Earthworm anti-platelet oligopeptide and application thereof

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to an earthworm anti-platelet oligopeptide and application thereof. The present application is an anti-platelet oligopeptide discovered by combining a Pheretima aspergillum (E. Perrier) transcriptome database with active-oriented separation. The oligopeptide can not only inhibit the activation and aggregation of platelets, but also effectively inhibit the adhesion and expansion of platelets, and can effectively inhibit various functions of platelets at an in-vitro level. In summary, the oligopeptide of the present application can significantly inhibit platelets, and is suitable for anti-thrombus drug research and development.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an oligopeptide for preventing platelet aggregation and application thereof. BACKGROUND

[0002] Cardiovascular diseases, including major cardiovascular events caused by thrombosis, such as myocardial infarction, ischemic stroke and pulmonary embolism, are one of the main causes of human death or disability. Platelets play a key role in hemostasis and thrombosis, and targeting platelets is essential for the treatment of cardiovascular diseases, including acute coronary syndrome, chronic coronary artery disease, and cerebrovascular and peripheral arterial diseases.

[0003] Pheretima aspergillum (E. Perrier) is one of the origins of the traditional Chinese medicine Pheretima, and contains a large amount of antithrombotic active ingredients such as eel kinase, which is widely used in antithrombotic therapy. Existing studies have shown that Pheretima aspergillum extract has anti-platelet activity, but no anti-platelet oligopeptide derived from Pheretima aspergillum has been found. SUMMARY

[0004] The purpose of the present application is to provide a novel oligopeptide for inhibiting platelet function.

[0005] TECHNICAL SOLUTION

[0006] An oligopeptide, characterized in that the oligopeptide is any one of the following amino acid sequences:

[0007] P1: AGDGLKRVGGADDALDK

[0008] P6: QQSQP.

[0009] A pharmaceutical composition, characterized in that it contains the oligopeptide and pharmaceutically acceptable excipients.

[0010] The oligopeptide or the pharmaceutical composition for use in the preparation of an anti-platelet or anti-thrombotic drug.

[0011] The oligopeptide or the pharmaceutical composition of claim 2 for use in the preparation of a product for improving microcirculation.

[0012] Specifically:

[0013] A series of anti-platelet oligopeptides are obtained by constructing a theoretical protein database from the transcriptome database of Pheretima aspergillum (E. Perrier) and combining active-oriented separation methods;

[0014] The oligopeptide has anti-platelet activity and is valuable for preventing and treating thrombotic diseases. The oligopeptide is derived from the transcriptome information of Pheretima aspergillum (E. Perrier) and is named P1-P7.

[0015] P1 and P6 with stronger anti-platelet activity are obtained through preliminary screening.

[0016] The amino acid sequences of P1-P7 provided by the application are as follows:

[0017]

[0018]

[0019] Beneficial effects

[0020] The oligopeptides P1 and P6 provided by the application have an amino acid sequence that has not been reported in the literature and belong to a brand-new sequence, and are tested to have a significant inhibitory effect on collagen-induced platelet aggregation in a dose-dependent manner. The oligopeptides P1 and P6 provided by the application are tested to inhibit the adhesion and spreading of platelets to collagen and fibrinogen. The oligopeptides P1 and P6 provided by the application are tested to inhibit collagen-induced platelet activation.

[0021] In summary, the oligopeptides provided by the application have advantages in anti-platelet activation, adhesion, spreading and aggregation. Therefore, the oligopeptides have a wide application prospect in products for improving microcirculation and anti-platelet drugs. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is the discovery of the active component of the water extract of P. coreana; wherein A is the liquid chromatogram of the <3KD component of the water extract of P. coreana; B-E are the effects of F1-F3 components on collagen or ADP-induced platelet aggregation, wherein the F2 component is the key component of the <3KD water extract of P. coreana for inhibiting platelet aggregation; F is the liquid chromatogram of the F2 component; G-J are the effects of F2.1-F2.5 components on collagen or ADP-induced platelet aggregation;

[0023] Figure 2 is the theoretical protein database combined with LC-MS analysis diagram of component F2.5, wherein A-G are the theoretical protein database combined with LC-MS analysis diagrams of P1-P7.

[0024] Figure 3 is the result diagram of the effects of oligopeptides P1-P7 on platelet aggregation, A is collagen induction, and B is ADP induction.

[0025] Figure 4 is the result diagram of the effects of oligopeptides P1 and P6 on platelet aggregation, A-D are collagen induction, and E-H are ADP induction.

[0026] Figure 5Figure is the result chart of the influence of oligopeptide P1 and P6 on platelet adhesion and spreading; A is the result of fluorescence microscope detection; B is the spreading area of platelet on collagen; C is the adhesion quantity of platelet on collagen; D is the result of laser confocal detection; F is the spreading area of platelet on fibrinogen; G is the adhesion quantity of platelet on fibrinogen.

[0027] Figure 6 Figure is the result chart of the influence of oligopeptide P1 and P6 on platelet activation; A is the result of flow cytometry detection, B is the statistical result. DETAILED DESCRIPTION

[0028] Oligopeptides P1-P7 were synthesized by Hangzhou Baiyike Biological Technology Co., Ltd.

[0029] Example 1 Discovery of active components of water extract of P. coretonotus

[0030] Ultrafiltration was used to separate the <3kD fraction (PAW <3KD) of the water extract of *Pheretima aspergillum*. Subsequently, the <3KD fractions were separated using a TSKgelAminde-80 HR liquid chromatography column to obtain fractions F1 (0-25 min), F2 (25-50 min), and F3 (50-75 min). The liquid chromatography conditions were: A: pure water and B: acetonitrile; 0-60 min: 95-60% B; 60-75 min: 60% B. F1, F2, and F3 were then lyophilized and concentrated, and diluted with water to a crude drug concentration of 1 g / mL. F1, F2, and F3 were added to platelet-rich plasma (PRP) at a final concentration of 5 mg / mL, and the effects of the three fractions on platelet aggregation were detected using a platelet aggregation analyzer (Helena Laboratories, USA). The specific method is as follows: Blood was collected from the abdominal aorta of rats using a 3.8% sodium citrate vacuum blood collection tube. The plasma was centrifuged at 70×g for 15 min to collect the upper layer of platelet-rich plasma (PRP). The remaining plasma was centrifuged at 700×g for 15 min to collect the upper layer of platelet-poor plasma (PPP). The PRP was then centrifuged again at 70×g for 10 min to remove as many residual white blood cells and red blood cells as possible. The supernatants were combined and immediately used for platelet aggregation experiments. After preheating the platelet aggregator to 37℃, several channels were first tested with double-distilled water, and then the instrument was zeroed using PPP. The PRP was incubated with F1, F2, and F3 (final concentration 5 mg / mL) at 37℃ for 5 min. Then, collagen or 50 μM ADP at a final concentration of 10 μg / mL was added to induce platelet aggregation. The stirring speed was set to 600 rpm. The degree of platelet aggregation was recorded by the platelet aggregator over a period of 5 min, and the maximum aggregation degree of platelets during the aggregation process was also recorded. The F2 fraction, which showed the best inhibitory effect on platelet aggregation, was further separated into F2.1-F2.5. After lyophilization and concentration, a stock solution with a crude drug concentration of 1 g / mL was prepared. The stock solution was then incubated with PRP at 37°C for 5 min at a final concentration of 5 mg / mL. Finally, collagen or 50 μMADP at a final concentration of 10 μg / mL was added to induce platelet aggregation, and the maximum aggregation degree of platelets during the aggregation process was recorded.

[0031] Results: The aqueous extract of *Pheretima aspergillum* with a concentration <3KD underwent fractional processing and analysis. Figure 1 As shown in Figure A, F1-F3 components were obtained; further verification was conducted on the effects of F1-F3 on collagen- or ADP-induced platelet aggregation. Figure 1 BE analysis showed that fraction F2 was a key component of *Pheretima aspergillum* water extract (<3KD) that inhibited platelet aggregation; to further analyze the composition of F2, fraction F2 was further separated, and the results are shown in […]. Figure 1 F; The effects of components F2.1-F2.5 on collagen- or ADP-induced platelet aggregation will be further investigated, and the results are shown in [the table below]. Figure 1 GJ, in which the F2.5 component is the key component of the F2 component that inhibits platelet aggregation.

[0032] In summary, F2.5 is the key component of P. coretonum water extract for inhibiting platelet aggregation.

[0033] Example 2: P. coretonum theoretical protein database combined with LC-MS to analyze F2.5 component

[0034] The P. coretonum transcriptome data was obtained from the SRA database ( https: / / www.ncbi.nlm.nih.gov / sra ), assembled using Trinity (Ver. 2.13.2), then translated using Transdecoder (Ver. 5.5.0), and finally redundant sequences were removed using CD-Hit (Ver. 4.8.1) to obtain the P. coretonum theoretical protein database.

[0035] Agilent 6530 QTOF mass spectrometer was used, ESI ion source, positive ion scanning mode, first mass spectrum scanning range m / z 100-3000; capillary voltage 4000V, dryer flow rate 10.0L / min, dryer temperature 320℃, atomization pressure 35psi, collision voltage 10-50V. Data processing was performed using pFind (Ver. 3.2.1) software. The P. coretonum theoretical protein database and mass spectrometry data were imported into pFind to obtain the sequence information of the F2.5 component.

[0036] Results: As shown in Figure 2 , a total of 7 sequences were obtained from the F2.5 component, named P1-P7. The amino acid sequence information is as follows:

[0037] Table 1: Amino acid sequences of P1-P7

[0038]

[0039] Example 3: Effect of oligopeptides P1-P7 on inhibiting platelet aggregation

[0040] The amino acid sequences P1-P7 obtained in Example 2 were entrusted to Nanjing Kingsrui Biological Technology Co., Ltd. for synthesis. The oligopeptides P1-P7 were pre-incubated at 37℃ for 5 min at 10μL 500μM and 180μL PRP, the baseline was adjusted, and then 10μg / mL collagen or 50μM ADP was added to induce platelet aggregation. The degree of platelet aggregation was recorded by a platelet aggregometer, and the recording time was 5 min, and the maximum aggregation degree of platelets during aggregation was recorded.

[0041] Results: As shown in Figure 3 , A-B is the effect of oligopeptides P1-P7 on the degree of collagen or ADP-induced platelet aggregation, and it was found that oligopeptides P1 and P6 have strong platelet aggregation inhibitory effect, and other oligopeptides have weak platelet aggregation inhibitory effect.

[0042] Example 4 Inhibitory effect of oligopeptides P1 and P6 on platelet aggregation

[0043] Further analysis of the dose-effect relationship of oligopeptides P1 and P6 on platelet aggregation:

[0044] Take 180 μL PRP into the aggregation tube, add 10 μL of 125 μM, 250 μM and 500 μM oligopeptides P1 or P6 respectively, pre-incubate at 37°C for 5 min, adjust the baseline, and then add collagen at a final concentration of 10 μg / mL or ADP at a final concentration of 50 μM to induce platelet aggregation. The stirring speed is set at 600 rpm. The degree of platelet aggregation is recorded by the platelet aggregometer, and the recording time is 5 min. The maximum degree of platelet aggregation during the aggregation process is recorded.

[0045] Results: Figure 4 A-D show that oligopeptides P1 and P6 at concentrations of 250 μM and 500 μM have significant inhibitory effects on collagen-induced platelet aggregation (P < 0.05). And with the increase of the concentration of oligopeptides, the inhibitory effect on platelet aggregation gradually increases. Figure 4 E-H show that oligopeptides P1 and P6 need to reach 500 μM to have a significant inhibitory effect on ADP-induced platelet aggregation.

[0046] Example 5 Inhibitory effect of oligopeptides P1 and P6 on platelet adhesion and spreading

[0047] Place a cover glass in a 24-well plate, coat with 250 μL of 5 μg / mL collagen or 50 μg / mL fibrinogen solution at 4°C overnight. Remove the coating solution and wash three times with pre-cooled PBS. Fresh PRP is centrifuged at 700 x g for 10 min, the supernatant is removed, and then washed three times with Ca 2+ free Tyrode's-HEPES buffer, and then resuspended in Tyrode's-HEPES buffer to wash the platelets. The platelet concentration is 2 x 10 7Subsequently, 10 μL of 250 μM oligopeptide P1 or P6 was added to 180 μL of washed platelets at 37 °C for 10 min, and the <3KD water extract of P. guillenii (PAW <3KD) was used as a positive control. Then, 10 μL of 20 mM CaCl2 solution was added to the 24-well plate coated with collagen or fibrinogen, and incubated at 37 °C for 1 h. The adherent platelets were washed three times with PBS. The adherent platelets were fixed with 4% (w / v) paraformaldehyde for 10 min and permeabilized with 0.1% (w / v) Triton X-100 for 5 min. After washing three times with PBS, the platelets were stained with FITC-labeled phalloidin (final concentration of 200 nM) for 30 min in the dark. After washing with PBS, the platelets were mounted with an anti-fluorescence quenching mounting medium. The platelets were observed under a fluorescence microscope, and 8 random fields were photographed. Image J software was used for quantification.

[0048] Results: During the thrombus formation process, platelet aggregation can be divided into three stages: platelet adhesion, spreading, and stabilization. Platelet adhesion refers to the process in which, after the endothelial cells are damaged, circulating platelets adhere to the wound site through the binding of GPVI-IX-V to the VWF-collagen complex. The adhesion process initiates the spreading process of platelets, and the conformation of αIIbβ3 on the platelets changes, thereby enhancing the affinity for fibrinogen. This process leads to rapid platelet aggregation.

[0049] Figure 5 A-C show that, compared with the control group, oligopeptides P1 and P6 can significantly reduce the number of platelets adhering to collagen and the spreading area of platelets on collagen. Figure 5 D-F show that, compared with the control group, the number of platelets adhering to fibrinogen and the spreading area of platelets on fibrinogen are significantly reduced after treatment with oligopeptides P1 and P6.

[0050] Example 6: Effect of oligopeptides P1 and P6 on inhibiting platelet activation

[0051] The concentration of washed platelets was adjusted to 2×10 7 The concentration of washed platelets was adjusted to 2×10

[0052] Results: CD62P, also known as P-selectin, is a cell adhesion molecule. CD62P is mainly expressed on the surface of activated platelets and endothelial cells, and plays a key role in the process of inflammation and thrombosis. If CD62P increases after platelet activation, this embodiment uses PE-conjugated CD62P antibody, which binds to CD62P, reflecting the degree of platelet activation.

[0053] Figure 6 A-B shows that compared with the control group, the fluorescence intensity of CD62P after the platelets are treated with oligopeptides P1 and P6 is significantly reduced, indicating that oligopeptides P1 and P6 significantly inhibit collagen-induced platelet activation.

Claims

1. An oligopeptide, characterized in that, The amino acid sequence of the oligopeptide is shown in SEQ ID NO:

1.

2. A pharmaceutical composition, characterized in that... Contains the oligopeptide of claim 1 and pharmaceutically acceptable excipients.

3. The use of the oligopeptide according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of an antiplatelet drug.

4. The use of the oligopeptide according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of an antithrombotic drug.

5. The application of an oligopeptide in the preparation of an antiplatelet drug, characterized in that, The amino acid sequence of the oligopeptide is shown in SEQ ID NO:

6.

6. The application of an oligopeptide in the preparation of an antithrombotic drug, characterized in that, The amino acid sequence of the oligopeptide is shown in SEQ ID NO:6.

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