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Platelet aggregation determination method based on high throughput detection and application thereof to drug screening

A technology for platelet aggregation and measurement method, which is applied in the field of drug research and development, and the field of platelet aggregation measurement, which can solve the problems of large variation in repeated detection, large blood collection volume, and long sample processing time.

Inactive Publication Date: 2015-10-14
TIANJIN INSTITUTE OF PHARMA RESEARCH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its main disadvantages include: blood sample processing needs to be completed in a short time, repeated detection has large variability, relatively large blood collection volume, and platelet-rich plasma needs to be separated, so the sample processing time is long, etc.
[0007] Currently, there is CN1866029A patent for clinical platelet aggregation instrument. The main limitations of this detection method are low throughput and high cost.
This model is not suitable for in vitro drug screening

Method used

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  • Platelet aggregation determination method based on high throughput detection and application thereof to drug screening
  • Platelet aggregation determination method based on high throughput detection and application thereof to drug screening
  • Platelet aggregation determination method based on high throughput detection and application thereof to drug screening

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 : Platelet Aggregation Determination in Healthy Volunteers

[0024] 1. Main reagents and instruments

[0025] Thrombin (sigma), thrombin receptor agonist peptide (TRAP-1) (PHOENLX PHARMA.INC.), L-500 centrifuge (Changsha Xiangyi), beaker, sterile syringe, sterile blood collection needle, blood collection tube, Adenosine disodium 5' diphosphate (Aladdin) purity: 95%, batch number: 34995, arachidonic acid, epinephrine, collagen (Aladdin), CMC-Na (Sinopharm Chemical Reagent Co., Ltd.), batch number: F20081015 96-well plate (Costar), Xiangyi low-speed centrifuge L-500 (Changsha Xiangyi), SpectraMax M5 microplate reader (TOP Biotek&Molecular Devices), LBY-NJ four-channel platelet aggregation analyzer (Beijing Prism).

[0026] 2. Experimental method

[0027] 2.1 Blood collection and platelet preparation

[0028] 4 healthy volunteers took blood from arm veins, each person drew 20mL of blood, the blood sample was mixed with 3.8% sodium citrate 9:1 for anticoagulat...

Embodiment 2

[0033] Example 2: Screening of anti-platelet aggregation drugs based on 96-well plate PAR-1 antagonists

[0034] 1. Main reagents and instruments

[0035]Thrombin receptor agonist peptide (PHOENLX PHARMA.INC.), L-500 centrifuge (Changsha Xiangyi), beaker, sterile syringe, sterile blood collection needle, blood collection tube. 96-well plate (Costar), Xiangyi low-speed centrifuge L-500 (Changsha Xiangyi), SpectraMax M5 microplate reader (TOP Biotek&Molecular Devices).

[0036] 2. Experimental method

[0037] 2.1 Blood collection and platelet preparation

[0038] The guinea pigs used the common carotid artery to collect blood. The blood sample was mixed with 3.8% sodium citrate 9:1 for anticoagulation. The blood sample was centrifuged at 200×g for 10 minutes at room temperature, and the upper layer of platelet-rich plasma (PRP) was sucked out. Bring its platelet count to 2 x 10 5 -6×10 5 / μl after drug screening. The experimental method is shown in Table 2, and the experim...

Embodiment 3

[0045] Example 3: Screening of anti-platelet aggregation drugs based on ADP receptor antagonists in 384-well plate

[0046] 1.1 Main reagents and instruments

[0047] Adenosine disodium 5' diphosphate (Aladdin) purity: 95%, batch number: 34995, L-500 centrifuge (Changsha Xiangyi), beaker, sterile syringe, sterile blood collection needle, blood collection tube. 384-well plate (Costar), Xiangyi low-speed centrifuge L-500 (Changsha Xiangyi), SpectraMax M5 microplate reader (TOP Biotek&Molecular Devices).

[0048] 2. Experimental method

[0049] 2.1 Blood collection and platelet preparation

[0050] Rats used the common carotid artery to collect blood. The blood sample was mixed with 3.8% sodium citrate 9:1 for anticoagulation. The blood sample was centrifuged at 200×g for 10 minutes at room temperature. The blood platelet count was 2×10 after mixing PRP with liquid 5 -6×10 5 / μl after drug screening. The experimental methods are shown in Table 4, and some screening result...

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Abstract

The invention discloses a platelet aggregation determination method based on high throughput detection. The method comprises the following steps: extracting a platelet sample with activity from an organism, and diluting a platelet suspension through a solvent; and transferring into a microplate for incubation, adding a stimulant of certain concentration, and determining with a dynamical method of a microplate reader, thereby obtaining the platelet aggregation rate. The method can be applied to rapid clinical evaluation on activity of platelets and high throughput screening of antithrombotic drugs at multiple targets, and is implemented through a cyclooxygenase inhibitor, an ADP receptor inhibitor, a protein activating enzyme receptor inhibitor, a platelet surface GPIIb / IIIa receptor inhibitor and the like.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for measuring platelet aggregation and the application of the method in the field of drug research and development. Background technique [0002] Platelets play an important role in physiological hemostasis, maintaining the integrity of blood vessel walls, and certain pathological processes, such as thrombosis, atherosclerosis, unstable angina, tumor metastasis, and inflammatory reactions. A large number of studies have shown that the activation and aggregation of platelets is the initiating factor of thrombus formation in vivo, and it is also one of the most critical components in the process of thrombus formation. Common clinical critical emergencies such as sudden cardiac death, myocardial infarction, stroke, and pulmonary infarction are all thrombotic diseases. Therefore, platelet function testing is of great significance for the early detection of thro...

Claims

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Application Information

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IPC IPC(8): G01N21/31
Inventor 周植星魏巍王玉丽潘婷婷于冰徐为人
Owner TIANJIN INSTITUTE OF PHARMA RESEARCH
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