The Antithrombotic Effects Of Dihydromyricetin And Its Mechanisms

Objection:Thrombus formation is a complex pathophysiological process,in which the activation of platelets and endothelial cells plays an important role.The platelet-independent fibrin generation by activated endothelium highlights the importance of vascular protection in addition to platelet inhibition in thrombosis prevention.At present,the commonly-used antithrombotic drugs in clinical practice mainly include antiplatelet and anticoagulant agents.However,these agents target either platelets or the coagulation cascades,but not both,and are often associated with adverse reactions such as bleeding.In addition,the therapeutic effects are not satisfactory.Therefore,the development of novel efficient antithrombotic drugs has important clinical significance.Dihydromyricetin(DHM)is a flavonoid with a wide range of protective effects on the cardiovascular system,but the effects of DHM on thrombosis and the underlying mechanisms are still unclear.This study aims to elucidate the effects of DHM on platelet and endothelial cells activation and its possible mechanisms.Methods:Washed human platelets or platelet-rich plasma were used to evaluate the effect of DHM on platelet aggregation induced by different agonists such as thrombin,collagen and ADP.The effect of DHM on P-selectin expression,PAC-1 binding and the intracellular Ca²⁺mobilization during platelet activation were measured by flow cytometry.The effect of DHM on platelet spreading on collagen surface was examined by confocal fluorescence microscope;The alkaline phosphatase method was utilized to detect the effect of DHM on platelet adhesion on collagen surface;Western blot was used to check the influence of DHM on the MAPK signal pathway during platelet and endothelial cells activation;Molecular docking analysis was used to confirm the possible binding of DHM with ERK and p38.The influence of DHM on the secretion of vWF and PDI by activated primary HUVECs were determined by ELISA.The effect of DHM on thrombin-induced phosphatidylserine exposure in primary HUVECs were examined using FITC-conjugated Annexin V on flowcytometry.The effect of DHM on TNF-?-induced TF expression in HUVECs were determined using f Xa generation assay.Thrombin-induced Ca²⁺mobilization,as determined by Fura-2 fluorescent signal,were monitored over time in primary HUVECs on fluorescence microscope.Cytotoxic effects of DHM on human platelets and primary HUVECs were measured by extracellular lactate dehydrogenase(LDH).The effects of DHM on thrombus formation in mouse carotid artery was measured in FeCl₃-induced thrombosis model,and Martius-Scarlet-Blue staining was used to assess fibrin deposition in the thrombus.Laser-induced thrombosis model was established to detect the effects of DHM on thrombus formation in the cremasteric arterioles.Platelet accumulation and fibrin generation was visualized using Dylight-649-labelled anti-CD42c antibody and Alexa-488-labelled59D8 monoclonal antibody,respectively.Tail bleeding assay was performed to evaluate the effects of DHM on mouse bleeding risk.The effects of DHM on plasma clotting time including prothrombin time(PT)and activated partial thromboplastin time(APTT)were examined on an automated coagulation analyzer.Results:(1)DHM concentration-dependently inhibits platelet aggregation induced by thrombin,collagen and ADP;(2)DHM concentration-dependently inhibits the expression of P-selectin and the binding of PAC-1 on activated platelets induced by thrombin;(3)DHM inhibits the spreading and adhesion of platelets on the surface of collagen;(4)DHM concentration-dependently inhibits calcium mobilization in thrombin-activated platelets and endothelial cells;(5)DHM significantly reduces the phosphorylation of ERK1/2 and p38 in the MAPK signaling pathway during platelet and endothelial cell activation;(6)DHM concentration-dependently inhibits the secretion of vWF and PDI and the expression of TF by activated endothelial cells;(7)DHM significantly inhibits phosphatidylserine(PS)exposure on activated endothelial cells;(8)DHM does not significantly increase the release of LDH from endothelial cells and platelets;(9)DHM significantly prolonged FeCl₃-induced mouse carotid arterial thrombosis,and the fibrin content in the thrombus was significantly reduced;(10)DHM significantly inhibited platelet aggregation and fibrin generation at the injury site in the cremasteric arterioles;(11)DHM has minimal effects on PT and APTT;(12)DHM has no effects on the tail bleeding time.Conclusions:In summary,we show that DHM is a potent antithrombotic agent whose effects involve both platelet inhibition and endothelial protection.Unlike classical antiplatelets,DHM also inhibits fibrin generation,an effect attributed to suppressed endothelial activation following vascular injury.At the molecular level,DHM targets MAPK signaling leading to attenuated Ca²⁺elevation,which is a central signaling node regulating granule secretion,integrin activation,and phosphatidylserine exposure.These combined events result in inhibited platelet activation and suppressed endothelial procoagulant activity.Furthermore,DHM does not present significant cytotoxic activity or increase the risks of bleeding.Thus,DHM is a promising candidate as a novel antithrombotic with improved efficacy and safety profiles.