Lumbricus antiplatelet oligopeptide and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to earthworm antiplatelet oligopeptide and application thereof. The invention relates to an anti-platelet oligopeptide, which is found by combining a transcriptome database of pheretima aspericum (E. Perriver) with activity-directed separation, and belongs to the technical field of anti-platelet oligopeptide and application of the anti-platelet oligopeptide in preparation of anti-platelet oligopeptide. The oligopeptide not only can inhibit activation and aggregation of platelets, but also can effectively inhibit adhesion and expansion of the platelets, and can effectively inhibit multiple functions of the platelets on the in-vitro level. In conclusion, the oligopeptide can significantly inhibit platelets, and is suitable for research and development of antithrombotic drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to earthworm antiplatelet oligopeptide and application thereof. Background Art

[0002] Cardiovascular disease, including major cardiovascular events caused by thrombosis, such as myocardial infarction, ischemic stroke, and pulmonary embolism, is one of the leading causes of death or disability in humans. 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 disease.

[0003] Pheretima sibiricum is one of the sources of the traditional Chinese medicine earthworm. It contains a large amount of antithrombotic active ingredients such as lumbrokinase, which are widely used in antithrombotic therapy. Existing studies have shown that Pheretima sibiricum extract has antiplatelet activity, but no antiplatelet oligopeptides derived from Pheretima sibiricum have been found. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a new oligopeptide that inhibits 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 by containing the oligopeptide and pharmaceutically acceptable excipients.

[0010] Use of the oligopeptide or the pharmaceutical composition in the preparation of antiplatelet or antithrombotic drugs.

[0011] Use of the oligopeptide or the pharmaceutical composition according to claim 2 in the preparation of a product for improving microcirculation.

[0012] Specifically:

[0013] A theoretical protein database was constructed using the transcriptome database of Pheretima aspergillum (E. Perrier) and a series of antiplatelet oligopeptides were obtained by combining the activity-guided separation method.

[0014] The oligopeptide has antiplatelet 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] Preliminary screening revealed P1 and P6 with stronger antiplatelet activity.

[0016] The amino acid sequences of P1-P7 provided by the present invention are:

[0017]

[0018]

[0019] Beneficial Effects

[0020] The oligopeptides P1 and P6 provided by the present invention have amino acid sequences that have not been reported in the literature and are completely new sequences. They have been tested to have a significant inhibitory effect on collagen-induced platelet aggregation, and are dose-dependent. The oligopeptides P1 and P6 provided by the present invention have been tested to inhibit the adhesion and expansion of platelets on collagen and fibrinogen. The oligopeptides P1 and P6 provided by the present invention have been tested to inhibit collagen-induced platelet activation.

[0021] In summary, the oligopeptide provided by the present invention has advantages in anti-platelet activation, adhesion, expansion and aggregation. Therefore, the oligopeptide has broad application prospects in products for improving microcirculation and anti-platelet drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the discovery of the active components of the water extract of Pheretima cyclophaga; A is the liquid chromatogram of the components of the water extract of Pheretima cyclophaga <3KD; BE is the effect of components F1-F3 on platelet aggregation induced by collagen or ADP, wherein component F2 is the key component of the water extract of Pheretima cyclophaga <3KD that inhibits platelet aggregation; F is the liquid chromatogram of component F2; GJ is the effect of components F2.1-F2.5 on platelet aggregation induced by collagen or ADP;

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

[0024] Figure 3 These are the results of the effects of oligopeptides P1-P7 on platelet aggregation, A is collagen-induced, and B is ADP-induced.

[0025] Figure 4 These are the results of the effects of oligopeptides P1 and P6 on platelet aggregation. AD is collagen-induced, and EH is ADP-induced.

[0026] Figure 5The figure shows the effect of oligopeptides P1 and P6 on platelet adhesion and expansion; A is the result of fluorescence microscopy; B is the expansion area of ​​platelets on collagen; C is the number of platelets adhered to collagen; D is the result of laser confocal microscopy; F is the expansion area of ​​platelets on fibrinogen; G is the number of platelets adhered to fibrinogen.

[0027] Figure 6 The results of the effects of oligopeptides P1 and P6 on platelet activation are shown in Figure 1; A is the flow cytometry test result, and B is the statistical result. DETAILED DESCRIPTION

[0028] Oligopeptides P1-P7 were commissioned to Hangzhou Baiyi Biotechnology Co., Ltd. for synthesis.

[0029] Example 1 Discovery of active components of Pheretima serrata aqueous extract

[0030] The <3kd fraction (PAW<3KD) of water extract of Pheretima striata was obtained by ultrafiltration separation, and then PAW<3KD was separated by liquid column TSKgelAminde-80 HR to obtain F1 (0-25min), F2 (25-50min), and F3 (50-75min) fractions. The liquid phase conditions were A: pure water and B: acetonitrile, 0-60min 95-60% B, 60-75min 60% B. Then, F1, F2, and F3 were freeze-dried and concentrated respectively and diluted with water to a crude drug amount of 1g / mL. F1, F2, and F3 were added to platelet-rich plasma (PRP) at a final concentration of 5mg / mL, and the effects of the three components on platelet aggregation were detected by platelet aggregation instrument (Helena Laboratories, USA). The specific method is as follows: 3.8% sodium citrate vacuum blood collection tubes were used to collect blood from the abdominal aorta of rats, and the upper platelet-rich plasma (PRP) was collected by centrifugation at 70×g for 15 minutes. The remaining plasma was centrifuged at 700×g for 15 minutes, and the upper platelet-poor plasma (PPP) was collected. PRP was centrifuged at 70×g for 10 minutes to remove residual white blood cells and red blood cells as much as possible, and the supernatant was combined and immediately subjected to platelet aggregation experiment. After the platelet aggregation instrument was preheated to 37°C, the optical path of several channels was first detected with double distilled water, and then the instrument was zeroed with PPP. After PRP was incubated with F1, F2, and F3 (final concentration 5 mg / mL) at 37°C for 5 minutes, a final concentration of 10 μg / mL collagen or 50 μM ADP was added to induce platelet aggregation. The stirring speed was set to 600 rpm. The degree of platelet aggregation was recorded by the platelet aggregation instrument for 5 minutes, and the maximum aggregation degree of platelets during the aggregation process was recorded. The F2 component with the best platelet aggregation inhibition effect was further separated into F2.1-F2.5, freeze-dried and concentrated to form a mother solution with a crude drug amount of 1 g / mL, and incubated with PRP at a final concentration of 5 mg / mL at 37°C for 5 minutes. After that, collagen with a final concentration of 10 μg / mL or 50 μM ADP was added to induce platelet aggregation, and the maximum aggregation degree of platelets during the aggregation process was recorded.

[0031] Results: The results of segmented analysis of water extract of Pheretima quinata<3KD were as follows: Figure 1 As shown in A, the F1-F3 components were obtained; the effects of F1-F3 on collagen or ADP-induced platelet aggregation were further verified. Figure 1 BE showed that the F2 component was the key component of the water extract of Pheretima cyclophaga <3KD that inhibited platelet aggregation; in order to analyze the composition of F2, the component F2 was further separated, and the results were shown in Figure 1 F; The obtained components F2.1-F2.5 were further studied for their effects on collagen or ADP-induced platelet aggregation. The results are shown in Figure 1 GJ, among which F2.5 component is the key component of F2 component to inhibit platelet aggregation.

[0032] In summary, F2.5 is the key component of the water extract of Pheretima cuniculus <3KD that inhibits platelet aggregation.

[0033] Example 2 Analysis of F2.5 component using the theoretical protein database of Pheretima sphaerocephala combined with LC-MS

[0034] From the SRA database ( https: / / www.ncbi.nlm.nih.gov / sra ) were used to obtain the transcriptome data of Pheretima pubescens, assembled using Trinity (Ver.2.13.2), translated using Transdecoder (Ver.5.5.0), and finally CD-Hit (Ver.4.8.1) was used to eliminate redundancy and obtain the theoretical protein database of Pheretima pubescens.

[0035] Agilent 6530QTOF mass spectrometer was used, ESI ion source, positive ion scan mode, primary mass spectrometry scan range m / z100-3000; capillary voltage was 4000V, dryer flow rate was 10.0L / min, dryer temperature was 320℃, atomization pressure was 35psi, collision voltage was 10-50V. Data processing was performed using pFind (Ver.3.2.1) software. The theoretical protein database of Pheretima annuli and mass spectrometry data were imported into pFind to obtain the sequence information of F2.5 component.

[0036] Results: Figure 2 As shown, 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 sequence P1-P7 obtained in Example 2 was commissioned to Nanjing GenScript Biotechnology Co., Ltd. for synthesis. The oligopeptides P1-P7 were pre-incubated at 37°C for 5 min at 10 μL of 500 μM and 180 μL of PRP, and the baseline was adjusted. After the baseline was stable, collagen with a final concentration of 10 μg / mL or 50 μM ADP was added to induce platelet aggregation. The platelet aggregation degree was recorded by a platelet aggregometer for 5 min, and the maximum aggregation degree of the platelets during the aggregation process was recorded.

[0041] Results: Figure 3 As shown, AB is the effect of oligopeptides P1-P7 on the platelet aggregation induced by collagen or ADP. The results show that oligopeptides P1 and P6 have strong platelet aggregation inhibitory effects, while other oligopeptides have weak inhibitory effects on platelet aggregation.

[0042] Example 4 Inhibitory effects 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] 180 μL PRP was added to the aggregation tube, and 10 μL 125 μM, 250 μM and 500 μM oligopeptide P1 or P6 were added respectively. The tube was pre-incubated at 37 °C for 5 min, the baseline was adjusted, and after the baseline was stable, a final concentration of 10 μg / mL collagen or 50 μM ADP was added to induce platelet aggregation. The stirring speed was set to 600 rpm. The platelet aggregation degree was recorded by a platelet aggregation instrument for 5 min, and the maximum aggregation degree of platelets during the aggregation process was recorded.

[0045] result: Figure 4 AD showed that 250μM and 500μM oligopeptides P1 and P6 had significant inhibitory effects on collagen-induced platelet aggregation (P<0.05), and with the increase of oligopeptide concentration, the inhibitory effect on platelet aggregation gradually increased. Figure 4 EH showed that the concentration of oligopeptides P1 and P6 needed to reach 500 μM to have a significant inhibitory effect on ADP-induced platelet aggregation.

[0046] Example 5 Effects of oligopeptides P1 and P6 on inhibiting platelet adhesion and expansion

[0047] Place the coverslip in a 24-well plate and coat with 250 μL of 5 μg / mL collagen or 50 μg / mL fibrinogen solution at 4°C overnight. Aspirate the coating solution and wash three times with pre-cooled PBS. Centrifuge the fresh PRP at 700 × g for 10 min, remove the supernatant, and add Ca-free 2+ After washing three times with Tyrode's-HEPES buffer, the platelets were resuspended in Tyrode's-HEPES buffer to obtain a platelet concentration of 2 × 10 7 / mL. Subsequently, 10μL of 250μM oligopeptide P1 or P6 and 180μL washed platelets were incubated at 37℃ for 10min. In addition, P. lumbricoides aqueous extract <3KD (PAW<3KD) was used as a positive control. Then 10μL of 20mM CaCl2 solution was added and dripped into a 24-well plate coated with collagen or fibrinogen. The platelets were incubated at 37℃ for 1h and washed three times with PBS to remove the unadherent platelets. The adherent platelets were fixed with 4% (w / v) paraformaldehyde for 10min and then permeated with 0.1% (w / v) Triton X-100 for 5min. After washing three times with PBS, the platelets were stained with FITC-labeled phalloidin (final concentration of 200nM) in the dark for 30min. After washing with PBS, the platelets were sealed with anti-fluorescence quenching sealing solution. The platelets were observed under a fluorescence microscope, and 8 fields of view were randomly photographed and quantified using Image J software.

[0048] Results: During thrombosis, platelet aggregation can be divided into three stages: platelet adhesion, expansion, and stability. The platelet adhesion process refers to the adhesion of circulating platelets to the wound site through the combination of GPVI-IX-V and VWF-collagen complex after endothelial cells are damaged. The adhesion process initiates the platelet expansion process, and the conformation of αIIbβ3 on platelets changes, thereby enhancing the affinity with fibrinogen, and platelets aggregate rapidly in this process.

[0049] Figure 5 AC showed that compared with the control group, oligopeptides P1 and P6 could significantly reduce the number and expansion area of ​​platelet adhesion on collagen. Figure 5 DF showed that compared with the control group, the number of platelets adhering to fibrinogen was significantly reduced after treatment with oligopeptides P1 and P6, and the expansion area of ​​platelets on fibrinogen was significantly reduced.

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

[0051] Adjust the concentration of washed platelets to 2 × 10 7 / mL, added with a final concentration of 1mM CaCl2 solution, and then incubated with oligopeptide P1 or P6 with a final concentration of 10μM and washed platelets at 37°C for 10min, and earthworm water extract <3KD (PAW<3KD) was used as a positive control, and then collagen with a final concentration of 10μg / mL was used to stimulate platelet activation at 37°C, and finally incubated with PE-conjugated CD62P antibody at room temperature in the dark for 10min, and samples were collected with a CytoFLEX machine (Beckman Coulter) and analyzed using FlowJo software (Tree Star, v10.6.2).

[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 inflammatory response and thrombosis. If CD62P increases after platelet activation, this example uses PE-coupled CD62P antibody, which reflects the degree of platelet activation after binding to CD62P.

[0053] Figure 6 AB showed that compared with the control group, the fluorescence intensity of CD62P was significantly reduced after platelets were treated with oligopeptides P1 and P6, which means that oligopeptides P1 and P6 significantly inhibited collagen-induced platelet activation.

Claims

1. An oligopeptide, characterized in that The oligopeptide is any one of the following amino acid sequences: P1:AGDGLKRVGGADDALDK P6:QQSQP.

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

3. Use of the oligopeptide according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of antiplatelet or antithrombotic drugs.

4. Use of the oligopeptide according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a product for improving microcirculation.

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