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Anticoagulant drug based on cobra venom PIII type metalloprotease and applications thereof

A technology of metalloprotease and cobra venom, applied in peptide/protein components, medical preparations containing active ingredients, enzymes, etc., can solve the problems that cannot be prepared with anticoagulant drugs, achieve no bleeding risk, reduce abnormal coagulation, Effect of lowering fibrinogen levels

Inactive Publication Date: 2016-05-11
THE KEY LAB OF CHEM FOR NATURAL PROD OF GUIZHOU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] SVMP isolated and prepared from Viperidae and Rattlesnake snake venoms cannot be used in the preparation of anticoagulants due to their inherent hemorrhagic toxicity

Method used

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  • Anticoagulant drug based on cobra venom PIII type metalloprotease and applications thereof
  • Anticoagulant drug based on cobra venom PIII type metalloprotease and applications thereof
  • Anticoagulant drug based on cobra venom PIII type metalloprotease and applications thereof

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Experimental program
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Effect test

Embodiment 1

[0047] The target protein is obtained by separating and purifying from the snake venom of Chinese cobra (Zhoushan species cobra) (classification name: Najaatra). The target protein is a single-chain glycoprotein, and its structural analysis shows that it has three structural domains: a metalloprotease domain, a disintegrin-like domain, and a cysteine-rich domain, belonging to the PIII type snake venom metalloprotease ( figure 1 ).

[0048] Table 1 The physical and chemical indicators of the target protein deduced from the primary structure

[0049]

[0050] As shown in Table 1: the target protein has the activity of hydrolyzing the alpha chain of fibrinogen, which can be completely inhibited by metal chelating agents EDTA, EGTA, 1,10-phenanthroline and reducing agent DTT. The actual molecular weights of the target proteins AtraseA and AtraseB are about 51.5 and 49.4kDa respectively, and their sequences are: AtraseA: SEQIDNo.1; AtraseB: SEQIDNo.2; but their apparent molecul...

Embodiment 2

[0066] Anticoagulant effect of cobra venom type PIII metalloprotease (taking AtraseA as an example)

[0067] 1. Anticoagulation in vitro

[0068] 1.1 In vitro experiments on the effects of anticoagulant drugs on the four coagulation indexes

[0069] 1.1.1 Determination method: standard human plasma (purchased from SIEMENS, Germany) was reconstituted in deionized water, shaken gently and allowed to stand at room temperature (25°C) for 15 minutes before use. The samples were diluted to 10 μg / ml, 50 μg / ml, and 100 μg / ml with PBS, respectively. The measurement system of each index is as follows:

[0070] TT: 200μl plasma + 40μl sample;

[0071] APTT: 100μl plasma + 20μl sample;

[0072] PT: 100μl plasma + 20μl sample;

[0073] FIB: 200 μl diluted plasma (standard plasma diluted 10 times) + 40 μl sample;

[0074] The mixture of the above sample and plasma was incubated in a water bath at 37°C for 5 min and 30 min, respectively, and experiments were carried out according to th...

Embodiment 3

[0092] 2. Anticoagulation in Rats

[0093] 2.10.3mg / kg dose anticoagulant anticoagulant effect

[0094] 2.1.1 Animals: SPF grade SD rats, weighing 250-280g. Animals were randomly divided into five groups, namely control group, 2h group, 6h group, 12h group and 24h group, with 10 rats in each group.

[0095] 2.1.2 Method: A sample was injected intravenously into the tail of rats at a dose of 0.3 mg / kg. Rat anesthesia and blood collection: according to the time requirements of different groups, the rats were anesthetized with 1% pentobarbital sodium 20 minutes before the experiment, and each rat was anesthetized with an average injection of 1.4ml pentobarbital sodium. After anesthesia, blood was collected at a ratio of 1:9 with 0.109 mol / L sodium citrate anticoagulant at 30 min, 2 h, 6 h, 12 h, and 24 h.

[0096] 2.1.3 Determination of anti-platelet aggregation

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Abstract

The invention discloses an anticoagulant drug based on cobra venom PIII type metalloprotease. The cobra venom PIII type metalloprotease is extracted from snake venom of naja atra and then purified. The metalloprotease is single chain glycoprotein. The structure analysis shows that metalloprotease has three structural domains: metalloprotease structural domain, disintegrin like structural domain, and cysteine enriched structural domain, and belongs to PIII type cobra venom metalloprotease. The metalloprotease is named as Atrase A and Atrase B; and the sequences of Atrase A and Atrase B are represented by SEQ ID No.1 and SEQ ID No.2 respectively. Target protein and anticoagulant drugs thereof have multiple anticoagulant effects: preventing multiple blood coagulation factors; reducing fibrinogen level; preventing platelet aggregation; and preventing complement so as to relieve abnormal blood coagulation caused by inflammation.

Description

technical field [0001] The invention relates to the field of application of cobra venom type PIII metalloprotease, in particular to an anticoagulant drug based on cobra venom type PIII metalloprotease and its application. Background technique [0002] Snake venom metalloproteinase (Snakevenom metalloproteinase, SVMP) belongs to the metalloproteinase M12 family Reprolysin member, mainly exists in Viperidae and Rattlesnake snake venoms, and its structure has a common feature of a conserved sequence of "HEXGHXXGXXHD" in the active center of the enzyme, zinc Ions bind to this conserved region. The significant toxicity characteristic of SVMP is hemorrhagic toxicity, and most of them have activities such as hydrolyzing cell basement membrane and extracellular matrix, anticoagulant, procoagulant, and antiplatelet aggregation, and play an important role in the toxic effect of blood circulation venomous snake venoms. According to the molecular weight and the number of structural dom...

Claims

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Application Information

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IPC IPC(8): C12N9/64A61K38/48A61P7/02
CPCA61K38/00C12N9/6418C12Y304/24001C12Y304/24041
Inventor 孙黔云王彩娥李亚男
Owner THE KEY LAB OF CHEM FOR NATURAL PROD OF GUIZHOU PROVINCE & CHINESE ACADEMY OF SCI
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