Octadecylated modified R18-7AA polypeptide and application of derivative polypeptide of octadecylated modified R18-7AA polypeptide

A technology of octadecyl and octadecyl, applied in the field of antithrombotic drug preparation, can solve the problems of granulocytopenia, increased bleeding risk, gastrointestinal irritation and the like

Active Publication Date: 2020-11-03
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the clinically used drugs for the prevention and treatment of thrombotic diseases mainly include anticoagulant drugs, thrombolytic drugs and antiplatelet aggregation drugs, ...

Method used

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  • Octadecylated modified R18-7AA polypeptide and application of derivative polypeptide of octadecylated modified R18-7AA polypeptide
  • Octadecylated modified R18-7AA polypeptide and application of derivative polypeptide of octadecylated modified R18-7AA polypeptide
  • Octadecylated modified R18-7AA polypeptide and application of derivative polypeptide of octadecylated modified R18-7AA polypeptide

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Experimental program
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Effect test

Embodiment 1

[0071] platelet aggregation inhibition test

[0072] Healthy human platelets were diluted with plasma to 2.5x10^8 / mL, 300 μL of diluted platelets were taken, and 10 μM, 30 μM and 100 μM of myrR18-7AA polypeptide were added respectively. After incubation at 37°C for 5 minutes, 30 μM of ADP and 2.25 μg / mL of Collagen induced platelet aggregation, and the aggregation curve within 5 minutes was drawn on a platelet aggregometer. Wash the platelets with bench-top solution, and finally dilute the platelets to 2.5x10^8 / mL with bench-top solution B, take 300 μL of washed platelets, add 0.21 μM, 0.63 μM, 1.85 μM and 5.56 μM myrR18 polypeptides, and incubate at 37 °C After 5 minutes, add 2.25 μg / mL collagen to induce platelet aggregation; add 1.85 μM, 5.55 μM, 16.67 μM, 50 μM and 150 μM myrR18 polypeptide respectively, and after incubation at 37°C for 5 minutes, add 0.18 U / mL collagen to induce platelet aggregation; Add 0.1 μM, 0.3 μM and 1 μM myrR18-7AA polypeptide respectively, incuba...

Embodiment 2

[0094] The co-localization of the octadecylated modified R18-7AA polypeptide with 14-3-3ζ;

[0095] Take the concentration as 1×10 4 / mL platelets were added with FITC-myrR18-7AA polypeptide (30 μM, amino acid sequence Myristoylated-SWLDLEA), and FITC-labeled myrR18-7AA disordered polypeptide (FITC-myrR18-7AAscr) was used as a negative control. After mixing, place in a 37°C incubator and incubate for 10 minutes. Add 1 mg / mL fibrinogen to the 12-well plate, air-dry for 2 hours, add the incubated platelets, add thrombin (0.12U / mL), mix well, place in an incubator, incubate at 37°C for 40 minutes, and remove the 12 wells platelet mixture in the plate, add PBS to remove the platelets that are not combined with fibrinogen and adhere to the wall, add 4% paraformaldehyde to fix, after 10min, remove the paraformaldehyde, wash with PBS three times, each After 5 minutes, use 0.3% Triton X-100 solution dissolved in PBS to rupture the membrane, remove after 10 minutes, add PBS to wash t...

Embodiment 3

[0098] The effect of myrR18-7AA polypeptide on the related proteins (c-Src protein, 14-3-3ζ, AKT protein) of integrin outside-in signaling pathway;

[0099] (1) Washed platelets were activated by collagen (1 μg / mL) using a platelet aggregator, and myrR18-7AA polypeptides of different concentrations (0 μM, 0.5 μM, 2.5 μM, 12.5 μM) were added to inhibit the activation of platelets. Platelet samples After obtaining, fully lyse;

[0100] (2) Take 20 μL of Dynabeads Protein A, absorb the magnetic beads with a magnetic stand, remove the solution, add 200 μL of PBS solution with 0.02% Tween-20, wash twice, add 2 μL of β3 protein antibody, and incubate at room temperature for 10 minutes.

[0101] (3) Use a magnetic stand to absorb the magnetic beads, remove the solution, and after the platelet sample is fully lysed, centrifuge at 10,000 g for 5 minutes, take the supernatant and add it to the magnetic beads, and incubate at room temperature for 10-30 minutes (adjust the incubation time...

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Abstract

The invention relates to octadecylated modified R18-7AA polypeptide and an application of derivative polypeptide of the octadecylated modified R18-7AA polypeptide, and belongs to the technical field of antithrombotic drug preparation. The invention provides an application of the polypeptide to preparation of drugs for resisting thrombus and inhibiting platelet aggregation. The polypeptide disclosed by the invention can realize an inhibition effect on collagen, thrombin and ADP (adenosine diphosphate) induced human platelet aggregation by inhibiting the combination of 14-3-3zeta proteins and integrin proteins; the polypeptide can significantly inhibit platelet extension without affecting the combination of platelets and fibrinogen, that is, the function of inhibiting platelet aggregation can be realized by inhibiting phosphorylation of related proteins of integrin external and internal signal channels and influencing the interaction of the 14-3-3zeta proteins, cSrc proteins and integrinbeta3 proteins; the aggregation of blood platelets can be remarkably inhibited, the preparation of antithrombotic drugs for resisting blood platelet aggregation and having no bleeding risk is realized, and a new thought is provided for the research and development of novel safe antithrombotic drugs in the future.

Description

technical field [0001] The invention relates to the technical field of preparation of antithrombotic drugs, in particular to the application of octadecylated modified R18-7AA polypeptides and derivative polypeptides thereof. Background technique [0002] A thrombus is a small clotted plaque formed by blood flow on the surface of a peeled or repaired vessel in the cardiovascular system. In the physiological process of coagulation, thrombus is the final product of the cascade reaction of the coagulation system, which is mainly composed of deposited platelets, insoluble fibrin, accumulated white blood cells and red blood cells. The formation of thrombus in the blood vessel will hinder the flow of blood flow, and even lead to the complete occlusion of the blood vessel. The thrombus peels off and detaches from the inner wall of the blood vessel and easily forms a migratory embolism. The resulting cardiovascular and cerebrovascular diseases include myocardial infarction, cerebral...

Claims

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Application Information

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IPC IPC(8): A61K47/54A61K38/16A61P7/02C07K14/00
CPCA61K38/16A61K47/54A61P7/02C07K14/00
Inventor 赖仞申传斌吕秋敏刘明李东升
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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