Application of ASGR1 in preparation of medicine for inhibiting abnormal activation of platelets

Abstract

The invention discloses application of ASGR1 in preparation of a medicine for inhibiting abnormal activation of platelets. The gene sequence of the ASGR1 is as shown in SEQ ID NO.1, and the amino acid sequence of the ASGR1 is as shown in SEQ ID NO.2. It is found for the first time that ASGR1 is expressed in platelets, and expression in activated platelets is increased compared with expression in resting platelets; the ASGR1 is taken as a drug target, and an antibody for inhibiting protein action is designed for neutralization, so that abnormal activation of platelets can be inhibited, and the purpose of preparing the drug for treating myocardial infarction is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an application of ASGR1 in preparing a drug for inhibiting abnormal platelet activation. Background Art

[0002] The prevalence of coronary atherosclerotic heart disease (CHD) in my country continues to rise. Atherosclerotic thrombosis is the ultimate mechanism of acute ischemic events such as myocardial infarction, and abnormal platelet activation is a key step in the formation of arterial thrombosis. The mechanism of why platelets are abnormally activated and prone to thrombosis is still not fully understood. Therefore, clarifying the mechanism of abnormal platelet activation and arterial thrombosis is of great significance for the prevention and treatment of CHD, especially acute ischemic events such as myocardial infarction.

[0003] Asialoglycoprotein receptor-1 (ASGR1) is a type II single-pass transmembrane glycoprotein, a member of the C-type lectin receptor (CLR) family. From the extracellular to the inward, it consists of a carbohydrate recognition domain (CRD), a C-terminal extracellular stem-loop domain (stalk), a single-pass transmembrane domain (TMD), and an N-terminal intracellular domain. The CRD of ASGR1 is the ligand recognition and binding domain.

[0004] Studies have found that the risk of coronary heart disease in carriers of ASGR1 functional loss-of-function mutants is about 34% lower than that in the normal population, suggesting that ASGR1 may be a potential therapeutic target for coronary heart disease. Recent studies have shown that ASGR1 can promote the occurrence and development of coronary heart disease by regulating cholesterol metabolism. However, clinical studies have confirmed that the degree of reduction in the risk of coronary heart disease, especially myocardial infarction (36%), caused by ASGR1 functional loss is much greater than the degree of reduction in circulating cholesterol levels (7%), suggesting that ASGR1 may have a non-lipid-regulating mechanism that promotes the development of coronary heart disease. There are currently no reports on the role and application of ASGR1 in platelet activation. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide an application of an asialoglycoprotein receptor ASGR1 in the preparation of a drug for inhibiting abnormal platelet activation.

[0006] In order to solve the above technical problems, the present invention also provides an application of ASGR1 in preparing a drug for inhibiting abnormal platelet activation, wherein the gene sequence of ASGR1 is shown in SEQ ID NO.1.

[0007] The amino acid sequence of ASGR1 is shown in SEQ ID NO.2.

[0008] The above application, further, the method of the application includes: taking ASGR1 as a drug target, neutralizing by designing antibodies that inhibit the action of the protein, and achieving the purpose of inhibiting abnormal platelet activation.

[0009] The above application, further, the method of the application includes: using ASGR1 as a drug target, using CRISPR / Cas9 technology, knocking out the ASGR1 gene, and achieving the purpose of inhibiting abnormal platelet activation. Further, the sgRNA used includes gRNA1 and gRNA2. The DNA sequence of the gRNA1 is shown in SEQ ID NO.5, and the DNA sequence of the gRNA2 is shown in SEQ ID NO.6.

[0010] Compared with the prior art, the advantages of the present invention are:

[0011] The present invention provides an application of ASGR1 in the preparation of a drug for inhibiting abnormal platelet activation. Abnormal platelet activation promotes arterial thrombosis and ultimately leads to the occurrence of acute ischemic events such as myocardial infarction. The present invention first discovered that ASGR1 is expressed in platelets, and that the expression in activated platelets is increased compared to that in resting platelets; ASGR1 whole-body knockout mice were constructed, and mouse platelets were extracted for detection, and it was found that ASGR1 knockout did not affect platelet generation, but ASGR1 knockout significantly weakened platelet activation induced by the activator arachidonic acid (AA); using the natural ligand of ASGR1, asialofetuin A, it was found that ASGR1 binding ligand asialofetuin A would not activate resting platelets, but would further enhance platelet activation induced by the activator AA. Therefore, the present invention provides the effect of ASGR1 in promoting abnormal platelet activation, and ASGR1 knockout can inhibit abnormal platelet activation, thereby achieving the purpose of preparing a drug for treating myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0013] Figure 1 Western Blot was used to detect the protein expression of ASGR1 in mouse liver, lung, spleen, heart, arterial tissue and platelets.

[0014] Figure 2The expression of ASGR1 protein in mouse platelets after activation by activator AA was detected by Western Blot, flow cytometry and laser confocal microscopy. Figure A is the Western Blot result, Figure B is the flow cytometry result, and Figure C is the laser confocal microscopy result.

[0015] Figure 3 The expression results of ASGR1 on platelet membrane were detected by Western Blot and flow cytometry. Figure A is the result of Western Blot detection; Figure B is the result of flow cytometry detection.

[0016] Figure 4 Western Blot detection of ASGR1 - / - Results of ASGR1 protein expression in liver and platelets of mice and WT mice.

[0017] Figure 5 These are the results of an investigation into the effect of ASGR1 on platelet volume distribution width and mean platelet volume. Figure A is the platelet count (PLT) result, Figure B is the platelet volume distribution width (MPV) result, and Figure C is the mean platelet volume (PDW) result.

[0018] Figure 6 The effect of ASGR1 on platelet spreading on fibrinogen was observed using laser confocal microscopy.

[0019] Figure 7 The results of flow cytometry detection of the expression of platelet membrane GPIIb / IIIa and P-selectin.

[0020] Figure 8 The results of flow cytometry detection of the effect of ASGR1 on the expression of GPIIb / IIIa and P-selectin on platelets. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with specific preferred embodiments, but the protection scope of the present invention is not limited thereby.

[0022] The materials, reagents and instruments used in the following examples can all be purchased from commercial channels. The experimental methods in the following examples are all conventional methods in the art unless otherwise specified.

[0023] Embodiment 1:

[0024] The asialoglycoprotein receptor ASGR1 belongs to the type II single-pass transmembrane glycoprotein, a member of the C-type lectin receptor (CLR) family, and is composed of 291 amino acids.

[0025] The gene sequence of human ASGR1 is shown in SEQ ID NO.1, specifically including:

[0026] ATGACCAAGGAGTATCAAGACCTTCAGCATCTGGACAATGAGGAGAGTGACCACCATCAGCTCAGAAAAGGGCCACCTCCTCCCCAGCCCCTCCTGCAGCGTCTCTGCTCCGGACCTCGCCTCCTCCTGCTCTCCCTGGGCCTCAGCCTCCTGCTGCTTGTGGTTGTCTGTGTGATCGGATCCCAAAACTCCCAGCTGCAGGAGGAGCTGCGGGGCCTGAGAGAGACGTTCAGCAACTTCACAGCGAGCACGGAGGCCCAGGTCAAGGGCTTGAGCACCCAGGGAGGCAATGTGGGAAGAAAGATGAAGTCGCTAGAGTCCCAGCTGGAGAAACAGCAGAAGGACCTGAGTGAAGATCACTCCAGCCTGCTGCTCCACGTGAAGCAGTTCGTGTCTGACCTGCGGAGCCTGAGCTGTCAGATGGCGGCGCTCCAGGGCAATGGCTCAGAAAGGACCTGCTGCCCGGTCAACTGGGTGGAGCACGAGCGCAGCTGCTACTGGTTCTCTCGCTCCGGGAAGGCCTGGGCTGACGCCGACAACTACTGCCGGCTGGAGGACGCGCACCTGGTGGTGGTCACGTCCTGGGAGGAGCAGAAATTTGTCCAGCACCACATAGGCCCTGTGAACACCTGGATGGGCCTCCACGACCAAAACGGGCCCTGGAAGTGGGTGGACGGGACGGACTACGAGACGGGCTTCAAGAACTGGAGGCCGGAGCAGCCGGACGACTGGTACGGCCACGGGCTCGGAGGAGGCGAGGACTGTGCCCACTTCACCGACGACGGCCGCTGGAACGACGACGTCTGCCAGAGGCCCTACCGCTGGGTCTGCGAGACAGAGCTGGACAAGGCCAGCCAGGAGCCACCTCTCCTTTAA。

[0027] Its amino acid sequence is shown in SEQ ID NO.2, specifically including:

[0028] MTKEYQDLQHLDNEESDHHQLRKGPPPPQPLLQRLCSGPRLLLLSLGLSLLLLVVVCVIGSQNSQLQEELRGLRETFSNFTASTEAQVKGLSTQGGNVGRKMKSLESQLEKQQKDLSEDHSSLLLHVKQFVSDLRSLSCQMAALQGNGSERTCCPVNWVEHERSCYWFSRSGKAWADADNYCRLEDAHLVVVTSWEEQKFVQHHIGPVNTWMGLHDQNGPWKWVDGTDYETGFKNWRPEQPDDWYGHGLGGGEDCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEPPLL。

[0029] The gene sequence of ASGR1 in mice is shown in SEQ ID NO.3, specifically including: ATGACAAAGGATTATCAAGATTTCCAGCACCTGGACAATGATAATGACCATCATCAACTCCGGAGAGGGCCGCCTCCCACTCCACGGCTCTTGCAGCGACTCTGCTCTGGATCCCGCCTCCTCCTGCTCTCCTCGAGCCTCAGCATTCTGTTGCTGGTGGTTGTCTGTGTGATCACATCCCAAAATTCCCAACTCCGGGAAGATCTGCTGGCTCTAAGGCAGAATTTCAGCAACCTCACTGTGAGCACTGAGGACCAGGTCAAGGCCCTGAGCACCCAGGGAAGTAGTGTGGGAAGAAAGATGAAGTTAGTGGAGTCGAAGCTGGAAAAACAGCAGAAGGATCTGACTGAAGATCACTCCAGTTTGCTACTGCACGTGAAGCAGTTAGTGTCTGACGTGCGAAGCTTGAGCTGCCAGATGGCTGCATTTCGGGGCAATGGCTCTGAAAGGACCTGCTGCCCCATCAACTGGGTGGAGTATGAAGGCAGCTGCTACTGGTTCTCCAGCTCTGTGAGGCCTTGGACTGAAGCTGACAAGTACTGCCAGCTGGAAAATGCCCATCTGGTGGTGGTGACCTCCAGGGATGAGCAGAACTTCCTCCAGCGCCACATGGGCCCCTTAAACACTTGGATTGGCCTAACTGACCAGAACGGGCCCTGGAAATGGGTGGATGGAACAGACTACGAGACAGGCTTCCAGAATTGGAGACCAGAGCAGCCAGATAACTGGTACGGACATGGGCTTGGAGGAGGCGAGGACTGTGCCCACTTCACGACGGATGGCCGCTGGAATGACGACGTCTGCAGGAGGCCCTACCGCTGGGTCTGTGAGACAAAGTTGGATAAGGCTAATTAG。

[0030] Its amino acid sequence is shown in SEQ ID NO.4, specifically including:

[0031] MTKDYQDFQHLDNDNDHHQLRRGPPPTPRLLQRLCSGSRLLLSSSLSILLLVVVCVITSQNSQLREDLLALRQNFSNLTVSTEDQVKALSTQGSSVGRKMKLVESKLEKQQKDLTEDHSSLLLHVKQLVSDVRSLSCQM AAFRGNGSERTCCPINWVEYEGSCYWFSSSVRPWTEADKYCQLENAHLVVVTSRDEQNFLQRHMGPLNTWIGLTDQNGPWKWVDGTDYETGFQNWRPEQPDNWYGHGLGGGEDCAHFTTDGRWNDDVCRRPYRWVCETKL.

[0032] The ASGR1 gene is located on chromosome 11 of mice, with a total of 9 exons, of which the start codon ATG is located in exon 2, and the stop codon TAG is located in exon 9. Exons 2-9 contain all coding regions, so exons 2-9 are used as the knockout target region, with a size of ~2741bp. If it is for humans, then as a drug target, the action of the protein can be inhibited, including the use of neutralizing antibodies.

[0033] Experiment 1: Investigating the expression of ASGR1 in mouse platelets

[0034] The mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (60 mg / kg), and blood was collected by removing the eyeballs and placed in anticoagulant tubes. Platelet rich plasma (PRP) was obtained by centrifugation at room temperature for 15 minutes, and PRP was centrifuged at 250g for 20 minutes, and the supernatant was removed to obtain platelet precipitate. The liver, lung, spleen, heart and arterial tissues of the mice were taken respectively, and the protein expression of ASGR1 in the liver, lung, spleen, heart, arterial tissue and platelets of the mice was detected by immunoblotting.

[0035] Figure 1 The results of Western Blot detection of ASGR1 protein expression in mouse liver, lung, spleen, heart, arterial tissue and platelets. It can be seen from the figure that ASGR1 is expressed on platelets.

[0036] Further, the platelet precipitate was resuspended in Tyrode'S buffer (purchased from Abiwei) to obtain a platelet suspension, which was then counted using three-classification platelets and adjusted to an appropriate density. 10 μL of AA was added and incubated in a 37°C water bath in the dark for 10 min. The expression of ASGR1 protein in platelets activated by AA was detected by immunoblotting, flow cytometry, and laser confocal microscopy.

[0037] Figure 2 The results of the expression of ASGR1 protein in mouse platelets after activation by the activator AA were detected by Western Blot, flow cytometry and laser confocal microscopy. Figure A is the result of Western Blot, Figure B is the result of flow cytometry, and Figure C is the result of laser confocal microscopy. It can be seen from the figure that the expression of ASGR1 on platelets activated by AA is significantly increased.

[0038] Experiment 2: Investigate the expression of ASGR1 in platelets of patients with coronary heart disease and healthy subjects.

[0039] 5 ml of fasting blood was collected from 3 patients with coronary heart disease and 3 healthy subjects in the morning in anticoagulation tubes. Platelet rich plasma (PRP) was obtained by centrifugation at room temperature for 15 minutes. PRP was centrifuged at 250g for 20 minutes, and the supernatant was removed to obtain platelet precipitate. The expression of ASGR1 on the platelet membrane was detected by immunoblotting.

[0040] Figure 3 The results of Western Blot and flow cytometry were used to detect the expression of ASGR1 on the platelet membrane. Figure A is the Western Blot test result; Figure B is the flow cytometry test result. It can be seen from the figure that the expression of ASGR1 on the platelets of patients with coronary heart disease is significantly higher than that on the platelets of healthy controls. Platelets in patients with coronary heart disease are usually abnormally activated, so the results further show that the expression of ASGR1 increases in activated platelets.

[0041] Example 2

[0042] An application of ASGR1 in the preparation of a drug for inhibiting abnormal platelet activation is to achieve the purpose of inhibiting abnormal platelet activation by designing a reaction system for knocking out the ASGR1 gene. The gene cannot be artificially knocked out in humans, and when designing a drug, an antibody against the protein can be designed to neutralize it, thereby eliminating the effect of the protein.

[0043] Experiment 3: By designing an ASGR1 gene knockout mouse model, whether the application method of the present invention can knock out ASGR1 is investigated.

[0044] 1. Vector construction:

[0045] 1.1. Synthesize gRNA primers. The ASGR1 gene is located on chromosome 11 of mice, with a total of 9 exons, of which the start codon ATG is located in exon 2, and the stop codon TAG is located in exon 9. Exons 2-9 contain all coding regions, so exons 2-9 are used as the knockout target region, with a size of ~2741bp. The applicant's research team and Cyagen (Cyagen) used C57BL / 6N mice as the background and CRISPR / Cas9 technology to design sgRNA (gRNA1: ccacctagtcagatgtaaaaggg (SEQ ID NO.5), gRNA2: ccagcgcgcagttctcttctgtc (SEQ ID NO.6).

[0046] 1.2. Primer annealing.

[0047] 1.3. The annealing product is connected to the basic carrier.

[0048] 1.4. Transform the ligation product into competent cells and culture on plates overnight.

[0049] 1.5. Pick a single colony and screen the positive clones by colony PCR.

[0050] 1.6. The identified positive clones were verified by sequencing.

[0051] 1.7. The sequencing was correct, indicating that the CRISPR vector had been successfully constructed.

[0052] 1.8. Obtain the final vector and proceed with subsequent plasmid preparation.

[0053] 2. Plasmid preparation:

[0054] 2.1. Prepare the reagents and items required for plasmid preparation.

[0055] 2.2. Transform Escherichia coli with vector plasmid.

[0056] 2.3. Extract appropriate amount of plasmid by shaking the bacteria.

[0057] 2.4. Purify the plasmid using a plasmid purification kit.

[0058] 2.5. Sequencing to verify the plasmid.

[0059] 3. Preparation of RNA:

[0060] 3.1. Prepare the reagents and items required for transcription.

[0061] 3.2. Linearization of PX330 plasmid.

[0062] 3.3. Use an in vitro transcription kit to transcribe the linearized DNA into RNA.

[0063] 3.4. PCR amplification of gRNA and gel recovery of PCR products.

[0064] 3.5. Use an in vitro transcription kit to transcribe the PCR product into RNA.

[0065] 3.6. Use electrophoresis to identify whether the transcribed RNA is degraded.

[0066] 3.7. Purify the RNA that passes the electrophoresis test using an RNA purification kit.

[0067] 3.8. Identify the purified RNA by electrophoresis.

[0068] 3.9. The purified RNA is used for subsequent fertilized egg injection.

[0069] 4. RNA injection:

[0070] 4.1. Obtain fertilized eggs from the uterus of donor female mice 0.5 days after mating.

[0071] 4.2. Install the microinjection equipment and prepare the necessary reagents and items.

[0072] 4.3. Select fertilized eggs with good morphology and moderate development status and transfer them to the culture medium of the injection dish. 4.4. Dilute the RNA to be injected.

[0073] 4.5. Aspirate the RNA into the injection needle and inject it into the cell nucleus one by one.

[0074] 5. Preparation of surrogate mice and embryo transplantation:

[0075] 5.1. Select female mice of appropriate age and place them in cages with neutered male mice to obtain surrogate female mice.

[0076] 5.2. Transplant the RNA-injected fertilized eggs into the uterus of a surrogate mouse.

[0077] 5.3. Place the surrogate mother mouse in a clean cage and keep it warm until it wakes up and then put it back in the cage for feeding. 5.4. After the fertilized egg transplant is completed, wait for the mice to be born.

[0078] 5.5. When the mice are about one week old, cut their toes and send them for PCR testing. Number them and separate them into cages after 3 weeks.

[0079] 6. Identification of F0 mice:

[0080] 6.1. Extract genomic DNA and use Triton X-100 and protease mixture lysis buffer to lyse the sample overnight.

[0081] 6.2. The next day, inactivate the protease by high temperature, collect the supernatant by centrifugation and use it as a PCR template.

[0082] 6.3. PCR amplification and electrophoresis detection; the wild ASGR1+ / + mice (WT mice) showed one band at 659bp; the homozygous ASGR1- / - mice showed one band at 896bp; the heterozygous ASGR1+ / - mice showed two bands at 659bp and 896bp.

[0083] Figure 4 The results of Western Blot detection of ASGR1- / - mice and WT mice showed that there was no expression of ASGR1 in the liver and platelets of ASGR1- / - mice, proving that ASGR1 was successfully knocked out.

[0084] 6.4. Gel recovery of PCR fragments and sequencing identification.

[0085] 6.5. Analyze the sequencing results and write down the positive mouse numbers.

[0086] 7. F1 mouse breeding:

[0087] 7.1. Positive mice were 8 weeks old and were placed in the same cage with 8-week-old mice.

[0088] 7.2. Put the newborn pups into new cages and number them.

[0089] 7.3. When the mice are about one week old, cut their toes and send them for PCR testing for identification.

[0090] 8. Identification of F1 mice:

[0091] 8.1. Genomic DNA extraction, Triton X-100 and protease mixture lysis buffer, lyse the sample overnight.

[0092] 8.2. The next day, inactivate the protease at high temperature, collect the supernatant by centrifugation and use it as a PCR template.

[0093] 8.3. PCR amplification and electrophoresis detection; the detection method is the same as above.

[0094] 8.4. Gel recovery of PCR fragments and sequencing identification.

[0095] 8.5. Analyze the sequencing results and write down the numbers of the positive mice (ASGR1- / - mice).

[0096] Experiment 4: Investigating the effect of ASGR1 on platelet volume distribution width and platelet mean volume

[0097] 4.1. Investigate the effect of ASGR1 on the bleeding time of mouse tails. The specific steps are as follows:

[0098] (1) Three ASGR1- / - mice and three WT mice were selected and anesthetized by intraperitoneal injection of 1% sodium pentobarbital (60 mg / kg). The mice were placed in a prone position with their tails exposed for disinfection and immersed in warm water at about 37°C for 5 minutes to allow the blood vessels in the tail to be filled.

[0099] (2) Use a scalpel to quickly cut off a 3-5 mm portion of the tail and immediately immerse the tail in a 50 ml EP tube containing isotonic saline preheated to 37°C in a water bath.

[0100] (3) The timing starts from the moment the bleeding starts from the tail and ends when the bleeding stops.

[0101] 4.2. Detection of platelet count, volume distribution width and mean volume: Take ASGR1 - / - Three mice and three WT mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (60 mg / kg), and blood was collected by removing the eyeballs and placed in anticoagulation tubes. The number, volume distribution width and average volume of platelets were detected using a fully automatic blood cell analyzer (model specification: BC-6900 / BC-6800 / SC-120).

[0102] Figure 5 The results of the investigation of the effect of ASGR1 on platelet volume distribution width and platelet mean volume. A in the figure is the platelet count (PLT) result, B in the figure is the platelet volume distribution width (MPV) result, and C in the figure is the platelet mean volume (PDW) result. It can be seen from the figure that ASGR1 does not affect the generation of platelets. However, ASGR1 knockout significantly prolongs the bleeding time of the mouse tail, suggesting that ASGR1 may play a certain role in platelet activation.

[0103] Experiment 5: Investigate the effect of ASGR1 on platelet spreading on fibrinogen.

[0104] Experimental steps: 5.1. Take ASGR1 - / - Two mice and two WT mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (60 mg / kg), and blood was collected by removing the eyeballs. The blood was diluted with Tyrode's buffer at a ratio of 1:1.

[0105] 5.2. Centrifuge at 1000r / 12min, and take the supernatant as PRP; centrifuge PRP at 4500r / 5min, and the precipitate obtained is platelets. Resuspend the platelets with Tyrode's buffer and centrifuge at 4500r / 5min. Repeat this operation once to obtain washed platelets.

[0106] 5.3. Counting: The number of experiments is initially determined to be 1*10 per well. 6 indivual.

[0107] 5.4. Resuspend the platelets in Tyrode's buffer and adjust the quantity. Place in a water bath at 37°C for 1 to 2 hours.

[0108] 5.5. Group processing:

[0109] Group 1: platelets from WT mice, incubated in a water bath at 37°C in the dark for 10 min;

[0110] Group 2: platelets from WT mice were added with 10 mL of AA and incubated in a 37°C water bath in the dark for 10 min;

[0111] Group 3: platelets from ASGR1- / - mice were added with 10 mL of AA and incubated in a 37°C water bath in the dark for 10 min;

[0112] Group 4: Platelets from ASGR1- / - mice were incubated in a 37°C water bath in the dark for 10 min.

[0113] 5.6. Place a round cover slip in each well of a 24-well plate in each group, add 400 μl of fibrinogen (50 μg / ml), incubate at 37°C for 2 h, and recover the fibrinogen.

[0114] 5.7. Wash three times with 1X PBS at 40-50r / 3min, blot dry with filter paper, add 500μl 1% BSA blocking solution to each well and incubate at 37℃ for 30min, discard the BSA blocking solution and wash three times with PBS at 40-50r / 3min, blot dry with filter paper.

[0115] 5.8. Incubate 400 μl of platelet suspension in a 24-well plate at 37°C for 30 min.

[0116] 5.9. After discarding the liquid, add 500 μl of preheated 4% paraformaldehyde fixative (purchased from Sevier Biotechnology) and let it stand at room temperature in the dark for 30 minutes for fixation.

[0117] 5.10. Aspirate the fixative solution, wash three times with 1X PBS by shaking slowly at 10 r / 1 min, and absorb the remaining liquid with filter paper.

[0118] 5.11. Add 0.5% TritonX-100 (purchased from Abiwei) to treat for 5 minutes, discard the liquid, add 1XPBS for 10r / 1min to wash three times, and absorb dry with filter paper.

[0119] 5.12. Take 5 μl of the stock solution of phalloidin (purchased from Wuhan Mitsubishi Pharmaceutical Co., Ltd.) and add it to 150 μl 1X PBS to dilute it to the working solution concentration. Take 100 μl of the prepared phalloidin on the sealing film, clip out the round cover glass, absorb the residual liquid, and slowly cover the phalloidin with the side with platelets attached facing down (to avoid bubbles), and incubate at room temperature away from light for 30 minutes.

[0120] 5.13. Take out the round cover glass and put it back into the 24-well plate with the platelet-attached side facing up. Wash it five times with PBS by shaking slowly at 10r / 3min.

[0121] 5.14. Take a clean glass slide, add 5-10 μl of anti-fluorescence quenching sealing medium (purchased from Abiwei) in a light-proof environment, take out the round cover glass and put the side with cells attached on the sealing medium.

[0122] 5.15. Use gelatin mounting medium (purchased from Abiwei) to mount the slides and dry the slides at room temperature 27°C away from light.

[0123] 5.16. Observe using a laser confocal microscope.

[0124] Figure 6 The effect of ASGR1 on platelet spreading on fibrinogen was observed using a laser confocal microscope. As can be seen from the figure, when platelets are activated, glycoproteins are expressed in large quantities on the platelet membrane, becoming specific molecular markers of platelet activation. Glycoprotein GPIIb / IIIa is a member of the integrin family and is the final "signal integration molecule" that causes platelet aggregation in response to different stimuli. It is also involved in the adhesion process of platelets. Glycoprotein P-selectin is only located on the α-granule membrane in resting platelets, and is distributed in large quantities to the surface of the platelet membrane when platelets are activated.

[0125] Figure 7 The results of flow cytometry detection of the expression of GPIIb / IIIa and P-selectin on the platelet membrane. A, B, C, and D in the figure are the flow cytometry detection results of groups 1, 2, 3, and 4, respectively. The results showed that ASGR1 knockout significantly reduced the expression of GPIIb / IIIa and P-selectin on the platelet membrane induced by the activator AA.

[0126] The above results suggest that ASGR1 knockout alleviates platelet activation induced by activator AA.

[0127] Experiment 6: Flow cytometry was used to detect the effect of ASGR1 on the expression of GPIIb / IIIa and P-selectin on platelets.

[0128] Experimental steps: 6.1. Take ASGR1 - / -Four WT mice and four WT mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (60 mg / kg). Blood was collected by removing the eyeballs and dripped into an EP tube to which sodium citrate anticoagulant (purchased from Aladdin) had been added in advance, and the blood was diluted with normal saline at a ratio of 1:1.

[0129] 6.2. Centrifuge at 1000r / 12min, and take the supernatant as PRP; centrifuge PRP at 4500r / 5min, and the precipitate obtained is platelets. Resuspend the platelets in 1X PBS and centrifuge at 4500r / 5min. Repeat this operation once to obtain washed platelets.

[0130] 6.3. Add 1 ml of Tyrode's buffer to resuspend the precipitate, and use three-class platelet counting to adjust the platelet count to 1*10 6 The diluted platelets were placed at 37°C for 1 hour.

[0131] 6.3. Group processing:

[0132] Group 1: platelets from WT mice, incubated in a water bath at 37°C in the dark for 10 min;

[0133] Group 2: platelets from WT mice, supplemented with asialofetuin A 10 μg ml -1 Incubate in a 37°C water bath in the dark for 1 h;

[0134] Group 3: platelets from WT mice were added with 10 mL of AA and incubated in a 37°C water bath in the dark for 1 h;

[0135] Group 4: 10 mL of AA was added to the platelets of WT mice, incubated in a 37°C water bath in the dark for 10 min, and then asialofetuin A 10 μg ml was added -1 Incubate in a 37°C water bath in the dark for 1 h;

[0136] Group 5: platelets from ASGR1- / - mice were added with 10 mL of AA and incubated in a 37°C water bath in the dark for 10 min;

[0137] Group 6: Platelets from ASGR1- / - mice were added with 10 mL of AA and incubated in a 37°C water bath in the dark for 10 min; then asialofetuin A 10 μg ml -1 Incubate in a 37°C water bath in the dark for 1 h.

[0138] 6.3. Add 5 μL of flow cytometry antibody PE-P-selectin (purchased from BD Biosciences) and 5 μL of FITC-GPIIb / IIIa (purchased from BioLegend), and incubate in a 37°C water bath in the dark for 30 min.

[0139] 6.3. Add 1X PBS to make up to 300 μL and detect using flow cytometer.

[0140] Figure 8 The results of flow cytometry detection of the effect of ASGR1 on the expression of GPIIb / IIIa and P-selectin on platelets. As can be seen from the figure: ASGR1's natural high-affinity ligand asialofetuin A enhances the expression of platelet integrins GPIIb / IIIa and P-selectin induced by activators. The results of flow cytometry detection showed that asialofetuin A alone did not affect the expression of platelet membrane GPIIb / IIIa and P-selectin (A and B in the figure); however, asialofetuin A could significantly increase the expression of GPIIb / IIIa and P-selectin on the platelet membrane induced by activator AA (C and D in the figure); while ASGR1 knockout eliminated asialofetuin A to increase the expression of platelet membrane GPIIb / IIIa and P-selectin induced by activator AA (E and F in the figure). The above results suggest that ASGR1 binds to the asialofetuin A ligand, asialofetuin A, to enhance activator-induced platelet activation.

[0141] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as above in the preferred embodiment, it is not used to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A use of ASGR1 in the preparation of a drug for inhibiting abnormal platelet activation, characterized in that: The gene sequence of ASGR1 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The application method comprises: taking ASGR1 as a drug target, neutralizing the ASGR1 by designing an antibody that inhibits the action of the protein, and achieving the purpose of inhibiting abnormal platelet activation.

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