Conotoxin-derived anticoagulant polypeptide Cb-13 and application thereof

Abstract

The invention provides conotoxin-derived anticoagulant polypeptide Cb-13 and application thereof, and belongs to the technical field of biological medicine. The invention provides an anticoagulant polypeptide Cb-13. The amino acid sequence of the anticoagulant polypeptide Cb-13 comprises SEQ ID No.1. The invention also provides a coding gene of the anticoagulant polypeptide Cb-13, and provides that the anticoagulant polypeptide Cb-13 does not have hemolytic activity and does not have cytotoxicity when the concentration is 400 [mu] M or below. The anticoagulant polypeptide Cb-13 disclosed by the invention has a prolonging effect on plasma recalcification and coagulation time, has an anticoagulant effect, has small influence on bleeding, and also has an obvious promoting effect on plasmin and uPa. Meanwhile, it is also proved that the anticoagulant polypeptide Cb-13 can inhibit thrombus formation, that is, the anticoagulant polypeptide Cb-13 has in-vivo and in-vitro fibrinolytic effect and anticoagulant effect, has small bleeding risk, and can be used as an antithrombotic or thrombolytic drug.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a conotoxin-derived anticoagulant polypeptide Cb-13 and an application thereof. Background Art

[0002] Cardiovascular and cerebrovascular diseases are a general term for cardiovascular and cerebrovascular diseases related to vascular lesions, mainly including heart failure, atherosclerosis, myocardial infarction, arrhythmia, hypertension and stroke. Although drugs and device technologies have improved in recent years, cardiovascular diseases are still the leading cause of death from non-communicable diseases worldwide, far higher than cancer and other diseases. There is a huge social demand and important social significance for the research of drugs to prevent and treat cardiovascular and cerebrovascular diseases. Cardiovascular and cerebrovascular diseases are diseases caused by vascular stenosis and occlusion caused by thrombus formation in the cardiovascular and cerebrovascular vessels, resulting in ischemia and infarction of the heart and brain, which leads to functional disorders. In a normal body, the coagulation pathway includes coagulation and anticoagulation. The interaction between procoagulant factors and anticoagulant factors maintains the dynamic balance of the coagulation system and the anticoagulation system, thereby maintaining the normal flow of blood in the body. Under normal physiological conditions, the anticoagulation mechanism has a slight advantage. When a blood vessel is injured, a hemostatic plate (a large number of platelets and fibrin) is rapidly induced to form at the bleeding site, which eventually stops the continued bleeding at the bleeding site. The formation of a thrombus is similar to that of a hemostatic plate, because factors such as inflammation lead to changes in the physiological environment in the body, and the expression level of procoagulant factors in the plasma increases, leading to a hypercoagulable tendency and forming a state prone to thrombosis. Ultimately, the coagulation cascade reaction leads to the production of a large amount of thrombin, which converts the soluble fibrinogen circulating in the body into insoluble fibrin, thereby leading to the formation of a thrombus.

[0003] Fibrinolysis is an important physiological component of hemostasis and its function is to limit clot formation. Fibrinolysis regulates fibrin formation during hemostasis as well as the degree of vascular occlusion. Following tissue and vascular injury, multiple hemostatic mechanisms are initiated, resulting in thrombin generation, platelet adhesion, platelet cross-linking, and fibrin formation. Fibrinolysis is initiated as part of the hemostatic response to limit clot formation. Fibrin, the end product of coagulation activation, is a cofactor for plasminogen activation by tissue-type plasminogen activator. Plasmin is the enzymatic factor in fibrinolysis, cleaving fibrin, but as a promiscuous enzyme with substrate specificity, it can also mediate proteolytic activation of multiple hemostatic and inflammatory components when its concentration exceeds that of local and systemic inhibitors, most notably α2-antiplasmin. When regulated, fibrinolysis is a protective physiological response that appropriately limits clot size. Fibrinolysis also contributes to coagulopathy through mechanisms other than fibrinogen and fibrin cleavage, but also cleaves glycoprotein Ib and IIb / IIIa receptors on platelets, thereby reducing platelet adhesion and aggregation.

[0004] Conotoxins or conopeptides are small peptides secreted by the marine mollusk Conus, usually containing 10-50 amino acids and 1-5 disulfide bonds. Conotoxins are promising neuropharmacological tools and drug candidates due to their high efficiency and specificity in targeting ion channels or neurotransmitter receptors. However, their role in anticoagulation is still unclear. Summary of the invention

[0005] The invention provides a conotoxin-derived anticoagulant polypeptide Cb-13 and application thereof. The anticoagulant polypeptide Cb-13 has significant fibrinolytic activity and anticoagulant activity.

[0006] The present invention provides an anticoagulant polypeptide Cb-13, comprising at least one of the following polypeptides:

[0007] (1) A polypeptide with an amino acid sequence as shown in SEQ ID No. 1;

[0008] (2) A polypeptide formed by chemically modifying one or more amino acid residues in the polypeptide shown in SEQ ID No. 1;

[0009] (3) The polypeptide described in (1) or the salt form of the polypeptide described in (2).

[0010] The present invention also provides a gene encoding the anticoagulant polypeptide Cb-13.

[0011] Preferably, the nucleotide sequence of the gene includes SEQ ID No.2.

[0012] The present invention also provides the use of the anticoagulant polypeptide Cb-13 or the gene in the preparation of anticoagulant drugs.

[0013] Preferably, the anticoagulant drug includes a preparation made by combining the active ingredient directly or with a pharmaceutical carrier;

[0014] The active ingredients include: anticoagulant polypeptide Cb-13 or a derivative of the anticoagulant polypeptide Cb-13 or a composition containing the anticoagulant polypeptide Cb-13.

[0015] The present invention also provides an anticoagulant drug, the active ingredients of which include the anticoagulant polypeptide Cb-13 and pharmaceutically acceptable excipients.

[0016] The present invention also provides the use of the anticoagulant polypeptide Cb-13 or the gene in preparing a drug for preventing and / or treating thrombotic diseases.

[0017] Preferably, the drug for preventing and / or treating thrombotic diseases includes a preparation made by combining the active ingredient directly or with a pharmaceutical carrier;

[0018] The active ingredients include: anticoagulant polypeptide Cb-13 or a derivative of the anticoagulant polypeptide Cb-13 or a composition containing the anticoagulant polypeptide Cb-13.

[0019] The present invention also provides a drug for preventing and / or treating thrombotic diseases, wherein the active ingredients include the anticoagulant polypeptide Cb-13 and pharmaceutically acceptable excipients.

[0020] Preferably, the working concentration of the anticoagulant polypeptide Cb-13 is not higher than 400 μM.

[0021] Beneficial effects: The present invention provides an anticoagulant polypeptide Cb-13, specifically a polypeptide derived from conotoxin, composed of 14 amino acids, and the sequence is shown in SEQ ID No. 1: SRAKMQNHKLFKLT. The anticoagulant polypeptide Cb-13 of the present invention has no hemolytic activity at a concentration of 400 μM and below, and has no cytotoxicity to HEK-293T cells and mouse spleen cells. The anticoagulant polypeptide Cb-13 of the present invention has the effect of significantly prolonging the plasma recalcification coagulation time at a concentration of 100 μM, and has an inhibitory effect on the plasma recalcification coagulation time; the administration of Cb-13 can significantly prolong the APTT coagulation time, has an anticoagulant effect, and Cb-13 has little effect on bleeding; Cb-13 has a significant promoting effect on plasma and uPa. At the same time, animal model experiments were also used to confirm that the anticoagulant polypeptide Cb-13 has a lower bleeding risk than sodium heparin. After administration, Cb-13 can inhibit the formation of rat tail thrombosis and carotid artery thrombosis. That is, the anticoagulant polypeptide Cb-13 of the present invention has in vitro and in vivo fibrinolytic and anticoagulant effects, and has a lower bleeding risk, and can be used as an antithrombotic or thrombolytic drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the result diagram of the effect of different concentrations of Cb-13 on plasma recalcification time;

[0023] Figure 2 This is the result diagram of the effect of different concentrations of Cb-13 on APTT coagulation time;

[0024] Figure 3 This is the result diagram of the effect of different concentrations of Cb-13 on PT coagulation time;

[0025] Figure 4 The reaction kinetics of Cb-13 with plasma and uPa are shown in the figure;

[0026] Figure 5 This is the result diagram of the hemolytic activity of Cb-13;

[0027] Figure 6The figure is a graph showing the cytotoxicity test results of Cb-13, wherein a: the cytotoxicity results of Cb-13 on human embryonic kidney cells (HEK-293T); b: the cytotoxicity results of Cb-13 on mouse spleen cells;

[0028] Figure 7 This is a graph showing the difference in tail bleeding risk between Cb-13 and positive drug sodium heparin;

[0029] Figure 8 The effect diagram of carrageenan rat tail model to verify the anti-thrombotic effect of Cb-13;

[0030] Fig. 9 The diagram shows the inhibitory effect of Cb-13 on the mouse carotid artery thrombosis model, in which a: 5-min and 10-min blood flow speckle imaging records of the mouse ferric chloride model; b: 5-min and 10-min blood flow value records of the mouse ferric chloride model; in the diagram, red represents the blood flow in the mouse carotid artery, blue is the background color, and also shows the situation of no blood flow, and the blood flow bar on the right shows that the blood flow span is (0-614). DETAILED DESCRIPTION

[0031] The present invention provides an anticoagulant polypeptide Cb-13, comprising at least one of the following polypeptides:

[0032] (1) A polypeptide with an amino acid sequence as shown in SEQ ID No. 1;

[0033] (2) A polypeptide formed by chemically modifying one or more amino acid residues in the polypeptide shown in SEQ ID No. 1;

[0034] (3) The polypeptide described in (1) or the salt form of the polypeptide described in (2).

[0035] The anticoagulant polypeptide Cb-13 of the present invention is selected from conotoxin and consists of 14 amino acids, and the sequence is shown in SEQ ID No. 1: SRAKMQNHKLFKLT. The present invention has no special restrictions on the preparation method of the polypeptide Cb-13, and the preparation method of the polypeptide well known in the art can be used, such as using a prokaryotic expression system or a eukaryotic expression system to express the anticoagulant polypeptide Cb-13, and the polypeptide Cb-13 can also be prepared by a chemical synthesis method.

[0036] The chemical modification of the present invention preferably includes chemical modification of the amino acid residue side chain groups, α-carbon atoms, terminal amino groups or terminal carboxyl groups of the polypeptide Cb-13, and the types of the chemical modification preferably include covalently linking one or more modifiers; the modifiers preferably include bovine serum albumin, human serum albumin, homologous IgG or polyethylene glycol, etc. The present invention has no special restrictions on the chemical modification method, and the chemical modification method known in the art can be used.

[0037] The salt form of the polypeptide Cb-13 of the present invention preferably includes hydrochloride, phosphate or acetate. The present invention has no particular limitation on the preparation method of the polypeptide salt form, and the preparation method of the polypeptide salt form known in the art can be used.

[0038] The present invention also provides a gene encoding the anticoagulant polypeptide Cb-13.

[0039] The nucleotide sequence of the gene of the present invention preferably includes SEQ ID No. 2: GTCACGAGCCAAGATGCAGAATCACAAGCTCTTCAAGTTGACCAA.

[0040] The present invention also provides the use of the anticoagulant polypeptide Cb-13 or the gene in the preparation of anticoagulant drugs.

[0041] In the embodiment of the present invention, the effect of Cb-13 on plasma recalcification time was verified by a plasma recalcification experiment. Cb-13 has the effect of significantly prolonging the plasma recalcification and coagulation time at a concentration of 100 μM, has an inhibitory effect on the plasma recalcification and coagulation time, and has a concentration-dependent effect from high concentration to low concentration.

[0042] In the present invention, the activated partial thromboplastin time APTT is a measure of the functionality and integrity of the intrinsic coagulation pathway and the final common pathway of the coagulation cascade reaction. In the embodiment, the APTT coagulation time test was performed on different concentrations of Cb-13 to observe the effect on the APTT coagulation time. The results showed that the addition of Cb-13 would prolong the APTT time, and there was a concentration-dependent effect from high concentration to low concentration. In summary, the administration of Cb-13 significantly prolongs the APTT coagulation time and has an anticoagulant effect.

[0043] In the examples of the present invention, different concentrations of Cb-13 were tested for PT time to observe the effect on PT coagulation time. The results showed that the addition of Cb-13 may not have a significant effect on the exogenous coagulation pathway, that is, Cb-13 has little effect on bleeding.

[0044] The anticoagulant drug of the present invention preferably includes a preparation prepared by directly or in combination with a pharmaceutical carrier the active ingredient; the active ingredient preferably includes: anticoagulant polypeptide Cb-13 or a derivative of the anticoagulant polypeptide Cb-13 or a composition comprising the anticoagulant polypeptide Cb-13.

[0045] The present invention also provides an anticoagulant drug, the active ingredients of which include the anticoagulant polypeptide Cb-13 and pharmaceutically acceptable excipients.

[0046] The present invention can use the anticoagulant polypeptide Cb-13 alone as the active ingredient of the anticoagulant drug, or can also use a composition containing the anticoagulant polypeptide Cb-13, such as other active compositions or combined drugs having anticoagulant activity.

[0047] The present invention has no particular limitation on the dosage form of the drug, and the drug can be prepared using conventional dosage forms in the art.

[0048] The present invention also provides the use of the anticoagulant polypeptide Cb-13 or the gene in preparing a drug for preventing and / or treating thrombotic diseases.

[0049] The drug for preventing and / or treating thrombotic diseases of the present invention preferably includes a preparation prepared by combining the active ingredient directly or with a pharmaceutical carrier; the active ingredient preferably includes: anticoagulant polypeptide Cb-13 or a derivative of the anticoagulant polypeptide Cb-13 or a composition comprising the anticoagulant polypeptide Cb-13.

[0050] The present invention also provides a drug for preventing and / or treating thrombotic diseases, wherein the active ingredients include the anticoagulant polypeptide Cb-13 and pharmaceutically acceptable excipients.

[0051] The working concentration of the anticoagulant polypeptide Cb-13 of the present invention is preferably not higher than 400 μM.

[0052] In order to further illustrate the present invention, a conotoxin-derived anticoagulant polypeptide Cb-13 and its application provided by the present invention are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0053] The animals, reagents and materials used in the examples of the present invention are all from commercially available materials, such as:

[0054] (1) All experimental animals were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. They were 6 to 8 weeks old, male, and weighed 18 to 22 g. After purchase, the mice were kept in an SPF animal room for one week, and then the experiments were carried out after the mice adapted to the environment.

[0055] (2) Physiological saline was purchased from Sichuan Kelun Pharmaceutical Co., Ltd., peptide Cb-13 was chemically synthesized by Hangzhou Gutuo Biosynthesis Co., Ltd., DMEM medium and FBS (Fetal Bovine Serum) were purchased from corningcellgo, penicillin / streptomycin were purchased from Solarbio, trypsin was purchased from Gibco, TritonX-100 was purchased from Tianjin Fengchuan Chemical Reagent Co., Ltd., Cell Counting Kit-8 was purchased from MCE, 2,3,5-triphenyltetrazolium chloride (TTC) and sodium heparin powder were purchased from Biofroxx, carrageenan powder was purchased from Beijing Coolaibo Technology Co., Ltd., FeCl3 powder was purchased from Tianjin Fengchuan Chemical Reagent Co., Ltd., Human Plasmin was purchased from Enzyme Research Laboratories, uPA was purchased from MedChemExpress, APTT kit was purchased from Beijing Regen Biotechnology Co., Ltd., and PT reagent was purchased from Nanjing Jiancheng Bioengineering Institute.

[0056] (3) Preparation of experimental solvent:

[0057] 50mL 10% DMEM medium: 5mL FBS, 0.5mL double antibody (penicillin-streptomycin), DMEM medium is fixed to 50mL;

[0058] 1L 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 make up to 1L;

[0059] 25mL 1% carrageenan rat tail solution: 0.25g carrageenan powder, dilute to 25mL with ddH2O;

[0060] 10mL 10% FeCl3 solution: 0.1g FeCl3 powder and 10mL normal saline;

[0061] 50mL 2% TTC solution: 1g TTC powder and 50mL ddH2O;

[0062] 100mL 25mM CaCl2: CaCl2·2H2O 0.37g and 25mM Hepes buffer 100mL;

[0063] 50mL HEPES buffer: HEPES 0.12g, NaCl 0.44g and ddH2O 50mL.

[0064] (4) Source of instruments used in the experiment:

[0065] The multifunctional microplate reader was purchased from Biotek, the vortex mixer was purchased from Labnet, the constant temperature oscillator was purchased from Shanghai Yiheng, the analytical balance was purchased from Sartorius, the biological safety cabinet was purchased from Memmert, the carbon dioxide constant temperature incubator was purchased from ThermoFisher, the advanced fluorescence imaging system was purchased from Sujing Antai, the clean bench was purchased from Chengdu Jinfeng, the liquid nitrogen tank was purchased from HIRAYAMA, the vertical high-pressure steam sterilizer was purchased from Shanghai Jinfu Experimental Instrument Equipment Co., Ltd., the circulating water vacuum pump and the -25°C low-temperature refrigerator were purchased from Zhongke Duling, the ultra-low temperature refrigerator was purchased from Wuhan Sewell Biotechnology Co., Ltd., and the inverted microscope was purchased from Yange Bio (agent).

[0066] Example 1

[0067] 1. Plasma recalcification (PRT)

[0068] Recalcification time is the time for plasma recalcification, which is the time when calcium is added back into the plasma from which calcium has been removed, so that the endogenous coagulation process can be restored.

[0069] a) First, blood was collected from the eyeball of the mouse and added to 3.8% sodium citrate anticoagulant, with the ratio of blood to sodium citrate anticoagulant being 3:1, followed by centrifugation at 3000 rpm for 15 min.

[0070] b) First, 30 μL of plasma, 10 μL of sample and 30 μL of 25 mM Hepes buffer were mixed and incubated in a 37°C constant temperature incubator for 10 min. The blank control group was supplemented with Hepes buffer.

[0071] c) Preheat 25 mM CaCl2 at 37°C, add 30 μL 25 mM CaCl2 and mix in an EP tube. The final concentrations of the small peptide administration groups are (100 μM, 50 μM, 25 μM, 12.5 μM), respectively.

[0072] d) Immediately place the plate in a microplate reader and perform kinetic monitoring at OD 650 nm absorbance for 30 min with 2 replicates.

[0073] e) The obtained data were plotted using GraphPadPrism 6.

[0074] The results are as follows Figure 1 As shown, Cb-13 has the effect of significantly prolonging the plasma recalcification and coagulation time at a concentration of 100 μM, has a prolonging effect on the plasma recalcification and coagulation time, and has a concentration-dependent effect from high concentration to low concentration.

[0075] 2. Prothrombin time (PT)

[0076] The prothrombin time (PT) is the most common coagulation time test, a screening method to determine the functionality and integrity of the extrinsic coagulation pathway and the common pathway. PT represents the time it takes for plasma to clot after the addition of CaCl2 and phospholipids, activators of the intrinsic pathway.

[0077] a) 50 μL of plasma and 5 μL of small peptides of different concentrations (100 μM, 50 μM, 25 μM, 12.5 μM) were mixed in an EP tube, and the blank control group was supplemented with 5 μL of normal saline. The final concentrations of the small peptide administration groups were (100 μM, 50 μM, 25 μM, 12.5 μM), respectively.

[0078] b) Preheat the PT (prothrombin) reagent at 37°C for 15 min, add 100 μL PT reagent into the EP tube, and test the coagulation time by monitoring 650 nm on a multifunctional microplate reader.

[0079] c) The obtained data were plotted using GraphPadPrism 6.

[0080] The results are as follows Figure 2 As shown, the coagulation start time of the NC group was about 16s, and the coagulation start time of Cb-13 at concentrations of 100μM, 50μM, 25μM, and 12.5μM was about 58s, 42s, 25s, and 22s, respectively, indicating that the addition of Cb-13 would delay the APTT time, and there was a concentration-dependent effect from high concentration to low concentration (100μM, 50μM, 10μM). In summary, the administration of Cb-13 significantly prolonged the APTT coagulation time and had an anticoagulant effect.

[0081] By testing the PT time of different concentrations of Cb-13, the effect on the PT coagulation time was observed. Figure 3 As shown, the concentration of the drug-dosing group was 100 μM. Compared with the blank group and the drug-dosing group, the coagulation starting time was about 16 seconds. The addition of Cb-13 did not prolong the coagulation time, and had little effect on the coagulation time, with no significant difference. Since PT is a screening method for detecting the functionality and integrity of the extrinsic coagulation pathway and the common pathway, exogenous coagulation is related to bleeding. The results show that the addition of Cb-13 may not have a significant effect on the exogenous coagulation pathway. In summary, Cb-13 has little effect on bleeding.

[0082] 3. Effects on enzymes related to the fibrinolytic system

[0083] Human Plasmin enzyme kinetic activity assay: Plasmin (200 nM), uPA (2.35 nM) and small peptides of different concentrations (100 μM, 50 μM, 25 μM, 12.5 μM) were incubated at room temperature for 5 min. After adding the corresponding substrate Human plasmin substrate (S = 0.5 mg / mL), the samples were immediately placed in an ELISA reader for kinetic detection at 405 nm absorption light and recorded continuously for 30 min.

[0084] The data were plotted using GraphPadPrism 6.

[0085] The reaction kinetics of the enzyme and substrate are shown in Figure 4 As shown, Cb-13 had a significant promoting effect on plasmin and uPa at concentrations of 100 μM, 50 μM, 25 μM, and 12.5 μM, and Cb-13 inhibited plasmin and uPa at lower concentrations, and the promoting activity could also be observed at 12.5 μM.

[0086] 4. Effects of cytotoxicity and hemolytic activity

[0087] (1) Cytotoxicity assay

[0088] a) Take out the frozen HEK-293T cells, turn on the water bath in advance, quickly thaw the frozen cells in a 37℃ water bath, transfer them to a new centrifuge tube, and centrifuge at 1000rpm for 5min. Discard the supernatant, add fresh DMEM medium containing 10% FBS to resuspend, divide into bottles, and culture the cells in a 37℃, 5% carbon dioxide incubator. When the cell density grows to about 80% of the bottom of the culture bottle, discard the medium and wash with sterile PBS 3 times.

[0089] b) Add 0.5 mL of trypsin to digest the adherent cells, and terminate the reaction with 2 mL of fresh DMEM medium containing 10% FBS.

[0090] c) Using a cell-grade sterile 96-well plate, add the above-mentioned HEK-293T and mouse spleen cell suspensions into the corresponding wells according to experimental requirements, 200 μL per well, and place in a cell culture incubator for overnight culture.

[0091] d) The next day, fresh culture medium was replaced, and the concentration gradient of Cb-13 in the treatment group was set to 400 μM, 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM, respectively. Sterile PBS was used in the negative control group. The culture was repeated three times and continued to be cultured in an incubator for 24 hours.

[0092] e) After 24 hours, 10 μL of CCK-8 solution was added to each well of the 96-well plate, and the plate was placed in an incubator in the dark for 2 hours. The light absorption value of each well was detected by an ELISA reader at a wavelength of 490 nm.

[0093] f) Calculation method: Cell survival rate = [(experimental well - blank well) / (control well - blank well)] x 100%.

[0094] g) The obtained data were plotted using GraphPadPrism 6.

[0095] The effect of Cb-13 on cell viability was determined by CCK-8 assay, and the cytotoxicity of Cb-13 was verified using human embryonic kidney HEK-293T cells and mouse spleen cells. Figure 6 As shown, the t-test method was used for statistical significance analysis, and the results are expressed as mean ± standard deviation, ns means that the difference is not statistically significant, *P<0.05. **P<0.01 means that the difference is statistically significant.

[0096] For HEK-293T cells, under the action of different concentrations of Cb-13 (400μM, 200μM, μM, 100μM, 50μM, 25μM, 12.5μM, 6.25μM), the cell survival rates were 111.1±9.214%, 100±2.439%, 104.9±0.407%, 100.9±1.084%, 100.5±1.897%, 101±2.574%, 100.4±3.252%, respectively, and the cell survival rate of the blank control group (PBS group) was 100.8±2.439%. The data showed that compared with the PBS control group, the Cb-13 administration group had no toxicity to HEK-293T cells, but at a concentration of 400μM, it also promoted cell proliferation, and there was no significant difference at other concentrations. This indicates that Cb-13 has no cytotoxicity to HEK-293T cells at a concentration below 400 μM.

[0097] For mouse spleen cells, under the action of different concentrations of Cb-13 (400μM, 200μM, μM, 100μM, 50μM, 25μM, 12.5μM, 6.25μM), the cell survival rates were 100.8±2.439%, 110.7±1.084%, 99.7±2.574%, 99.05±1.084%, 104.0±4.065%, 102.6±1.490%, respectively. The cell survival rate of the blank control group (PBS group) was 100.0±2.439%. The data showed that compared with the PBS control group, the Cb-13 administration group had no toxicity to mouse spleen cells, but at 200μM and 100μM concentrations, it also promoted cell proliferation, and there was no significant difference at other concentrations. It shows that Cb-13 has no cytotoxicity to mouse spleen cells at a concentration below 400μM. In summary, it was shown that Cb-13 had no cytotoxicity at a concentration below 400 μM.

[0098] (2) Cell hemolysis experiment

[0099] a) Mix the collected blood with saline in a ratio of 1:1, centrifuge at 3500 rpm for 5 minutes, discard the supernatant, wash the red blood cells with saline 3 times until the supernatant no longer shows red color, and discard the supernatant.

[0100] b) Use physiological saline to control the density of the washed red blood cells to 10 7 ~10 8 / mL (OD value range at 540nm is between 0.8 and 1).

[0101] c) The test sample Cb-13 was prepared into a concentration gradient as follows: 400 μM, 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM and 6.25 μM, with three parallel wells for each concentration.

[0102] d) Add the diluted red blood cell saline suspension and the sample to be tested to a 1 mL EP tube at a ratio of 1:1, incubate for 30 minutes, and centrifuge at 1000 rpm for 5 minutes. Pipette the supernatant into a sterile 96-well plate and measure the light absorption value of the supernatant at a wavelength of 540 nm on an ELISA reader.

[0103] e) In this experiment, the negative control was normal saline, and the positive control was 10% TritonX-100. The substances in ruptured red blood cells have the best ultraviolet absorption, and the absorption value was measured at 540nm by an ELISA reader.

[0104] f) The obtained data were plotted using GraphPadPrism 6.

[0105] The hemolysis test described in the present invention uses fresh mature human red blood cells. Since the hemolytic activity is proportional to the light absorption value at 540nm, the amount of hemoglobin leakage in the red blood cells is detected by using an ELISA instrument, and the OD value indicates the size of the hemolytic activity. The results are as follows Figure 5 As shown, under the action of different concentrations of Cb-13 (400μM, 200μM, μM, 100μM, 50μM, 25μM, 12.5μM, 6.25μM, 3.125μM), there was no significant increase in the absorbance at 540nm after administration of Cb-13. Compared with the positive control group, the OD value was generally lower than that of the NC blank control group, and there was no significant difference between the results of the drug-treated group and the blank control group. The positive control Trinton X-100 had obvious hemolytic activity, which was significantly different from the blank control group. In summary, it shows that Cb-13 has no hemolytic activity at a concentration of 400μM and below.

[0106] 5. Effects of bleeding and in vivo coagulation activity assay

[0107] (1) Tail bleeding experiment

[0108] a) Place the mouse in the designed device, clean the mouse tail with anhydrous ethanol, and adjust the water bath temperature to 37°C.

[0109] b) Inject drugs of each group through the tail vein. The concentrations of the small peptide administration groups were (4, 2, 1 mg / kg), the negative control group was injected with the same volume of normal saline, and the positive control group was injected with sodium heparin (1000 U / kg).

[0110] c) Prepare a conical flask, pour in physiological saline, mark each group of mice 2 mm away from the tail tip with a marker pen, and use a sterilized blade to cut the marked area evenly.

[0111] d) After cutting, carefully immerse the tail in 20 mL of normal saline that has been pre-warmed to 37°C. The bleeding time is counted from the beginning until no more fibrous blood appears.

[0112] e) The bleeding time of each group of mice was recorded.

[0113] f) The obtained data were plotted using GraphPadPrism 6.

[0114] In order to detect the difference between Cb-13 and positive drug sodium heparin on the risk of tail bleeding, the tail bleeding time of mice was tested. The positive control was set as sodium heparin, which is a commonly used anticoagulant in clinical practice. Figure 7As shown, under the action of different concentrations of Cb-13 (4mg / kg, 2mg / kg, 1mg / kg), the bleeding time was 169±21s, 157.667±9s, 145.667±11s, respectively, the bleeding time of the NC group (normal saline group) was 123.0±5s, and the bleeding time of the positive control (heparin sodium group) group was 237.0±9s. Tail vein injection of 1mg / kg, 2mg / kg and 4mg / kg of Cb-13 prolonged the tail bleeding time of mice compared with the blank control group, but the risk of bleeding was lower than that of heparin sodium.

[0115] (2) Carrageenan black tail experiment

[0116] By injecting carrageenan into the mouse peritoneum to induce tail thrombosis, the scope and degree of thrombosis can be observed based on the significant changes in the color of the mouse tail skin. This method is simple, fast, intuitive, highly operable, and reproducible. As an in vivo model, the carrageenan-induced mouse tail thrombosis model does not require traumatic surgical exposure to cause thrombosis, which reduces the interference of many human factors in the thrombosis process and is also minimally traumatic to animals.

[0117] a) Kunming mice (22-26 g), male, 4 weeks old.

[0118] b) The concentrations of the experimental peptide administration groups were (4, 2, 1 mg / kg), the negative control group was injected with the same volume of normal saline, and the positive control group was injected with sodium heparin (1000 U / kg). Each group of drugs was injected through the tail vein.

[0119] c) Mice were intraperitoneally injected with 120 μL (0.8% w / v) of carrageenan (type I) dissolved in PBS.

[0120] d) The mice were given sufficient water and food and placed in an environment with a temperature of 16°C to induce thrombosis in the mouse tail.

[0121] e) After 48 h, the mice were euthanized and the length of the tail thrombus was measured with a ruler.

[0122] f) The obtained data were plotted using GraphPadPrism 6.

[0123] The carrageenan mouse tail model was used to evaluate whether the drug has an anti-thrombotic effect. The groups were divided into Cb-13 (4mg / kg, 2mg / kg, 1mg / kg) administration group, NC group (normal saline group), and positive control group (heparin sodium group 1000U / kg). The carrageenan-induced mouse tail thrombosis model was established, and the length of the tail thrombus in each group of mice was measured with a ruler after 48 hours. The results are shown in Figure 8As shown, compared with the negative control group, the Cb-13 administration group has the effect of inhibiting rat tail thrombosis, and there is a significant difference. Different concentrations of Cb-13 (4mg / kg, 2mg / kg, 1mg / kg) reduced the length of carrageenan rat tail thrombosis, showing a dose-effect relationship.

[0124] (3) FeCl3 carotid artery thrombosis model experiment

[0125] a) C57 BL / 6J mice (male, 8 weeks old) were anesthetized with 2.0% isoflurane and fixed in a supine position after complete anesthesia. The core body temperature was maintained at 37°C during the entire operation.

[0126] b) Prepare the skin of the neck and disinfect with iodine. Cut the epidermis of 1 cm in length in the middle of the neck triangle to expose the subcutaneous tissue. Use elbow forceps to bluntly separate the sternocleidomastoid muscle and cut the anterior belly of the digastric muscle to expose the mouse common carotid artery. Gently peel off the tissue adhering to the blood vessel and fully peel off the blood vessel. Separate one of the carotid arteries from the adherent tissue and the adjacent vagus nerve. Place a semicircular paper with a diameter of 5 mm under the separated blood vessel as a background board to eliminate blood flow interference.

[0127] c) Each group of drugs were injected through the tail vein 5 minutes before ferric chloride induction. The concentrations of the small peptide administration groups were (4, 2, 1 mg / kg), the negative control group was injected with the same volume of normal saline, and the positive control group was injected with sodium heparin (1000 U / kg).

[0128] d) Pre-soak a filter paper (2×2 mm) with 10% (w / v) FeCl3 solution and stick it on the exposed carotid artery. Remove the filter paper after 30 seconds and wash away the residual FeCl3 in the treated area with PBS. FeCl3 promotes thrombosis at the injured site by inducing damage to vascular endothelial cells.

[0129] e) After FeCl3 induction, the blood flow was continuously recorded. Laser speckle perfusion imaging was used to record the blood flow of the carotid artery in each group. When there was no blood flow, the recording was stopped and the blood flow value of each group of mice was recorded.

[0130] f) Use the analysis software provided with the instrument to perform data analysis.

[0131] The inhibitory effect of Cb-13 on thrombosis was observed by establishing a mouse carotid artery thrombosis model. The Cb-13 inhibitor was administered in a dose gradient of (4 mg / kg, 2 mg / kg, 1 mg / kg), and the positive control group was heparin sodium (dose of 1000 U / kg). The mice were administered via the tail vein 10 minutes before ferric chloride induced thrombosis, and the laser speckle blood flow detector was used to record the carotid blood flow of different groups at 5 minutes and 10 minutes. The data was analyzed using the analysis software provided by the instrument. At 10 minutes, the NC (normal saline group) group mice showed almost no blood flow; while the Cb-13 administration group had the effect of prolonging carotid artery thrombosis under the action of 1 mg / kg, which was in sharp contrast to the NC group. In the blood flow value recording chart, it can also be seen that at 5 minutes and 10 minutes, the blood flow values ​​of the NC group and the Cb-13 administration group were significantly different. In summary, compared with the NC control group, Cb-13 concentrations of 4 mg / kg, 2 mg / kg, and 1 mg / kg had an anti-carotid thrombosis effect, and there was a certain dose-effect relationship from high concentration to low concentration ( Fig. 9 ), and in Fig. 9 The t-test method was used for statistical significance analysis, and the results were expressed as mean ± standard deviation (n = 6). *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001 indicated that the difference was statistically significant.

[0132] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. An anticoagulant polypeptide Cb-13, characterized in that: Includes at least one of the following polypeptides: (1) A polypeptide with 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; (3) The polypeptide described in (1) or the salt form of the polypeptide described in (2).

2. A gene encoding the anticoagulant polypeptide Cb-13 according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene includes SEQ ID No.

2.

4. Use of the anticoagulant polypeptide Cb-13 according to claim 1 or the gene according to claim 2 or 3 in the preparation of anticoagulant drugs.

5. The application according to claim 4, characterized in that: The anticoagulant drug includes a preparation made by combining the active ingredient directly or with a pharmaceutical carrier; The active ingredients include: anticoagulant polypeptide Cb-13 or a derivative of the anticoagulant polypeptide Cb-13 or a composition containing the anticoagulant polypeptide Cb-13.

6. An anticoagulant drug, characterized in that: The active ingredients include the anticoagulant polypeptide Cb-13 according to claim 1 and pharmaceutically acceptable excipients.

7. Use of the anticoagulant polypeptide Cb-13 according to claim 1 or the gene according to claim 2 or 3 in the preparation of a drug for preventing and / or treating thrombotic diseases.

8. The use according to claim 7, characterized in that: The drug for preventing and / or treating thrombotic diseases includes a preparation made by combining the active ingredient directly or with a pharmaceutical carrier; The active ingredients include: anticoagulant polypeptide Cb-13 or a derivative of the anticoagulant polypeptide Cb-13 or a composition containing the anticoagulant polypeptide Cb-13.

9. A drug for preventing and / or treating thrombotic diseases, characterized in that: The active ingredients include the anticoagulant polypeptide Cb-13 according to claim 1 and pharmaceutically acceptable excipients.

10. The anticoagulant drug according to claim 6 or the drug according to claim 9, characterized in that: The working concentration of the anticoagulant polypeptide Cb-13 is not higher than 400 μM.

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