator and start the aggregation experiment when the platelet aggregator incubation temperature reaches 37°C; add 300 μL of the platelets diluted in step 2 to each of the four cuvettes, and add one rotor to each cuvette. Add three different concentrations (diluted with PBS buffer) of peptide SR14 solution to three cuvettes, namely 100 μM, 20 μM and 4 μM, respectively. Add an equal volume of PBS to the fourth cuvette as a control group.

[0057] 4. Place the four cuvettes from step 3 into the incubation tank of the platelet aggregator and incubate at 37°C for 2 minutes. Use an equal volume of benchtop solution B as a blank control before aggregation. Add 1 μL of thrombin to each of the four cuvettes to activate the platelets, with a final thrombin concentration of 0.01 U / mL. Observe the platelet aggregation in each group using the platelet aggregator and record the maximum aggregation rate. The results are as follows: Figures 1-2 As shown.

[0058] Depend on Figure 1 It can be concluded that, compared with the control group, the platelet aggregation rate gradually decreased with the increase of peptide SR14 concentration, especially after mixing 100 μM peptide SR14 with platelets, platelet aggregation was basically completely inhibited.

[0059] Depend on Figure 2 It can be concluded that: Figure 1 Statistical analysis of the maximum aggregation rate showed that the inhibitory effects of the three different concentrations of SR14 peptide solutions on platelet aggregation were significantly different from those of the control group (P < 0.01).

[0060] comprehensive Figures 1-2 This demonstrates that the polypeptide SR14 described in this invention can interfere with thrombin-activated platelet aggregation and has significant anti-platelet aggregation activity.

[0061] Example 3

[0062] The effects of peptide SR14 on thrombin-activated platelet downstream pathways and downstream calcium ion release were investigated using the following steps:

[0063] 1. Sample preparation

[0064] After separating platelets as in Example 2, the platelet concentration was adjusted to 10 using Benzoate B solution. 5Thrombin was used as an agonist, with a final concentration of 0.01 U / mL. The experimental group received SR14 peptide, while the control group received an equal volume of physiological saline, and both were incubated with platelets. In the control group, platelets were activated and aggregated after thrombin was added. The reaction was terminated by adding 300 μL of RIPA lysis buffer at different time points (50 s, 100 s, 200 s). In the experimental group, the reaction was terminated by adding 300 μL of RIPA lysis buffer at 200 s after thrombin was added. The samples were placed on ice for 30 min to fully lyse, then transferred to centrifuge tubes and centrifuged at 10000 g for 10 min at 4 °C. The supernatant was aliquoted and stored at -20 °C.

[0065] 2. BCA determination of total protein content in each sample

[0066] (1) According to the instructions, mix Solution A and Solution B in a volume ratio of 50:1 to prepare an appropriate amount of working solution, and place it on ice to avoid light for later use.

[0067] (2) Take 20 μL of protein standard (5 mg / mL BSA) and dilute it to 100 μL to make the final concentration 1.0 mg / mL.

[0068] (3) Add the diluted standard (1.0 mg / mL BSA) to the microplate at concentrations of 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL and 20 μL respectively, and add standard diluent to bring the total amount of standard to 20 μL.

[0069] (4) Add an appropriate volume of sample to the microplate and dilute with standard diluent to 20 μL.

[0070] (5) Add 200 μL BCA working solution to each well, mix the sample and working solution, and place at 37°C for 30 min.

[0071] (6) After cooling to room temperature, the absorbance at 562 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the protein concentration in each sample was calculated based on the standard curve.

[0072] 3. Western-Blot experiment

[0073] (1) After determining the total protein concentration in each sample according to the BCA method, 20 μg of total protein from each sample was taken for SDS-PAGE electrophoresis with a gel concentration of 12.5%. Each protein sample was added to SDS loading buffer (5×), treated in boiling water or a 95℃ PCR instrument for 5 min, then removed, cooled slightly on ice, and loaded; stacking gel, 60V; separating gel, 120V.

[0074] (2) After electrophoresis, protein bands in the 10 kDa-55 kDa range were cut and transferred to a 0.22 μm PVDF membrane using wet transfer. Transfer conditions: constant current 200 mA, 40 min, kept on ice or at 4°C throughout.

[0075] (3) After the transfer is completed, rinse the PVDF membrane with TBST for 5 min.

[0076] (4) Incubate in the blocking solution for 1 hour at room temperature.

[0077] (5) Rinse with TBST for 5 minutes.

[0078] (6) Incubate with primary antibody overnight at 4°C.

[0079] (7) TBST rinse, 4×5min.

[0080] (8) Incubate with secondary antibody for 1 hour at room temperature.

[0081] (9) TBST rinse, 4×5min.

[0082] (10) The results of color development using ECL chemiluminescence method, darkroom exposure or gel imaging system are photographed and developed.

[0083] 4. Flow cytometry detection of intracellular calcium ion release in platelets

[0084] (1) The platelet separation method is as shown in Example 2. The washed platelets are resuspended using benchtop solution A, and the platelet concentration is adjusted to 10. 8 / mL, wrap the centrifuge tube with aluminum foil, add Flou-3AM calcium ion fluorescent probe dye under light-protected conditions, the final dye concentration is 5μM, mix by inversion, and incubate at 37℃ in the dark for 20min.

[0085] (2) After incubation, the platelets were centrifuged at 400g for 5 minutes at room temperature, then resuspended in Benchmark A solution and washed 2-3 times to remove excess dye; the platelets were then resuspended in Benchmark B solution and the platelet concentration was adjusted to 10. 6 / mL, the peptide SR14 and the positive control drug bivalirudin were added and incubated separately.

[0086] (3) The intracellular calcium ion concentration of platelets before activation was detected by the instrument. After 100 seconds of instrumentation, the sample was removed, thrombin was added and shaken quickly, and the intracellular calcium ion concentration of platelets after activation was detected by the instrument.

[0087] Depend on Figure 3 It can be concluded that as thrombin activates platelets, the P44 / 42MAPK and P38 MAPK signaling pathways within platelets are gradually activated, while peptides SR14 and bivalirudin inhibit this process.

[0088] Depend on Figure 4 It can be concluded that when thrombin activates platelets, a large amount of calcium ions are released from the platelets, while polypeptide SR14 and bivalirudin inhibit the release of calcium ions from the platelets.

[0089] Example 4

[0090] The verification of the anticoagulant function and low bleeding risk of peptide SR14 was performed using the following steps:

[0091] 1. Mouse carotid artery thrombosis model

[0092] Thirty-six C57 mice (20-22g, male and female) were randomly divided into six groups (n=6): a saline group, a polypeptide administration group (0.2mg / kg, 1mg / kg, and 5mg / kg), a bivalirudin tail vein injection group (5mg / kg), and a clopidogrel gavage group (5mg / kg). Twenty minutes after tail vein injection or one hour after gavage, the mice were anesthetized with sodium pentobarbital via intraperitoneal injection. The mice were then fixed to a surgical table, and the neck skin was longitudinally incised along the midline using sterile equipment. The muscle tissue was bluntly dissected with hemostatic forceps to expose the trachea. The bilateral common carotid arteries were separated using a glass needle, and a pad soaked in 10% FeCl3 (w / v) was placed over the exposed carotid arteries to induce thrombus formation. Laser Doppler ultrasound was used to detect blood flow and thrombus formation in the carotid arteries at 5 and 10 minutes.

[0093] 2. Mouse tail hemorrhage model

[0094] Twenty-four C57 mice (20-22g, male and female) were randomly divided into four groups (n=6): a saline group, a polypeptide administration group (5mg / kg), a bivalirudin tail vein injection group (5mg / kg), and a clopidogrel gavage group (5mg / kg). Measurements were taken 20 minutes after tail vein injection or 1 hour after gavage administration. To measure tail hemorrhage time, mice were restrained, and approximately 2mm of the tail tip was cut off. The tail was kept vertical and immediately immersed in 37℃ saline. The time from tail avulsion to cessation of bleeding was observed and recorded. Results are as follows: Figures 5-6 As shown.

[0095] Depend on Figure 5 It can be concluded that peptide SR14 significantly inhibited the reduction in intravascular blood flow induced by ferric chloride and prolonged the time to thrombus formation. Consistent with positive control drugs, it exhibits a significant anticoagulant effect.

[0096] Depend on Figure 6It can be concluded that compared with the two positive drugs, the risk of bleeding is significantly reduced by polypeptide SR14, and this is statistically significant.

[0097] As can be seen from the above embodiments, the polypeptide SR14 of the present invention can effectively bind to platelet receptors, interfere with thrombin-activated platelet aggregation, and thus achieve an antithrombotic effect; at the same time, the polypeptide SR14 has a low bleeding risk.

[0098] 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. A polypeptide SR14, characterized in that, The amino acid sequence of the polypeptide SR14 is SCEPC QTLAVRSYR.

2. Use of the polypeptide SR14 of claim 1 in the preparation of an antithrombotic medicament.

3. Use according to claim 2, characterized in that, The antithrombotic medicament is a medicament having the efficacy of anti-platelet aggregation and / or inhibiting the activation of thrombin.

4. An antithrombotic agent, characterized by comprising a compound of the formula (I) or a salt thereof. The antithrombotic medicament comprises an effective dose of the polypeptide SR14 of claim 1 and a pharmaceutically acceptable carrier.

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