An antithrombotic polypeptide Cb17037 and its application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-thrombotic polypeptide Cb17037 and its application. The anti-thrombotic polypeptide Cb17037 provided by the present invention is screened from cone snail toxin, has no bleeding risk at a concentration of 20 mg / kg and below, and has a good improvement effect on carotid artery injury and ischemic stroke. The anti-thrombotic polypeptide Cb17037 of the present invention has a significant inhibitory effect on platelet activation and aggregation induced by PAR‑1 AP, but has no significant effect on basic coagulation indicators such as APTT, PT and TT. The anti-thrombotic polypeptide Cb17037 of the present invention has an anti-thrombotic effect in vivo and in vitro, can be used to prepare products that inhibit platelet activation and / or aggregation, anti-thrombotic drugs, prevent and / or treat thrombotic diseases, and has a lower risk of bleeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an anti-thrombotic polypeptide Cb17037 and an application thereof. Background Art

[0002] The main cause of cardiovascular and cerebrovascular diseases and death is thromboembolic disease. Vascular endothelial damage activates platelets in the corresponding area, further promoting thrombosis, causing vascular occlusion and multiple organ damage. Thrombosis formation depends on platelet activation and aggregation. The activation signal induced by platelet activation can trigger the release of platelet granules, resulting in the release of Ca 2+ The activation process is amplified by the release of integrin αIIbβ3, which in turn recruits more platelets to aggregate and stabilizes the formation of thrombi. Inhibiting platelet activation and aggregation is one of the mainstays of antithrombotic therapy, so structural and functional studies of platelet-related enzymes and receptors, as well as the development of related drugs, are of great significance.

[0003] Protease-activated receptor 1 (PAR1) is the primary thrombin receptor on platelets, playing a crucial role in platelet activation, aggregation, and thrombosis, making it a well-established target for antithrombotic drugs. While endogenous agonists for G protein-coupled receptors (GPCRs) are typically free molecules, PAR family receptors employ a unique activation mechanism: thrombin recognizes and specifically cleaves the receptor's N-terminus, exposing a new N-terminus. This newly exposed N-terminus then acts as a tethered ligand, binding to and activating the receptor through intramolecular interactions. Because the activated state structure of this family of receptors remains unknown, the mechanisms of tethered ligand binding, receptor activation, and G protein binding remain to be elucidated, hindering the research and development of drugs targeting this family of receptors and hindering the development of antithrombotic drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide an antithrombotic polypeptide Cb17037 and its application, which inhibits PAR-1 AP-induced platelet activation and aggregation, has antithrombotic effects in vivo and in vitro, has a lower risk of bleeding, and is used to prepare products that inhibit platelet activation and / or aggregation, antithrombotic drugs, and prevent and / or treat thrombotic diseases.

[0005] The present invention provides an antithrombotic polypeptide Cb17037, comprising at least one of the following polypeptides:

[0006] (1) A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1;

[0007] (2) A polypeptide formed by chemically modifying one or more amino acid residues in the polypeptide shown in SEQ ID NO: 1;

[0008] (3) A salt form of the polypeptide described in (1) or (2).

[0009] The present invention also provides a gene encoding the anti-thrombotic polypeptide Cb17037 described in the above technical solution.

[0010] Preferably, the nucleotide sequence of the gene is shown in SEQ ID NO: 2.

[0011] The present invention also provides the use of the anti-thrombotic polypeptide Cb17037 described in the above technical solution or the polypeptide encoded by the gene described in the above technical solution in the preparation of a product that inhibits platelet activation and / or aggregation, wherein the platelet activation and / or aggregation is platelet activation and / or aggregation caused by activation of protease-activated receptor 1.

[0012] The present invention also provides an inhibitor for inhibiting platelet activation and / or aggregation, comprising the antithrombotic polypeptide Cb17037 described in the above technical solution or the polypeptide encoded by the gene described in the above technical solution;

[0013] The platelet activation and / or aggregation is platelet activation and / or aggregation caused by activation of protease-activated receptor 1.

[0014] Preferably, the working concentration of the antithrombotic polypeptide Cb17037 or the polypeptide encoded by the gene is ≤20 mg / kg.

[0015] The present invention also provides the use of the antithrombotic polypeptide Cb17037 described in the above technical solution, or the polypeptide encoded by the gene described in the above technical solution, or the inhibitor for inhibiting platelet activation and / or aggregation described in the above technical solution in the preparation of antithrombotic drugs.

[0016] The present invention also provides the use of the antithrombotic polypeptide Cb17037 described in the above technical solution, or the polypeptide encoded by the gene described in the above technical solution, or the inhibitor of platelet activation and / or aggregation described in the above technical solution in the preparation of drugs for preventing and / or treating thrombotic diseases.

[0017] The present invention also provides an antithrombotic drug comprising an active ingredient and pharmaceutically acceptable excipients;

[0018] The active ingredient includes the antithrombotic polypeptide Cb17037 described in the above technical solution, or the polypeptide encoded by the gene described in the above technical solution, or the inhibitor for inhibiting platelet activation and / or aggregation described in the above technical solution.

[0019] Preferably, the working concentration of the antithrombotic polypeptide Cb17037 or the polypeptide encoded by the gene is ≤20 mg / kg.

[0020] Beneficial effects:

[0021] The present invention provides an antithrombotic polypeptide Cb17037, comprising at least one of the following polypeptides: (1) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1; (2) a polypeptide formed by chemically modifying one or more amino acid residues in the polypeptide shown in SEQ ID NO: 1; and (3) a salt form of the polypeptide (1) or (2). The antithrombotic polypeptide Cb17037 of the present invention is screened from cone snail toxin, does not have a bleeding risk at a concentration of 20 mg / kg or less, and has a good improvement effect on carotid artery injury and ischemic stroke. The antithrombotic polypeptide Cb17037 of the present invention has a significant inhibitory effect on platelet activation and aggregation induced by PAR-1 AP, but has no significant effect on basic coagulation indicators such as APTT, PT and TT. The antithrombotic polypeptide Cb17037 of the present invention has an antithrombotic effect in vivo and in vitro, can be used to prepare products that inhibit platelet activation and / or aggregation, antithrombotic drugs, and prevent and / or treat thrombotic diseases, and has a lower bleeding risk. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0023] Figure 1 The results of the platelet aggregation induced by different agonists with different concentrations of peptide Cb17037 are shown in the figure; A is the agonist PAR-1 AP (protease-activated receptor 1), B is the agonist U46619 (TXA2 mimetic), C is the agonist Collagen, and D is the agonist ADP (adenosine diphosphate);

[0024] Figure 2 The figure shows the results of flow cytometry detection of the effect of different concentrations of peptide Cb17037 on PAR-1 AP-induced platelet integrin activation and P-selectin release; A is the flow cytometry detection result of platelet integrin activation; B is the statistical result of platelet integrin activation level; C is the flow cytometry detection result of platelet P-selectin release; D is the statistical result of platelet P-selectin release level;

[0025] Figure 3 This is a statistical result of the mean fluorescence intensity of peptide Cb17037 on the effect of thrombus formation in vitro over time;

[0026] Figure 4Figure 1 shows the effects of different concentrations of peptide Cb17037 on platelet adhesion and spreading; A is a representative confocal image of platelet spreading on fibrinogen; B is a quantitative statistical result of platelet spreading area; C is a quantitative statistical result of platelet spreading number;

[0027] Figure 5 The results of the effects of different concentrations of peptide Cb17037 on APTT (A), PT (B) and TT (C) are shown;

[0028] Figure 6 Figure 1 shows the inhibitory effect of peptide Cb17037 on the mouse FeCl3 carotid artery thrombosis model; A is a speckle imaging recording of blood flow in the mouse ferric chloride model at different times, with red indicating carotid artery blood flow and blue indicating no blood flow; B is a real-time monitoring recording of changes in carotid artery blood flow; C is a quantitative statistical graph of occlusion time in the ferric chloride-induced carotid artery thrombosis model;

[0029] Figure 7 Figure 1 is a graph showing the activity of peptide Cb17037 on photochemically induced cerebral ischemia; A is a schematic diagram of TTC staining results of mouse brain sections; B is the statistical results of mouse cerebral ischemia volume; C is the statistical results of mouse cerebral ischemia volume ratio;

[0030] Figure 8 This is a graph showing the test results of peptide Cb17037 on the risk of tail bleeding;

[0031] Figures 1 to 8 In the middle, * represents P <0.05, ** represents P <0.01, *** represents P <0.001, the difference was statistically significant. DETAILED DESCRIPTION

[0032] The present invention provides an antithrombotic polypeptide Cb17037, comprising at least one of the following polypeptides: (1) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1; (2) a polypeptide formed by chemically modifying one or more amino acid residues in the polypeptide shown in SEQ ID NO: 1; and (3) a salt form of the polypeptide of (1) or (2).

[0033] The antithrombotic polypeptide Cb17037 of the present invention is screened from cone snail toxin. There is no special limitation on the preparation method of the antithrombotic polypeptide Cb17037. The preparation method of the polypeptide well known in the art can be used, such as expressing the antithrombotic polypeptide Cb17037 using a prokaryotic expression system or a eukaryotic expression system. The antithrombotic polypeptide Cb17037 can also be prepared by chemical synthesis.

[0034] In one embodiment, the chemical modification described herein includes chemically modifying amino acid residue side chain groups, α-carbon atoms, terminal amino groups, or terminal carboxylic acid groups of the antithrombotic polypeptide Cb17037. In one embodiment, the chemical modification described herein includes covalently linking one or more modifiers. In one embodiment, the modifier described herein includes bovine serum albumin, human serum albumin, homologous IgG, or polyethylene glycol. The present invention does not specifically limit the method of chemical modification; any chemical modification method known in the art may be employed.

[0035] As an embodiment, the salt form of the antithrombotic polypeptide Cb17037 of the present invention includes hydrochloride, phosphate or acetate. The present invention has no particular limitation on the preparation method of the antithrombotic polypeptide salt form, and any method known in the art for preparing polypeptide salt forms can be used.

[0036] The present invention also provides a gene encoding the antithrombotic polypeptide Cb17037 described in the above technical solution. As an embodiment, the nucleotide sequence of the gene of the present invention is shown in SEQ ID NO: 2, specifically: 5'-ATAACCCACAAACCAGAAACTGTGCAAACAGCGAAAATGCTTTCCATGCATCAAATT-3'.

[0037] The present invention also provides the use of the anti-thrombotic polypeptide Cb17037 described in the above technical solution or the polypeptide encoded by the gene described in the above technical solution in the preparation of a product that inhibits platelet activation and / or aggregation, wherein the platelet activation and / or aggregation is platelet activation and / or aggregation caused by activation of protease-activated receptor 1.

[0038] As an embodiment, the platelet activation and / or aggregation caused by activation of protease-activated receptor 1 in the present invention is PAR-1 AP-induced platelet activation and / or aggregation.

[0039] The present invention also provides an inhibitor for inhibiting platelet activation and / or aggregation, comprising the anti-thrombotic polypeptide Cb17037 described in the above technical solution or the polypeptide encoded by the gene described in the above technical solution; the platelet activation and / or aggregation is platelet activation and / or aggregation caused by activation of protease-activated receptor 1.

[0040] As an embodiment, the platelet activation and / or aggregation caused by activation of protease-activated receptor 1 in the present invention is PAR-1 AP-induced platelet activation and / or aggregation.

[0041] In one embodiment, the working concentration of the antithrombotic polypeptide Cb17037 of the present invention or the polypeptide encoded by the gene described in the above technical solution is ≤20 mg / kg; in another embodiment, the working concentration of the antithrombotic polypeptide Cb17037 of the present invention or the polypeptide encoded by the gene described in the above technical solution is ≤20 mg / kg.

[0042] The present invention also provides the use of the antithrombotic polypeptide Cb17037 described in the above technical solution, or the polypeptide encoded by the gene described in the above technical solution, or the inhibitor for inhibiting platelet activation and / or aggregation described in the above technical solution in the preparation of antithrombotic drugs.

[0043] The present invention also provides the use of the antithrombotic polypeptide Cb17037 described in the above technical solution, or the polypeptide encoded by the gene described in the above technical solution, or the inhibitor of platelet activation and / or aggregation described in the above technical solution in the preparation of drugs for preventing and / or treating thrombotic diseases.

[0044] As an embodiment, the thrombotic disease of the present invention includes ischemic stroke.

[0045] The present invention also provides an antithrombotic drug comprising an active ingredient and pharmaceutically acceptable excipients;

[0046] The active ingredient includes the antithrombotic polypeptide Cb17037 described in the above technical solution, or the polypeptide encoded by the gene described in the above technical solution, or the inhibitor for inhibiting platelet activation and / or aggregation described in the above technical solution.

[0047] In one embodiment, the working concentration of the antithrombotic polypeptide Cb17037 of the present invention or the polypeptide encoded by the gene described in the above technical solution is ≤20 mg / kg; in another embodiment, the working concentration of the antithrombotic polypeptide Cb17037 of the present invention or the polypeptide encoded by the gene described in the above technical solution is 20 mg / kg.

[0048] To further illustrate the present invention, an antithrombotic polypeptide Cb17037 and its applications provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0049] The animals, reagents, and materials used in the examples of the present invention were all commercially available, such as:

[0050] (1) All experimental animals were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. They were 6 to 8 weeks old, male, and weighed 18 to 22 g. After purchase, the mice were housed in an SPF-grade animal room for one week. Experiments were then conducted after the mice had adapted to the environment.

[0051] (2) Physiological saline was purchased from Shandong Qidu Pharmaceutical Co., Ltd., 3,3'-dihexyloxacarbocyanine iodide (DiOC6) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., collagen was purchased from HYPHEN BioMed, isoflurane was purchased from Reward Life Sciences Co., Ltd., Rose bengal was purchased from Sigma, Triton X-100 was purchased from Tianjin Fengchuan Chemical Reagent Co., Ltd., 2,3,5-triphenyltetrazolium chloride (TTC) was purchased from Shanghai Taoshu Biotechnology Co., Ltd., FeCl3 powder was purchased from Tianjin Fengchuan Chemical Reagent Co., Ltd., and APTT / PT / TT kits were purchased from Beijing Zhongchi Weiye Technology Development Co., Ltd.

[0052] (3) Preparation of experimental solvent:

[0053] 1 L PBS phosphate buffer: KH2PO4 0.24 g, Na2HPO4 1.44 g, NaCl 8 g, KCl 0.2 g, ddH2O 8 mL, adjust pH to 7.4 with concentrated HCl, and dilute to 1 L;

[0054] 10 mL 10% FeCl3 solution: 0.1 g FeCl3 powder and 10 mL normal saline;

[0055] 50 mL 2% TTC solution: 1 g TTC powder and 50 mL ddH2O;

[0056] 100 mL 25 mM CaCl2: CaCl2·2H2O 0.37 g and 25 mM Hepes buffer 100 mL;

[0057] 50 mL HEPES buffer: 0.12 g HEPES, 0.44 g NaCl, and 50 mL ddHO.

[0058] (4) Sources of instruments used in the experiment:

[0059] A multifunctional microplate reader was purchased from Biotek, a vortex mixer was purchased from Labnet, a platelet aggregometer (LBY-NJ4) was purchased from Telecom, an electronic balance (AX124ZH / E) was purchased from Ohaus Instruments (Changzhou) Co., Ltd., an anesthesia machine (R5001E) was purchased from Reward Life Sciences Co., Ltd., a laser Doppler microscope (RFLSI ZW) was purchased from Reward Life Sciences Co., Ltd., a mouse skull drill (QLD-W50S) was purchased from Quanlidao (Changzhou) Tools Co., Ltd., a laboratory syringe pump (TYD01-01) was purchased from Baoding Refu Fluid Technology Co., Ltd., an inverted fluorescence microscope (NIB 900) was purchased from Ningbo Yongxin Optics Co., Ltd., Jiangsu Province, a laser speckle blood flow imager was purchased from Shenzhen Reward Life Sciences Co., Ltd., an electric constant temperature water bath was purchased from Shanghai Yuyan Scientific Instrument Co., Ltd., and a confocal microscope was purchased from Leica.

[0060] Example 1

[0061] A short sequence of 19 amino acids (ITHKPETVQTAKMLSMHQI, SEQ ID NO: 1) was extracted from cone snail toxin and the amino acid sequence described in SEQ ID NO: 1 was chemically synthesized by Hangzhou Gutuo Biosynthesis Co., Ltd. to obtain a linear polypeptide named polypeptide Cb17037 (abbreviated as 17037).

[0062] Example 2

[0063] Platelet aggregation assay

[0064] 1. Agonists: 20 μM PAR-1 AP (proteinase-activated receptor 1), 0.3 μM U46619 (TXA2 mimetic), 1 μg / mL collagen, 10 μM ADP (adenosine diphosphate), 0.06 U / mL thrombin.

[0065] 2. Polypeptide: Polypeptide Cb17037 of Example 1.

[0066] 3. Platelet aggregation was detected using a semi-automatic platelet aggregation instrument. The principle is to measure the change in the light transmittance of the platelet suspension during platelet activation. When a platelet agonist is added to the platelet suspension, along with platelet activation, the platelet cytoskeleton changes or adheres to each other, aggregating into flocculent precipitates, reducing the turbidity of the overall solution, resulting in a significant change in light transmittance that can be detected by the platelet aggregation instrument. This experiment can effectively reflect the effect of the peptide Cb17037 on platelet aggregation, and the experimental results will provide an important reference for subsequent in vitro and in vivo experiments. The specific detection steps are as follows:

[0067] a) Whole blood was drawn from the cubital vein of healthy volunteers into blood collection tubes containing anticoagulant (A012393, Jiangsu Kangjian Medical Supplies Co., Ltd.);

[0068] b) Set the centrifuge conditions as follows: 5 / 9 speed, 100 × g, room temperature for 10 min;

[0069] c) Gently pipette the yellowish-white platelet-rich plasma (PRP) layer from the upper layer into a 1.5 mL centrifuge tube (MCT-150-C, Axygen). Add 20 μL of 125 mM EDTA (10234620250A, Sinopharm Chemical Testing) per 1 mL of PRP and centrifuge at 400 × g for 5 min at room temperature.

[0070] d) Discard the supernatant and resuspend the platelets in 0.5 mL of benchtop buffer A per tube. Combine the resuspended platelets from both tubes into a new 1.5 mL conical centrifuge tube, mix thoroughly, and centrifuge at 400 × g for 5 min at room temperature.

[0071] e) Discard the supernatant and resuspend the platelets in 1 mL of benchtop buffer A per tube. Transfer the platelets to a new 1.5 mL conical centrifuge tube and centrifuge at 400 × g for 5 minutes at room temperature.

[0072] f) Discard the supernatant and resuspend the platelets in 500 μL of benchtop buffer B per tube, and adjust the platelet concentration to 3 × 10 8 / mL; use benchtop solution B to calibrate 100% clarity, add a magnetic rotor to the aggregation dish, and add 250 μL of washed platelets to the aggregation dish to avoid bubbles that affect transmittance detection;

[0073] g) Add the corresponding concentration of peptide 17037 and place the aggregation dish in the incubation well for 5 minutes;

[0074] h) After incubation, place the aggregation dish into the detection well, turn on the instrument, and carefully add the corresponding agonist (PRP is used as the ADP agonist in this experiment) to begin recording the aggregation curve.

[0075] The results are as follows Figure 1 As shown. Figure 1 It can be seen that the peptide Cb17037 has a significant inhibitory effect on platelet aggregation induced by PAR-1 AP, and has a concentration-dependent effect from high concentration to low concentration ( Figure 1 A), but had no effect on platelet aggregation induced by agonists such as collagen, ADP, and U46619 ( Figure 1 Middle B~D).

[0076] Example 3

[0077] Flow cytometry detection of peptide Cb17037 on platelet activation-related indicators

[0078] In order to detect platelet activation related indicators, flow cytometry was used to detect the effect of peptide Cb17037 on integrin activation. The specific method is as follows: the washed platelets were adjusted to a platelet concentration of 1×10 6 / mL; the polypeptide Cb17037 obtained in Example 1 was added to 50 μL of platelet solution, and 15 μM Par-1 was added as a platelet agonist. In this system, PAC-1 antibody and P-Selectin antibody were used to detect platelet integrin activation and P-selectin release, respectively. The platelet suspension was then incubated at 37°C in a dark environment for 30 minutes and diluted before loading. The PAC-1 binding level was assessed using flow cytometry, and the data were analyzed using FlowJo™ v10 software. The results are as follows: Figure 2 As shown. Figure 2 It can be seen that the peptide Cb17037 inhibited PAR-1 AP-induced PAC-1 binding and platelet P-selectin release, which means that the peptide Cb17037 can inhibit the activation of platelet integrins and the secretion of α-granules, which is of great significance for the prevention and treatment of thrombotic diseases.

[0079] Example 4

[0080] In vitro thrombosis assay

[0081] The experiment evaluated the ability of the polypeptide Cb17037 in Example 1 to inhibit platelet adhesion and thrombosis under low shear stress by fluorescently labeling blood cells anticoagulated with sodium citrate and using the mean fluorescence intensity of platelets adhered to the collagen surface as an evaluation indicator of the antithrombotic effect. The specific steps are as follows:

[0082] Angiogenesis slides (μ-Slide VI 0.1, Ibidi) were coated with 100 μg / mL collagen at room temperature and incubated overnight at 4°C. Sodium citrate anticoagulated whole blood was collected from healthy volunteers and fluorescently labeled with 1 μM 3,3'-diethyloxacarbocyanine iodide (DiOC6, Sigma). Excess collagen on the slides was rinsed with saline and then a syringe pump was used for 300 s. -1 The co-incubated test sample (peptide Cb17037) and fluorescently labeled whole blood were perfused onto the angiogenesis slide for 5 minutes at a shear rate. Platelet aggregation and thrombus formation were recorded in real time using an inverted fluorescence microscope. Platelet mean fluorescence intensity data were analyzed using Image J software. Figure 3 As shown. Figure 3 It can be seen that the polypeptide Cb17037 of Example 1 can inhibit the adhesion and aggregation of platelets on the collagen surface under low shear force.

[0083] Example 5

[0084] Platelet extension assay

[0085] The effect of the polypeptide Cb17037 in Example 1 on platelet adhesion and extension was determined by a platelet extension experiment. The specific steps are as follows:

[0086] The washed platelets were suspended in Tyrode's buffer. The concentration of the washed platelets in Tyrode's buffer was 1×10 5 After mixing platelets at different concentrations of peptide Cb17037 (50 / 100 / 200 μM), they were added to coverslips coated with 100 μg / mL fibrinogen and incubated at 37°C for 40 min. Subsequently, the platelets were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 10 min, and then blocked with PBS solution containing 3% BSA for 1 h. They were then stained with FITC-labeled phalloidin overnight at 4°C in a dark environment and observed under a confocal microscope using a 63x oil objective. The number and area of ​​platelets were calculated using Image J software version 1.35h, and the results are shown in Figure 2. Figure 4 As shown. Figure 4 It can be seen that the polypeptide Cb17037 has a significant inhibitory effect on the extension of platelets on fibrinogen.

[0087] Example 6

[0088] In vitro anticoagulation of healthy human blood

[0089] Extrinsic coagulation is associated with bleeding. Activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) are screening methods for detecting the functionality and integrity of the extrinsic coagulation pathway and the common pathway. The effects of the polypeptide Cb17037 of Example 1 on the extrinsic coagulation pathway were evaluated by measuring APTT, PT, and TT. The specific detection steps are as follows:

[0090] Blood was collected from healthy volunteers using venous blood and the anticoagulant was 10 9 mmol / L sodium citrate, the blood volume and anticoagulant volume ratio is 9:1, immediately inverted to mix thoroughly, centrifuged at 3000 rpm for 15 minutes, and the upper plasma is taken as the test plasma sample. The test plasma sample is incubated with different concentrations (25μM, 50μM, 100μM) of Example 1 polypeptide Cb17037 at 37℃ for 5 minutes, and the following indicators are detected on the machine: (1) activated partial thromboplastin time (APTT); (2) prothrombin time (PT); (3) thrombin time (TT). The data are plotted using GraphPad Prism 8, and the results are as follows: Figure 5 shown.

[0091] according to Figure 5 It can be seen that different concentrations of peptide Cb17037 had no effect on the APTT time. Peptide Cb17037 does not have an anticoagulant effect; the addition of peptide Cb17037 at different concentrations did not prolong the coagulation time (PT and TT), and had little effect on the coagulation time, with no significant differences. This indicates that the addition of peptide Cb17037 may not significantly affect the extrinsic coagulation pathway.

[0092] Example 7

[0093] FeCl3 carotid artery thrombosis model experiment

[0094] a) Male C57 BL / 6J mice (8 weeks old) were selected and anesthetized with isoflurane. After full anesthesia, the mice were fixed in a supine position and their core body temperature was maintained at 37°C throughout the surgery.

[0095] b) Prepare the neck skin and disinfect with iodine. Cut a 1 cm section of epidermis in the middle of the neck triangle to expose the subcutaneous tissue. Use curved forceps to bluntly dissect the sternocleidomastoid muscle and cut the anterior belly of the digastric muscle to expose the mouse's common carotid artery. Gently peel away any tissue adhering to the vessel, thoroughly dissecting the vessel. Separate one of the carotid arteries from the adherent tissue and adjacent vagus nerve. Place a 5 mm diameter semicircular paper sheet beneath the isolated vessel as a background to eliminate interference with blood flow.

[0096] c) Drugs were injected through the tail vein within 1 min before ferric chloride (FeCl3) induction. The experimental group (17037) was injected with the peptide Cb17037 of Example 1 at a concentration of 20 mg / kg, and the negative control group (control, NC) was injected with the same volume of normal saline.

[0097] d) A nylon thread (6-0) pre-soaked in 10% (w / v) FeCl3 solution was attached to the exposed carotid artery and removed 2 minutes later. FeCl3 promotes thrombosis at the injured site by inducing endothelial cell damage.

[0098] e) After FeCl3 induction, blood flow was continuously recorded. Laser speckle perfusion imaging was used to record the carotid artery blood flow in each group. Recording was stopped when blood flow ceased, and the blood flow value of each group of mice was recorded. Data were analyzed using the instrument's own analysis software. Statistical significance was analyzed using the t-test. Results are expressed as mean ± standard deviation (n = 5). Figure 6 shown.

[0099] according to Figure 6As can be seen, at 500 seconds, the NC (normal saline) group showed almost no blood flow. However, the Cb17037 peptide group, administered at a concentration of 20 mg / kg, prolonged carotid artery thrombosis, in stark contrast to the NC group. Significant differences in blood flow values ​​were observed between the NC and Cb17037 groups. Compared to the NC control group, the Cb17037 peptide at a concentration of 20 mg / kg demonstrated a significant anti-carotid artery thrombotic effect.

[0100] Example 8

[0101] Photochemically induced cerebral ischemia model experiment

[0102] a) Male C57 BL / 6J mice (8 weeks old) were selected and anesthetized with isoflurane. After full anesthesia, the mice were fixed in a supine position and their core body temperature was maintained at 37°C throughout the surgery.

[0103] b) Prepare the skin, disinfect with iodine, and perform a longitudinal incision (2.0-2.5 cm) to expose the skull. To avoid wound complications, skull exposure should be performed with a single incision. Gently scrape the skull membrane to expose the skull surface, clearly visualizing the coronal plane, the midline, and the bregma.

[0104] c) Apply vaseline to keep the surface moist. Use a punch to create a shallow depression to make the blood vessels clearly visible. Inject an appropriate volume of 10 mg / mL rose bengal into the tail vein at a dose of 20 mg / kg per mouse. Immediately irradiate with a laser beam for 20 min.

[0105] d) After laser irradiation, 20 mg / kg of the peptide Cb17037 obtained in Example 1 was subcutaneously injected. 1.5 hours later, another 20 mg / kg of the peptide Cb17037 was subcutaneously injected. The negative control group (NC) was injected with the same volume of normal saline.

[0106] e) 24 hours after laser irradiation, the brain was harvested and cut into 10 slices of uniform thickness (approximately 1 mm per slice). The slices were stained with 2% TTC solution in a 37°C incubator protected from light for 15 min.

[0107] f) After staining, remove the brain tissue from the 37°C incubator. Infarcted tissue appears pale, while normal tissue appears dark red. Then, photograph the sections with a digital camera. Image J software calculates the relative infarct volume. Infarct volume is measured using the area method (% of total volume). The results are shown below. Figure 7 As shown. Figure 7As can be seen, the cerebral infarction volume and volume ratio of the Cb17037-treated mice were significantly reduced compared to the NC (normal saline) group. In summary, compared to the NC control group, the Cb17037 peptide at a concentration of 20 mg / kg has an anti-photochemical thrombotic effect.

[0108] Example 9

[0109] Effect of bleeding (tail bleeding test)

[0110] a) Place the mouse in the designed apparatus, clean the mouse's tail with anhydrous ethanol, and adjust the water bath temperature to 37°C.

[0111] b) The polypeptide Cb17037 obtained in Example 1 was injected via the tail vein at a concentration of 20 mg / kg in the administration group and the negative control group was injected with the same volume of normal saline.

[0112] c) Prepare a conical flask and pour physiological saline into it. Mark each group of mice 2 mm from the tail tip with a marker pen. Use a sterilized blade to cut evenly at the marked area.

[0113] d) After cutting, carefully immerse the tail in 20 mL of normal saline that has been pre-warmed to 37°C. Bleeding time is measured from the beginning until no more fibrous blood appears.

[0114] e) Record the bleeding time of each group of mice and plot the data using GraphPad Prism 8. Figure 8 As shown. Figure 8 As can be seen, at a 20 mg / kg concentration of peptide Cb17037, the bleeding time was 58.5 ± 21.58 s, compared to 57 ± 21.89 s in the control group (normal saline group). Tail vein injection of 20 mg / kg of peptide Cb17037 did not significantly prolong the tail bleeding time of mice compared to the blank control group, indicating a lower risk of bleeding.

[0115] Based on the above, it can be seen that the antithrombotic polypeptide Cb17037 provided by the present invention has a significant inhibitory effect on PAR-1 AP-induced platelet activation and aggregation, has an antithrombotic effect in vivo and in vitro, and has a lower bleeding risk.

[0116] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. An antithrombotic polypeptide Cb17037 or a salt thereof, characterized in that: The amino acid sequence of the antithrombotic polypeptide Cb17037 is a polypeptide shown in SEQ ID NO:

1.

2. A gene encoding the antithrombotic polypeptide Cb17037 according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

4. An inhibitor for inhibiting platelet activation and / or aggregation, characterized in that The invention comprises the antithrombotic polypeptide Cb17037 or a salt thereof according to claim 1 or the polypeptide encoded by the gene according to claim 2 or 3; The platelet activation and / or aggregation is platelet activation and / or aggregation caused by activation of protease-activated receptor 1.

5. The inhibitor for inhibiting platelet activation and / or aggregation according to claim 4, characterized in that The working concentration of the antithrombotic polypeptide Cb17037 or the polypeptide encoded by the gene is ≤20 mg / kg.

6. Use of the antithrombotic polypeptide Cb17037 or a salt thereof according to claim 1, the polypeptide encoded by the gene according to claim 2 or 3, or the inhibitor for inhibiting platelet activation and / or aggregation according to claim 4 or 5 in the preparation of an antithrombotic drug.

7. Use of the antithrombotic polypeptide Cb17037 or a salt thereof according to claim 1, the polypeptide encoded by the gene according to claim 2 or 3, or the inhibitor of platelet activation and / or aggregation according to claim 4 or 5 in the preparation of a medicament for preventing and / or treating thrombotic diseases.

8. The use according to claim 6 or 7, characterized in that The antithrombotic drug is an antithrombotic drug that inhibits platelet activation and / or aggregation. The platelet activation and / or aggregation is platelet activation and / or aggregation caused by activation of protease-activated receptor 1.

9. An antithrombotic drug, characterized in that: including active ingredients and pharmaceutically acceptable excipients; The active ingredient includes the antithrombotic polypeptide Cb17037 or a salt thereof according to claim 1, the polypeptide encoded by the gene according to claim 2 or 3, or the inhibitor for inhibiting platelet activation and / or aggregation according to claim 5.

10. The antithrombotic drug according to claim 9, characterized in that The working concentration of the antithrombotic polypeptide Cb17037 or the polypeptide encoded by the gene is ≤20 mg / kg.

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