Anti-platelet aggregation polypeptide or active fragment thereof, and preparation method therefor and use thereof

Abstract

Disclosed are an anti-platelet aggregation polypeptide or an active fragment thereof, and a preparation method therefor and a use thereof. On the basis of a large number of screening experiments, it has been found that certain polypeptides can bind to PEAR1 on the surfaces of platelets, thereby inhibiting or antagonizing a platelet aggregation function. Thus, the anti-platelet aggregation polypeptide can be used as an anti-platelet drug for preventing and treating thrombus and embolism diseases.

Description

Antiplatelet aggregation polypeptide or its active fragment, preparation method and application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to application number 2023114340402, filed on November 1, 2023, entitled “Anti-platelet aggregation polypeptide, preparation method and application thereof” and application number 2023114338455, filed on November 1, 2023, entitled “Platelet aggregation inhibitory peptide, preparation method and application thereof”, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the field of anti-platelet aggregation, and in particular to an anti-platelet aggregation polypeptide targeting PEAR1 receptor, and a preparation method and application thereof. Background Art

[0004] Antiplatelet drugs can inhibit platelet aggregation and are one of the important drugs for the prevention and treatment of thromboembolic diseases. In patients with transient ischemic attack or ischemic stroke, the use of antiplatelet drugs in the acute and recovery phases can significantly improve patient prognosis. In patients with acute coronary syndrome after percutaneous coronary intervention, antiplatelet drug treatment can reduce the risk of adverse cardiovascular events. Therefore, antiplatelet drugs are not only important therapeutic drugs for thromboembolic diseases, but also secondary preventive drugs for many cardiovascular diseases, playing an important role in clinical practice.

[0005] At present, the antiplatelet drugs used in clinical practice in my country mainly target three receptors: cyclooxygenase, P2Y 12 Receptor, glycoprotein IIb / IIIa (GPIIb / IIIa) receptor. Aspirin is a cyclooxygenase antagonist that blocks the synthesis of thromboxane A2 by antagonizing cyclooxygenase, thereby inhibiting platelet aggregation. However, since cyclooxygenase is widely distributed in blood vessels, stomach and kidney tissues, aspirin use can cause adverse reactions such as gastrointestinal tract and asthma. Clopidogrel, prasugrel, and ticagrelor are P2Y 12 P2Y receptor antagonist. 12 Receptor antagonists inhibit the interaction of adenosine diphosphate with P2Y 12 Binding to GPIIb / IIIa receptors inhibits platelet aggregation. However, clinical application varies widely, and bleeding or ischemic adverse events can occur during treatment, impacting patient prognosis. GPIIb / IIIa receptor antagonists include abciximab, tirofiban, and eptifibatide. Inhibiting GPIIb / IIIa can block its binding to ligands such as fibrinogen and inhibit platelet aggregation. However, the antiplatelet effect of GPIIb / IIIa receptors is so potent that they are limited to short-term intravenous use.

[0006] Therefore, existing drugs have disadvantages such as limited options, large individual differences, adverse reactions and intolerance. There is an urgent need to discover new targets related to platelet function and develop drugs targeting new targets.

[0007] The information in the background technology is only intended to illustrate the general background of the invention and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art known to a person skilled in the art.

[0008] Summary of the Invention

[0009] To solve at least some of the technical problems in the prior art, the present invention provides a polypeptide having the activity of binding to platelets and inhibiting platelet aggregation. Specifically, the present invention includes the following contents.

[0010] In a first aspect, the present invention provides a polypeptide or an active fragment thereof, which has the activity of targeting and binding to the PEAR1 receptor and inhibiting platelet aggregation.

[0011] In certain embodiments, the polypeptide or active fragment thereof according to the present invention has a structure represented by the following formula: V1-LKYX1YENHX2-V2 Formula (I);

[0012] Wherein, X1 represents W or H, X2 represents A or N; V1 does not exist or represents the first variable region, and V2 does not exist or represents the second variable region.

[0013] In certain embodiments, according to the polypeptide or active fragment thereof of the present invention, the first variable region is selected from A, EA or GEA, and the second variable region is selected from I, IS, ISI.

[0014] In certain embodiments, the polypeptide or active fragment thereof according to the present invention has the activity of inhibiting platelet aggregation and has the structure described by the following formula: KVIYY-V3 Formula (II);

[0015] Among them, V3 represents a variable region with a length of 0-4 amino acids.

[0016] In certain embodiments, according to the polypeptide or active fragment thereof according to the second aspect of the present invention, the variable region is selected from K, KD, KDG, and KDGE.

[0017] In certain embodiments, the polypeptide or active fragment thereof according to the present invention comprises a polypeptide or active fragment thereof having 80%, preferably 85%, and preferably 90% homology with the polypeptide or active fragment thereof represented by formula (I) and (II) and having the same function.

[0018] In certain embodiments, the polypeptide or active fragment thereof according to the present invention comprises a polypeptide or active fragment thereof having the same function as the above-mentioned polypeptide after substitution and / or deletion and / or addition of one or several amino acid residues.

[0019] In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide of the present invention or an active fragment thereof.

[0020] The third aspect of the present invention provides a vector molecule comprising the nucleic acid molecule of the present invention.

[0021] The fourth aspect of the present invention provides a host cell comprising the nucleic acid molecule or the vector molecule of the present invention.

[0022] The fifth aspect of the present invention provides a method for preparing the polypeptide or active fragment thereof according to the present invention, which comprises preparing the polypeptide by artificial synthesis or by genetic engineering.

[0023] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the polypeptide or active fragment thereof and / or nucleic acid molecule described in the present invention, and a pharmaceutically acceptable carrier.

[0024] The seventh aspect of the present invention provides use of a polypeptide or an active fragment thereof or a nucleic acid molecule thereof in the preparation of an anti-platelet aggregation drug.

[0025] In an eighth aspect, the present invention provides an in vitro method for inhibiting platelet aggregation, comprising the step of contacting the polypeptide of the present invention or an active fragment thereof with platelets in vitro.

[0026] The ninth aspect of the present invention provides a method for preventing, treating or ameliorating embolic diseases, which comprises the step of administering a therapeutically effective amount of the polypeptide or its active fragment and / or nucleic acid molecule, or pharmaceutical composition according to the present invention to a subject in need.

[0027] In certain embodiments, the method for preventing, treating or ameliorating embolic diseases according to the present invention further comprises the step of combining with other drugs or therapeutic therapies.

[0028] The tenth aspect of the present invention provides the use of a polypeptide or its active fragment or its nucleic acid molecule in the preparation of a drug for preventing, treating or improving embolic diseases, wherein the polypeptide or its active fragment is the polypeptide or its active fragment described in the first aspect of the present invention, and the nucleic acid molecule is the nucleic acid molecule described in the second aspect of the present invention.

[0029] In view of the current situation that there are no drug molecules that target the PEAR1 receptor inhibitory function, the present invention, based on a large number of screening experiments, found that specific polypeptides can bind to the PEAR1 receptor on the platelet surface, thereby inhibiting or antagonizing platelet aggregation function, and thus can be used as antiplatelet drugs for the prevention and treatment of thrombosis and embolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG1 shows the platelet function inhibition corresponding to different polypeptide sequences having the structure represented by formula (I).

[0031] FIG2 is a binding curve of exemplary polypeptides to PEAR1.

[0032] FIG3 shows the platelet function inhibition corresponding to different polypeptide sequences having the structure represented by formula (II).

[0033] FIG4 is a binding curve of exemplary polypeptides to PEAR1. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0037] As used herein, the term "polypeptide" refers to a polymer consisting of a plurality of amino acid residues, or a variant, synthetic or naturally occurring analog thereof. Thus, polypeptides are applicable to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids (e.g., chemical analogs of corresponding naturally occurring amino acids), as well as to naturally occurring amino acid polymers and naturally occurring chemical derivatives thereof. Examples of such derivatives include, but are not limited to, post-translational modifications and degradation products, including pyroglutamyl, isoaspartyl, proteolytic, phosphorylated, glycosylated, oxidized, isomerized, and deaminated variants.

[0038] Herein, the term "active fragment" refers to a portion of the polypeptide sequence of the present invention. A "fragment" or "active fragment" comprises a number of consecutive amino acid residues sufficient to maintain biological activity (i.e., targeting and binding to the PEAR1 receptor and inhibiting platelet aggregation activity).

[0039] As used herein, the term "platelet aggregation inhibitory activity" may sometimes be referred to as "antiplatelet aggregation activity" or "antiplatelet activity", and refers to the activity of reducing, decreasing, inhibiting or blocking platelet aggregation, preferably with an inhibition rate of 5% or more, 10% or more, preferably 12% or more, such as 14% or more, 15% or more. The inhibition rate refers to the aggregation amount of the control platelets and the aggregation amount of the test platelets obtained under the same conditions / the aggregation amount of the control platelets. The determination of the inhibition rate is not particularly limited, and includes determinations of platelet aggregation inhibition recorded within 5 minutes using a platelet aggregometer (such as Helena AggRAM).

[0040] peptides

[0041] The polypeptides provided herein are short peptides that inhibit platelet aggregation. Because they can target and bind to the PEAR1 receptor, they are sometimes referred to as "PEAR1 receptor inhibitors." Generally, the polypeptides of the present invention are 20 or fewer amino acids in length, such as 18 or fewer amino acids, 15 or fewer amino acids, or 12 or fewer amino acids.

[0042] In certain embodiments, the polypeptides of the present invention have the structure shown in Formula (I): V1-LKYX1YENHX2-V2 Formula (I).

[0043] In formula (I), X1 represents W or H, X2 represents A or N, V1 does not exist or represents the first variable region, and V2 does not exist or represents the second variable region.

[0044] The first variable region is selected from A, EA or GEA, and the second variable region is selected from I, IS or ISI.

[0045] In formula (I), when V1, V2, and V3 do not exist, "-" does not exist. When V1 and V2 represent different amino acid residues, "-" represents a covalent bond, such as a peptide bond.

[0046] Without being bound by any theory, the polypeptide of the present invention is selected from at least one of the following sequences:

[0047] In certain embodiments, the polypeptide of the present invention has a structure represented by formula (II): KVIYY-V3 Formula (II);

[0048] Wherein, V3 represents a variable region of 0-4 amino acids in length, preferably, V3 is selected from K, KD, KDG, KDGE;

[0049] In formula (II), when V3 is absent, "-" is absent. When V3 represents a different amino acid residue, "-" represents a covalent bond, such as a peptide bond.

[0050] Without being bound by any theory, the polypeptide of the present invention is selected from at least one of the following sequences:

[0051] It is understood that modified polypeptides, polypeptides having certain homology with the polypeptides of the present invention, and / or variants of the polypeptides are all within the scope of protection of the present invention. A modified polypeptide means that the polypeptide of the present invention may be a modified polypeptide, and the modification site may be one or more of N-terminal modification, C-terminal modification, side chain modification, amino acid modification, and backbone modification. The type of modification or the method of modification is not particularly limited, and examples include but are not limited to one or more of amino modification, acetylation modification, biotinylation modification, fluorescent labeling modification, polyethylene glycol modification, isoprenylation modification, myristoylation and palmitoylation modification, phosphorylation modification, glycosylation modification, polypeptide conjugate modification, and special amino acid modification. The polypeptide having the above modifications essentially has the same function or activity as the polypeptide of the amino acid sequence of SEQ ID No.: 1-11, that is, the polypeptide having the above modifications still maintains or has an improved function or activity of inhibiting platelet aggregation and / or targeting and binding to the PEAR1 receptor.

[0052] In the present invention, the terms "homology" and "identity" are used interchangeably. Homologous sequences include amino acid sequences that are at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequences of the present invention, such as SEQ ID Nos.: 1-11. Similarly, polypeptides having the aforementioned homologies essentially have the same function or activity as the polypeptides of the amino acid sequences of SEQ ID Nos.: 1-11, i.e., polypeptides having the aforementioned homologies still maintain or have enhanced platelet aggregation inhibition activity and / or targeted binding to the PEAR1 receptor function or activity.

[0053] To determine sequence identity, a sequence alignment can be performed, which can be performed in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters for the alignment, including any algorithms needed to achieve optimal alignment over the full-length sequences being compared.

[0054] Variants of polypeptides include those in which the amino acid sequences of SEQ ID No.: 1-11 have undergone substitution and / or deletion and / or addition of one or several amino acid residues and have the same function. In the present invention, the term "substitution" refers to the replacement of one or more amino acids by different amino acids. "Deletion" refers to the reduction of one or more amino acids in an amino acid sequence. "Addition" refers to a change in an amino acid sequence that results in the addition of one or more amino acids compared to a specified sequence. It should be noted that these variants of polypeptides obtained by the above-mentioned substitutions and / or deletions and / or additions essentially have the same function or activity as the polypeptides of the amino acid sequences of SEQ ID No.: 1-11, that is, these variants of polypeptides obtained by the above-mentioned substitutions and / or deletions and / or additions still maintain or have improved platelet aggregation inhibition activity and / or targeted binding function or activity of the PEAR1 receptor.

[0055] Nucleic acid molecules

[0056] In one aspect of the present invention, a nucleic acid molecule is provided, which comprises a nucleotide sequence encoding the polypeptide of the present invention.

[0057] As long as the present invention can produce polypeptide of the present invention, then for nucleic acid molecule, it is not particularly limited, and it comprises the nucleotide sequence of coding polypeptide and optional other sequences.Exemplary other sequences are connected with the nucleotide sequence of coding polypeptide in an operable manner.The nucleotide sequence of coding polypeptide can be the nucleotide sequence comprising a polypeptide gene, or it can be the nucleotide sequence comprising a plurality of tandem polypeptide genes.The example of other sequences includes but is not limited to the control sequence for gene expression, such as promoter, any leader, terminator etc. with function in host cell.The nucleotide sequence of coding polypeptide comprises natural coding sequence (for example, by the sequence that obtainable database is easily determined) or degenerate sequence, for example the codon optimized coding sequence designed for specific host cell, particularly the codon optimized version carried out for host.

[0058] Carrier molecules

[0059] One aspect of the present invention provides a vector molecule comprising the nucleic acid molecule of the present invention.

[0060] The vector of the present invention refers to an artificial construct that can deliver and preferably express one or more target genes or sequences in a host cell. The vector of the present invention is not limited and can be an expression vector, a viral vector, etc. In certain embodiments, the vector comprises a target gene encoding a polypeptide gene of the present invention or a precursor thereof, a promoter, a terminator, or optionally further comprises a marker gene. The vector can use a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0061] host cells

[0062] In one aspect of the present invention, a host cell is provided, which comprises the polypeptide, nucleic acid molecule or vector molecule of the present invention.

[0063] The host cells of the present invention refer to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. Host cells include any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication.

[0064] Preparation method

[0065] One aspect of the present invention provides a method for preparing the polypeptide of the present invention. The preparation method is not particularly limited and includes preparation by artificial synthesis or genetic engineering.

[0066] In certain embodiments, the polypeptides of the present invention are obtained by artificial synthesis. Methods for artificially synthesizing polypeptides are known in the art. Specifically, they include liquid-phase and solid-phase methods. Liquid-phase peptide synthesis methods include those employing BOC protection and Z protection. These methods are preferably used for the synthesis of short peptides, particularly the polypeptides of the present invention. Compared to solid-phase peptide synthesis, these methods offer numerous advantages, including a wide selection of protecting groups, low costs, and ease of scale-up. Liquid-phase peptide synthesis is generally employed for the polypeptides of the present invention.

[0067] In certain embodiments, the polypeptides of the present invention are synthesized by solid phase peptide synthesis methods, which include two schemes, FMOC protection and BOC protection, which have the advantages of convenient and rapid synthesis and easy automation.

[0068] In certain embodiments, the polypeptides of the present invention are obtained by genetic engineering expression. Genetic engineering expression systems include prokaryotic expression systems, eukaryotic expression systems, and cell-free expression systems. Examples of prokaryotic expression systems include Escherichia coli expression systems. Eukaryotic expression systems include zymocyte expression systems, insect cell expression systems, and mammalian cell expression systems.

[0069] Pharmaceutical composition

[0070] One aspect of the present invention provides a pharmaceutical composition comprising the polypeptide and / or nucleic acid molecule described herein and a pharmaceutically acceptable carrier. The polypeptide and nucleic acid molecule have been described in detail above and will not be further elaborated upon here. The pharmaceutically acceptable carrier is described below.

[0071] In the present invention, pharmaceutically acceptable carriers are well known in the art and can be determined by a person skilled in the art to meet clinical standards. Pharmaceutically acceptable carriers include diluents and excipients.

[0072] Examples of suitable pharmaceutically acceptable carriers include, but are not limited to: (1) Dulbecco's phosphate buffered saline, pH about 7.4, with or without about 1 mg / ml to 25 mg / ml human serum albumin; (2) 0.9% saline (0.9% w / v sodium chloride), and (3) 5% (w / v) glucose; antioxidants such as tryptamine and stabilizers such as Tween 20 may also be included.

[0073] The pharmaceutical composition of the present invention can be in any suitable dosage form. For example, an injection, a suspension, an emulsifier, etc. The pharmaceutical composition of the present invention can be administered to the body in a known manner. For example, it can be delivered to the tissue of interest by intramuscular injection, optionally administered intravenously, transdermally, intranasally, orally, through the mucosa, or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be administered herein can vary to a large extent depending on a variety of factors, such as the target cell, the type of organism or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0074] Treatment

[0075] One aspect of the present invention provides a method for preventing, treating, or ameliorating embolic diseases. According to one embodiment of the present invention, the method comprises administering a therapeutically effective amount of the polypeptide and / or nucleic acid molecule, or pharmaceutical composition, of the present invention to a subject in need thereof.

[0076] As used herein, the terms "administer" and "give" or "administer" are used interchangeably and refer to the introduction of a predetermined amount of a substance (e.g., a polypeptide of the present invention or a fragment thereof) into a subject by some suitable means. The term "therapeutically effective amount" refers to an amount sufficient to significantly improve certain symptoms associated with a disease or condition, that is, an amount that provides a therapeutic effect for a given condition and dosing regimen. The term "treat" is used to refer to obtaining a desired pharmacological and / or physiological effect. "Treatment" as used herein encompasses administering the polypeptides, nucleic acids, vectors, host cells, and pharmaceutical compositions of the invention to an individual to achieve treatment.

[0077] Those skilled in the art will appreciate that the treatments described herein may also include combining other drugs or therapeutic therapies to achieve combined treatment of embolic diseases. The other drugs may or may not be therapeutic agents for embolic diseases, and are not particularly limited thereto. The other therapeutic therapies may be selected from respiratory and circulatory support therapy, anticoagulant therapy, thrombolytic therapy, interventional therapy, and the like.

[0078] In the present invention, embolic diseases mainly refer to thromboembolic diseases, examples of which include but are not limited to venous thromboembolism, pulmonary thromboembolism, deep vein thrombosis, etc.

[0079] As used herein, the term "subject" or "patient" refers to a vertebrate, preferably a mammal, and more preferably a human. Mammals include, but are not limited to, rodents, apes, and livestock. Specific mammals include rats, mice, cats, dogs, monkeys, and humans. Non-human mammals include all mammals other than humans.

[0080] use

[0081] One aspect of the present invention provides use of the polypeptide or nucleic acid molecule thereof in the preparation of an anti-platelet aggregation drug.

[0082] Currently available antiplatelet drugs generally target cyclooxygenase, P2Y 12 Receptor, glycoprotein IIb / IIIa (GPIIb / IIIa) receptor. Unlike existing drugs, the polypeptides of the present invention can bind to PEAR1, thereby inhibiting or blocking the activation of the PEAR1 receptor on the platelet surface. This activation can cause tyrosine phosphorylation of the PEAR1 intracellular segment, leading to phosphorylation of phosphatidylinositol-3-kinase, activation of GPIIb / IIIa and platelet aggregation.

[0083] In one aspect, the present invention further provides use of the polypeptide or nucleic acid molecule thereof in the preparation of a drug for preventing, treating or ameliorating embolic diseases.

[0084] Methods for inhibiting platelet aggregation

[0085] One aspect of the present invention provides a method for inhibiting platelet aggregation, comprising contacting platelets with a polypeptide of the present invention. The method of the present invention can be an in vitro method or an in vivo method. Inhibiting platelet aggregation in vitro can be used to analyze or study platelet function, and further, for example, to screen for useful compounds, particularly high-energy compounds.

[0086] Example 1

[0087] 1. Experimental instruments and reagents

[0088] Experimental instruments: 1 / 10,000 electronic balance; vertical reactor (20*25mm, No. 1 sand core); centrifuge tube; 100-1000μl pipette; 1ml pipette; nitrogen; circulating water vacuum pump; washing bottle; test tube; long-necked pipette;

[0089] Experimental reagents: 2-Cl resin; Fmoc-AA-OH; anhydrous DCM; DIEA; methanol; DCM; technical-grade DMF; analytical-grade DMF; 20% piperidine / DMF; detection reagents A (5 g ninhydrin-100 ml anhydrous ethanol) and B (analytical-grade pyridine); HOBT; DIC;

[0090] 2. Experimental steps

[0091] (1) Weigh 500 mg of 2-Cl-Trt resin using an electronic balance, place it in a 25 ml reactor, add DCM and soak for 30 minutes. Then wash it with DMF three times and DCM once.

[0092] (2) Weigh 0.15 mmol of Fmoc-AA-OH (C-terminal) into a centrifuge tube, add 8 ml of anhydrous DMF and 800 μl of DIEA, and shake well. Pipette the solution into the reactor from the previous step and allow to react with nitrogen bubbling for 1.5 h. After the reaction, wash the resin with DMF three times for 30 s each, then add a mixture of 0.5 ml of DIEA, 0.5 ml of methanol, and 2 ml of DCM and react for 20 min.

[0093] (3) Washing: After the liquid in the reactor is drained with a circulating water vacuum pump, industrial-grade DMF is added to the reactor with a wash bottle to completely immerse the resin in the solution. Wash for 30 seconds, and then drain the liquid in the reactor with a circulating water vacuum pump. Repeat this operation 4 times.

[0094] (4) Fmoc removal: Add 20% piperidine / DMF solution to the reactor using a wash bottle. The volume of the reagent should be about 3 times the volume of the resin. The resin should be completely soaked in the solution. Nitrogen bubbling should be used for the reaction for 20 minutes.

[0095] (5) Washing: Follow step (3), and replace the industrial-grade DMF with analytical-grade DMF during the fifth washing process;

[0096] (6) Resin detection: Use a long-necked pipette to take 10-20 resins from the reactor and place them at the bottom of the test tube. Then use a rubber-tipped dropper to take two drops of detection reagents A and B and drop them into the test tube to ensure that the resin and the detection reagents are in full contact. Then place the test tube in a constant temperature of 100°C and heat for 2 minutes.

[0097] (7) Condensation: Weigh 0.5 mmol Fmoc-AA-OH (second position from the C-terminus) and 0.5 mmol HOBT (0.075 g) into a centrifuge tube and dissolve them thoroughly with 1 ml DMF. Then add 100 μl DIC and mix for 1 min. Add the mixture to the dried resin and allow to react with nitrogen bubbling for 1 h.

[0098] (8) Repeat steps (5) to (7) until the peptide connection is completed.

[0099] (9) Finally, Fmoc was removed and then washed. The resin was then washed three times with methanol according to the washing method. Finally, the liquid in the reactor was drained with a circulating water vacuum pump and vacuum dried until the resin was in granular form.

[0100] Example 2

[0101] 1. Experimental methods:

[0102] Sixteen-week-old C57BL / 6J mice were anesthetized with 20% urethane and the abdominal aorta was extracted using 3.8% sodium citrate anticoagulant (anticoagulant:blood = 1:9). The upper plasma layer was centrifuged at 90g for 9 minutes and then centrifuged at 150g for 15 minutes. The supernatant was discarded and washed platelets were added to the platelet buffer. The platelet count in the washed platelet was adjusted to 3-4 × 10 8 / mL. 240 μL of washed platelets were removed, and 5 μL of normal saline was added to the control group, while 5 μL of peptide (final concentration 1 mg / mL) was added to the experimental group. The plates were incubated at 37°C for 5 minutes. After the plates were placed in a magnetic rotor, 5 μL of ADP solution (01905, Sigma: Darmstadt, Germany; final concentration 50 μM) was added. Platelet aggregation was recorded over 5 minutes using a platelet aggregometer (Helena AggRAM). The maximum platelet aggregation rates of the experimental and control groups were compared.

[0103] 2. Experimental results:

[0104] The results are shown in Figure 1. Figure 1 shows the platelet function inhibition corresponding to different polypeptide sequences having the structure shown in formula (I). If the platelet inhibition rate is higher than 15%, it is considered that the polypeptide has an inhibitory effect on platelet aggregation and has antiplatelet activity. As shown in Figure 1, based on the results of polypeptides 1, 2, 3, 4, and 5, LKYWYENHA was found to be a sequence that maintains antiplatelet activity. In order to further determine the extension of its front and rear amino acids, polypeptides 6, 7, and 8 were constructed. The results showed that the antiplatelet activity of the polypeptide gradually decreased with the extension of the amino acid, and the front end could be extended by up to three amino acids GEA. 13, 14, and 15 were constructed. The results showed that the antiplatelet activity of the peptide gradually decreased with the extension of the amino acid, and the back end could be extended by up to three amino acids ISI. In order to confirm the variability of amino acids, amino acids at different positions were replaced to form polypeptides 9, 10, 11, and 12. The results showed that only the W amino acid could be replaced by the H amino acid and still had antiplatelet activity. Therefore, based on the above experimental results, it was concluded that the polypeptide with the following formula is the active polypeptide: (G)+(E)+(A)+L+K+Y+W(H)+Y+E+N+H+A(N)+(I)+(S)+(I) Formula (III).

[0105] Among them, "+" represents a covalent bond, and "()" indicates that the corresponding amino acid residue in the brackets is an optional amino acid residue, that is, it may exist or not.

[0106] Example 3

[0107] This example is used to test whether the peptide can bind to the PEAR1 protein. The following uses peptide 1 in Figure 1 as an example for verification.

[0108] 1. Experimental methods:

[0109] Recombinant human PEAR1 protein was immobilized on a Cytiva CM5 chip using surface plasmon resonance (SPR) using an amino-coupling reagent. Peptide 1 was serially diluted in PBS-P, then the protein solution was passed through the chip at a flow rate of 30 μL / min for 120 seconds, followed by dissociation for 480 seconds. The chip surface was regenerated with glycine hydrochloride (pH 2.0), and kinetic data were generated using a 1:1 binding model in the Biaevaluation program.

[0110] 2. Experimental results:

[0111] As shown in FIG2 , polypeptide 1 in FIG1 can bind to PEAR1 protein with a binding constant Kd of 0.17 μM.

[0112] Example 4

[0113] This example is used to verify that the polypeptide can inhibit platelet function in mice.

[0114] 1. Experimental methods:

[0115] Sixteen-week-old C57BL / 6J mice and Pear1 knockout mice were prepared. For the negative control group, C57BL / 6J mice were intravenously injected with 0.05 ml / 10 g of saline solution. For the experimental group, C57BL / 6J mice were intraorbitally injected with 1 mg / kg of peptide 1 (Figure 1) at 0.05 ml / 10 g. For the positive control group, Pear1 knockout mice were intravenously injected with 0.05 ml / 10 g of saline solution. After 30 minutes, ADP (250 mg / kg) was intraorbitally injected at 0.05 ml / 10 g. The mice were observed for survival within 10 minutes.

[0116] 2. Experimental results:

[0117] Mice in the negative control group rapidly developed tachypnea and dyspnea, with 90% (9 / 10) of the mice dying within 10 minutes, leaving a survival rate of 10%. Peptide 1 in Figure 1 increased the survival rate of mice with ADP-induced pulmonary embolism to 75% (3 / 4) 30 minutes after intravenous administration, while the survival rate of Pear1 knockout mice in the positive control group was 75% (9 / 12).

[0118] Example 5

[0119] 1. Experimental instruments and reagents

[0120] Experimental instruments: 1 / 10,000 electronic balance; vertical reactor (20*25mm, No. 1 sand core); centrifuge tube; 100-1000μl pipette; 1ml pipette; nitrogen; circulating water vacuum pump; washing bottle; test tube; long-necked pipette;

[0121] Experimental reagents: 2-Cl resin; Fmoc-AA-OH; anhydrous DCM; DIEA; methanol; DCM; technical-grade DMF; analytical-grade DMF; 20% piperidine / DMF; detection reagents A (5 g ninhydrin-100 ml anhydrous ethanol) and B (analytical-grade pyridine); HOBT; DIC;

[0122] 2. Experimental steps

[0123] (1) Weigh 500 mg of 2-Cl-Trt resin using an electronic balance, place it in a 25 ml reactor, add DCM and soak for 30 minutes. Then wash it with DMF three times and DCM once.

[0124] (2) Weigh 0.15 mmol of Fmoc-AA-OH (C-terminal) into a centrifuge tube, add 8 ml of anhydrous DMF and 800 μl of DIEA, and shake well. Pipette the solution into the reactor from the previous step and allow to react with nitrogen bubbling for 1.5 h. After the reaction, wash the resin with DMF three times for 30 s each, then add a mixture of 0.5 ml of DIEA, 0.5 ml of methanol, and 2 ml of DCM and react for 20 min.

[0125] (3) Washing: After the liquid in the reactor is drained with a circulating water vacuum pump, industrial-grade DMF is added to the reactor with a wash bottle to completely immerse the resin in the solution. Wash for 30 seconds, and then drain the liquid in the reactor with a circulating water vacuum pump. Repeat this operation 4 times.

[0126] (4) Fmoc removal: Add 20% piperidine / DMF solution to the reactor using a wash bottle. The volume of the reagent should be about 3 times the volume of the resin. The resin should be completely soaked in the solution. Nitrogen bubbling should be used for the reaction for 20 minutes.

[0127] (5) Washing: Follow step (3), and replace the industrial-grade DMF with analytical-grade DMF during the fifth washing process;

[0128] (6) Resin detection: Use a long-necked pipette to take 10-20 resins from the reactor and place them at the bottom of the test tube. Then use a rubber-tipped dropper to take two drops of detection reagents A and B and drop them into the test tube to ensure that the resin and the detection reagents are in full contact. Then place the test tube in a constant temperature of 100°C and heat for 2 minutes.

[0129] (7) Condensation: Weigh 0.5 mmol Fmoc-AA-OH (second position from the C-terminus) and 0.5 mmol HOBT (0.075 g) into a centrifuge tube and dissolve them thoroughly with 1 ml DMF. Then add 100 μl DIC and mix for 1 min. Add the mixture to the dried resin and allow to react with nitrogen bubbling for 1 h.

[0130] (8) Repeat steps (5) to (7) until the peptide connection is completed.

[0131] (9) Finally, Fmoc was removed and then washed. The resin was then washed three times with methanol according to the washing method. Finally, the liquid in the reactor was drained with a circulating water vacuum pump and vacuum dried until the resin was in granular form.

[0132] Example 6

[0133] 1. Experimental methods:

[0134] Sixteen-week-old C57BL / 6J mice were anesthetized with 20% urethane and the abdominal aorta was extracted using 3.8% sodium citrate anticoagulant (anticoagulant:blood = 1:9). The upper plasma layer was centrifuged at 90g for 9 minutes and then centrifuged at 150g for 15 minutes. The supernatant was discarded and washed platelets were added to the platelet buffer. The platelet count in the washed platelet was adjusted to 3-4 × 10 8 / mL. 240 μL of washed platelets were removed, and 5 μL of normal saline was added to the control group, while 5 μL of peptide (final concentration 1 mg / mL) was added to the experimental group. The plates were incubated at 37°C for 5 minutes. After the plates were placed in a magnetic rotor, 5 μL of ADP solution (01905, Sigma: Darmstadt, Germany; final concentration 50 μM) was added. Platelet aggregation was recorded over 5 minutes using a platelet aggregometer (Helena AggRAM). The maximum platelet aggregation rates of the experimental and control groups were compared.

[0135] 2. Experimental results:

[0136] The results are shown in Figure 3. Figure 3 shows the platelet inhibition effects of different polypeptide sequences with the structure represented by Formula (II). As shown in the figure, the results for the 1', 2', 3', 4', and 5' peptides revealed that KVIYY is the sequence that maintains the peptide's antiplatelet activity. To further confirm the extension of the front and back amino acids, constructs were made at 6', 7', 8', and 9'. The results showed that the peptide's antiplatelet activity gradually decreased with amino acid extension, with the back end extending up to four amino acids, KDGE. Constructions at 10', 11', 12', 13', and 14' revealed that the front end extension abolished the peptide's antiplatelet activity. To confirm amino acid variability, amino acids were substituted at different positions to form peptides at 15', 16', and 17'. These substitutions abolished the peptide's antiplatelet activity. Therefore, based on these experimental results, the peptide with the following formula is considered active: K+V+I+Y+Y+(K)+(D)+(G)+(E) Formula (IV).

[0137] Among them, "+" represents a covalent bond, and "()" indicates that the corresponding amino acid residue in the brackets is an optional amino acid residue, that is, it may exist or not.

[0138] Example 7

[0139] This example is used to test whether the polypeptide can bind to the PEAR1 protein. The following uses polypeptide 1' in Figure 3 as an example for verification.

[0140] 1. Experimental methods:

[0141] Recombinant human PEAR1 protein was immobilized on a Cytiva CM5 chip using surface plasmon resonance (SPR) using an amino-coupling reagent. Peptide 1' (Figure 3) was serially diluted with PBS-P. The protein solution was then passed through the chip at a flow rate of 30 μL / min for 120 seconds, followed by dissociation for 480 seconds. The chip surface was regenerated with glycine hydrochloride (pH 2.0), and kinetic data were generated using a 1:1 binding model in the Biaevaluation program.

[0142] 2. Experimental results:

[0143] As shown in FIG4 , the polypeptide 1′ in FIG3 can bind to the PEAR1 protein with a binding constant Kd of 0.31 μM.

[0144] Example 8

[0145] This example is used to verify that the polypeptide can inhibit platelet function in mice.

[0146] 1. Experimental methods:

[0147] Sixteen-week-old C57BL / 6J mice and Pear1 knockout mice were prepared. For the negative control group, C57BL / 6J mice were intravenously injected with 0.05 ml / 10 g of saline. For the experimental group, C57BL / 6J mice were intraorbitally injected with 1 mg / kg of peptide 1' (Figure 3) at 0.05 ml / 10 g. For the positive control group, Pear1 knockout mice were intravenously injected with 0.05 ml / 10 g of saline. After 30 minutes, ADP (250 mg / kg) was intraorbitally injected at 0.05 ml / 10 g. The mice were observed for survival within 10 minutes.

[0148] 2. Experimental results:

[0149] Mice in the negative control group rapidly developed shortness of breath and dyspnea, with 90% (9 / 10) of the mice dying within 10 minutes, leaving a survival rate of 10%. Intravenous administration of polypeptide 1' in Figure 3 increased the survival rate of mice with ADP-induced pulmonary embolism to 60% (6 / 10) 30 minutes after administration, while the survival rate of Pear1 knockout mice in the positive control group was 75% (9 / 12).

[0150] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications and variations may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims is to be given the broadest interpretation so as to encompass all such modifications and equivalent structures and functions.

Claims

1. A polypeptide or an active fragment thereof, characterized in that: It can target and bind to the PEAR1 receptor and inhibit platelet aggregation.

2. The polypeptide or active fragment thereof according to claim 1, characterized in that: It includes a polypeptide selected from any one of the following (1)-(3): (1) A polypeptide having a structure represented by the following formula (I) and / or (II): V1-LKYX1YENHX2-V2 Formula (I), Wherein, X1 represents W or H, X2 represents A or N; V1 does not exist or represents the first variable region, V2 does not exist or represents the second variable region; KVIYY-V3 formula (II); Wherein, V3 represents the third variable region of 0-4 amino acids in length; (2) a polypeptide having 80%, preferably 85%, and more preferably 90% homology with the polypeptide of (1) and having the same function; (3) A polypeptide having the same function as the polypeptide described in (1) or (2) by substitution and / or deletion and / or addition of one or more amino acid residues.

3. The polypeptide or active fragment thereof according to claim 2, characterized in that: The first variable region is selected from A, EA or GEA, and the second variable region is selected from I, IS or ISI.

4. The polypeptide or active fragment thereof according to claim 2, characterized in that: The third variable region is selected from K, KD, KDG or KDGE.

5. A nucleic acid molecule, characterized in that The method comprises a nucleotide sequence encoding a polypeptide according to any one of claims 1 to 4 or an active fragment thereof.

6. A carrier molecule, characterized in that It comprises the nucleic acid molecule according to claim 5.

7. A host cell, characterized in that It comprises the nucleic acid molecule according to claim 5 or the vector molecule according to claim 6.

8. The method for preparing the polypeptide according to any one of claims 1 to 4, characterized in that: Prepared by artificial synthesis or genetic engineering.

9. A pharmaceutical composition, characterized in that It comprises the polypeptide or active fragment thereof according to any one of claims 1 to 4 and / or the nucleic acid molecule according to claim 5, and a pharmaceutically acceptable carrier.

10. A method for preventing, treating or ameliorating embolic diseases, characterized in that: The method comprises administering to a subject in need thereof a therapeutically effective amount of the polypeptide or active fragment thereof according to any one of claims 1 to 4, or the nucleic acid molecule according to claim 5, or the pharmaceutical composition according to claim 9.

11. The method for preventing, treating or ameliorating embolic diseases according to claim 10, characterized in that: Further included are the steps of combining with other drugs or therapeutic therapies.

12. Use of a polypeptide or its active fragment or its nucleic acid molecule in the preparation of an antiplatelet aggregation drug, characterized in that: The polypeptide or active fragment thereof is the polypeptide or active fragment thereof according to any one of claims 1 to 4, and the nucleic acid molecule is the nucleic acid molecule according to claim 5.

13. Use of a polypeptide or its active fragment or its nucleic acid molecule in the preparation of a drug for preventing, treating or ameliorating embolic diseases, characterized in that: The polypeptide or active fragment thereof is the polypeptide or active fragment thereof according to any one of claims 1 to 4, and the nucleic acid molecule is the nucleic acid molecule according to claim 5.

14. A method for inhibiting platelet aggregation in vitro, characterized in that: The method comprises the step of contacting the polypeptide or active fragment thereof according to any one of claims 1 to 4 with platelets.

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