The Antithrombotic Effects Of Asebogenin And The Mechanisms

Objective:Thrombosis is a major cause of high morbidity and mortality in patients with cardiovascular diseases.Platelet activation by exposed collagen through glycoprotein Ⅵ（GPⅥ）and formation of neutrophil extracellular traps（NETs）are critical pathogenic factors for arterial and venous thrombosis.Both events are regulated by spleen tyrosine kinase（Syk）-mediated signaling events.Asebogenin is a dihydrochalcone whose pharmacological effects remain largely unknown.This study aims to investigate the antithrombotic effects of asebogenin and the underlying molecular mechanisms.Methods:The effects of asebogenin on platelet aggregation induced by different agonists were assessed by light transmission aggregometry.The effects of asebogenin on platelet P-selectin exposure,fibrinogen binding,and calcium mobilization-induced by collagen-related peptide（CRP）were determined by flow cytometry.Alkaline phosphatase method was used to detect the effect of asebogenin on platelet adhesion.The effect of asebogenin on platelet spreading was examined using confocal fluorescence microscopy.Quantitative phosphoproteomics,microscale thermophoresis,in vitro kinase assay and molecular docking combined with dynamics simulation were performed to characterize the targets of asebogenin.Immunoblotting was applied to measure the effect of asebogenin on the phosphorylation of Syk and related proteins in activated platelets,neutrophils,and the lysates of deep vein thrombosis（DVT）.Ultrasonic Doppler blood flowmeter was utilized to determine the effect of asebogenin on Fe Cl₃-induced carotid artery thrombosis.The effect of asebogenin on laser-induced cremaster arteriole thrombosis was examined using intravital microscopy.The effects of asebogenin on neutrophil recruitment and NETs formation during DVT were determined by immunohistochemistry.Murine tail bleeding assay was performed to evaluate the effect of asebogenin on bleeding risk.The cytotoxicity of asebogenin in platelets and neutrophils was examined by a commercial lactate dehydrogenase（LDH）assay kit.Results:（1）Asebogenin inhibited collagen-and CRP-induced platelet aggregation.（2）Asebogenin inhibited CRP-induced platelet P-selectin exposure,fibrinogen binding,and calcium mobilization.（3）Asebogenin suppressed platelet adhesion and spreading.（4）Asebogenin directly interacted with Syk through hydrogen bonds that may interfere with the phosphorylation at Tyr525/526,which is important for Syk activation.（5）Asebogenin reduced phosphorylation of Syk,PLCγ2,and ERK1/2 in CRP-stimulated platelets.（6）Asebogenin prolonged the time to thrombotic occlusion induced by Fe Cl₃in the carotid artery and inhibited laser injury-induced microvascular thrombosis in the cremaster arterioles.（7）Asebogenin suppressed DVT induced by inferior vena cava（IVC）stenosis.（8）Asebogenin inhibited Syk phosphorylation in activated neutrophils and decreased NETs formation in vitro.（9）Asebogenin reduced neutrophil accumulation,NETs formation,and Syk phosphorylation in DVT.（10）Asebogenin did not prolong the tail bleeding time.（11）Asebogenin did not show cytotoxicity in platelets and neutrophils at the therapeutic doses.Conclusions:Asebogenin exhibits potent antithrombotic effects by targeting Syk-mediated pathways and is a potential lead compound for the development of efficient and safe antithrombotic agents.