Compounds having antithrombotic activity and pharmaceutical use thereof

Abstract

The present application belongs to the technical field of biological medicine, and relates to a compound with antithrombotic activity and pharmaceutical use thereof. Experimental results show that the compound can significantly and stably inhibit the PLTP transfer activity in a safe concentration range without cytotoxicity; the compound can effectively inhibit the adhesion, spreading, activation and clot retraction function of platelets by blocking the integrin alpha IIb beta 3 mediated bidirectional signal transduction; the compound has good pharmacokinetic characteristics in mice, and can effectively inhibit the formation of arterial thrombosis and inflammation-driven venous thrombosis without significantly increasing the risk of bleeding.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a compound with antithrombotic activity and its pharmaceutical uses. Background Technology

[0002] Myocardial infarction has become a major global public health challenge, with extremely high morbidity and mortality rates. The core pathological basis of this disease lies in the rupture or erosion of atherosclerotic plaques in the arterial wall, which in turn induces the formation of acute occlusive thrombi. Platelets play a central role in both physiological hemostasis and pathological thrombosis. Once the vascular endothelium is damaged and the subendothelial matrix is ​​exposed, local platelet adhesion, "inside-to-outside" and "outside-to-inside" transmembrane signal transduction, degranulation and release, and eventual aggregation are rapidly initiated. Overactivation of this process directly leads to the occurrence of pathological thrombosis.

[0003] Antiplatelet therapy targeting the aforementioned pathways is currently the cornerstone of clinical prevention and treatment of cardiovascular events. However, existing traditional drugs face serious clinical limitations. Commonly used drugs, such as P2Y12 receptor antagonists like aspirin and ticagrelor, as well as integrin αIIbβ3 antagonists, generally exhibit varying degrees of drug resistance, gastrointestinal damage, and side effects. More importantly, these drugs excessively and singularly block platelet function, completely disrupting the physiological hemostasis barrier, leading to a significantly increased risk of massive bleeding and severe thrombocytopenia. Therefore, exploring novel mechanistic targets that balance potent antithrombotic efficacy with extremely low bleeding risk is particularly urgent for providing safer clinical antithrombotic intervention strategies.

[0004] The molecular formula is C 25 H 29 ClN2O4, chemically named 4-[(2-chlorophenyl)methyl]-1-cyclohexyl-3-(3,4-dimethoxyphenyl)-2,5-piperazinedione, has a molecular weight of 456.96. Currently available literature only discloses the physicochemical properties and synthetic methods of this compound; no reports on its pharmacological efficacy have been found. Summary of the Invention

[0005] The first objective of this invention is to provide a compound with antithrombotic activity, the compound having the following structural formula: .

[0006] A second object of the present invention is to provide pharmaceutical uses of the above-mentioned compounds, including: The application of the above compounds in the preparation of PLTP inhibitors.

[0007] The application of the above compounds in the preparation of drugs for treating myocardial infarction.

[0008] The above compounds are used in the preparation of drugs that inhibit platelet adhesion, spreading, activation, and clot retraction.

[0009] The platelet adhesion, spreading, activation, and clot retraction functions are achieved by blocking bidirectional signal transduction mediated by integrin αIIbβ3.

[0010] The application of the above compounds in the preparation of drugs for treating arterial thrombosis.

[0011] The application of the above compounds in the preparation of drugs for treating inflammation-driven venous thrombosis.

[0012] A third object of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned compound having antithrombotic activity, and a pharmaceutically acceptable carrier.

[0013] The dosage of the compound or its pharmaceutically acceptable salt described in this invention may vary depending on the route of administration, the patient's age, weight, the type and severity of the disease being treated, etc. The dosage range for treating the disease may be 0.001-20 mg / kg / day / person, preferably 0.003-10 mg / kg / day / person, and may be administered once or multiple times.

[0014] The carriers include common pharmaceutical diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc.

[0015] The pharmaceutical composition can be used orally or non-orally, such as by injection, spray, nasal drops, eye drops, penetration, absorption, or physical or chemically mediated methods to introduce it into the body, such as into muscles, intradermis, subcutaneous tissue, veins, or mucous membranes; or by being mixed with or encapsulated by other substances before being introduced into the body.

[0016] When used orally, it can be formulated into conventional solid dosage forms, such as tablets, powders, granules, capsules, ointments, creams, etc.; or into liquid dosage forms, such as water or oil suspensions or other liquid preparations, such as oral liquids. When used non-orally, it can be formulated into injections, etc.

[0017] A fourth object of the present invention is to provide pharmaceutical uses of the above-described pharmaceutical compositions, including: The application of the above-mentioned pharmaceutical composition in the preparation of PLTP inhibitors.

[0018] The above-mentioned pharmaceutical composition is used in the preparation of antithrombotic drugs.

[0019] The above-mentioned pharmaceutical composition is used in the preparation of drugs for treating myocardial infarction.

[0020] The above-mentioned pharmaceutical composition is used in the preparation of drugs that inhibit platelet adhesion, spreading, activation, and clot retraction.

[0021] The above-mentioned compounds with antithrombotic activity are used in the preparation of drugs for treating arterial thrombosis.

[0022] The above-mentioned compounds with antithrombotic activity are used in the preparation of drugs for treating inflammation-driven venous thrombosis.

[0023] Compared with the prior art, the present invention has the following beneficial effects: Experimental results show that: 1. Clinical plasma samples and non-targeted lipidomics results showed that the activity and content of PLTP in the plasma of patients with untreated first-time myocardial infarction (FMI) were significantly increased. P< 0.0001), and not linearly correlated with myocardial injury markers; this process is accompanied by a significant upregulation of a large number of glycerophospholipid metabolites, primarily lysophosphatidylcholine (LysoPC). PLTP - / - Further plasma lipidomics in mice confirmed that knockout of PLTP led to a significant downregulation of the abundance of several key glycerophospholipid molecules (including PC and PE derivatives) in vivo.

[0024] 2. Subcellular localization and cell separation and extraction analysis showed that PLTP is mainly specifically stored in the α-granules and dense granules of platelets, and is released in large quantities via the arachidonic acid pathway upon platelet activation. In the resting state, PLTP exhibits significant co-localization with cPLA2 and sPLA2; after activation, sPLA2 translocates and accumulates in membrane components. P= 0.0129), while the content of PLTP in both the membrane and cytoplasm decreased significantly.

[0025] 3. The novel inhibitor 5015, obtained through virtual screening and in vitro activity verification, can significantly inhibit the lipid transactivation activity of PLTP within a non-cytotoxic concentration range (2.5-80 μM), and reaches peak plasma concentration within 5 minutes after a single intravenous injection, achieving in vivo inhibition.

[0026] 4. In vitro functional experiments showed that 5015 significantly inhibited platelet adhesion to collagen surfaces and the expansion of fibrinogen surface area. Flow cytometry confirmed that pre-incubation with 5015 significantly reduced thrombin-induced P-selectin membrane expression and high-affinity conformation activation of integrin αIIbβ3. P< It reduced the platelet count by 0.0001 and significantly inhibited the late-stage clot retraction rate, effectively blocking the bidirectional signal transduction of platelets "from inside to outside" and "from outside to inside".

[0027] 5. In vitro efficacy and safety assessments showed that, compared with the positive control drug aspirin, 5015 did not significantly prolong the tail-cutting hemorrhage time in mice (P>0.05). Without a significant risk of bleeding, 5015 significantly reduced the FeCl3-induced pulsatility index (PI) of the common carotid artery in mice. P< 0.0001), and inhibited carrageenan-induced tail vein thrombosis (the proportion of thrombosis in the high-dose group decreased to 36.32%±1.71%). Attached Figure Description

[0028] Figure 1 The structural formulas are for four small molecule compounds (5015, 6292, 6639, 7035). Figure 2 The effect of four small molecule compounds (5015, 6292, 6639, 7035) on plasma PLTP activity under different pre-incubation times is shown in the figure. Figure 3 The effect of four compounds on plasma PLTP activity at different concentration gradients is shown in the figure. Figure 4 Representative images and quantitative analysis diagrams showing the effects of four small molecule compounds on the maximum amplitude (MA) index of coagulation function; Figure 5 Figure showing the effect of different concentrations of 5015 on platelet activity in humans and mice using the AlamarBlue assay; Figure 6 The figure shows the cytotoxic effects of different concentrations of 5015 on human and mouse platelets, as determined by a lactate dehydrogenase (LDH) release assay. Figure 7 Representative fluorescent images of human platelet adhesion stained with fluorescein isothiocyanate-labeled phalloidin; Figure 8 Representative fluorescent images of mouse platelet adhesion; Figure 9 A statistical analysis chart of the number of adherent platelets (left image: human platelets; right image: mouse platelets). Figure 10 Representative fluorescent images of human platelets stained with fluorescein isothiocyanate-labeled phalloidin; Figure 11 Representative fluorescent images of mouse platelets spread out; Figure 12 A quantitative statistical analysis chart of platelet spreading surface area (left image: human platelets; right image: mouse platelets). Figure 13The effect of 5015 on the expression of human platelet activation markers αIIbβ3 and JON / A and on clot retraction is shown in the flow cytometry graph for detecting the expression level of platelet surface P-selectin (CD62P). The left figure is a representative flow cytometry histogram, and the right figure is a quantitative statistical analysis graph of mean fluorescence intensity (MFI). Figure 14 The effect of 5015 on the expression of human platelet activation markers αIIbβ3 and JON / A and clot retraction is shown in the experimental cytology detection of integrin αIIbβ3 (labeled with JON / A antibody). The left figure is a representative histogram, and the right figure is a quantitative statistical analysis graph. Figure 15 Representative photographs of human platelet-rich plasma (PRP) clot retraction experiment, where the state of the blood clot was observed and photographed at regular intervals (0-60 min) under 37℃ conditions; Figure 16 A graph showing the quantitative statistical analysis of clot retraction rate; Figure 17 This is a pharmcokinetic profile of plasma drug concentration-time following a single intravenous injection (IV-dose) of compound 5015. Figure 18 The scatter plot shows the bleeding time of mice in each group. The aspirin group showed a significantly prolonged bleeding time, while the 5015 treatment group showed no significant difference (ns) compared with the control group. Figure 19 The graph shows the detection of PLTP activity in mouse plasma: the top graph is the kinetic curve of PLTP activity over time, and the bottom graph is the quantitative statistical graph of PLTP activity at 30 min and 60 min time points. The results show that 5015 significantly inhibited the lipid transconversion activity of PLTP in plasma. Figure 20 Doppler ultrasound images of carotid artery blood flow in each group (WT, Sham, Model, Vehicle, Aspirin, 5015-LD, 5015-HD) of mice in the experiment on the effect of 5015 on FeCl3-induced carotid artery thrombosis. Figure 21 This is a quantitative statistical analysis of the carotid artery pulsatility index in the experiment on the effect of 5015 on FeCl3-induced carotid artery thrombosis in mice. Figure 22 The images show representative images of tail thrombosis (manifested as blackening, necrosis and shriveling of the tail) in each group of mice during the experiment on the effect of 5015 on carrageenan-induced tail vein thrombosis in mice. The red scale indicates the length of the thrombus. Figure 23 This is a quantitative statistical analysis chart showing the black tail rate (thrombus length as a percentage of tail length) in mice during the experiment on the effect of 5015 on carrageenan-induced tail vein thrombosis in mice.

[0029] In the above figures, experimental results are expressed as mean ± standard error (Mean ± SEM), and P < 0.05 is considered statistically significant. P> 0.05 indicates no significant difference (ns). Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments, but these are not intended to limit the scope of the invention.

[0031] Unless otherwise specified, the contents of each component used below are weight percentages; all experimental methods used are conventional methods; and all reagents and biological materials used are commercially available.

[0032] In the technical solution of this invention, the compound Compound 5015 is also known as compound 5015.

[0033] Experimental methods 1.1 Clinical Sample Acquisition and Animal Experiments 1.1.1 Acquisition of Clinical Samples This study included healthy volunteers, patients newly diagnosed with acute myocardial infarction (AMI) who had not yet received antiplatelet therapy, and patients with AMI who had received dual antiplatelet therapy (aspirin and an ADP receptor antagonist), anticoagulation, and lipid-lowering therapy. This study has been approved by the Chinese Clinical Trial Registry, registration number: ChiCTR2400091043.

[0034] 1.1.2 Blood sample collection from patients with myocardial infarction before treatment (1) Samples were taken after the patient was diagnosed with AMI and before drug treatment was started. The median cubital vein was selected, and a disposable venous blood collection needle was used to puncture the vein and connect it to a purple-capped vacuum blood collection tube (EDTA anticoagulant tube) to collect 2-3 mL of venous whole blood. (2) Centrifuge at 1200×g for 5min to separate plasma and red blood cells. After separation, transfer plasma and red blood cells to EP tubes and immediately freeze them at -80℃ for detection of PLTP-related indicators.

[0035] 1.1.3 Blood sample collection from patients with myocardial infarction after treatment After completing a standardized treatment regimen for 5 consecutive days, and once the blood drug concentration has stabilized, collect 2-3 ml of whole blood from the patient, following the same procedure as in 1.1.2.

[0036] 1.1.4 Blood Sample Collection from Healthy Individuals Undergoing Physical Examinations Blood samples were collected from healthy individuals undergoing routine physical examinations, and the collection and preservation methods were as described above.

[0037] 1.1.5 Sources of human platelet-rich plasma Platelet-rich plasma is prepared using a blood cell apheresis machine, with a platelet concentration of 800-1000 × 10⁻⁶. 9 / L.

[0038] 1.1.6 Laboratory Animals C57 mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.; KM mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. PLTP Gene knockout PLTP - / - Mice were purchased from Cyagen Biosciences Co., Ltd. Under pathogen-free conditions, the animal room temperature was controlled at 24-26℃, the relative humidity at 50-60%, and mice were allowed free access to food and water, with a 12-hour day-night cycle.

[0039] 1.1.7 Genotyping of Laboratory Animals (1) Extraction of mouse DNA: The experimental mice were numbered and marked, and tissue samples were collected using standard toe clipping or ear piercing methods. DNA extraction was performed using a modified alkaline lysis method. The specific reagent preparation and operation steps are as follows: Alkaline lysis buffer (solution A): Weigh 0.01g NaOH and 0.029g EDTA, dissolve them in deionized water and bring the volume to 50mL to prepare a mixed solution containing 5mM NaOH and 2mM EDTA.

[0040] Neutralization buffer (Solution B): Measure 500 μL of 1M Tris-HCl solution, add deionized water to bring the volume to 50 mL, and adjust the final concentration to 10 mM Tris-HCl.

[0041] (2) Tissue lysis: Place mouse tissue in a 1.5 mL sterile centrifuge tube and mince it thoroughly with ophthalmic scissors. Add 100 μL of alkaline lysis buffer (Solution A) to the tube and incubate in a 95 °C metal bath or constant temperature water bath for 1 h to ensure complete lysis of tissue DNA.

[0042] (3) Neutralization and separation: After heat treatment, add 100 μL of neutralization buffer (Solution B) to the centrifuge tube, shake vigorously to mix and terminate the lysis reaction, centrifuge at 2000g, 4℃ for 10 min; (4) DNA collection: Carefully aspirate the supernatant and transfer it to a new 1.5 mL centrifuge tube (avoid aspirating tissue residue at the bottom). Store the obtained DNA sample at -20°C for subsequent PCR detection.

[0043] (5) Mouse genotyping Using extracted mouse genomic DNA as a template, a specific PCR reaction system was constructed based on the target gene sequence. After adding upstream and downstream primers and amplification reagents, PCR cycling was performed. A 1% agarose gel was constructed to separate the PCR amplification products. 10 μL of PCR amplification product was added to each well, and electrophoresis was performed at 120V for 25 min. After electrophoresis, the band migration was observed using a gel imaging system, and the genotype was identified (wild-type mice: a band was present at 530 bp;). PLTP - / - A band was observed at 340 bp in the mouse.

[0044] 1.2 Platelet Test 1.2.1 Extraction of human washed platelets (1) Transfer platelet-rich plasma (PRP) to a clean centrifuge tube, add prostaglandin E1 (PGE1) to a final concentration of 1 μM to inhibit activation, and centrifuge at 800 rpm for 10 min. (2) Discard the supernatant, add an appropriate amount of calcium-free Tyrode's buffer, gently blow and resuspend the precipitate, centrifuge at 2000 rpm at room temperature for 10 min, and repeat the above steps twice. (3) Discard the supernatant, gently resuspend the platelets at the bottom of the centrifuge tube using Tyrode's buffer (calcium-free), determine the platelet concentration, and adjust the platelet count to 500 × 10⁻⁶ using Tyrode's buffer (calcium-free). 9 Use per ml, let stand at room temperature for one hour.

[0045] 1.2.2 Preparation of washed mouse platelets Preparation of PGE1 working solution: Add 1 mg of PGE1 powder to 200 μL of anhydrous ethanol and dissolve thoroughly to obtain PGE1 stock solution. Before use, take an appropriate amount of stock solution and dilute with anhydrous ethanol at an appropriate ratio to obtain the working solution.

[0046] (1) Blood was collected from the inner canthal vein of mice using capillary blood collection tubes. The blood and sodium citrate were thoroughly mixed at a ratio of 9:1. An equal volume of calcium-free Tyrode's Buffer was added and mixed. PGE1 working solution with a final concentration of 50 ng / mL was added and gently mixed. The blood from two mice was combined into a 10 mL centrifuge tube. (2) Centrifuge at 200×g at room temperature for 20 min, aspirate the upper light white liquid, add PGE1 with a final concentration of 50 ng / mL, and mix well; (3) Centrifuge at 800×g at room temperature for 10 min, discard the supernatant, add EDTA to a final concentration of 1 mM and PGE1 to 50 ng / mL, gently pipette and resuspend, combine the platelet suspensions from each tube, the total volume is about 6 mL. (4) Centrifuge at 800×g at room temperature for 10 min, discard the supernatant, retain the precipitate at the bottom of the tube, and resuspend it with an appropriate amount of calcium-free Tyrode's Buffer according to the cell density requirements of subsequent experiments, so that the platelets are evenly distributed in the buffer. The prepared platelet suspension can be used after standing at room temperature for 30 min.

[0047] 1.2.3 Thromboelastography detection (1) System startup and preparation: Turn on the power of the thromboelastography (TEG) power analyzer and start the supporting software. Complete the system login and self-test procedures to ensure that the instrument is ready.

[0048] (2) Sample processing: After the kaolin reagent has returned to room temperature, take 1 mL of platelet-rich plasma (PRP) and inject it into the reagent bottle. Tighten the cap and gently invert the bottle 5 times to mix. Strictly avoid violent shaking during the operation.

[0049] (3) Loading the test cup: Install the micropipette kit into the instrument cup slot, tighten the cup cap, and press the cup body into the cup slot to ensure a tight fit without any looseness.

[0050] (4) Construction of reaction system: Add 20 μL of 0.2M calcium chloride and 330 μL of blood sample to the preheated test cup.

[0051] (5) Data acquisition: Raise the cup holder to the fixed position and move the level lever to the Test position to start the detection. The system automatically records the coagulation process until a stable maximum amplitude (MA) parameter is recorded.

[0052] 1.2.4 Platelet membrane extraction Platelet membranes were extracted using a modified repeated freeze-thaw method, and the specific steps are as follows: (1) PRP purification: In order to further remove residual red blood cells, the collected PRP was centrifuged at low speed of 100-200×g for 20min at room temperature, and the supernatant was retained. (2) Platelet (PLT) isolation: PBS buffer containing 1 mM EDTA and 2 µM prostaglandin E1 (PGE1) was added to the purified PRP to inhibit platelet activation during preparation. After mixing, the mixture was centrifuged at 800-900×g for 20 min at room temperature, the supernatant was discarded, and the white platelet precipitate at the bottom was collected. (3) Extraction of PLM: The platelet precipitate was resuspended in PBS containing protease inhibitor and 1 mM EDTA and frozen at -80°C for 1 h, then thawed at room temperature. The thawed suspension was centrifuged at 5000×g for 5 min to collect the precipitate. To ensure the acquisition of purified platelet membrane, the above "freeze-thaw-centrifuge" cycle should be repeated at least 3 times. The final product was stored at -80°C for later use.

[0053] 1.2.5 Platelet motility test Platelet metabolic activity was determined using the AlamarBlue fluorescence method. The density of the prepared washed platelet suspension was adjusted to 3 × 10⁻⁶. 7 The sample was then mixed with inhibitors or solvents of varying concentrations at a concentration gradient, and pre-incubated at 37°C for 10 min to ensure sufficient interaction between the drug and platelets. After pretreatment, AlamarBlue reagent was added to the reaction system, and the samples were incubated at 37°C in the dark for another 4 h. After the reaction, the fluorescence intensity of each well was detected using a microplate reader (excitation wavelength 550 nm, emission wavelength 590 nm); the intensity of this fluorescence signal directly corresponds to the metabolic activity of platelets, thus characterizing their survival level.

[0054] 1.2.6 Drug cytotoxicity assay To assess whether the small molecule inhibitor 5015 is toxic to platelets, this study used the lactate dehydrogenase (LDH) release assay. Washed platelets were incubated with different concentrations of 5015 or an equal volume of lysozyme at 37°C for 30 minutes. After incubation, the supernatant was collected by centrifugation to determine the extracellular LDH level. Simultaneously, an equal volume of platelet suspension was completely lysed using 0.3% Triton X-100 as a positive control for total LDH activity. Quantitative analysis of LDH was strictly performed according to the kit instructions (based on the 2,4-dinitrophenylhydrazine colorimetric method). In the reaction system, the sample was incubated sequentially with matrix buffer and coenzyme I at 37°C for 15 minutes, followed by the addition of 2,4-dinitrophenylhydrazine for another 15 minutes. After the colorimetric reaction was complete, the reaction was terminated with 0.4 mM NaOH solution, and the readings were stabilized by standing at room temperature for 5 minutes. Finally, the optical density (OD) value at a wavelength of 450 nm was measured using a microplate reader, and the LDH release rate was calculated according to the following formula to quantify cytotoxicity: N: Dilution factor of the sample before testing; C standard: standard solution concentration, 0.2 μmol / mL; 1000: unit conversion, mL→L; 1.2.7 Platelet Adhesion Test (1) Collagen coating: A 14 mm round coverslip was placed in a 24-well plate. 250 μL of collagen solution (5 μg / mL) and 1% BSA (boiled and cooled) were added to the experimental group and the negative control group, respectively, and incubated overnight at 4°C. (2) Blocking: Wash thoroughly three times with PBS buffer to remove residual collagen and BSA. Block with pre-cooled 1% BSA at room temperature for 1 hour to effectively block non-specific binding sites; (3) Platelet adhesion: After blocking, add 250 μL of washed platelet suspension pretreated with different concentrations of 5015 or solvent (30 min) to the wells washed with PBS. Transfer the culture plate to a 37°C incubator and incubate for 1 hour to allow it to fully contact the collagen and adhere. Wash with PBS preheated to 37°C to remove unadhered platelets. (4) Fixation: Add 4% paraformaldehyde (PFA) and fix for 15 minutes; (5) Membrane permeation: After washing with PBS, permeabilize the membrane with 0.5% Triton X-100 for 15 minutes; (6) Staining: Immediately add fluorescein isothiocyanate-labeled phalloidin to a final concentration of 100 nM, stain for 1 hour at room temperature in the dark, and mount with anti-fluorescence quenching mounting medium. (7) Image acquisition and data analysis: Five fields of view were randomly selected under a fluorescence microscope to acquire images, and the number of adhered platelets was quantitatively counted using ImageJ software.

[0055] 1.2.8 Platelet spreading test (1) Fibrinogen coating: Fibrinogen was used to construct a simulated damage surface. After placing a 14 mm slide in a 24-well plate, a fibrinogen solution diluted with NaHCO3 (50 μg / mL) was added and the plate was coated overnight at 4°C. (2) Blocking: After washing with PBS, block with 1% BSA for 1 hour; (3) Platelet spreading: Washed platelets were pre-incubated with 5015 at 37°C for 30 minutes. Then, the pretreated platelet suspension was mixed with 1mM calcium chloride and thrombin and added to the well plate, and incubated at 37°C for 1 hour. (4) Fixation: After the reaction, remove the non-adherent cells and fix them with 4% PFA for 15 min; (5) Membrane permeation: After washing with PBS, permeabilize the membrane with 0.5% Triton X-100 for 15 minutes; (6) Staining: Immediately add fluorescein isothiocyanate-labeled phalloidin to a final concentration of 100 nM, stain for 1 hour at room temperature in the dark, and mount with anti-fluorescence quenching mounting medium. (7) Image acquisition and data analysis: Observe under a fluorescence microscope, randomly acquire 5 field-of-view images for each group, and statistically analyze the spreading area (Surface Area) of a single platelet.

[0056] 1.2.9 Expression of platelet p-selectin and JON / A (1) Platelet pretreatment and drug intervention: Washed platelets were resuspended in calcium-free 1×Tyrode Buffer and the cell density was adjusted to 5x10⁻¹. 7 / mL. Take 250μL of cell suspension and place it in a 1.5mL EP tube. Add solvent control (Vehicle) or different concentrations of 5015 respectively, and incubate at 37℃ for 5min. (2) Agonist activation: Take 50 μL of sample from each of the above pretreatment systems into a new EP tube, add thrombin (final concentration 0.02 U / mL) to induce platelet activation, and incubate at 37°C for 5 min; (3) Fluorescent antibody labeling: CD62P antibody labeled with fluorescein isothiocyanate or JON / A antibody labeled with PE were added respectively, and the mixture was reacted at room temperature in the dark for 15 min. (4) Detection and analysis: After the reaction was completed, 250 μL of PBS was added to each tube to stop the reaction and mix well, then transferred to flow cytometry tubes. Data were collected using an Agilent flow cytometer and statistically analyzed using FlowJo 7.0 software.

[0057] 1.2.10 Blood clot retraction experiment (1) Extract PRP and PPP, and dilute PRP with PPP to maintain the final platelet concentration at 250 × 10⁻⁶. 9 / mL; (2) Take 400 μL of PRP after concentration adjustment and add it to the system containing 5015 solution of different concentrations, and incubate at 37°C for 10 minutes. (3) Add calcium chloride (final concentration 2mM) to the mixture and mix well, then add 1U / mL thrombin and gently invert to mix. Take 300μL of the mixture and place it in a transparent reaction vessel to stand; (4) Take a picture of the reaction cup every 5-10 minutes to continuously observe the contraction and size of the blood clot.

[0058] (5) Area measurement and analysis: The area of ​​the blood clot was extracted and calculated using ImageJ image processing software. The formula for calculating the shrinkage rate is: shrinkage rate (%) = 100 - [(area of ​​blood clot after shrinkage / initial total area of ​​sample) × 100].

[0059] 1.2.11 Immunofluorescence detection of the localization of PLTP and PLA2 in platelets (1) The steps for extracting washed platelets are as described above. After incubating the washed platelets in a 24-well plate containing a 14 mm round coverslip for 1 h, fix them at room temperature for 10 min with 300 μL of 4% paraformaldehyde solution. (2) Grouping: 1) PLTP and cPLA2 co-positioning detection group; 2) PLTP and sPLA2 co-positioning detection group; (3) Perforation: Add 0.5% Triton X-100 solution into the well and allow it to permeate for 15 minutes to fully perforate the platelet membrane; (4) Blocking: Add 5% BSA blocking solution and block for 1 hour; (5) According to the purpose of detection, dilute the primary antibody with antibody diluent (anti-PLTP antibody working concentration 1:1000, anti-cPLA2 antibody working concentration 1:500, anti-sPLA2 antibody working concentration 1:500), add blocking solution without any antibody to the platelet area of ​​the shadow control smear, and incubate overnight at 4°C; (6) Wash three times with PBS buffer, five minutes each time; (7) Use antibody dilution buffer to dilute the fluorescently labeled secondary antibody solution to the optimal working concentration, add 100 μL of fluorescent secondary antibody to each well, and incubate at room temperature in the dark for 1 h; (8) Discard the secondary antibody and wash three times with PBS buffer, 5 minutes each time, in the dark; (9) Wash with PBS for 5 min, repeat 3 times; (10) Mounting: Add one drop of anti-fluorescence attenuation mounting medium to each smear, cover with a coverslip, and ensure that the coverslip and slide are tightly fitted without air bubbles. After drying, observe under an inverted fluorescence microscope and randomly select 5 fields of view to take pictures.

[0060] 1.3 Animal Experiments 1.3.1 Carrageenan-induced mouse tail thrombosis model (1) Grouping and administration Twenty-five male KM mice were randomly divided into five groups (n=5): a blank control group, a solvent control group (Vehicle), an aspirin positive control group (50 mg / kg), a low-dose 5015 group (20 mg / kg), and a high-dose 5015 group (40 mg / kg). During the pretreatment phase before modeling, all mice received intraperitoneal injections once daily for seven consecutive days. The blank control group and the solvent control group received an equal volume of physiological saline or solvent, while the other groups received the corresponding dose of the drug solution.

[0061] (2) Modeling and testing One hour after the last administration, except for the blank control group, all other groups of mice were intraperitoneally injected with 1% carrageenan solution to induce acute thrombosis. To further promote stable thrombus formation, the injected mice were housed in a constant temperature environment at 18°C. The observation period lasted until 24 hours after modeling. The black tail length (i.e., thrombus formation length) and total tail length of the mice were measured, and the black tail rate (%) was calculated to quantify the antithrombotic activity of the drug.

[0062] 1.3.2 FeCl3-induced mouse carotid artery thrombosis model (1) Grouping and administration of experimental animals Thirty male C57 mice (18-22g) were selected and fasted the night before the experiment, with free access to water. After weighing, the mice were randomly divided into 6 groups: blank control group, sham-operated group, model group, solvent control group, and low-dose (2mg / kg) and high-dose (5mg / kg) 5015 groups. All drugs were administered via tail vein injection. The solvent control group and model group were injected with the corresponding solvent or physiological saline, respectively.

[0063] (2) Modeling Thirty minutes after drug administration, mice were anesthetized by inhalation with 2% isoflurane. Once anesthesia took effect, the mice were fixed in a supine position on the operating table. The neck area was prepared and incised along the midline to expose the subcutaneous tissue. Using forceps, the carotid artery was dissected until it was fully exposed, and the fascia and connective tissue attached to the vessel were carefully dissected. A 1.5cm × 0.5cm plastic film (cling film) was placed under the freed carotid artery to protect the surrounding tissue. Subsequently, strips of filter paper (2mm × 1mm) were soaked in a 10% ferric chloride (FeCl3) solution, removed, and directly applied to the surface of the artery for 3 minutes to induce thrombus formation.

[0064] (3) Doppler ultrasound detection of carotid artery blood flow Immediately after 3 minutes, remove the filter paper strip and place the Doppler ultrasound probe at the lower edge of the carotid artery to be tested. Carefully insert the blood vessel into the probe's detection groove. Then, apply an appropriate amount of ultrasound coupling agent to the contact gap between the probe groove and the blood vessel wall, ensuring no air bubbles obstruct signal transmission. Start the recording software's "Start" program to monitor and record the dynamic changes in mouse carotid artery blood flow in real time. Recording continues until a significant attenuation of the blood flow signal is observed and it no longer fluctuates (indicating vascular occlusion); if no obvious downward trend in blood flow is observed within 45 minutes, this time point is used as the cutoff point for recording.

[0065] 1.3.35015 Effect on Tail Bleeding Time in Mice (1) Grouping and administration: Twenty male C57 mice (18-22g) were selected, weighed, and randomly divided into four groups: a blank control group, an aspirin positive control group, and low-dose (2mg / kg) and high-dose (5mg / kg) 5015 groups. All drugs were administered via tail vein injection, while the control group received an equal volume of physiological saline.

[0066] (2) Modeling and testing: Thirty minutes after administration, the mouse tail was swiftly cut transversely 3 mm from the tip using a surgical scalpel, and the timing was immediately started. Subsequently, every 15 seconds, the cut was gently aspirated with the edge of filter paper until complete cessation of bleeding was observed (defined as no rebleeding within 1 minute). To avoid excessive blood loss in the animals, if bleeding lasted longer than 30 minutes, data were recorded consistently over 30 minutes.

[0067] 1.4 Cell Experiments 1.4.1 Cell resuscitation (1) Take out the MEG-01 cell cryopreservation tube and immediately immerse it in a 37°C constant temperature water bath for rapid thawing, ensuring that the cryopreservation solution is completely thawed within 1-2 minutes; (2) Transfer the thawed cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 5 min at room temperature. After centrifugation, carefully remove the supernatant and keep only the cell pellet at the bottom. (3) Add 15 mL of fresh MEG-01 cell culture medium to the cell pellet and gently pipette along the tube wall to prepare a uniform single-cell suspension. Then, inoculate the suspension into a sterile culture dish and gently shake the bottle in a "cross" motion to distribute the cells evenly. Incubate in a 5% CO2 37℃ constant temperature incubator.

[0068] 1.4.2 Cell passage (1) When the cell density reaches 80%, the cells are passaged. Use a pipette to aspirate the cell suspension from the culture flask, gently blow on the flask wall to allow the cells to detach completely, transfer the cell suspension to a sterile centrifuge tube, and centrifuge at 1000 rpm for 5 min at room temperature. After centrifugation, carefully remove the upper layer of old culture medium, retaining the cell pellet at the bottom of the tube.

[0069] (2) Add 10 mL of fresh MEG-01 culture medium to the cell pellet and gently pipette to prepare a cell suspension. At a passage ratio of 1:3, inoculate equal volumes of the suspension into three new sterile culture dishes, and replenish each dish with 15 mL of the medium. Gently shake the culture dishes horizontally using the "cross-shading method" to distribute the cells evenly, label them, and place them in a 5% CO2 incubator at 37°C for incubation.

[0070] 1.4.3 Cell cryopreservation Cells in the logarithmic growth phase with a cell density greater than 80% were selected for cryopreservation. Following the previously described method, the cells were gently pipetted and prepared into a suspension, transferred to centrifuge tubes, and centrifuged at 1000 rpm for 5 min at room temperature. The supernatant was carefully aspirated, taking care not to aspirate the sediment at the bottom of the tube, and 1 mL of cell cryopreservation medium was added to resuspend the cells. The cell suspension was aliquoted into sterile cryovials and stored in an ultra-low temperature freezer for later use.

[0071] 1.4.4 Phorbolone (PMA)-induced MEG-01 cell differentiation into platelet microparticles model (1) Preparation of PMA solution 1) PMA storage solution: Dissolve 1 mg PMA in 1 mL of cell culture grade dimethyl sulfoxide (DMSO), aliquot it into 1.5 mL EP tubes (100 μL / tube), and store at -20 °C protected from light.

[0072] 2) PMA working solution: Dilute the above PMA stock solution with RPMI 1640 basal medium to prepare a PMA working solution with a concentration of 1000 ng / mL. Prepare and use immediately.

[0073] (2) Cell grouping and pretreatment 1) Cell seeding: Take MEG-01 cells in the logarithmic growth phase, count them, adjust the density, and seed at 5 × 10⁶ cells / year. 5 The inoculum was evenly seeded into a 6-well cell culture plate at a rate of 1 cell per well. 2) Cell starvation treatment: Before PMA induction, MEG-01 cells were starved in serum-free RPMI 1640 medium for 6 hours, and then different concentrations of PMA were added to induce MEG-01 cells for 72 hours. Cell grouping: MEG-01+0ng / mLPMA MEG-01 + 10ng / mLPMA MEG-01 + 20ng / mLPMA MEG-01 + 40ng / mLPMA MEG-01 + 80ng / mLPMA 1.5 PLTP Activity Assay 1.5.1 Preparation of fluorescent donors Dissolve 10 mg NBD-PE and 2 mg PC together in 2 mL chloroform and dry under nitrogen to remove organic solvent. Resuspend the NBD-PE and PC mixture in 10 mL LTSE buffer (components: 10 mM Tris, 0.15 M NaCl, 2 mM EDTA). To obtain a homogeneous system, sonicate the mixture at room temperature for 30 min until the solution becomes clear and transparent pale yellow, then store it at room temperature in the dark for later use.

[0074] 1.5.2 Receptor Preparation The density-adjusted emulsion was used as the lipid receptor in the experiment. A KBr solution with a density of 1.21 g / mL was prepared by fully dissolving 333.394 g of KBr in 1000 mL of ddH2O. Subsequently, milk and the KBr solution were mixed evenly at a volume ratio of 1:4, and the resulting suspension was used as the standard receptor in the PLTP activity assay.

[0075] 1.5.3 Assay for lipid transconverting activity The transliposome activity of PLTP was monitored by fluorescence kinetics using black flat-bottomed microplates. The reaction system consisted of 3 μL of liposome donor, 3 μL of acceptor, and 91 μL of TSE buffer. 3 μL of fresh plasma sample was added to each experimental well, while an equal volume of TSE buffer was used as a blank control. After mixing, the mixture was incubated at 37°C. The excitation wavelength was set to 460 nm and the emission wavelength to 530 nm. Fluorescence readings were collected every 10 min for at least 1 h. After the reaction, 100 μL of 100% isopropanol was injected into each well to disrupt the liposome structure and release total fluorescence. The final fluorescence intensity was measured for data analysis.

[0076] PLTP activity calculation formula: PLTP activity (pmol / well) = (m×F) s ×Vdonor) / (M r ×F t (×Vtotal)×100%.

[0077] In the above PLTP activity calculation formula, m is the total mass of NBD-PE in the donor solution; Mr is the relative molecular weight of NBD-PE; Vdonor is the donor volume added to the single-well reaction system; Vtotal is the total volume of the donor solution; Fs is the fluorescence value of the sample to be tested; and Ft is the total fluorescence value after isopropanol treatment.

[0078] 1.6 Molecular Experiments 1.6.1 Total RNA extraction and platelet count adjustment (1) Centrifuge to collect cells, discarding the supernatant. Repeat every 5-10 × 10⁻⁶ cells. 6 Add 1 ml of lysis buffer RZ to the cells.

[0079] (2) Place the homogenized sample at room temperature for 5 minutes to allow the nucleic acid protein complex to be completely separated.

[0080] (3) Add 200 μl of chloroform, cover the tube, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes.

[0081] (4) Centrifuge at 12,000 rpm for 10 min at 4℃, aspirate the RNA from the aqueous phase and transfer it to a new tube.

[0082] (5) Add 0.5 times the volume of anhydrous ethanol and mix well. Transfer the resulting solution and precipitate together into an adsorption column and centrifuge at 12,000 rpm for 30 s at 4°C; (6) Add 500 μl of protein removal solution RD to the adsorption column, centrifuge at 12000 rpm for 30 s at 4℃, and discard the waste liquid.

[0083] (7) Add 500 μl of wash buffer RW to the adsorption column, let stand at room temperature for 2 min, and centrifuge at 12000 rpm for 30 s at 4℃. Repeat the above operation once.

[0084] (8) Place the adsorption column into the collection tube and centrifuge at 12,000 rpm for 2 min at 4°C to remove residual liquid.

[0085] (9) Transfer the adsorption column into a new 1.5 mL centrifuge tube, add 30 μL LNase-Free ddH2O, place at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min at 4℃.

[0086] (10) The obtained RNA was aliquoted and stored. 5 μL was used for concentration detection, and the rest was stored in a -80℃ freezer for later use.

[0087] 1.6.2 RNA Reverse Transcription RNA reverse transcription will be performed, followed by real-time quantitative PCR (qPCR) to detect the expression of megakaryocyte / platelet-specific surface markers CD41 and CD61.

[0088] 1.6.4 PLTP Plasma Concentration Detection (ELISA) (1) Centrifuge the standard at 12000×g for 30s, add 1mL of diluent to dissolve it completely, and set aside. Prepare six gradient standard samples of different concentrations using the serial dilution method, with pure diluent as blank control well and sample diluent as blank control.

[0089] (2) Dilute the biotin-labeled antibody, horseradish peroxidase-labeled avidin, etc. according to the dilution ratio provided in the kit instructions; (3) Before the formal test, the plasma sample needs to be diluted 1:200. All subsequent sample addition and incubation steps must be strictly performed in accordance with the standard steps of the kit.

[0090] 1.6.5 Western blotting assay (1) Platelet protein extraction: The concentration of washed platelets was adjusted to 3×10⁻⁶. 8 After incubating the plate under the aforementioned experimental conditions for 1 hour, remove the supernatant and wash three times with PBS buffer. Then, add 100 μL of lysis buffer containing the protease and phosphatase inhibitor RIPA to each well. Use a cell scraper to evenly scrape platelets from the bottom of the plate, lysing them as you scrape, and lyse on ice for 30 min. After lysis, centrifuge at 12000×g for 30 min at 4°C and collect the supernatant.

[0091] (2) Protein denaturation: Add 5× Loading buffer to the platelet lysis buffer at a ratio of 1:4, mix well, centrifuge, 95℃ for 15 min - 4℃ for 30 min to completely denature the protein, aliquot the denatured protein and store it at -80℃.

[0092] (3) Polyacrylamide gel electrophoresis (SDS-PAGE): Gel preparation: Align and fix clean glass plates of different lengths on the gel casting base, ensuring a good seal. After gently mixing the separating gel components, slowly pour them along the edge of the glass plate to the predetermined height, and immediately cover the liquid surface with a layer of isopropanol to isolate air and flatten the gel surface. After the separating gel solidifies, remove the top covering liquid, pour in the stacking gel solution and vertically insert the sample comb of the corresponding size, and let it stand until the gel is completely solidified for later use.

[0093] (4) SDS-PAGE electrophoresis separation: After the separating gel and stacking gel solidify, fix the glass plate to the electrophoresis tank core and inject the electrophoresis buffer. Remove the comb and add 10 μL of denatured protein sample and 5 μL of standard protein marker. The electrophoresis program is set in two steps: first, run at a constant voltage of 80V for 20 min to allow the sample to accumulate into a line in the stacking gel, then increase the voltage to 120V and continue electrophoresis for about 1 h until the bromophenol blue indicator migrates to the bottom of the gel, and then end the electrophoresis.

[0094] (5) Transfer: Immediately after separation, transfer is performed. The PVDF membrane is pre-activated by immersing it in methanol for 5 minutes, and a pre-cooled transfer buffer is prepared. The gel plate is removed to remove the stacking gel, and a transfer sandwich structure of "sponge pad-filter paper-gel-PVDF membrane-filter paper-sponge pad" is constructed. Air bubbles must be strictly eliminated during the operation. The transfer clamp is placed in the tank with the black side facing the negative electrode. Under ice-water bath cooling conditions, the membrane is transferred at a constant current of 300mA for 1 hour.

[0095] (6) Immunoblot reaction and detection: The transferred PVDF membrane was immersed in protein-free rapid blocking solution and blocked on a shaker at room temperature for 1 h to block non-specific binding sites. Subsequently, the membrane was placed in a proportionally diluted primary antibody incubation box and incubated overnight on a shaker at 4°C. The next day, the primary antibody was recovered and the membrane was washed 4 times (5 min each time) with TBST buffer, and the corresponding secondary antibody was added and incubated at room temperature for 1 h. After washing with TBST (4 × 5 min), the membrane surface was wetted with a 1:1 prepared ECL chemiluminescent reagent. Band signals were acquired in a gel imaging system, and the gray values ​​of each protein band were quantitatively statistically analyzed using ImageJ software.

[0096] 1.6.6 Dotblotting (1) Spotting: Cut the nitrocellulose membrane to a suitable size and draw a 1cm × 1cm square grid on the membrane surface. Take 2 μL of sample and drop it vertically into the center of each grid. (2) Air drying: Transfer the membrane after spotting to a 37°C constant temperature incubator and let it stand until the sample droplets are completely air dried; (3) The blocking and antibody incubation methods are the same as those used in Western blotting; (4) Use ImageJ software to calculate the gray values ​​of each band and perform statistical analysis.

[0097] 1.7 Statistical Methods Experimental data were statistically analyzed using GraphPadPrism 8.0 software. Independent samples t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons of three or more groups. All experimental results are expressed as mean ± standard error (Mean ± SEM), and p < 0.05 was considered statistically significant.

[0098] Experimental results 1. In patients with their first myocardial infarction, elevated plasma PLTP originates from platelets, and its mediated glycerophospholipid metabolism is related to thrombus formation. Troponin is a classic and specific biomarker widely used clinically to assess the degree of myocardial ischemia and necrosis. Correlation analysis of plasma PLTP concentration and corresponding troponin levels in patients with acute myocardial infarction (FMI) showed no significant linear correlation. This key result suggests that the dramatic increase in PLTP in the peripheral blood of myocardial infarction patients is not due to passive release from necrotic cardiomyocytes, but is more likely closely related to the initiating step in the pathogenesis of acute myocardial infarction—namely, acute thrombosis triggered by plaque rupture in the coronary arteries and massive platelet activation.

[0099] Untargeted lipidomics analysis was performed on plasma samples from patients in the normal control group, the FMI group, and the antiplatelet-treated group (FMI-t). Metabolite classification revealed a high enrichment of lipid molecules, with glycerophospholipids being the dominant group. Numerous differentially expressed metabolites were observed in patient plasma before and after antiplatelet therapy. Analysis of differentially expressed glycerophospholipids revealed a significant upregulation of the expression of multiple lysophosphatidylcholine (LysoPC) molecules during the untreated acute phase of myocardial infarction (FMI group). LysoPC is known to be an important intermediate metabolite in the hydrolysis of cell membrane phospholipids to produce arachidonic acid (AA), a key pro-inflammatory substance and a platelet-activating factor. Its significant reversion after antiplatelet therapy further demonstrates the close relationship between plasma lipid metabolic remodeling and the highly activated state of platelets in vivo.

[0100] Using PLTP whole gene knockout (PLTP) - / - In mice, DNA was extracted from tail tissue and subjected to PCR amplification and agarose gel electrophoresis. Homozygous individuals with the target fragment of 313 bp (PLTP) were successfully identified and screened. - / - PLTP knockout was performed on wild-type (WT) control mice and wild-type (WT) control mice containing the 211 bp target fragment. Subsequently, plasma lipidomics sequencing was performed on the plasma of both genotypes of mice. KEGG topology analysis showed that significantly differentially expressed metabolites induced by PLTP knockout were highly enriched in the glycerophospholipid metabolism pathway. Quantitative analysis confirmed that, compared with the wild-type control, PLTP... - / - The relative abundance of several key glycerophospholipid molecules (including various PC and PE derivatives) in mouse plasma was significantly downregulated. These in vivo data fully demonstrate the crucial role of PLTP in maintaining glycerophospholipid metabolism, providing a basis for its participation in regulating platelet lipid membrane signaling and subsequent thrombus formation.

[0101] 2. Colocalization of PLTP with cPLA2 and sPLA2 in platelets and changes in subcellular distribution after activation. The spatial relationship study of PLTP with cytoplasmic phospholipase A2 (cPLA2, PLA2G4A) and secretory phospholipase A2 (sPLA2, PLA2G2A) showed that, under resting conditions, PLTP, cPLA2 and sPLA2 all had significant colocalization signals in platelets, suggesting that they may have functional interactions.

[0102] The protein changes under different thrombin stimulation times were detected using Dotblot technology. The results showed that PLTP protein expression was most significant and stable at 5 min of thrombin stimulation. Therefore, 5 min was selected as the standard stimulation time for subsequent experiments.

[0103] In experiments on the separation and extraction of platelet membrane proteins and cytoplasmic proteins, Western blotting results showed that PLTP, cPLA2, and sPLA2 were distributed in both membrane and cytoplasmic components under resting conditions. However, after activation by thrombin stimulation, the distribution of these proteins changed significantly.

[0104] In the membrane components, compared with the resting group, the levels of cPLA2 (PLA2G4A) and PLTP proteins on the platelet membrane of the activated group were significantly reduced. P =0.0313 and P =0.0120), while the film distribution of sPLA2 (PLA2G2A) increased significantly ( P =0.0129). In the cytoplasmic components, the protein level of PLTP also showed a significant decrease after activation ( P =0.0269); while the cytoplasmic content of cPLA2 and sPLA2 showed no statistically significant difference before and after activation (ns). These results suggest that platelet activation is accompanied by the translocation and enrichment of sPLA2 onto the membrane, as well as the consumption of PLTP in the membrane and cytoplasm, which may be closely related to the release of PLTP-mediated lipid transport signals.

[0105] 3. Establishment of a PMA-induced MEG-01 cell differentiation model into platelet granules and subcellular localization analysis of PLTP and PLA2. The effects of different concentrations of PMA on the growth and morphology of MEG-01 cells were investigated. The results showed that the control group (Control) MEG-01 cells exhibited round, suspended growth with strong refractive properties. After PMA induction, the cells gradually adhered to the cell wall, increased in size, extended pseudopodia, and some cells fragmented, producing granular material. Within the time range of 24 to 72 hours, cell differentiation characteristics became more pronounced with increasing PMA concentration (10-80 ng / mL), but excessive cell debris was observed at high concentrations (40-80 ng / mL), indicating significant cytotoxicity.

[0106] Further observation of cell morphology after 72 hours using Wright-Giemsa staining revealed that MEG-01 cells significantly increased in volume and decreased in nucleocytoplasmic ratio under induction at 10 ng / mL and 20 ng / mL LPMA, exhibiting nuclear lobulation and numerous platelet-like anucleate cytoplasmic fragments in the field of view. Considering both cell state and differentiation efficiency, subsequent experiments selected 10-20 ng / mL LPMA treatment for 72 hours as the optimal induction condition.

[0107] The expression of megakaryocyte / platelet-specific surface markers CD41 and CD61 was detected by qPCR. The results showed that, compared with the blank control group, the expression levels of CD41 and CD61 mRNA in the PMA-induced group were significantly increased. P< The result was 0.001, confirming that MEG-01 cells had successfully differentiated into megakaryocytes / platelets. Furthermore, the transcriptional level of phospholipid transfer protein (PLTP) was detected, and the results showed that PLTP mRNA expression increased after PMA treatment, but the difference did not reach a statistically significant level. P =0.1023).

[0108] Following induction stimulation, the abundance of PLTP protein in both the membrane and cytoplasmic components of MEG-01 precursor cells decreased significantly. Considering the physiological characteristics of platelets, this simultaneous decrease in the levels of proteins in both intracellular and extracellular components indicates that the large amount of PLTP stored intracellularly is not being degraded, but rather is being continuously secreted into the extracellular environment during cell activation and granule release.

[0109] Meanwhile, the subcellular localization of secretory phospholipase A2 (sPLA2) differs from that of PLTP. Cell activation promotes the accumulation of sPLA2 in large quantities in the cell membrane, resulting in a significant increase in its expression on membrane components, suggesting that the enzyme is being targeted and recruited to the phospholipid bilayer to establish a catalytic reaction interface.

[0110] 4. Screening and identification of PLTP small molecule inhibitors Four small molecule compounds were selected (numbers: 5015, 6292, 6639, and 7035; structural formulas of the four compounds are shown in [link to sample]). Figure 1 In vitro PLTP lipid-transfer activity was validated.

[0111] To optimize experimental conditions, the effects of four compounds at different concentrations (10 μM, 5 μM, 2.5 μM, and 1.25 μM) and different pre-incubation times on plasma PLTP conversion activity were first investigated. The results showed that when the pre-incubation time reached 40 min, the inhibitory effect of the compounds on PLTP conversion activity tended to stabilize, indicating that 40 min was the optimal reaction time. Figure 2 , Figure 3 Therefore, 40 minutes was used as the standard pre-incubation time for all subsequent in vitro experiments.

[0112] The effects of the above compounds on coagulation function were assessed using thromboelastography (TEG). TEG comprehensively reflects the dynamic process of blood coagulation, where the maximum amplitude (MA) value represents the maximum strength of the blood clot, mainly reflecting platelet aggregation function and fibrinogen activity. Experimental results showed ( Figure 4Compared with the control group, the MA values ​​of the treatment groups of compounds 5015, 6292 and 6639 were significantly reduced. P <0.01 or P <0.001 indicates that these three compounds can effectively inhibit the formation intensity of blood clots; while compound 7035 has no significant effect on the MA value ( P >0.05). Based on multiple experimental verifications, and given that compound 5015 exhibits the strongest and most stable inhibitory effect, we ultimately selected 5015 as the target molecule for subsequent experimental studies.

[0113] 5. Platelet toxicity assessment and PLTP lipid-transfer activity verification of compound 5015 The molecular formula of compound K784-5015 (hereinafter referred to as 5015) is C25H29ClN2O4. To evaluate the effect of this compound on platelet activity in humans and mice, the potential effect on platelet activity in humans and mice was first detected using the AlamarBlue assay. The results showed that even at the highest tested concentration (80 μM), platelet activity in both humans and mice was not significantly inhibited. Figure 5 ).

[0114] Further, the toxicity of 5015 to platelets was assessed by detecting the release of lactate dehydrogenase (LDH). After co-incubating different concentrations of 5015 with human and mouse platelets, the results showed that there was no statistically significant difference in LDH release between the experimental group and the blank control group. P >0.05)( Figure 6 This demonstrates that 5015 has no cytotoxic effect on human and mouse platelets. Within this safe concentration range, PLTP translipidation activity verification experiments showed that 5015 effectively inhibited the translipidation activity of PLTP in the concentration range of 2.5-80 μM.

[0115] 6.5015 inhibits the adhesion of human and mouse platelets to collagen surfaces. Platelets possess vascular repair capabilities under physiological conditions. When vascular endothelium is damaged and sloughs off, the exposed subendothelial matrix (primarily collagen) induces platelet adhesion. Under high shear stress conditions in arterial blood, platelets "anchor" themselves to collagen and vWF via their surface glycoproteins GP1b-IX and integrin α2β1, leading to platelet aggregation and the release of more platelets to adhere to the damaged site for repair. However, excessive platelet adhesion is also a significant risk factor for thrombosis.

[0116] To assess the effect of compound 5015 on this process, human and mouse washed platelets were pre-incubated with different concentrations of 5015 and then placed on glass slides coated with cured collagen for adhesion. Subsequently, the platelet cytoskeleton was stained with fluorescently labeled phalloidin, and the adhered platelets were observed and counted using a fluorescence microscope to evaluate the platelet adhesion.

[0117] Fluorescence imaging results showed ( Figure 7 , Figure 8 As the concentration of 5015 increased, the number of red fluorescently labeled platelets adhering to the field of view gradually decreased. Quantitative statistical analysis showed that ( Figure 9 Within the concentration range of 10-80 μM, 5015 significantly inhibited platelet adhesion to collagen surfaces. After treatment with 5015, the number of human platelets decreased from 25.67±2.08 in the solvent control group to 16.03±3.51. P <0.05); mouse platelets decreased from 146.7±15.5 in the solvent control group to 44.41±4.08 ( P (<0.05) Therefore, 5015 significantly inhibits platelet adhesion on the collagen surface.

[0118] 7.5015 inhibits the spreading of human and mouse platelets on fibrinogen. Upon stimulation and activation, platelets trigger an intracellular signaling cascade, inducing inside-out signaling that causes a conformational change in integrin αIIbβ3 and its binding to fibrinogen. Subsequently, the binding of the ligand to αIIbβ3 further initiates outside-in signaling, leading to platelet spreading, granule release, and clot retraction—a process crucial for thrombus stability.

[0119] Thrombin-activated human and mouse platelets were incubated on the surface of fibrinogen, then permeabilized using a Triton X-100 membrane, and the platelet cytoskeleton was stained with fluorescently labeled phalloidin for observation. Fluorescence microscopy imaging showed... Figure 10 , Figure 11 In the blank control group and the solvent control group, platelets spread extensively on the fibrinogen surface; however, on the BSA surface, platelets showed almost no adhesion or spreading. The results indicate that ( Figure 12 The surface area of ​​human platelets on fibrinogen after incubation with 5015 was 1.96 ± 0.53 μm. 2 The value was significantly smaller than that of the control group (3.25±0.62μm). 2The area of ​​platelet expansion on the surface of fibrinogen in mice after incubation with 5015 (1.11 ± 0.15 μm). 2 The value was significantly smaller than that of the control group (3.5±0.19μm). 2 In summary, 5015 can significantly inhibit the platelet spreading process on fibrinogen, indicating that it effectively inhibits the "outside-inside" signal transduction mediated by integrin αIIbβ3.

[0120] 8. Effects of compound 5015 on the expression of platelet activation markers αIIbβ3 and JON / A and on clot retraction Upon stimulation by agonists such as thrombin, platelets undergo degranulation, causing P-selectin (CD62P) to rapidly translocate from the α-granule membrane to the cell membrane surface. Simultaneously, integrin αIIbβ3 undergoes a conformational change and becomes activated, mediating fibrinogen binding and platelet aggregation. Therefore, the expression level of CD62P on the membrane and the degree of activation of αIIbβ3 (detected by JON / A antibody) are the "gold standard" for evaluating platelet activation status.

[0121] Flow cytometry results showed ( Figure 13 , Figure 14 In the resting state, platelets express only trace amounts of CD62P and αIIbβ3. After stimulation with thrombin, the positivity rates of CD62P and JON / A on the platelet surface in the solvent control group were significantly increased. P <0.0001 indicates that platelets are fully activated. However, pre-incubation with 5015 significantly inhibited the expression of the above activation markers induced by thrombin. Compared with the control group, the mean fluorescence intensity (MFI) of CD62P and the binding rate of activated αIIbβ3 in the 5015 treatment group were significantly decreased ( P <0.0001 to P =0.0295), indicating that 5015 effectively blocked the "inside-out" signal transduction of platelets.

[0122] Upon platelet activation, integrin αIIbβ3 on its membrane surface changes from a low-affinity conformation to a high-affinity conformation, significantly enhancing its binding ability to ligands such as vWF, fibronectin, and hylocinin exposed at vascular injury sites. Furthermore, activated integrin αIIbβ3 can mediate platelet aggregation and inter-platelet connections through fibrinogen bridging. The clot retraction assay is a classic method for evaluating the outside-in signal transduction function between platelets.

[0123] Compared with the solvent control group, platelets pre-incubated with 5015 showed a significantly reduced degree of clot retraction 20 minutes after the onset of clot retraction. Figure 15Further quantitative analysis results showed that () Figure 16 The clot retraction rate in the 5015 treatment group was significantly reduced, and the difference was statistically significant. These results indicate that 5015 not only inhibited the early platelet activation process but also significantly weakened the late clot retraction function, suggesting that it has a regulatory role in the bidirectional signal transduction process mediated by integrin αIIbβ3.

[0124] 9. Pharmacokinetic characteristics and bleeding risk assessment of compound 5015 in vivo To further clarify the metabolism and distribution patterns of 5015 in vivo, we examined the time-dependent changes in drug concentration in mouse plasma after a single intravenous injection (IVdose). The results are shown below. Figure 17 Pharmacokinetic curves showed that after entering the bloodstream, the plasma concentration of 5015 reached a peak of approximately 1000 ng / mL 5 minutes (0.083 h) after administration. Subsequently, the drug exhibited typical two-compartment model characteristics, with a rapid decline in plasma concentration within 0-2 h, indicating rapid distribution or metabolism in the body; after 4 h, the plasma concentration remained at a low level, and by 8 h, it was essentially completely metabolized. Combined with PLTP activity assay results (… Figure 19 ), 5015 significantly inhibited plasma PLTP activity within 60 minutes after administration ( P <0.0001), indicating that its effective blood concentration in vivo is maintained for a duration sufficient to cover the activity window required for antithrombosis.

[0125] Bleeding risk is a major side effect of antiplatelet drugs (such as aspirin and clopidogrel) in clinical application. Severe coagulation dysfunction can lead to life-threatening bleeding events. Therefore, when developing novel antithrombotic drugs, minimizing bleeding risk while ensuring antithrombotic efficacy is a key indicator for drug safety evaluation. The mouse tail-cutting hemorrhage model, which physically transcribes the tail tip to impair vascular integrity and triggers the body's physiological hemostasis process, is highly dependent on platelet adhesion and aggregation, vasoconstriction, and the cascade activation of coagulation factors. This model is a classic method for evaluating whether a drug disrupts basic coagulation function in the body.

[0126] In this experiment, we evaluated the effect of compound 5015 on bleeding time in mice. Figure 18 The results showed that the bleeding time in mice treated with the positive control drug aspirin was significantly prolonged, increasing from 1196.0±69.03s in the solvent control group to 1760.0±28.64s. P=The blood loss was 0.0026, indicating a significant bleeding tendency. In contrast, 5015 demonstrated superior safety. Although the mean bleeding time was slightly increased in the low-dose group (1330.0±170.0s) and high-dose group (1545.0±217.0s) of 5015, statistical analysis showed no significant difference compared to the control group. P >0.05, ns). This result suggests that 5015 can effectively inhibit thrombus formation within the tested dose range, while also having a low risk of bleeding.

[0127] 10.5015 inhibits FeCl3-induced carotid artery thrombosis in mice. To evaluate the antithrombotic efficacy of 5015 in vivo, we used the classic FeCl3-induced right common carotid artery (RCCA) thrombosis model in mice. In this model, FeCl3 causes endothelial cell damage through oxidative stress, which in turn induces platelet aggregation and coagulation cascade reactions, ultimately leading to occlusive thrombus formation.

[0128] In the experiment, 10% FeCl3 filter paper strips were applied to the outside of the right common carotid artery (RCCA) of mice to induce injury. Subsequently, Doppler flow imaging was used to monitor the hemodynamic changes of the carotid artery in real time. The ultrasound images showed ( Figure 20 The carotid artery blood flow in the WT and Sham groups of mice showed a continuous and full normal waveform; while in the Model and Vehicle groups, the blood flow waveform showed obvious narrowing and peak characteristics after injury, indicating a sharp increase in distal vascular resistance and obstruction of blood flow.

[0129] To further quantify the degree of thrombosis, we statistically analyzed the carotid artery pulsatility index (PI). Figure 21 The results showed that compared with the Sham group, the PI values ​​of mice in the Model and Vehicle groups were significantly increased, indicating thrombosis. However, after treatment with low-dose (LD) and high-dose (HD) 5015, the PI values ​​of the mouse carotid arteries were significantly decreased. P The levels of FeCl3 in the 5015 group decreased to approximately 0.85 and 0.7, respectively, and the high-dose group showed effects close to those in the positive control group (aspirin). These results indicate that FeCl3 can effectively improve the hemodynamic state of damaged blood vessels and significantly inhibit FeCl3-induced arterial thrombosis.

[0130] 11.5015 inhibits carrageenan-induced tail vein thrombosis in mice. Carrageenan is a sulfated polysaccharide that acts as a potent inflammatory agent, inducing a severe inflammatory response upon entering the body, leading to endothelial cell damage and dysfunction. This endothelial damage further exposes collagen and releases tissue factor, activating extrinsic coagulation pathways and platelets, ultimately resulting in hypercoagulability and the formation of fibrin-rich mixed thrombi in the extremities. The carrageenan-induced mouse tail vein thrombosis model effectively mimics the clinical pathological process of inflammation-mediated venous thrombosis.

[0131] Six- to eight-week-old KM mice were randomly divided into three groups and administered aspirin (positive control), low-dose aspirin, and high-dose 5015 via intraperitoneal injection, respectively. A solvent control group was also included. After seven consecutive days of administration, an equal volume of carrageenan was injected intraperitoneally to induce tail vein thrombosis. The thrombosis in the tail veins of the mice was observed and measured 24 hours after modeling.

[0132] The results show that ( Figure 22 In the solvent control group, the tails of mice exhibited typical deep reddish-brown or even blackened necrosis, indicating widespread venous thrombosis. Quantitative analysis was performed by measuring the length of the tail thrombus. Figure 23 In the solvent-controlled group, the percentage of thrombus length to tail length was as high as 80.89% ± 3.16%. In contrast, thrombus formation was significantly inhibited in the aspirin-positive control group, with the percentage of thrombus length decreasing to 18.87% ± 1.28%. The 5015-treated groups also showed significant antithrombotic effects, with the percentages of thrombus length in the low-dose and high-dose groups being 53.97% ± 2.92% and 36.32% ± 1.71%, respectively. These results indicate that 5015 can significantly inhibit carrageenan-induced tail vein thrombosis in a dose-dependent manner.

[0133] In short, the above experimental results show that: 1. First-time acute myocardial infarction is accompanied by the release of a large amount of PLTP from platelets. PLTP can participate in the thrombus formation process by regulating glycerophospholipid metabolism.

[0134] 2. Platelet activation promotes the continuous secretion of PLTP, which is accompanied by the translocation and enrichment of secretory phospholipase A2 (sPLA2) to the cell membrane, and together they participate in lipid transport signal transduction.

[0135] 3. The small molecule compound of the present invention can significantly and stably inhibit the lipid transactivation activity of PLTP within a safe concentration range without cytotoxicity.

[0136] 4. The compounds of the present invention can effectively inhibit platelet adhesion, spreading, activation, and clot retraction by blocking the bidirectional signal transduction (Inside-out and Outside-in) mediated by integrin αIIbβ3.

[0137] 5. The compounds of the present invention exhibit good pharmacokinetic characteristics in mice and can effectively inhibit arterial thrombosis and inflammation-driven venous thrombosis without significantly increasing the risk of bleeding.

[0138] The English abbreviations involved in the technical solution of this invention and their corresponding full English and Chinese names are as follows: .

Claims

1. A compound with antithrombotic activity, characterized in that, The structural formula of the compound is: 。 2. The use of the compound of claim 1 in the preparation of PLTP inhibitors.

3. The use of the compound of claim 1 in the preparation of a drug for treating myocardial infarction.

4. The use of the compound of claim 1 in the preparation of a drug that inhibits platelet adhesion, spreading, activation, and clot retraction.

5. The application according to claim 4, characterized in that, The platelet adhesion, spreading, activation, and clot retraction functions are achieved by blocking bidirectional signal transduction mediated by integrin αIIbβ3.

6. The use of the compound of claim 1 in the preparation of a drug for treating arterial thrombosis.

7. Use of the compound of claim 1 in the preparation of a drug for treating inflammation-driven venous thrombosis.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains a therapeutically effective amount of the compound of claim 1 that has antithrombotic activity, and a pharmaceutically acceptable carrier.

9. Use of the pharmaceutical composition of claim 8 in the preparation of a PLTP inhibitor.

10. Use of the pharmaceutical composition of claim 8 in the preparation of a medicament for treating myocardial infarction.

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