Study On The Mechanism Of Inhibition Of Isoflavone Compounds On Platelet Integrin Signaling

Cardiovascular disease(CVD)has the highest mortality rate in China,accounting for more than 40%of the total deaths of residents..Thrombus is responsible for the high-mortality cardiovascular complications such as myocardial infarction and stroke.Platelets play important roles in the pathogenesis of these diseases and integrins are critical in cell adhesion,migration,survival and differentiation,and they are essential for platelet activation and aggregation.The conduction of integrin signaling is dependent on the interaction of its intracellular portion with multiple signaling proteins.Developing anti-platelet drugs based on integrin signaling pathways is a hot spot in the field of thrombosis research.Existing clinical anti-platelet drugs inevitabily increases the risk of bleeding in patients,which greatly limits the use of these drugs,and brings inconvenience to thromboprophylaxis and treatment of patients with cardiovascular diseases.Although isoflavoniods can effectively inhibit platelet activation induced by various factors,their related targets and molecular mechanisms remain unclear.We found that 20 μM isoflavoniods inhibit platelet aggregation excited by various agonist,including integrin outside-in signal activator.Using surface plasmon resonance,we found that THO,CAL,GEN and DAI isoflavoniods can directly interact and bind to different 14-3-3 proteins,and the binding ability to 14-3-3ζis the strongest with KD values of 2.7×10-5,1.8×10-5,5.2×10-5 and 2.1×10-4 M,respectively.Through co-immunoprecipitation techniques,we found that 100 μM isoflavoniods blocks the interaction of integrin β3 intracellular domain with 14-3-3,thereby inhibiting the early intrinsic signaling pathway of platelet integrin,which in turn inhibited platelet activation,aggregation or spreading.During this process,100 μM isoflavoniods did not inhibit the binding integrins to fibrinogen,suggesting that the effects of isoflavones on the intrinsic signaling pathway are limited.Isoflavoniods significantly inhibited carrageenan induced tail thrombus length without affecting the tail bleeding time and liver blood losses after the transection.Moreover,,100 mg/kg isoflavoniods did not affect platelet clot retration.These mean that isoflavoniods inhibit thrombosis without increasing the risk of bleeding.By studying the effect of isoflavonoids on 14-3-3 proteins,this paper reveals the mechanism which isoflavonoids exert antithrombotic effects by inhibiting the intrisic signal transduction pathway of platelet integrin..This work provides potential drug molecules for the prevention and treatment of thrombotic diseases,and also reveals that 14-3-3 can be a potential target for antithrombotic therapy without bleeding side-effect.atelet clot retration.These mean that isoflavoniods inhibit thrombosis without increasing the risk of bleeding.By studying the effect of isoflavonoids on 14-3-3 proteins,this paper reveals the mechanism which isoflavonoids exert antithrombotic effects by inhibiting integrin outside-in signaling transduction of platelet.This work provides potential drug molecules for the prevention and treatment of thrombotic diseases,and also reveals that 14-3-3 can be a potential target for antithrombotic therapy without bleeding side-effect.