Clam worm polypeptide antithrombotic preparation

Abstract

The invention provides a nereis polypeptide antithrombotic preparation, and belongs to the technical field of biological pharmacy. In-vitro tests show that the nereis fibrinolytic protease polypeptide can inhibit thrombin and fibrinogen from reacting to generate fibrin, so that the blood coagulation reaction is inhibited. In-vitro anti-platelet aggregation activity determination is carried out on the nereis fibrinolytic protease polypeptide, and the inhibition rate of 640 [mu] g / ml of nereis fibrinolytic protease polypeptide for resisting platelet aggregation is 75.68%, which is close to the inhibition effect of 1 mg / ml of aspirin. A nereis fibrinolytic protease polypeptide antithrombotic effect test shows that the nereis fibrinolytic protease polypeptide group has the pharmacodynamic effect of reducing the weight of thrombus and the weight-length ratio of the thrombus, has a good antithrombotic effect and can be used as a nereis polypeptide antithrombotic preparation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to a nereid polypeptide antithrombotic preparation. Background Art

[0002] Arterial thrombosis is one of the important complications of atherosclerotic plaque rupture, an acute vascular occlusion, and the main pathogenic factor triggering serious cardiovascular and cerebrovascular events such as myocardial infarction and stroke. Most arterial thrombi are formed on the basis of atherosclerosis. Since the blood flow velocity in arteries is high, the coagulation process is activated, and sufficient thrombin cannot accumulate locally; only when atherosclerotic plaques rupture and endothelial cells are damaged will platelets adhere and aggregate, causing lumen stenosis, enabling the local accumulation of an effective concentration of thrombin, and thrombin converts fibrinogen into fibrin to form a thrombus. Atherosclerosis is mostly related to dietary and lifestyle habits in addition to some congenital factors. An unreasonable diet structure and excessive intake of high-fat and high-sugar foods will increase the probability of getting the disease. In addition, smoking, excessive drinking, and lack of exercise are also important factors in the formation of arterial thrombi.

[0003] Arterial thrombus is formed on the basis of atherosclerosis and has become an extremely important public health problem. There are many deficiencies in the clinical application of existing antithrombotic drugs. All antithrombotic drugs may cause serious bleeding, especially in elderly or patients with renal insufficiency; affected by genes, age, and co-existing diseases, the efficacy of some patients is not good; the dose needs to be monitored or adjusted, such as warfarin requires regular measurement of INR, and heparin requires measurement of APTT; and long-term use has safety problems, such as gastrointestinal damage caused by aspirin.

[0004] Antithrombotic peptides are a class of short-chain amino acid sequences with thrombus formation inhibitory activity, usually derived from natural organisms such as nereids, snake venoms, leeches, etc., or designed through artificial synthesis. They show significant potential in anticoagulation, thrombolysis, or antiplatelet aggregation and may become candidate molecules for a new generation of antithrombotic drugs. Exploring antithrombotic peptides from natural sources is expected to obtain antithrombotic drugs with good safety. Summary of the Invention

[0005] The purpose of the present invention is to provide a nereid polypeptide antithrombotic preparation, belonging to the technical field of biopharmaceuticals.

[0006] To achieve the above purpose, the present invention provides the following technical solutions: First, the present invention provides a nereid polypeptide antithrombotic preparation, wherein the polypeptide is Perinereis aibuhitensis fibrinolytic protease polypeptide ACL12061.1, and the amino acid sequence of the Perinereis aibuhitensis fibrinolytic protease polypeptide ACL12061.1 is CASGYAGVYARVS.

[0007] Second, the present invention provides a thrombin activity inhibitory polypeptide, which is the nereis fibrinolysin polypeptide ACL12061.1, and the amino acid sequence of the nereis fibrinolysin polypeptide ACL12061.1 is CASGYAGVYARVS.

[0008] Third, the present invention provides an antiplatelet aggregation polypeptide, which is the nereis fibrinolysin polypeptide ACL12061.1, and the amino acid sequence of the nereis fibrinolysin polypeptide ACL12061.1 is CASGYAGVYARVS.

[0009] Beneficial effects: Through in vitro experiments, the present invention found that the nereis fibrinolysin polypeptide can inhibit the reaction of thrombin and fibrinogen to generate fibrin, thereby inhibiting the precipitation of blood clots and fibrin, and has good anticoagulant activity. At the same time, the in vitro antiplatelet aggregation activity of the nereis fibrinolysin polypeptide was measured. The inhibition rate of 640 μg / ml nereis fibrinolysin polypeptide on antiplatelet aggregation was 75.68%, which was close to the inhibitory effect of 1 mg / ml aspirin. Further, through the anti-thrombosis effect experiment of the nereis fibrinolysin polypeptide, the present invention found that the nereis fibrinolysin polypeptide group has the pharmacological effects of reducing thrombus weight, reducing thrombus length, and the ratio of thrombus weight to length, and has good anti-thrombosis effect, and can be used as an anti-thrombosis preparation of nereis polypeptide. As an anti-thrombosis preparation, natural active polypeptides may reduce the bleeding risk and become safe and effective anti-thrombosis drugs. Description of the Drawings

[0010] Figure 1 Mass spectrometry detection results of nereis fibrinolysin polypeptide; Figure 2 Effect of nereis fibrinolysin on thrombus weight; Figure 3 Effect of nereis fibrinolysin on thrombus length; Figure 4 Effect of nereis fibrinolysin on the ratio of thrombus weight to length. Detailed Embodiments

[0011] Example 1: Design of the anticoagulant active polypeptide sequence of nereis fibrinolysin Analyze the amino acid sequence of nereis fibrinolysin ACL12061.1, select 13 amino acid peptides at positions 234-246, and its amino acid sequence is: CASGYAGVYARVS, and entrust Nanjing Genscript Biotech Co., Ltd. to synthesize. Figure 1 It is the mass spectrometry detection result of the synthesized polypeptide.

[0012] Example 2: Detection of the anticoagulant activity of nereis fibrinolysin polypeptide 1.1 Reagents (1) Dilute the freeze-dried human fibrinogen solution with 0.9% sodium chloride solution to a solution containing 6 mg of fibrinogen per 1 ml.

[0013] (2) Dilute the human thrombin solution with physiological sodium chloride solution containing 1% human albumin to 5 IU / ml.

[0014] (3) Dilute the freeze-dried nereis fibrinolytic protease polypeptide with 0.9% sodium chloride solution to solutions containing 0 μg, 20 μg, 40 μg, 80 μg, 160 μg, 320 μg, and 640 μg per 1 ml.

[0015] 1.2 Determination: Take 0.1 ml of the human thrombin solution with a concentration of 5 IU / ml, incubate at 37 °C for 2 minutes, add 0.1 ml of the fibrinogen solution at 6 mg / ml, add 0.2 ml of the nereis fibrinolytic protease polypeptide solution at different dilution ratios, and record the clotting time with an automatic blood coagulation analyzer. Detect 3 tubes in parallel.

[0016] 1.3 Results: As can be seen from Table 1, the human thrombin solution without the addition of nereis fibrinolytic protease polypeptide solution (0 μg) and the fibrinogen solution coagulated 14 seconds after mixing; the clotting time of the group added with 20 μg - 160 μg of nereis fibrinolytic protease polypeptide solution increased with the increase of the added concentration, and no blood clot or fibrin precipitation was detected in the group added with 320 μg and 640 μg of nereis fibrinolytic protease polypeptide solution after 1 hour of detection. It can be seen that nereis fibrinolytic protease polypeptide can inhibit the reaction of thrombin and fibrinogen to generate fibrin, thereby inhibiting the coagulation reaction.

[0017] Table 1 Detection of the anticoagulant activity of nereis fibrinolytic protease polypeptide

[0018] Note: "-" indicates that no blood clot or fibrin precipitation was detected after 1 hour of detection.

[0019] Example 3: In vitro antiplatelet aggregation activity detection of nereis fibrinolytic protease polypeptide Randomly select 10 SD rats, anesthetize them with 0.12 mol / L sodium pentobarbital (60 mg / kg), collect blood from the abdominal aorta, and anticoagulate with 3.8% sodium citrate at a ratio of 1:9. Centrifuge the anticoagulated blood sample for 10 min (160×g), and take the supernatant to obtain platelet-rich plasma (PRP). Centrifuge the remaining blood after aspirating PRP for 10 min (2000×g), and take the supernatant to obtain platelet-poor plasma (PPP). The PRP and PPP of 10 rats are mixed respectively and used for the in vitro antiplatelet aggregation activity detection of nereis fibrinolytic protease polypeptide, which is completed within 3 h. Preheat the platelet aggregometer to 37 °C. The reaction system is 300 μL. Precisely add 240 μL of PRP and PPP into the corresponding double-cup respectively. Add 60 μL of nereis fibrinolytic protease polypeptide solution containing 20 μg, 40 μg, 80 μg, 160 μg, 320 μg, 640 μg per 1 ml into the PRP and PPP channels respectively. The blank group is added with an equal volume of deionized aqueous solution of 0.1% DMSO. Incubate in a 37 °C water bath for 10 min, then put it into the test channel, add 10 μL of ADP inducer, measure the maximum aggregation rate of rat platelets within 5 min, and calculate the inhibition rate of the polypeptide on platelet aggregation according to the inhibition rate formula.

[0020] Inhibition rate = (maximum aggregation rate of the blank group - maximum aggregation rate of the drug group) / maximum aggregation rate of the blank group As can be seen from the results in Table 2, using aspirin as a positive control, the in vitro anti-platelet aggregation activity of nereis fibrinolytic protease polypeptide was determined. The results showed that the anti-platelet aggregation effect of the 20 - 320 μg / ml nereis fibrinolytic protease polypeptide solution group increased with the increase of polypeptide concentration. The inhibition rate of 640 μg / ml nereis fibrinolytic protease polypeptide on anti-platelet aggregation was 75.68, close to the inhibition effect of 1 mg / ml aspirin.

[0021] Table 2 Detection of in vitro anti-platelet aggregation activity of nereis fibrinolytic protease polypeptide

[0022] Example 4: Anti-thrombotic effect test of nereis fibrinolytic protease polypeptide 1.1 Experimental animals SD male rats, 25, weighing 200 - 250 g, SPF grade.

[0023] 1.2 Experimental protocol The SD rats were randomly divided into 5 groups equally, namely the normal control group, the sham operation group, the model group, and the nereis fibrinolytic protease polypeptide group (320 mg / kg), with 5 rats in each group. All rats in each group were fasted 12 h before surgery, anesthetized with isoflurane, then shaved and disinfected on the abdomen in the supine position. In the model group and the drug administration groups, a longitudinal incision of 2 - 3 cm was made along the midline of the abdomen, the inferior vena cava and the abdominal aorta were separated 2 mm below the left renal vein, ligated together with a glass tube of the same diameter of 0.3 mm, and then slowly withdrawn; finally, the branch veins between the renal vein and the iliac vein were ligated, and the abdomen was sutured with 3-0 suture and disinfected; after the operation, the rats were kept warm with a warm pad until they woke up; in the sham operation group, only the inferior vena cava and the abdominal aorta were separated without ligation. On the first day after the operation, each drug administration group was continuously gavaged for 1 week, once a day; the normal control group, the sham operation group, and the model group were all gavaged with an equal volume of solvent. One week after the modeling and drug administration, the rats were anesthetized with isoflurane, a longitudinal incision was made along the midline of the abdomen, the abdominal aorta and the inferior vena cava were separated, the formed thrombus site was taken out with scissors, the residual blood on the thrombus was blotted with filter paper, and the weight and length of the inferior vena cava thrombus formed in the rats were measured and recorded.

[0024] 1.3 Results After collecting blood from the abdominal aorta of rats, the abdominal aorta and inferior vena cava were isolated. Thrombus formation at the surgical site in the model group was clearly observable with the naked eye, while no thrombus formation was seen in the normal control group and sham operation group. The thrombus weights and lengths of rats in each group are shown in Figure 2 and Figure 3 . Figure 4 These are the analysis results of the thrombus weight / length ratio. Compared with the model group, the nereistoxin polypeptide group had a pharmacological effect of reducing thrombus weight and the thrombus weight / length ratio.

Claims

1. A nereid polypeptide antithrombotic preparation, characterized in that, The polypeptide is the Perinereis aibuhitensis fibrinolytic protease polypeptide ACL12061.1, and the amino acid sequence of the Perinereis aibuhitensis fibrinolytic protease polypeptide ACL12061.1 is CASGYAGVYARVS.

2. A thrombin activity inhibitory polypeptide, characterized in that, The thrombin activity inhibitory polypeptide is the Perinereis aibuhitensis fibrinolytic protease polypeptide ACL12061.1, and the amino acid sequence of the Perinereis aibuhitensis fibrinolytic protease polypeptide ACL12061.1 is CASGYAGVYARVS.

3. An antiplatelet aggregation polypeptide, characterized in that, The antiplatelet aggregation polypeptide is the Perinereis aibuhitensis fibrinolytic protease polypeptide ACL12061.1, and the amino acid sequence of the Perinereis aibuhitensis fibrinolytic protease polypeptide ACL12061.1 is CASGYAGVYARVS.

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