Encoding gene of a coral-derived anticoagulant polypeptide and its application

Abstract

This invention provides a gene encoding a coral-derived anticoagulant polypeptide and its applications, belonging to the field of bioactive peptide technology. The invention provides an anticoagulant polypeptide GcKuz1, which includes the mature peptide segment shown in SEQ ID No. 1. The polypeptide GcKuz1 of this invention exerts antithrombotic and anticoagulant effects by binding to KLKB1 in the thrombin system. This polypeptide does not cause hemolysis or cytotoxicity, and has no bleeding risk, exhibiting good biocompatibility. The anticoagulant polypeptide of this invention has therapeutic effects on thrombotic diseases, such as arterial thrombotic diseases, venous thrombotic diseases, and capillary thrombotic diseases.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to the encoding gene of a coral-derived anticoagulant polypeptide and its application. Background Technology

[0002] Cardiovascular and cerebrovascular diseases are a collective term for diseases of the heart and brain blood vessels. Their pathogenesis is complex and diverse, including ischemic or hemorrhagic diseases of the heart, brain, and other tissues caused by risk factors such as hyperlipidemia, blood viscosity, atherosclerosis, and hypertension. Cardiovascular and cerebrovascular diseases have seriously threatened human health and quality of life, and have imposed a huge burden on the socio-economic system. Therefore, researching drugs to treat cardiovascular and cerebrovascular diseases has enormous social demand and significant social importance.

[0003] Under normal physiological conditions, the body's hemostatic, anticoagulant, and fibrinolytic systems interact and restrain each other, and are regulated by neurohumoral mechanisms, preventing blood from overflowing the blood vessel walls and causing bleeding, as well as from clotting within the vessels and leading to thrombosis. However, under pathological conditions, hypercoagulability and decreased anticoagulant or fibrinolytic activity can lead to a pre-thrombotic state or thrombosis. Conversely, these imbalances can trigger a hypocoagulable state or bleeding. The coagulation system is closely related to thrombotic diseases; thrombosis is the most serious complication of cardiovascular disease and a leading cause of death worldwide. Platelets and the coagulation cascade are effective targets for antithrombotic therapy, but these methods carry an inherent risk of bleeding.

[0004] Bioactive peptides are a class of short peptides or polypeptide molecules with specific biological activities. They regulate cell signaling, promote physiological responses, and influence the physiological functions of organisms by binding to specific receptors. Animal-derived bioactive peptides have been a hot topic in novel drug development in recent years. However, due to factors such as high toxicity and bleeding risk, very few peptide drugs for cardiovascular diseases have been applied clinically to date, far from meeting the growing demand in the anti-cardiovascular drug market. Therefore, how to obtain a peptide drug with high activity and low toxicity for the prevention and treatment of cardiovascular diseases to meet the need for the large-scale development of drugs for the treatment of cardiovascular diseases is an urgent problem to be solved. Summary of the Invention

[0005] This invention provides a gene encoding a coral-derived anticoagulant polypeptide and its application. The anticoagulant polypeptide has the ability to specifically bind to plasma kallikrein and does not pose a bleeding risk, thus having the potential use in treating thrombotic diseases.

[0006] The present invention provides an anticoagulant polypeptide comprising the mature peptide segment shown in SEQ ID No. 1.

[0007] In one embodiment of the present invention, the anticoagulant polypeptide comprises the amino acid sequence shown in SEQ ID No. 2.

[0008] This invention provides a gene encoding the aforementioned anticoagulant polypeptide.

[0009] In one embodiment of the present invention, the nucleotide sequence of the gene encoding the mature peptide segment of the anticoagulant polypeptide is shown in SEQ ID No. 3.

[0010] In one embodiment of the present invention, the nucleotide sequence of the gene encoding the anticoagulant polypeptide is shown in SEQ ID No. 4.

[0011] This invention provides the application of the above-mentioned anticoagulant polypeptide in the preparation of biomaterials with anticoagulant and / or antithrombotic activities.

[0012] In one embodiment of the present invention, the biomaterial includes a drug.

[0013] This invention provides a drug with anticoagulant and / or antithrombotic activity, the active ingredient of which includes the above-mentioned anticoagulant polypeptide, and also includes pharmaceutically acceptable excipients.

[0014] In one embodiment of the present invention, the dosage of the drug, calculated as an anticoagulant polypeptide, is not less than 2 mg / kg mouse.

[0015] Beneficial effects: This invention provides an anticoagulant polypeptide GcKuz1, which includes the mature peptide segment shown in SEQ ID No. 1. It is a novel Kunitz-type polypeptide predicted from the transcriptome of reef-building corals, targeting plasma kallikrein in the coagulation system. The anticoagulant polypeptide exerts antithrombotic and anticoagulant effects by binding to KLKB1. This polypeptide does not cause hemolysis or cytotoxicity, and has no bleeding risk, exhibiting good biocompatibility. The GcKuz1 polypeptide of this invention exhibits anticoagulant activity in vitro. Specifically, GcKuz1 concentration-dependently prolongs the fibrin formation time in plasma recalcification and prolongs the activation time of partial thromboplastin, but has no effect on prothrombin time. The GcKuz1 polypeptide can concentration-dependently prolong the time of FeCl3-induced carotid artery thrombosis in mice; the GcKuz1 polypeptide can concentration-dependently inhibit carrageenan-induced tail thrombosis in mice; and the GcKuz1 polypeptide can concentration-dependently inhibit and alleviate damage caused by stroke reperfusion, including reducing the ischemic area of ​​the mouse brain, improving grip strength, and improving the neurobehavioral phenotype of mice. The anticoagulant polypeptide of this invention has therapeutic effects on thrombotic diseases, such as arterial thrombotic diseases, venous thrombotic diseases, and capillary thrombotic diseases. Attached Figure Description

[0016] Figure 1The figure shows the sequence, base sequence, and multiple sequence alignment results of the GcKuz1 peptide. In the figure, A represents the full-length cDNA and amino acid sequence of GcKuz1, the green background represents the signal peptide sequence, and the red background represents the mature peptide sequence; B represents the multiple sequence alignment of the GcKuz1 peptide with Kunitz-type peptides from different species.

[0017] Figure 2 The figure shows the binding and interaction results of GcKuz1 with Plasma Kallikrein to verify enzyme kinetic detection and SPR analysis. In the figure, A represents the enzyme kinetic detection results; B represents the surface plasmon resonance (SPR) analysis results.

[0018] Figure 3 The figure shows the results of the in vitro anticoagulant activity validation of GcKuz1 peptide. In the figure, A and D represent PRT analysis; B and E represent APTT analysis; C and F represent PT analysis; the data represent the mean ± standard error of three independent experiments (n>=3); **p<0.001, ***p<0.001 compared with the control group;

[0019] Figure 4 The image shows the therapeutic effect of GcKuz1 on ferric chloride-induced carotid artery thrombosis in mice. In the figure, A represents the imaging results of blood flow in the mouse carotid artery; B represents the carotid artery embolism time in mice; C represents the ROI value analysis results of blood flow in the mouse carotid artery; data are expressed as mean ± standard error (n=6); ****p<0.0001 compared with the control group;

[0020] Figure 5 The image shows the therapeutic effect of GcKuz1 on carrageenan-induced tail thrombosis in mice; A in the image represents a representative image of a mouse tail thrombosis; B represents the length of the mouse tail thrombosis; data are expressed as mean ± standard error (n = 6); ****p < 0.0001 compared with the model group;

[0021] Figure 6 The image shows the therapeutic effect of GcKuz1 on cerebral ischemia-reperfusion injury in a tMCAO mouse model; A and B in the figure represent TTC staining images and statistical graphs of brain sections of tMCAO mice; C represents the neurological injury scores of mice in different treatment groups; D represents the grip strength of mice in different treatment groups; data are expressed as mean ± standard error (n=6); *p<0.05, ***p<0.001, ****p<0.0001 compared with the control group;

[0022] Figure 7The results show that GcKuz1 does not cause bleeding, hemolysis, or cytotoxicity. A represents the hemolytic activity of GcKuz1 on erythrocytes, with Triton X-100 as the positive control; B represents the bleeding risk of GcKuz1 assessed using a mouse tail hemorrhage assay (with heparin sodium as the positive control); CCK8 analysis results of cell viability in SH-SY5Y cells (C), mouse spleen cells (D), and mouse peritoneal macrophages (E) 24 hours after GcKuz1 administration; data are from three independent experiments and are presented as mean ± standard error (n = 3–5). #### p<0.0001, ****p<0.0001, NS indicates no significant difference compared to the control group. Detailed Implementation

[0023] The present invention provides an anticoagulant polypeptide comprising the mature peptide segment shown in SEQ ID No. 1.

[0024] The anticoagulant polypeptide of this invention is derived from reef-building coral and named GcKuz1. The mature peptide of GcKuz1 has three disulfide bonds, a molecular weight of 6095.83 Daltons, and its amino acid sequence is shown in SEQ ID No. 1: SICHLPKDVGFCKALIRRYFYNTSKGKCEMFHYGGCYGNKNNFKTLVSCKRA C. The complete primary structure sequence of the polypeptide GcKuz1 of this invention is shown in SEQ ID No. 2: MKSTFLVLVILLYYHAVQASICHLPKDVGFCKALIRRYFYNTSKGKCEMFHYGGCYGNKNNFKTLVSCKRACERNKRSCPRRETTSGQ.

[0025] This invention provides a gene encoding the aforementioned anticoagulant polypeptide.

[0026] In one embodiment of the present invention, the nucleotide sequence of the gene encoding the mature peptide segment of the anticoagulant polypeptide is as shown in SEQ ID No. 3: TCATCTGCCACCTACCTAAAGATGTCGGATTCTGCAAGGCCCTCATTCGCCGGTATTTTTACAACACTTCCAAGGGGAAATGTGAGATGTTTCACTACGGTGGATGCTATGGTAATAAAAATAACTTCAAGACACTTGTGAGTTGCAAGAGAGCGTGC; and the nucleotide sequence of the gene encoding the anticoagulant polypeptide is as shown in SEQ ID No. 4: 5'-ATGAAGTCCACCTTCCTAGTTTTGGTTATATTGTTGTACTATCACGCAGTT CAGGCTTCCAATCTGCCACCTACCTAAAGATGTCGGATTCTGCAAGGCCCTCATTCGCCGGTATTTTTACAACACTTCCAAGGGGAAATGTGAGATGTTTCACTACGGTGGATGCTATGGTAATAAAAATAACTTCAAGACACTTGTGAGTTGCAAGAGAGCGTGCGAACGTAACAAGCGGTCTTGCCCTCGACGGGAGACAACATCTGGTCAA-3'.

[0027] This invention provides the application of the above-mentioned anticoagulant polypeptide in the preparation of biomaterials with anticoagulant and / or antithrombotic activities.

[0028] In this invention, enzyme kinetics and surface plasmon resonance methods were used to confirm that the peptide GcKuz1 can bind to Plasma kallikrein / KLKB1, with a binding KD constant of 8.63 μM. In vitro experiments also confirmed that GcKuz1 concentration-dependently prolongs fibrin formation time in plasma recalcification and extends the activated partial thromboplastin time, but has no effect on prothrombin time. In vivo experiments confirmed that GcKuz1 concentration-dependently prolongs FeCl3-induced carotid artery thrombosis time in mice, with an effective dose not less than 2 mg / kg. Furthermore, GcKuz1 concentration-dependently inhibits carrageenan-induced tail thrombosis in mice. GcKuz1 concentration-dependently inhibits and mitigates stroke reperfusion injury, including reducing ischemic brain area, improving grip strength, and improving neurobehavioral phenotypes in mice. GcKuz1 does not cause significant hemolysis or hemorrhage and is non-cytotoxic, exhibiting high biocompatibility. In summary, the polypeptide GcKuz1 described in this invention has potential application value in the treatment of thrombotic diseases.

[0029] This invention provides a drug with anticoagulant and / or antithrombotic activity, the active ingredient of which includes the above-mentioned anticoagulant polypeptide, and also includes pharmaceutically acceptable excipients.

[0030] In one embodiment of the present invention, the dosage of the drug, calculated as an anticoagulant polypeptide, is not less than 2 mg / kg mouse.

[0031] To further illustrate the present invention, the coding gene of a coral-derived anticoagulant polypeptide and its application are described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1: Discovery of the GcKuz1 polypeptide sequence and its encoding gene

[0033] Blast annotation and Pfam analysis identified a transcript fragment (ID: c102034_g1_i1) with a typical Kunitz domain from the transcript sequence of Goniopora columna. Multiple sequence alignment analysis with Kunitz peptide homologs from different species confirmed that GcKuz1 is a novel GcKuz1 peptide from corals.

[0034] The results are as follows Figure 1 As shown, GcKuz1 is a novel coral-derived Kunitz polypeptide with the mature peptide sequence shown in SEQ ID No. 1 and the complete amino acid sequence shown in SEQ ID No. 2.

[0035] Example 2: Determination of the target site of GcKuz1 peptide

[0036] 1. Enzyme kinetics detection:

[0037] 110 nM KLKB1 and different final concentrations of GcKuz1 peptide (2–16 μM) were added to 50 μL of PBS buffer and incubated at room temperature for 5 min. Then, 50 μL of the substrate Chromogenix S-2302 was added. TM (S820340, Chromogenix, Ita) was immediately analyzed using a microplate reader at 450 nm for continuous absorption over 30 minutes. Enzyme kinetics results are as follows: Figure 2 As shown in Figure A, the GcKuz1 peptide can inhibit the activity of Plasma Kallikrein.

[0038] 2. Surface plasmon resonance (SPR):

[0039] A CM5 sensor chip was activated for 20 minutes at a flow rate of 5 μL / min using 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mM N-hydroxysuccinimide. KLKB1 protein was diluted to 20 μg / mL with 1 mL of sodium acetate (10 mM, pH 5.0) and flowed through the activated surface to achieve the target response value (RU). Remaining activation sites on the chip were blocked with 45 μL of hexanolamine (1 M, pH 8.5). Real-time detection results were recorded using a Biacore S200 instrument (USA) at a flow rate of 30 μL / min. Subsequently, in order to determine the equilibrium dissociation constant (KD) of GcKuz1 and KLKB1, sequentially diluted GcKuz1 (10, 5, 2.5 and 1.25 μM) was treated in a 150 mM phosphate buffer solution at pH 7.4, and its interaction with KLKB1 immobilized on the chip surface was analyzed using the BIA evaluation program.

[0040] Surface plasmon resonance (SPR) analysis results of the interaction between GcKuz1 and Plasma Kallikrein are as follows: Figure 2 As shown in Figure B, GcKuz1 can bind to KLKB1, and the binding KD constant is 8.63 μM.

[0041] Example 3: GcKuz1 polypeptide exhibits anticoagulant activity in vitro.

[0042] In this embodiment, the anticoagulant activity of the polypeptide GcKuz1 provided by this invention was detected using plasma recalcification time (PRT), activated partial thromboplastin time (APTT), and prothrombin time (PT). The polypeptide GcKuz1 described in this embodiment was synthesized by Goodtop Biotechnology Co., Ltd. using a chemical solid-phase synthesis method.

[0043] 1. Plasma recalcification time (PRT) assay: In a 96-well plate, 19 μL of healthy human plasma, 1 μL of GcKuz1 peptide at different final concentrations (2 μM, 4 μM, 8 μM, 16 μM), and 40 μL of HEPES buffer (150 mM Hepes, 20 mM NaCl, pH 7.4) were added to each well and incubated at 37°C for 5 min. After incubation, 40 μL of CaCl2 solution preheated at 37°C for 10 min was added to each well and the mixture was immediately mixed. The absorbance was continuously measured at 650 nm wavelength over 30 min using a microplate reader, and the fibrin formation time was recorded.

[0044] 2. Activated Partial Thromboplastin Time (APTT) Assay: The APTT assay was performed according to the instructions of the Activated Partial Thromboplastin Time (APTT) kit (TC0306100T, LEAGENE, China). 49 μL of healthy human plasma, 1 μL of different final concentrations of GcKuz1 peptide (2 μM, 4 μM, 8 μM, 16 μM), and 50 μL of LAPTT ellagic acid solution were incubated at 37°C for 5 min. After incubation, 50 μL of CaCl2 solution preheated to 37°C for 10 min was added, and the mixture was immediately mixed. The absorbance was continuously measured at 650 nm over 3 min using a microplate reader, and the fibrin formation time was recorded.

[0045] 3. Prothrombin Time (PT) Measurement: Performed according to the PTASSAYKIT (GMS10176, GENMED, USA) instructions. 49 μL of healthy human plasma and 1 μL of different final concentrations of GcKuz1 peptide (2 μM, 4 μM, 8 μM, 16 μM) were incubated at 37°C for 5 min. Immediately after incubation, 50 μL of preheated PT reagent was added, and the absorbance was measured within 3 min. Fibrin formation time was also recorded.

[0046] The results are as follows Figure 3 As shown, GcKuz1 concentration-dependently prolongs the fibrin formation time in plasma recalcification and prolongs the activation time of partial thromboplastin, but has no effect on prothrombin time.

[0047] Example 4: GcKuz1 peptide inhibits ferric chloride-induced carotid artery thrombosis in mice.

[0048] In this embodiment, the GcKuz1 polypeptide provided by the present invention has an inhibitory effect on ferric chloride-induced carotid artery thrombosis in mice.

[0049] Six-week-old C57 BL / 6J male mice (approximately 20g) were used in this experiment and divided into six groups: a saline group, a GcKuz1 peptide treatment group (1mg / kg, 2mg / kg, and 4mg / kg), and a positive control group (2500U / kg). Ten minutes after intravenous injection of the above drugs, the mice were anesthetized intraperitoneally with 1.5% sodium pentobarbital (40mg / kg), fixed in a supine position, and their necks were disinfected with 75% alcohol. A midline cervical incision was made, and the skin and subcutaneous tissue were dissected layer by layer. The sternocleidomastoid muscle was separated, and the anterior belly of the digastric muscle was cut to expose the left common carotid artery (CCA). Filter paper was perforated using a 5mm diameter punch, and a piece of saline-soaked filter paper was placed under the common carotid artery to remove background interference. A piece of filter paper soaked in 10% FeCl3 was placed over the artery. After FeCl3 stimulation, the filter paper was removed, and residual FeCl3 was washed away with saline. Then, the blood flow changes were observed under a laser speckle flowmeter, and the vascular flow values ​​at 5 min and 10 min were recorded to evaluate the effect of GcKuz1 peptide on the time of carotid artery thrombosis in mice.

[0050] The results are as follows Figure 4 As shown, the peptide GcKuz1 can prolong the time of FeCl3-induced carotid artery thrombosis in mice in a concentration-dependent manner, with effective doses of 2 mg / kg and 4 mg / kg.

[0051] Example 5: GcKuz1 peptide inhibits carrageenan-induced tail thrombosis in mice.

[0052] In this embodiment, the GcKuz1 polypeptide provided by the present invention has an inhibitory effect on carrageenan-induced tail thrombosis in mice.

[0053] Six-week-old male Balb / C mice were intraperitoneally injected with 1% carrageenan (type I) (CC3171, coolaber, China). Thirty minutes later, they were injected via the tail vein with saline, different concentrations of GcKuz1 peptide (1 mg / kg, 2 mg / kg and 4 mg / kg), and the positive control drug heparin sodium (2500 U / kg). The length of the tail thrombus was observed and recorded 24 hours later to evaluate the effect of GcKuz1 peptide on tail thrombus formation in mice.

[0054] The therapeutic effect of GcKuz1 on carrageenan-induced tail thrombosis in mice was as follows: Figure 5 As shown, the peptide GcKuz1 can inhibit carrageenan-induced tail thrombosis in mice in a concentration-dependent manner, with effective doses of 2 mg / kg and 4 mg / kg.

[0055] Example 6: GcKuz1 peptide reduces stroke-induced reperfusion injury in mice.

[0056] Male C57 BL / 6J mice (approximately 20g) were anesthetized for 6 weeks with 1.5% sodium pentobarbital (40mg / kg). After moderate-depth anesthesia, the skin and subcutaneous tissue of the rats were incised layer by layer through a midline cervical incision. The sternocleidomastoid muscle was dissected, and the anterior belly of the digastric muscle was cut to expose the right common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA). The thyroid artery and pharyngeal artery on the ECA were coagulated using an electrocoagulator and then cut. The distal end of the ECA was ligated, and a suture was attached to its proximal end to temporarily clamp the CCA and ICA. The ECA was cut, and a suture plug was inserted into the ICA from the ECA stump. The ECA stump was ligated, the arterial clamp of the ICA was removed, and the suture plug was slowly advanced from the ICA inward and upward. The direction was adjusted appropriately, and the suture plug was inserted to the marked position. Timing was started, and the suture plug was removed after 60 minutes. After observing no active bleeding, the incision was sutured. The entire process was kept warm with an electric blanket at a temperature of 36-37℃. After recovery, the mice were returned to their cages and allowed free access to food. 24 hours after the stroke, neurological deficit scores and muscle strength tests were assessed and recorded. Brain tissue was then removed, placed in a brain mold (on ice), and sliced ​​posteriorly along the optic chiasm into 2mm thick sections. These sections were then placed in 2% TTC phosphate buffer and stained at 37°C for 30 minutes, followed by overnight fixation with 4% paraformaldehyde and photographed.

[0057] Percentage of ischemic volume = [Ischemic volume - (Left hemisphere volume - Right hemisphere volume)] / Right hemisphere volume × 100. Drugs were administered via the tail vein 10 minutes before ischemia-reperfusion. The experimental groups were: saline group, groups with different concentrations of GcKuz1 peptide (1 mg / kg, 2 mg / kg, and 4 mg / kg), and the positive control drug icarlan peptide group.

[0058] The therapeutic effect of GcKuz1 on cerebral ischemia-reperfusion injury in tMCAO mouse model is as follows: Figure 6 As shown, the peptide GcKuz1 can inhibit and reduce stroke-induced reperfusion injury in a concentration-dependent manner, including reducing the ischemic area of ​​the brain in mice, increasing grip strength in mice, and improving the neurobehavioral phenotype of mice, at effective doses of 2 mg / kg and 4 mg / kg.

[0059] Example 7: GcKuz1 peptide has no cytotoxicity, hemolysis, or bleeding risk.

[0060] 1. Cell hemolysis experiment

[0061] Gently pour 50 mL of red blood cells into the centrifuge pan, add physiological saline along the pan wall, and centrifuge at 3500 rpm for 10 min at room temperature. Repeat this step three times. For the final centrifuge, add 20 mL of physiological saline to adjust the final blood cell concentration to 1 × 10⁻⁶. 8Cells / mL. 100 μL of GcKuz1 (6.25, 12.5, 25 and 50 μM) was added to each well, followed by 100 μL of blood cells in each well. The cells were then incubated at 37 °C for 30 min, centrifuged horizontally at 3500 rpm for 10 min at room temperature, and 150 μL of the supernatant was collected for detection at 540 nm.

[0062] GcKuz1's hemolytic activity against erythrocytes, such as Figure 7 As shown in Figure A, the results of the erythrocyte hemolysis experiment show that there is no significant difference in absorbance at 540 nm between 0 and 25 μM GcKuz1, indicating that GcKuz1 does not damage blood cells. Since GcKuz1 is administered via the tail vein, it is the first thing to enter the bloodstream, so determining whether GcKuz1 has a destructive effect on erythrocytes is a very important factor.

[0063] 2. Cytotoxicity test

[0064] 100 μL of human neuroblastoma cells (SH-SY5Y), mouse peritoneal macrophages, and mouse spleen cells were added at a concentration of 10 μL. 4 Cells were seeded at a density of cells / mL in 96-well plates. After culturing for 24 hours at 5% CO2 and 37°C, different final concentrations of GcKuz1 (3.125–50 μM) were added and incubated for another 24 hours. Then, 10 μL of Cell Counting Kit-8 was added and incubated for another 90 minutes; absorbance was measured at 450 nm.

[0065] This experiment was conducted to evaluate the safety of the GcKuz1 peptide, and the results showed that GcKuz1 was not cytotoxic (see...). Figure 7 (CE).

[0066] 3. Mouse tail bleeding experiment

[0067] Ten minutes after intravenous injection of GcKuz1 (1, 2, and 4 mg / kg) and heparin sodium (positive control), the mouse tails were disinfected with anhydrous ethanol. A mark was made 0.2 mm from the tail tip, and a 2 mm long tail was removed from 6-week-old C57 BL / 6J mice using a sterilized scalpel. The tails were then placed in physiological saline at 37°C. The time of tail bleeding was recorded to assess the risk of bleeding associated with GcKuz.

[0068] See results Figure 7 In the study of mouse tail bleeding time, the risk of bleeding associated with GcKuz1 was determined. The results showed that GcKuz1 did not pose a bleeding risk.

[0069] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

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

2.

2. The gene encoding the anticoagulant polypeptide of claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence of the gene encoding the anticoagulant polypeptide is shown in SEQ ID No.

4.

4. The use of the anticoagulant polypeptide of claim 1 in the preparation of a medicament having anticoagulant and / or antithrombotic activity.

5. A drug with anticoagulant and / or antithrombotic activity, characterized in that, The active ingredient is the anticoagulant polypeptide of claim 1, and also includes pharmaceutically acceptable excipients.

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