Earthworm anticoagulant oligopeptide and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to earthworm anticoagulant oligopeptide and application thereof. The invention relates to an anticoagulant oligopeptide, which is found by combining the activity-directed separation of pheretima asper (E. Perriver) with the screening of an AI (artificial intelligence) prediction model. The amino acid sequence of the oligopeptide is LMWFSDRNK; the oligopeptide regulates thrombin in an allosteric manner, enhances the combination of thrombin and antithrombin, and can effectively inhibit blood coagulation and thrombus formation at in-vivo and in-vitro levels; the oligopeptide has a remarkable anticoagulant effect and is suitable for research and development of antithrombotic drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to earthworm anticoagulant oligopeptides and their applications. Background Technology

[0002] Cardiovascular diseases, including major cardiovascular events caused by thrombosis such as myocardial infarction, ischemic stroke, and pulmonary embolism, are among the leading causes of death and disability in humans. Anticoagulation therapy is an important means of preventing and treating these diseases, including deep vein thrombosis, cerebrovascular diseases, and peripheral artery diseases.

[0003] The emergence of artificial intelligence (AI), particularly machine learning and deep learning, has demonstrated remarkable potential in drug development. Its advantages include autonomously extracting features and identifying patterns from large datasets, thereby accelerating research time, reducing costs, and increasing success rates compared to traditional methods. In the context of bioactive peptide discovery, AI-driven methods can rapidly predict, screen, and optimize candidate sequences from complex protein hydrolysates or genomic data. This significantly improves the efficiency and accuracy of identifying promising therapeutic peptides from natural sources, providing a powerful complementary strategy to overcome the limitations of traditional activity-guided isolation.

[0004] Earthworm (Pheretima asiatica) Pheretima aspergillum Earthworm (Pheretima aspergillum) is one of the sources of the traditional Chinese medicine earthworm, which contains a large number of antithrombotic active ingredients such as lumbrokinase, and is widely used in antithrombotic therapy. Existing studies have shown that extracts of *Pheretima aspergillum* have anticoagulant activity; however, no anticoagulant oligopeptides derived from *Pheretima aspergillum* have been found. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a novel oligopeptide with anticoagulant function.

[0006] Technical solution: An oligopeptide, characterized in that the oligopeptide has the following amino acid sequence: LK9: LMWFSDRNK.

[0007] A pharmaceutical composition characterized by comprising the oligopeptide and pharmaceutically acceptable excipients.

[0008] The use of the oligopeptide or the pharmaceutical composition in the preparation of anticoagulant, antithrombotic, or microcirculation-improving drugs.

[0009] The use of the oligopeptide or the pharmaceutical composition in the preparation of a medicament for treating coagulation disorders, thrombosis, or microcirculatory disturbances, characterized in that the diseases are ischemic cardiovascular and cerebrovascular diseases, thromboembolic diseases, peripheral vascular ischemic diseases, diabetic vascular complications, and fundus microcirculatory disturbances.

[0010] The application is characterized in that the diseases include coronary heart disease, angina pectoris, myocardial infarction, cerebral infarction, transient ischemic attack, cerebral ischemia, arteriosclerosis, deep vein thrombosis of the lower extremities, arteriosclerosis obliterans of the lower extremities, thromboangiitis obliterans, diabetic foot, pulmonary embolism, renal artery thrombosis or embolism, retinal artery occlusion, retinal vein occlusion, atrial fibrillation with thromboembolism, post-thrombotic syndrome of deep veins, or peripheral artery thrombotic disease.

[0011] The use of the oligopeptide or the pharmaceutical composition in the preparation of drugs or cosmetics that improve skin microcirculation.

[0012] Specifically: via *Pheretima asiatica* Pheretima aspergillum An anticoagulant oligopeptide was obtained by activity-directed separation combined with an AI prediction model using *E. perrier*. This oligopeptide exhibits anticoagulant activity and has value in preventing and treating thrombotic diseases. The oligopeptide was named LK9.

[0013] The amino acid sequence of LK9 provided by this invention is LMWFSDRNK, as shown in SEQ ID NO:1.

[0014] Beneficial Effects: The oligopeptide LK9 provided by this invention has a novel amino acid sequence not reported in the literature. Testing showed that it significantly prolonged plasma PT and APTT in a dose-dependent manner. The oligopeptide LK9 provided by this invention also promoted the binding of thrombin and antithrombin. Furthermore, the oligopeptide LK9 provided by this invention inhibited thrombin-induced pulmonary microthrombus formation in mice. It also prolonged FeCl3-induced carotid artery occlusion time in mice. Finally, the oligopeptide LK9 provided by this invention showed no significant bleeding risk at doses of 5 mg / kg and 2.5 mg / kg.

[0015] In summary, the oligopeptide provided by this invention has anticoagulant activity, can intervene in thrombus formation in vivo, and poses no risk of bleeding at certain doses. Therefore, this oligopeptide has broad application prospects in products that improve microcirculation and in anticoagulant drugs. Attached Figure Description

[0016] Figure 1 The discovery of active components in the aqueous extract of *Pheretima aspergillum*; AC represents the effect of different molecular weights of *Pheretima aspergillum* on coagulation, with the <3KD aqueous extract being the key anticoagulant component; D is the liquid chromatogram of the <3KD component of the aqueous extract of *Pheretima aspergillum*; EG represents the effect of components F1-F3 on coagulation, with component F3 being the key anticoagulant component of the <3KD aqueous extract of *Pheretima aspergillum*. Figure 2This diagram illustrates the establishment of an anticoagulant peptide prediction model. A shows the machine learning model for predicting anticoagulant peptides. BD represents the results of three-fold cross-validation optimization. E represents the training accuracy (train_acc) and validation accuracy (val_acc) of the final prediction model. Figure 3 This is a validation of the anticoagulant peptide activity predicted by AI, where AC represents the predicted effect of anticoagulant peptide on plasma coagulation, DF represents the effect of different concentrations of LK9 on plasma coagulation, G represents the effect of different concentrations of LK9 on PT, H represents the effect of different concentrations of LK9 on APTT, I represents the relative activity of thrombin at 1 h to assess the effect of LK9 on promoting the binding of thrombin and antithrombin and reducing thrombin activity, J represents the formation of LK9-mediated thrombin-antithrombin complex over time, and K represents the inactivation kinetics of LK9-mediated thrombin binding to antithrombin. Figure 4 This study demonstrates how the anticoagulant peptide LK9 protects mice from thrombin-induced pulmonary embolism. Figure A shows the macroscopic morphology of lung tissue after modeling; Figure B shows a typical HE-stained section of lung tissue (black arrows indicate pulmonary thrombosis); Figure C shows a quantitative statistical graph of microthrombi in lung tissue (n=10); Figures DE show the survival analysis of the model mice (n=8); and Figures FG show the peripheral blood coagulation parameters PT and APTT of the model mice. Bars represent the mean ± standard deviation. P-values ​​(* p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001) were determined using one-way ANOVA. Figure 5 This section describes the antithrombotic effect of LK9 on a FeCl3-induced carotid artery injury model. Image A represents a representative image of FeCl3-induced carotid artery injury, images BC show the quantitative statistics of LK9's inhibitory effect on thrombus formation, and images DE show the effect of LK9 on bleeding time and amount in mice. Bars represent mean ± standard deviation. P-values ​​(* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001) were determined using one-way ANOVA. Detailed Implementation

[0017] The oligopeptide LK9 was synthesized by Hangzhou Baiyi Biotechnology Co., Ltd.

[0018] Example 1: Discovery of the active components in the water extract of *Pheretima aspergillum*: The aqueous extract of *Pheretima aspergillum* was centrifuged at 5000 × g at 20℃ using ultrafiltration centrifuge tubes with molecular weight cutoffs of 10 kDa and 3 kDa. The extract was separated into four fractions based on relative molecular mass: PA>10 kDa, PA<10 kDa, PA 3~10 kDa, and PA<3 kDa. Subsequently, the PA<3 kDa fraction was separated using a TSKgel Aminde-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 plasma at a final concentration of 5 mg / mL, and their effects on plasma coagulation were detected using an ELISA reader (Thermo Fisher Scientific, USA). The specific method was as follows: Blood was collected from the abdominal aorta of rats using a 3.8% sodium citrate vacuum blood collection tube. The plasma layer was collected after centrifugation at 700 ×g for 15 min. The plasma and sample were pre-incubated at 37°C for 10 min, and then CaCl2 (1 mM) was added, and plasma coagulation was monitored at 37°C for 15 min.

[0019] Results: The clotting time was significantly prolonged and the degree of clotting was significantly reduced in the PA <10 kDa group. Further investigation revealed that the PA <3 kDa fraction exhibited significant anticoagulant activity, comparable to that of the PA <10 kDa fraction. Figure 1 AC). The PA>10 kDa and PA 3~10 kDa fractions showed no significant anticoagulant activity. Therefore, we selected the PA<3 kDa fraction, which exhibited the strongest anticoagulant activity, for further investigation. Subsequently, the PA<3 kDa fraction was separated by preparative liquid chromatography, and three subfractions were collected based on elution time: F1 (0~25 min), F2 (25~50 min), and F3 (50~75 min). Figure 1 D). Among them, such as Figure 1 EG showed that only the F3 fraction exhibited significant anticoagulant activity, significantly prolonging clotting time and reducing plasma coagulation.

[0020] These results indicate that the F3 moiety constitutes the main anticoagulant bioactive component of *Eriocaulon ginseng*.

[0021] Example 2, Construction of an anticoagulant peptide prediction model: An anticoagulant peptide prediction model was constructed using a directed information transfer neural network (Chemprop). This model can directly predict molecular properties based on the molecular graph structure, where atoms are represented as nodes and chemical bonds as edges. For each target molecule, the corresponding molecular graph is reconstructed from its simplified molecule-input line-input system (SMILES) string. Since the target properties in this study correspond to a binary classification task (i.e., whether a molecule has anticoagulant activity), we trained the model to output a value between 0 and 1, which represents the predicted anticoagulant potential of the input molecule. The Chemprop software is open source and can be obtained at https: / / github.com / chemprop / chemprop. To complete the binary classification task, we constructed a corresponding dataset containing 115 known anticoagulant peptides and 3308 non-anticoagulant peptides. First, the complete dataset was divided into training and test sets in a 9:1 ratio. To enhance the model's generalization ability and optimize parameters, a three-fold cross-validation strategy was implemented in the training set: the training set was divided into three subsets; in each iteration, two subsets were used as sub-training sets for model training, and the remaining subset was used as a sub-validation set for evaluating model performance. After three cycles of training and evaluation, the optimal model parameters were determined by combining the results of all iterations. The optimized model was then used to predict potential anticoagulant peptides in the active site peptide library of earthworms.

[0022] Results: To effectively screen for potential anticoagulant peptides from *Eriocaulon buergerianum*, we optimized a machine learning prediction model. A training dataset was constructed by collecting 115 known anticoagulant peptides and 3308 non-anticoagulant peptides from published literature and established databases. Figure 2 A) The peptide sequence was converted into SMILES symbols and used as model input. A prediction model was built using the Chemprop deep learning framework for molecular property prediction. A three-fold cross-validation strategy was employed to optimize model performance and ensure robustness. The training process for each fold is as follows: Figure 2 As shown in Figure BD, the graph illustrates the progress of model accuracy and loss at different training periods. Figure 2 As shown in E, the final model achieved an average accuracy of 94.53%. This validated model was subsequently used to screen for potential bioactive peptides in the previously identified F3 bioactive fraction of *Eriocaulon buergerianum*. The model predicted a probability score for each peptide, reflecting its likelihood of possessing anticoagulant activity (Table 1). Based on a classification threshold of 0.5, peptides with predicted scores higher than this value were prioritized for peptide synthesis and selected for further experimental validation in subsequent stages of this study.

[0023] Table 1. Amino acid sequences of oligopeptides with an anticoagulant activity score >0.5 in component F3. .

[0024] Example 3, in vitro efficacy validation of potential anticoagulant peptides: The amino acid sequence obtained in Example 2 was synthesized by Hangzhou Baiyi Biotechnology Co., Ltd. Oligopeptides were pre-incubated in 10 μL of 500 μM and 180 μL of plasma at 37 °C for 5 min, followed by the addition of 10 μL of 20 mM CaCl2. The degree of plasma coagulation was recorded using an ELISA reader for 15 min, including the coagulation time and maximum coagulation degree. Different concentrations of LK9 were added to 50 μL of plasma and pre-incubated at 37 °C for 5 min, followed by the addition of PT or APTT detection reagents, and the PT or APTT was recorded using an ELISA reader. 50 μM LK9 was pre-incubated with 5 U of thrombin at 37 °C for 30 min, followed by the addition of 2.5 U of antithrombin and incubation at 37 °C for different times. Finally, 0.25 mM of thrombin substrate S-2238 was added, and the relative thrombin activity was recorded using an ELISA reader.

[0025] Results: Based on the prediction model, 10 peptides with a score greater than 0.5 were synthesized from the F3 fraction, and their anticoagulant activity was evaluated experimentally. Among them, LMWFSDRNK (LK9) showed the strongest anticoagulant effect. Initial screening showed that LK9 significantly prolonged clotting time and reduced plasma coagulation intensity. Figure 3 AC). This anticoagulant activity is dose-dependent; higher LK9 concentrations lead to prolonged clotting time and a more significant reduction in plasma coagulation. Figure 3 Consistent with previous findings, we further demonstrated using in vitro coagulation assays that LK9 dose-dependently prolonged PT and APTT. Figure 3 LK9 prolonged PT and APTT, indicating its inhibitory effect on both intrinsic and extrinsic coagulation pathways. We then examined whether LK9 affected the binding between thrombin and antithrombin. Compared to the thrombin-antithrombin control, pre-incubation of thrombin with LK9 followed by the addition of antithrombin resulted in a significant decrease in thrombin activity (GH). Figure 3 I). Furthermore, LK9 accelerates the binding kinetics between thrombin and antithrombin (I). Figure 3 J), thereby increasing the inactivation rate of thrombin ( Figure 3 K).

[0026] Example 4, Inhibitory effect of oligopeptide LK9 on pulmonary embolism in mice: Further analysis of the in vivo antithrombotic efficacy of oligopeptide LK9: Mice were randomly divided into 6 groups (n=8): sham-operated group (0.9% saline), model group (0.9% saline), heparin group (1000 U / kg), and low, medium, and high dose LK9 groups (2.5, 5, and 10 mg / kg, respectively). Five minutes after administration of the drug via the tail vein, mice in each group were injected via the tail vein with the modeling agents (thrombin 250 U / kg and adrenaline 50 μg / kg). The survival time of mice in each group was recorded after modeling, with 30 minutes as the humane endpoint.

[0027] Results: Compared with the sham-operated group, the model group mice showed significant pathological changes in lung tissue, confirming successful model establishment. The model group exhibited marked pulmonary congestion and hemorrhage foci compared to the sham-operated group. Figure 4 A). HE staining further revealed alveolar structure destruction, alveolar collapse, and multiple thromboembolisms in the model group. Figure 4 B, indicated by the arrow). LK9 exhibits a dose-dependent protective effect. Both macroscopic and microscopic observations showed a slight reduction in hemorrhage and structural damage in the low-dose (LK9-L) group. The medium-dose (LK9-M) group showed more significant improvement, with reduced congestion and better preservation of alveolar structure. The high-dose LK9 (LK9-H) group demonstrated the most significant protective effect, with lung morphology similar to that of sham surgery, comparable to the positive control group (Heparin). Figure 4 Consistent with these pathological observations, quantitative analysis showed that LK9 dose-dependently reduced the number of microthrombi in lung tissue (AB). Figure 4 C). Furthermore, LK9 significantly prolonged survival in a dose-dependent manner ( Figure 4 D) and improving survival rate Figure 4 E). Analysis of peripheral blood coagulation parameters in model mice confirmed the systemic anticoagulant effect of LK9. Compared with the model group, high-dose LK9 treatment significantly prolonged PT and APTT. Figure 4 (FG). In summary, these results indicate that LK9 has an effective protective effect against thrombin-induced pulmonary embolism in mice, exhibiting dose-dependent protection against pathological damage in vivo, reducing thrombus formation, improving survival outcomes, and prolonging key coagulation parameters.

[0028] Example 5: Oligopeptide LK9 delays FeCl3-induced carotid artery thrombosis in mice: Mice were randomly divided into 5 groups: control group, LK9-L (2.5 mg / kg) group, LK9-M (5 mg / kg) group, LK9-H (10 mg / kg) group, and heparin (1000 U / kg) group. Eight animals were in each group. Mice were first injected intraperitoneally with tetrabromoethanol (400 mg / kg), followed by a tail vein injection: the treatment group received the specified dose of LK9, the positive control group received heparin, and the control group received saline. After administration, the mice were fixed in a supine position on the operating table, neck hair was removed, and a skin incision was made in the carotid artery region. The carotid artery was exposed with blunt forceps, and 1×2 mm filter paper soaked in 8% (w / v) FeCl3 was applied to the carotid artery for 3 min. The carotid artery was then flushed three times with saline. Blood flow imaging was performed using a full-field laser perfusion imaging system (moorFLIP2), and the blood flow occlusion time 30 min after exposure of the carotid artery for humanitarian reasons was recorded.

[0029] Five minutes after anesthesia, mice were injected via the tail vein with LK9-L (2.5 mg / kg), LK9-M (5 mg / kg), LK9-H (10 mg / kg), and heparin (1000 U / kg). The control group received an equal volume of 0.9% saline (n=8). Ten minutes later, the distal end of the severed tail tip was removed 1.5 mm with a sharp scalpel, and the bleeding tail was then immersed in 0.9% saline at 37°C. The time to cessation of bleeding was recorded.

[0030] Results: To further validate the antithrombotic effect of LK9, we established FeCl3-induced carotid artery thrombosis. After surgical exposure of the carotid artery, blood flow was monitored in real time using a MoorFLPI 2 camera. After recording baseline blood flow, filter paper saturated with 8% FeCl3 was applied to the artery and vein for 3 min to induce endothelial injury and thrombus formation. Representative images of perfusion units (PUs) over time showed that in the control group, the blood flow leading to vascular occlusion progressively decreased, while LK9 treatment attenuated this process in a dose-dependent manner. Figure 5 A). Further analysis confirmed that, compared with the solvent control group, LK9 significantly prolonged the time to complete occlusion ( Figure 5 (BC), indicating its ability to reduce arterial thrombosis in vivo. In addition to efficacy, we also assessed the potential bleeding risk of LK9 using a tail hemorrhage detection method. Compared with the control group, low and medium doses of LK9 did not significantly prolong tail hemorrhage time or increase blood loss (BC). Figure 5 (DE). In summary, low and medium doses of LK9 can effectively inhibit FeCl3-induced carotid artery thrombosis without increasing the risk of bleeding, supporting its potential as a promising antithrombotic drug.

Claims

1. An oligopeptide, characterized in that, The oligopeptide has the following amino acid sequence, LK9:LMWFSDRNK, as shown in SEQ ID NO:

1.

2. A pharmaceutical composition, characterized in that... It 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 anticoagulant, antithrombotic or microcirculation-improving drugs and functional products.

4. The use of the oligopeptide according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a medicament for the prevention or treatment of diseases related to coagulation disorders, thrombosis, or microcirculatory disturbances, characterized in that, The diseases mentioned are ischemic cardiovascular and cerebrovascular diseases, thromboembolic diseases, peripheral vascular ischemic diseases, diabetic vascular complications, and fundus microcirculation disorders.

5. The application according to claim 4, characterized in that, The diseases mentioned include coronary heart disease, angina pectoris, myocardial infarction, cerebral infarction, transient ischemic attack, cerebral ischemia, arteriosclerosis, deep vein thrombosis of the lower extremities, arteriosclerosis obliterans of the lower extremities, thromboangiitis obliterans, diabetic foot, pulmonary embolism, renal artery thrombosis or embolism, retinal artery occlusion, retinal vein occlusion, atrial fibrillation with thromboembolism, post-thrombotic syndrome of deep veins, or peripheral artery thrombotic disease.

6. The use of the oligopeptide according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a drug or cosmetic for improving skin microcirculation.

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