| Background:Cardiovascular disease(CVD)is a major disease that seriously endangers human health.According to the annual report released by the World Health Organization in 2020,about 9.6 million people die from acute myocardial infarction(AMI)and heart failure each year worldwide.The cardiovascular disease situation in China is even less optimistic,as the 2020 China Cardiovascular Health and Disease Report point out that China’s cardiovascular disease mortality rate is the highest of all types of diseases,and the incidence is increasing year by year in both rural and urban populations.Acute myocardial infarction is one of the major causes of cardiovascular disease death,and coronary artery thrombosis leading to tissue ischemia and necrosis is an important pathological basis.Antiplatelet therapy is one of the main therapeutic measures for cardiovascular diseases such as coronary artery disease,and it is important to reduce the incidence of thrombotic disease.However,antiplatelet therapy may lead to bleeding complications,and elderly patients with cardiovascular disease,who often have the multisystem disease,have a significantly increased chance of thrombotic events and the risk of bleeding during treatment.Therefore,finding safer and more effective antithrombotic targets to reduce the risk of bleeding during antithrombotic therapy in elderly patients with cardiovascular disease is an urgent clinical challenge.How to achieve rapid diagnosis and precise intervention in the early stages of thrombosis is important to reduce tissue necrosis,the incidence of adverse clinical events,and mortality due to thrombotic disease.Pharmacological and genetic evidence suggests that mitogen-activated protein kinases(MAPKs)signaling pathways play an important role in hemostasis and thrombosis.Apoptosis Signal Regulating Kinase 1(ASK1),an upstream kinase of the MAPKs signaling pathway,can be activated by physiological agonists during platelet activation and plays a central role in regulating TXA2 production and release,as well as in platelet granule secretion and integrin signaling.It has been shown that GS-4997,a specific inhibitor of ASK1,can effectively inhibit platelet TXA2 release and platelet aggregation in vitro,but there are still no reports on whether the same effect can be produced in the in vivo environment.Therefore,using ASK1 as a therapeutic target,a new technique to inhibit thrombus formation at a holistic level can be explored in-depth and help to suggest new strategies and rationale for antiplatelet therapy.Arterial thrombosis is mainly composed of activated platelets and fibrin.The composition,structure and function of platelet membrane proteins are altered upon platelet activation.Integrin αⅡbβ3,the most expressed membrane protein on the platelet surface and the ultimate pathway for platelet activation induced by different agonists,undergoes conformational changes during platelet activation and increases its affinity for fibrinogen and fibrin.Thus,αⅡbβ3 is an important molecular target on the platelet surface.It has been shown that peptides containing RGD sequences can specifically bind conformationally altered αⅡbβ3,and the cyclic RGD peptide(c-RGDfK)can bind firmly to αⅡbβ3 under high arterial shear stress.Therefore,using c-RGDfK to identify activated platelets using conformationally altered αⅡbβ3 as a target may enable early and accurate diagnosis of arterial thrombosis.Current clinical methods for thrombosis diagnosis are mainly based on anatomical imaging,mainly including angiography,ultrasound imaging,computer tomography(CT)and magnetic resonance imaging(MRI).In recent years,molecular imaging techniques can accurately assess the pathophysiological processes in arterial thrombosis,guiding for early diagnosis and precise treatment of thrombosis.Ultrasound vascular imaging(Ultrasound,US)stands out among the various imaging methods due to its advantages of non-invasive,realtime and convenience.By constructing targeted ultrasound microbubble contrast agents encapsulated with specific drugs and combining ultrasound-targeted microbubble destruction(UTMD),non-invasive diagnosis and precise drug delivery can be achieved for arterial thrombosis.Based on the above research background,this study uses ultrasound microbubbles as the main material,platelet activation marker αⅡbβ3 as the target and the ASK 1 inhibitor GS-4997 as the encapsulated target,and constructs an ultrasound-targeted microbubble contrast agent to target platelet activation,and establishes the assessment of arterial thrombosis and targeted therapeutic techniques through cellular and animal thrombosis models,providing theoretical basis and methods for early diagnosis and treatment of thrombosis.objectives:1.platelet-rich plasma and washed platelets were isolated from whole blood by centrifugation,platelet activation was induced by thrombin and ADP,and the regulatory effects of GS-4997 on platelet ASK1 phosphorylation,TXA2 release,α granule secretion andαⅡbβ3 activation were verified using molecular biology,and platelet aggregation assays were performed to verify the effects of ASK1 inhibitors on platelet aggregation function.2.To construct ultrasonic microbubbles encapsulating GS-4997 by thin-film dispersion method,characterize the physical and chemical properties of microbubble morphology,particle size distribution and concentration,and determine the drug encapsulation rate and stability.3.To verify the targeting of ultrasound microbubble on activated platelets and their ability to inhibit platelet aggregation and TXA2 release at the cellular level.4.In vivo imaging was performed to assess the imaging ability of ultrasound microbubble on carotid artery thrombosis in mice,and UTMD was used to burst the microbubble to release the drug and verify the inhibitory effect of the system on carotid artery thrombosis.Methods:1.Venous blood was obtained by venipuncture and anti coagulation,and platelet-rich plasma was separated by centrifugation.Platelet activation was induced by 0.1U/ml thrombin or 20μM ADP,and the effect of GS-4997 on the regulation of ASK1 phosphorylation in activated platelets was analyzed by Western Blot;the regulation of platelet TXA2 release by GS-4997 was verified by ELISA;the effect of GS-4997 on platelet α-granule secretion and integrin αⅡbβ3 activation was analyzed by flow cytometry.The effect of GS-4997 on platelet aggregation induced by thrombin or ADP was examined by platelet aggregation assay.2.Coupled with cRGDfK and DSPE by michael addition reaction,thin-film dispersion method was used to prepare ultrasonic microbubble contrast agent encapsulated with GS4997.Use optical and fluorescence microscopy to observe morphology of microbubble;Use dynamic light scattering and Coulter particle size meter to analyze microbubble particle size and number;Use zeta potential to analyze microbubble dispersion in solution;Use UV absorption spectroscopy analysis to calculate drug encapsulation rate;low-frequency ultrasound to burst microbubble and calculate drug release efficiency;Use latex tube imaging to verify the in vitro imaging ability of microbubbles.3.20μM ADP induced platelet activation and labeled activated platelets with antiCD62p-FITC;flow cytometry and laser confocal microscopy were used to observe and detect the targeting of microbubble on activated platelets;different concentrations of drug-loaded microbubble were co-incubated with activated platelets,and the effects of microbubble on activated platelet aggregation and TXA2 release were analyzed with/without ultrasonic breakdown.4.The FeCl3 chemical injury method was used to construct a mouse carotid artery thrombosis model.In vivo fluorescence imaging and ultrasound imaging were used to verified the ability of microbubble to target arterial thrombus;UTMD burst the microbubble to release the drug,confocal intravital microscopy and ultrasound Doppler flow analysis were used to verified the inhibitory effect of the system on arterial thrombus.Results:1.platelet-rich plasma was separated by centrifugation of whole blood at 200 g for 10 min.The method was reproducible,and the platelets were counted microscopically with a cell counting plate,and approximately 6x108 platelets per ml of platelet-rich plasma were calculated.(1)After platelet activation induced by 0.1 U/ml thrombin or 20μM ADP,ASK1 was rapidly phosphorylated,and 10μM GS-4997 significantly reduced the level of ASK1 phosphorylation,indicating that GS-4997 could effectively inhibit ASK1 phosphorylation during platelet activation.(2)10μM GS-4997 significantly inhibited TXA2 release induced by 0.1 U/ml thrombin(175.7±5.2 pg/ml vs 93.8±6.4 pg/ml)or 20μM ADP(201.3±4.7 pg/ml vs 114.2±6.8 pg/ml).(3)10μM GS-4997 significantly inhibited the expression of CD62p and αⅡbβ3 on the surface of activated platelets,suggesting that inhibition of ASK1 phosphorylation may affect platelet α granule secretion and integrin αⅡbβ3 activation.(4)10μM GS-4997 significantly inhibited platelet aggregation induced by 0.1 U/ml thrombin(69.1±2.8%vs 42.1±3.1%)and 20μM ADP(66.3±3.6%vs 28.1±1.8%).Below 10μM,the inhibitory effect was enhanced with increasing drug concentration.2.Construction and characterization of targeted ultrasound microbubble contrast agents encapsulating ASK1 inhibitor GS-4997.(1)Targeted ultrasound microbubble encapsulating GS-4997 were successfully constructed using thin-film dispersion.Under optical microscopy,the microbubble were in the shape of uniformly dispersed spheres with a particle size of about 1-3μm.Under fluorescence microscopy,the red fluorescence was uniformly distributed on the surface of the microbubble,suggesting that the lipid-soluble dye DID was successfully bound to the surface of the microbubble.(2)The results of dynamic light scattering and Coulter particle size analysis showed that the average particle size of microbubble was 1.3±0.24μm,and the average number was 2.02.5x107/ml,which remained stable under 4° conditions.(3)The UV-Vis absorption spectroscopy results showed that GS-4997 was successfully encapsulated inside the microbubbles with a drug loading rate of about 52.36%,and there was no significant drug leakage within 24 hours at 4°.The drug release rate reached 70%after 2 min of low-frequency ultrasound breakage of the microbubble.3.The targeting of ultrasound microbubble on activated platelets and the inhibition of platelet aggregation and TXA2 release were verified at the cellular level.1)Laser confocal microscopy showed that microbubble could effectively bind activated platelets,and flow cytometry demonstrated that the binding capacity of microbubble to activated platelets was significantly higher than that of resting platelets.2)ELISA and platelet aggregation assays showed that in vitro breakdown of microbubble to release drugs significantly inhibited activated platelet TXA2 release and platelet aggregation.4.In vivo imaging methods to assess drug-loaded targeted ultrasound microbubble inhibition of arterial thrombosis.(1)The FeCl3 chemical injury method was successfully constructed in a mouse carotid artery thrombosis model,and the successful model construction was verified using ultrasound Doppler flow analysis.(2)In vivo fluorescence imaging suggested that microbubble effectively bound to the site of arterial thrombosis,suggesting that microbubble have good targeting in vivo.(3)Confocal intravital microscopy imaging showed that using UTMD to burst the microbubble at the thrombus site could effectively release the drug and significantly inhibit the formation of arterial thrombus.(4)Ultrasound imaging showed that microbubble significantly enhanced the echogenic signal of arterial thrombus,and the release of drug by breaking microbubble significantly increased the intensity of blood flow at the thrombus site compared with the control group.(5)Immunofluorescence staining showed that microbubble were enriched at the thrombus site,and the thrombus area was significantly reduced in the treatment group.HE staining showed no significant inflammation and necrosis in major organs,suggesting good biocompatibility of microbubble.Conclusion:1.ASK1 is phosphorylated during platelet activation,GS-4997 significantly inhibits ASK1 phosphorylation,platelet TXA2 release,α granule secretion,αⅡbβ3 activation and platelet aggregation,and may serve as a new target for anti-platelet therapy.2.Ultrasound microbubble have good stability and high drug loading rate.Breaking the microbubble and releasing drugs can significantly inhibit platelet aggregation and TXA2 release.3.Ultrasound microbubble can target arterial thrombi in vivo,and the release of drugs through ultrasound targeted microbubble destruction can significantly inhibit the formation of arterial thrombi,and this system may provide a new method and basis for the early diagnosis and treatment of arterial thrombi. |
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