An antithrombotic preparation of a polypeptide from a nereid

Abstract

The present application provides a kind of nereid polypeptide antithrombotic preparation, belong to the technical field of biopharmaceuticals.The present application finds that nereid plasmin polypeptide can inhibit the reaction of thrombin and fibrinogen to generate fibrin, thereby inhibiting the blood clotting reaction through in vitro test.Nereid plasmin polypeptide is determined for in vitro anti-platelet aggregation activity, and the inhibition rate of 640 μg / ml nereid plasmin polypeptide for anti-platelet aggregation is 75.68%, close to the inhibition effect of 1 mg / ml aspirin.The present application finds that nereid plasmin polypeptide group has the pharmacodynamic effect of reducing the weight of thrombus and the ratio of thrombus weight and length through nereid plasmin polypeptide antithrombotic effect test, has good antithrombotic effect, and can be used as nereid polypeptide antithrombotic preparation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological pharmacy, and particularly relates to a polypeptide anti-thrombus preparation of nereis. BACKGROUND

[0002] Arterial thrombosis is one of the important complications of atherosclerotic plaque rupture, is acute vascular obstruction, and is a major pathogenic factor of serious cardiovascular and cerebrovascular events such as myocardial infarction and stroke. Arterial thrombosis is mostly formed on the basis of atherosclerosis. Because the blood flow velocity in the artery is high, the coagulation process is activated, and enough thrombin cannot be accumulated locally; only when the atherosclerotic plaque is broken and the endothelial cells are damaged, can platelets adhere and aggregate, causing lumen stenosis, so that the local thrombin is accumulated to an effective concentration, and the thrombin converts fibrinogen into fibrin to form a thrombus. Atherosclerosis is mostly related to dietary and living habits in addition to some congenital factors. Unreasonable dietary structure and large intake of high-fat and high-sugar food can increase the probability of disease. In addition, smoking, alcoholism and lack of exercise are also important factors for the formation of arterial thrombosis.

[0003] Arterial thrombosis is formed on the basis of atherosclerosis, and it has become an extremely important public health problem. There are many deficiencies in the clinical application of existing anti-thrombus drugs. Anti-thrombus drugs can all cause serious bleeding, especially in elderly or renal dysfunction patients; the efficacy of some patients is poor due to the influence of genes, age and combined diseases; the dose needs to be monitored or adjusted, such as INR for warfarin and APTT for heparin; and long-term use has safety problems, such as gastrointestinal damage of aspirin.

[0004] Anti-thrombus peptides are a class of short-chain amino acid sequences with thrombosis inhibition activity, which are usually derived from natural organisms such as nereis, snake venom, leech, etc., or are designed by artificial synthesis. They show significant potential in anticoagulation, thrombolysis or anti-platelet aggregation, and can become candidate molecules for a new generation of anti-thrombus drugs. Mining natural anti-thrombus peptides is expected to obtain anti-thrombus drugs with good safety. SUMMARY

[0005] The present application belongs to the technical field of biological pharmacy, and particularly relates to a polypeptide anti-thrombus preparation of nereis.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0007] Firstly, the present application provides a polypeptide anti-thrombus preparation of nereis, wherein the polypeptide is a Perinereis aibuhitensis plasmin polypeptide ACL12061.1, and the amino acid sequence of the Perinereis aibuhitensis plasmin polypeptide ACL12061.1 is CASGYAGVYARVS.

[0008] Secondly, the present application provides a thrombin activity inhibiting polypeptide, which is the Perinereis aibuhitensis fibrinolysin polypeptide ACL12061.1, and the amino acid sequence of the Perinereis aibuhitensis fibrinolysin polypeptide ACL12061.1 is CASGYAGVYARVS.

[0009] Thirdly, the present application provides an anti-platelet aggregation polypeptide, which is the Perinereis aibuhitensis fibrinolysin polypeptide ACL12061.1, and the amino acid sequence of the Perinereis aibuhitensis fibrinolysin polypeptide ACL12061.1 is CASGYAGVYARVS.

[0010] Beneficial effects: The present application finds that the Perinereis aibuhitensis 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 through in-vitro tests. Meanwhile, the in-vitro anti-platelet aggregation activity of the Perinereis aibuhitensis fibrinolysin polypeptide is determined, and the inhibition rate of the Perinereis aibuhitensis fibrinolysin polypeptide on anti-platelet aggregation is 75.68% at 640 g / ml, which is close to the inhibition effect of 1 mg / ml aspirin. Further, the present application finds that the Perinereis aibuhitensis fibrinolysin polypeptide group has the pharmacodynamic effects of reducing the weight of thrombus, reducing the length of thrombus, and reducing the weight-length ratio of thrombus through the anti-thrombus effect test of the Perinereis aibuhitensis fibrinolysin polypeptide, and has good anti-thrombus effect, and can be used as an anti-thrombus preparation of Perinereis aibuhitensis polypeptide. The natural active polypeptide as an anti-thrombus preparation can reduce the risk of bleeding and become a safe and effective anti-thrombus drug. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 Mass spectrum detection result of the Perinereis aibuhitensis fibrinolysin polypeptide;

[0012] Figure 2 Influence of the Perinereis aibuhitensis fibrinolysin polypeptide on the weight of thrombus;

[0013] Figure 3 Influence of the Perinereis aibuhitensis fibrinolysin polypeptide on the length of thrombus;

[0014] Figure 4 Influence of the Perinereis aibuhitensis fibrinolysin polypeptide on the weight-length ratio of thrombus. DETAILED DESCRIPTION

[0015] Example 1: Sequence design of the Perinereis aibuhitensis fibrinolysin anticoagulant polypeptide

[0016] The amino acid sequence of the Perinereis aibuhitensis fibrinolysin polypeptide ACL12061.1 is analyzed, and the 13-amino-acid peptide segment at positions 234-246 is selected, and the amino acid sequence is CASGYAGVYARVS, and the synthesis is entrusted to Nanjing Kingsrui Biological Technology Co., Ltd., Figure 1 the mass spectrum detection result of the synthesized polypeptide.

[0017] Example 2: Anti-coagulation activity of Arenicola fibrinolysin polypeptide

[0018] 1.1 Reagents

[0019] (1) Fibrinogen solution: The lyophilized human fibrinogen solution was diluted with 0.9% sodium chloride solution to a solution containing 6 mg per 1 ml.

[0020] (2) Human thrombin solution: The human thrombin solution was diluted with physiological sodium chloride solution containing 1% human blood albumin to 5 IU / ml.

[0021] (3) Arenicola fibrinolysin polypeptide solution: The lyophilized Arenicola fibrinolysin polypeptide was diluted with 0.9% sodium chloride solution to a solution containing 0 μg, 20 μg, 40 μg, 80 μg, 160 μg, 320 μg, 640 μg per 1 ml.

[0022] 1.2 Measurement 0.1 ml of the human thrombin solution with a concentration of 5 IU / ml was incubated at 37°C for 2 minutes, 0.1 ml of the fibrinogen solution with a concentration of 6 mg / ml was added, 0.2 ml of the Arenicola fibrinolysin polypeptide solution with different dilutions was added, and the coagulation time was recorded by an automatic blood coagulation instrument. Three tubes were detected in parallel.

[0023] 1.3 Results As can be seen from Table 1, the human thrombin solution and the fibrinogen solution without the addition of the Arenicola fibrinolysin polypeptide solution (0 μg) produced coagulation after 14 seconds; the coagulation time of the group with the addition of 20 μg to 160 μg of the Arenicola fibrinolysin polypeptide solution was prolonged with the increase of the added concentration; the coagulation time of the group with the addition of 320 μg and 640 μg of the Arenicola fibrinolysin polypeptide solution was not detected for 1 hour. It can be seen that the Arenicola fibrinolysin polypeptide can inhibit the reaction of thrombin and fibrinogen to generate fibrin, thereby inhibiting the coagulation reaction.

[0024] Table 1 Anti-coagulation activity detection of Arenicola fibrinolysin polypeptide

[0025]

[0026] Note: "-" indicates that no coagulation or fibrin precipitation was detected for 1 hour.

[0027] Example 3: Anti-platelet aggregation activity of Arenicola fibrinolysin polypeptide in vitro

[0028] Randomly take 10 SD rats, 0.12 mol / L sodium pentobarbital (60 mg / kg) anesthesia, abdominal aortic blood, with 3.8% sodium citrate 1:9 anticoagulation. Take the anticoagulated blood sample centrifugation 10 min (160 x g), take the upper liquid, that is, the platelet-rich plasma (PRP). After taking PRP, the remaining blood was centrifuged for 10 min (2 000 x g), and the upper liquid was taken, that is, the platelet-poor plasma (PPP). The PRP and PPP of 10 rats were mixed respectively and used for the in vitro anti-platelet aggregation activity detection of Arenicola fibrinolysin polypeptide, which was completed within 3 h.

[0029] Turn on the preheating of the platelet aggregometer to 37℃, the reaction system is 300 μL, and 240 μL of PRP and PPP are precisely added into the corresponding double cups, 60 μL of 1 ml of Arenicola fibrinolysin polypeptide solution containing 20 μg, 40 μg, 80 μg, 160 μg, 320 μg and 640 μg is added into the PRP and PPP channels respectively, and the same volume of 0.1% DMSO deionized water solution is added into the blank group. After incubation at 37℃ water bath for 10 min, it is placed into the test channel, 10 μL of ADP inducer is added, and the maximum aggregation rate of rat platelets within 5 min is determined. The inhibition rate of polypeptide on platelet aggregation is calculated according to the inhibition rate formula.

[0030] Inhibition rate = (maximum aggregation rate of blank group - maximum aggregation rate of drug group) / maximum aggregation rate of blank group

[0031] As shown in Table 2, using aspirin as a positive control, the in vitro anti-platelet aggregation activity of Arenicola fibrinolysin polypeptide was determined, and the results showed that the anti-platelet aggregation effect of 20-320 μg / ml Arenicola fibrinolysin polypeptide solution group increased with the increase of polypeptide concentration. The inhibition rate of 640 μg / ml Arenicola fibrinolysin polypeptide on platelet aggregation was 75.68, close to the inhibition effect of 1 mg / ml aspirin.

[0032] Table 2 In vitro anti-platelet aggregation activity detection of Arenicola fibrinolysin polypeptide

[0033]

[0034] Example 4: Anti-thrombosis effect test of Arenicola fibrinolysin polypeptide

[0035] 1.1 Test animals SD male rats, 25, 200-250 g, SPF level.

[0036] 1.2 Test scheme SD rats were randomly divided into 5 groups, namely normal control group, sham operation group, model group, Nereid fibrinolysin polypeptide group (320 mg / kg), 5 rats in each group. Each group was fasted for 12 h before operation, and after isoflurane anesthesia, the rats were placed in a supine position and the abdomen was disinfected. The model group and the drug administration group were longitudinally cut 2-3 cm along the midline of the abdomen, and the inferior vena cava and abdominal aorta were separated 2 mm below the left renal vein, and a glass tube with the same diameter of 0.3 mm was ligated together, and then slowly extracted; finally, the branch veins between the renal vein and the iliac vein were ligated, and the abdomen was sutured with 3-0 suture line and disinfected; After operation, the rats were warmed with a warm pad until they were awake; The sham operation group only separated the inferior vena cava and abdominal aorta, and did not ligate. On the first day after operation, each drug administration group was given intragastric administration for 1 week, once a day; The normal control group, sham operation group and model group were all given the same amount of solvent. After the modeling was completed and the drug administration was given for 1 week, isoflurane anesthesia was performed, the abdominal aorta and inferior vena cava were separated along the midline of the abdomen, the thrombus site was removed with scissors, and the blood remaining on the thrombus was absorbed with filter paper. The weight and length of the inferior vena cava thrombus formed in the rats were measured and recorded.

[0037] 1.3 Results After the abdominal aorta of the rats was taken out, the abdominal aorta and inferior vena cava were separated, and the thrombus was obviously observed at the operation site in the model group under naked eye. No thrombus was formed in the normal control group and the sham operation group, and the thrombus weight and length of each group of rats were as follows: Figure 2 and Figure 3 . Figure 4 The analysis results of the ratio of thrombus weight to weight length. Compared with the model group, the Nereid fibrinolysin polypeptide group had a pharmacodynamic effect of reducing the weight of the thrombus and the ratio of the weight of the thrombus to the length of the thrombus.

Claims

1. A sandworm polypeptide antithrombotic agent, characterized in that, The amino acid sequence of the polypeptide is CASGYAGVYARVS.

2. A thrombin activity-inhibiting polypeptide, characterized in that, The amino acid sequence of the thrombin activity inhibiting polypeptide is CASGYAGVYARVS.

3. An anti-platelet aggregation polypeptide, characterized in that, The amino acid sequence of the anti-platelet aggregation polypeptide is CASGYAGVYARVS.

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