Antithrombotic effect of Meteorin protein or gene
By using Meteorin protein or gene and its potentiators to inhibit platelet aggregation and clot formation, the balance between efficacy and bleeding risk of existing antithrombotic drugs has been resolved, achieving safe and effective antithrombotic therapy and blood sample stability, which has research and clinical application value.
Patent Information
- Application Number
- CN202511231256.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-30
- Publication Date
- 2026-05-05
AI Technical Summary
Existing antithrombotic drugs struggle to maintain a balance between efficacy and bleeding risk during use, and there is a lack of novel therapeutic targets to enhance antithrombotic efficacy without increasing bleeding risk.
Meteorin protein or gene and its potentiators are used to prepare antithrombotic drugs and in vitro inhibitory reagents to inhibit platelet aggregation and clot formation by upregulating Meteorin protein expression or by using agonists and stabilizers to enhance its activity.
It effectively inhibits platelet aggregation and clot formation, maintains the stability of blood samples, extends shelf life, improves the accuracy and safety of drug development, reduces false positive or false negative results, and provides a safe antithrombotic treatment option.
Smart Images
Figure CN121971589A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of molecular biology and biomedicine, and more specifically, to the antithrombotic effects of Meteorin protein or gene. Background Technology
[0002] Meteorin (Metrn) is a neurotrophic factor encoded by the Metrn gene on human chromosome 16 and mouse chromosome 17. It is a secreted protein composed of 293 amino acids with a molecular weight of approximately 31 kDa. Structurally, it is reported to consist of 10 conserved Cys residues forming 5 disulfide bonds. Discovered in 2004, it is primarily expressed in the central nervous system and plays a neuroprotective role.
[0003] Thrombotic diseases are a group of serious cardiovascular and cerebrovascular diseases, such as ischemic stroke and myocardial infarction, posing a significant threat to human life and health. For these diseases, existing antiplatelet drugs, such as aspirin and clopidogrel, are available clinically. However, maintaining a balance between their efficacy and adverse bleeding reactions has always been a key challenge in the use of these drugs. Therefore, there is an urgent need to develop novel therapeutic targets to enhance antithrombotic efficacy without increasing the risk of bleeding.
[0004] Existing technologies disclose various effects of Meteorin. For example, patent application number 2022109162665 discloses a lentiviral vector of shRNA with the Metrn gene knocked out and its application, specifically disclosing that the lentiviral vector of shRNA with the Metrn gene knocked out has significant effects in promoting the mobilization of hematopoietic stem / progenitor cells and promoting the proliferation and migration of bone marrow hematopoietic stem / progenitor cells. Patent publication number CN115948544B discloses the application of CITED4 and / or METRN in the differential diagnosis of the degree of intervertebral disc degeneration, specifically disclosing that METRN shows significant differences in tissues with different degrees of intervertebral disc degeneration, and has high diagnostic value for the degree of intervertebral disc degeneration, and can be used for the differential diagnosis of intervertebral disc degenerative lesions. Patent application number 201810021543X discloses the use of nickel streak protein in the treatment of anomalous pain, hyperalgesia, spontaneous pain, and phantom pain. As a novel secreted protein, no research reports have been found on the use of Meteorin in thrombotic diseases. Summary of the Invention
[0005] The first object of the present invention is to provide pharmaceutical use of the Meteorin protein or gene and its synergists.
[0006] A second objective of this invention is to provide the use of the Meteorin protein or gene and its synergists in the preparation of in vitro inhibitory reagents.
[0007] A third objective of this invention is to provide a method for screening potential antithrombotic substances.
[0008] To achieve the first objective mentioned above, the technical solution adopted by the present invention is as follows:
[0009] The use of Meteorin protein or gene and its potentiators in the preparation of antithrombotic drugs.
[0010] In the pharmaceutical applications described above, the synergist is further selected from agonists, upregulators, or stabilizers.
[0011] In the pharmaceutical use described above, the amino acid sequence of the Meteorin protein is further shown in SEQ ID NO.1.
[0012] In the pharmaceutical applications described above, the drug may further comprise a conventional pharmaceutical carrier and be prepared into various dosage forms in the form of a pharmaceutical composition. For example, it may be prepared into conventional solid dosage forms such as tablets, powders, or capsules; when used for injection, it may be prepared into an injection solution.
[0013] In the pharmaceutical use described above, the drug further upregulates the expression of Meteorin protein.
[0014] To achieve the second objective mentioned above, the technical solution adopted by the present invention is as follows:
[0015] The use of Meteorin protein or gene and its enhancers for the preparation of reagents that inhibit platelet aggregation or platelet clot contraction in isolated blood samples.
[0016] In the use of the in vitro inhibitory reagent as described above, the ex vivo blood sample further refers to a platelet suspension.
[0017] Reagents that inhibit platelet aggregation or platelet clot contraction in isolated blood samples directly function to maintain the stability of blood samples in the in vitro environment, preventing platelet activation or aggregation. Therefore, Meteorin protein or gene and its synergists can be used as additives for blood sample collection tubes. Such in vitro inhibitory reagents not only have certain research value in scientific research laboratories, but also have good commercial value in clinical practice, testing, and drug development.
[0018] In the fields of clinical testing and in vitro diagnostics, the aforementioned in vitro inhibitory reagent can be used for the preservation and testing of blood samples, preventing premature activation of platelets during collection and testing, thereby ensuring the accuracy of clinical test data such as platelet function testing, coagulation experiments, and flow cytometry analysis, and can be further applied to the development of platelet function testing kits.
[0019] In drug development and screening, in vitro inhibitory reagents can serve as an auxiliary tool in the development of antiplatelet and antithrombotic drugs, maintaining the stability of blood samples and avoiding false positive or false negative results caused by sample aggregation, thereby helping to establish a reliable pharmacodynamic evaluation system.
[0020] In blood storage and transfusion applications, in vitro inhibitors can be used to delay premature platelet activation during storage and transportation, thus extending the shelf life of platelet products. Simultaneously, in the quality control processes of blood bank storage companies, these reagents can also be used to ensure the stability and scientific rigor of the testing procedures.
[0021] To achieve the third objective mentioned above, the technical solution adopted by the present invention is as follows:
[0022] A method for screening potential antithrombotic substances includes the following steps:
[0023] a) Contact the candidate material with a system containing Meteorin protein or gene;
[0024] b) Observe the effects of candidate substances on the expression and activity of Meteorin protein or gene, wherein if the candidate substance can promote Meteorin gene expression or increase Meteorin protein activity, then the candidate substance is a potential antithrombotic substance.
[0025] This invention discloses for the first time the antithrombotic use of Meteorin protein or gene and its potentiators. Experiments have demonstrated that incubation with Meteorin protein significantly inhibits platelet aggregation, reduces platelet spreading area, delays clot contraction, and simultaneously inhibits platelet ATP release, α-granule release, and activation of surface integrin αⅡbβ3. In whole-body thrombosis experiments, Meteorin protein effectively inhibits arterial thrombus formation. Because Meteorin protein is an endogenous human protein with fewer side effects, it possesses high safety as a potential drug and shows promising prospects for industrialization. Attached Figure Description
[0026] Appendix Figure 1 Meteorin (Metrn) protein inhibited platelet aggregation induced by Thrombin (0.012 U / mL) and Collagen (0.3 μg / mL). n = 4. *P < 0.05, **P < 0.01.
[0027] Appendix Figure 2 Meteorin (Metrn) protein inhibited platelet spreading area at 20, 40, and 60 minutes. n = 4. **P < 0.01, ****P < 0.0001. ns, no significance.
[0028] Appendix Figure 3 Meteorin (Metrn) protein inhibits blood clot retraction. n=4. **P<0.01.
[0029] Appendix Figure 4 Meteorin (Metrn) protein inhibits platelet ATP release. n=4. **P<0.01.
[0030] Appendix Figure 5 Meteorin (Metrn) protein inhibits the release of platelet α-granules and the activation of surface integrin αIIbβ3. n=4. *P<0.05,***P<0.001,****P<0.0001.
[0031] Appendix Figure 6 Meteorin (Metrn) protein inhibits arterial thrombosis. n = 14-15. ***P < 0.001. Detailed Implementation
[0032] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.
[0033] Meteorin protein and gene
[0034] In this invention, the terms "Meteorin" and "Metrn" are used interchangeably. Human, rat, and mouse Meteorin gene and protein sequences are publicly available on the NCBI website. The Meteorin protein used in this invention can be naturally occurring, for example, it can be isolated and purified from mammals. Furthermore, the Meteorin protein can also be artificially prepared, for example, by preparing recombinant proteins using conventional genetic engineering techniques. Any suitable Meteorin protein is applicable to this invention. The Meteorin protein includes the full-length Meteorin protein or its bioactive fragment. As one specific embodiment, the Meteorin protein can be a human Meteorin protein, the amino acid sequence of which is shown in NP_076947.1. As another specific embodiment, the Meteorin protein can be a rat or mouse Meteorin protein; the amino acid sequence of the rat Meteorin protein is shown in NP_001009962.1, and the amino acid sequence of the mouse Meteorin protein is shown in NP_598480.1. The amino acid sequences of Meteorin proteins formed by substitution, deletion, or addition of one or more amino acid residues are also included in this invention. Meteorin proteins or their bioactive fragments comprise a subset of conserved amino acid substitution sequences, the substitutions of which do not affect their activity or retain some of their activity. Appropriate amino acid substitutions are well-known techniques in the art, which can be readily implemented and ensure that the biological activity of known molecules is not altered. These techniques have made it clear to those skilled in the art that, generally, changing a single amino acid in a non-essential amino acid region of a polypeptide does not change its biological activity. Preferably, the amino acid sequence (SEQ ID NO:1) of the Meteorin protein used in specific embodiments of the present invention is as follows:
[0035] gysedrcswrgsgltqepgsvgqltldctegaiewlypagalrltlggpdpgtrpsivclrperpfagaqvfaermtgnlelllaegpdlaggrcmrwgprerralflqatphrdisrrvaafrfelhedqraem spqaqglgvdgacrpcsdaelllaactsdfvihgtihgvahdtelqesvitvvvarvirqtlplfkegssegqgrasirtllrcgvrpgpgsflfmgwsrfgeawlgcaprfqefsrvysaaltthlnpcemald
[0036] Meteorin synergist and its uses
[0037] The term "Meteorin enhancer" includes agonists, upregulators, and stabilizers, referring to any substance that can increase the activity and stability of Meteorin, upregulate its expression, or extend its effective duration. These substances can all be used in this invention. They can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA), the protein level, or viral products that upregulate Meteorin expression.
[0038] Example 1: Metrn protein inhibits platelet aggregation.
[0039] I. Experimental Methods
[0040] C57BL / 6J mice were anesthetized by intraperitoneal injection of an excessive amount of sodium pentobarbital (100 mg / kg). Approximately 100 μL of 2% sodium citrate solution was drawn into a 1 mL syringe. After complete anesthesia, the mice's limbs were immobilized, and the abdominal aorta was exposed. Blood was slowly drawn from the abdominal aorta using a 1 mL syringe needle (approximately 1 mL). Immediately after blood collection, the blood and sodium citrate solution were gently mixed. An equal volume of physiological saline was added to the whole blood, followed by the addition of Apyrase (final concentration 1 U / mL), and the mixture was inverted and mixed thoroughly. The mixture was centrifuged at 700 rpm for 20 minutes at room temperature. The supernatant platelet-rich plasma (PRP) was carefully transferred to a new centrifuge tube using a Pasteur pipette. Centrifuge at 1700 rpm for 10 minutes at room temperature (5 minutes for mouse platelets), discard the supernatant platelet-free plasma (PPP), resuspend the platelets in a suitable amount of preheated (37°C) Tyrode's buffer using a Pasteur pipette, and count the platelets using an animal whole blood analyzer to adjust the platelet concentration to 2-3 × 10⁻⁶. 8 After processing, the platelet suspension was set aside. The platelet agglutinator was preheated to 37°C with a rotor stirring speed of 1200 rpm. 300 μL of platelet suspension was added to a platelet aggregation tube, the stirring rotor was added, and the tube was placed into the platelet agglutinator channel. After the baseline stabilized, different types of agonists were added to the platelets, and the aggregation curves and aggregation rates were recorded. The treatment group was pre-incubated with Metrn for 5 minutes.
[0041] II. Results
[0042] like Figure 1 As shown, in the platelet aggregation assay, 1 μg / mL of Metrn protein was found to inhibit platelet aggregation induced by thrombin (0.012 U / mL) and collagen (0.3 μg / mL).
[0043] Example 2: Metrn protein reduces platelet spreading area
[0044] I. Experimental Methods
[0045] The platelet suspension prepared by the method in Example 1 was incubated on a glass plate pre-coated with collagen (concentration of 40 μg / mL). After 20, 40, and 60 minutes, the platelets were fixed with paraformaldehyde, blocked with serum, and subjected to lysis buffer to rupture the membrane. Then, the platelets were incubated with 488-phalloidin to stain the platelet cytoskeleton proteins. Subsequently, five fields of view were randomly selected for microscopic photography, and the average spreading area of platelets in each field of view was calculated using ImageJ software.
[0046] II. Results
[0047] like Figure 2 As shown, in the platelet spreading experiment, it was found that 1 μg / mL of Metrn protein could inhibit the spreading area of platelets on collagen (40 and 60 minutes), but the spreading area was not affected at 20 minutes.
[0048] Example 3: Metrn protein inhibits blood clot retraction response
[0049] I. Experimental Methods
[0050] The platelet suspension (250 μL) prepared by the method in Example 1 was mixed with 10 μL of platelet-free plasma (PPP) and 1 μL of thrombin (concentration of 10 U / mL) in a glass tube, and photographed at the corresponding time points. The area of contraction in the field of view was calculated using ImageJ software.
[0051] II. Results
[0052] like Figure 3 As shown, in the blood clot retraction experiment, it was found that 1 μg / mL of Metrn protein could inhibit the blood clot retraction rate (20, 30 minutes).
[0053] Example 4: Metrn protein inhibits platelet ATP release
[0054] I. Experimental Methods
[0055] Take 300 μL of the platelet suspension prepared by the method in Example 1, add it to a platelet aggregation tube, add a stirring rotor, add ATP-Lume fluorescent dye, place the tube into the well channel of a platelet aggregator, and after the baseline stabilizes, add different types of agonists to the platelets and record the ATP release curve. The drug-treated group was incubated with Metrn for 5 minutes beforehand.
[0056] II. Results
[0057] like Figure 4As shown, 1 μg / mL of Metrn protein was found to inhibit the release of ATP from activated platelets induced by thrombin (0.012 U / mL) and collagen (0.3 μg / mL).
[0058] Example 5: Metrn protein inhibits platelet α-granule release and integrin αIIbβ3 activation.
[0059] I. Experimental Methods
[0060] Platelets prepared by the method in Example 1 were diluted 10-fold and incubated with Metrn for 5 minutes beforehand. Then, they were stimulated with thrombin and simultaneously incubated with CD62P-FITC and JON / A-PE antibodies for 30 minutes. After fixation with paraformaldehyde, the platelets were analyzed by flow cytometry.
[0061] II. Results
[0062] like Figure 5 As shown, 1 μg / mL of Metrn protein was found to inhibit the release of α-particles from activated platelets and the activation of surface integrin αⅡbβ3.
[0063] Example 6: Metrn protein inhibits arterial thrombosis
[0064] I. Experimental Methods
[0065] Before modeling, mice were injected intravenously with 6g of rhodamine (for thrombus observation) and 3μg of Metrn protein. After the injection, mice were anesthetized with sodium pentobarbital (80mg / kg) via intraperitoneal injection. An incision was made along the midline of the abdomen, starting below the manubrium of the sternum and moving towards the abdomen (to avoid damaging blood vessels). The mice were placed in a left lateral decubitus position in a culture dish (approximately 10cm in diameter). An inverted glass dish (approximately 3cm in diameter) was placed next to the mouse, level with the abdominal incision. The small intestine was carefully pulled out using forceps (to avoid pinching blood vessels and causing bleeding) and spread evenly on the dish. Warm saline was added to maintain the temperature and humidity of the intestinal tract. Under a microscope, a suitable field of view was selected for the mesenteric artery. A piece of filter paper (1.5mm × 3mm) soaked in 10% FeCl3 solution was carefully placed on the mesenteric artery. After 1 minute, the filter paper was removed, and thrombus formation was observed under a microscope. The entire process was documented using a computer-controlled microscope with photographs and video recordings.
[0066] II. Results
[0067] like Figure 6 As shown, Metrn protein was found to inhibit arterial thrombus formation and significantly delay the time it takes for thrombi to block blood vessels.
[0068] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.
Claims
1. The use of Meteorin protein or gene and its potentiators, characterized in that, Used to prepare antithrombotic drugs.
2. The use according to claim 1, characterized in that, The synergist is selected from agonists, upregulators, or stabilizers.
3. The use according to claim 1, characterized in that, The amino acid sequence of the Meteorin protein is shown in SEQ ID NO.
1.
4. The use according to claim 1, characterized in that, The drug may also contain conventional pharmaceutical carriers.
5. The use according to claim 1, characterized in that, The drug upregulates the expression of Meteorin protein.
6. The use of Meteorin protein or gene and its potentiators, characterized in that, This reagent is used to prepare a reagent that inhibits platelet aggregation in isolated blood samples.
7. The use according to claim 6, characterized in that, The ex vivo blood sample refers to a platelet suspension.
8. The use of Meteorin protein or gene and its potentiators, characterized in that, This reagent is used to prepare a solution that inhibits the contraction of platelet clots in isolated blood samples.
9. The use according to claim 8, characterized in that, The ex vivo blood sample refers to a platelet suspension.
10. A method for screening potential antithrombotic substances, characterized in that, Includes the following steps: a) Contact the candidate material with a system containing Meteorin protein or gene; b) Observe the effects of candidate substances on the expression and activity of Meteorin protein or gene, wherein if the candidate substance can promote Meteorin gene expression or increase Meteorin protein activity, then the candidate substance is a potential antithrombotic substance.