| Background:Thrombotic diseases are the important pathological basis of cardiovascular events,and the abnormal activation of platelets is one of the main causes.There are still many deficiencies in current antiplatelet drugs,such as drug resistance and bleeding risk,so it is urgent to find new antiplatelet drugs.Cytoskeletal protein rearrangement plays an important role in platelet activation.In recent years,the biological function of SEPTIN,known as the fourth novel cytoskeletal protein,has attracted much attention,but the mechanism of its action in the process of platelet activation is still unclear.Purpose:In this study,SEPTINS specific inhibitor Forchlorfenuron(FCF)was used to study the effects of SEPTINS on platelet function and to explore the possible mechanism of action.Method:Immunofluorescence technique was used to observe the distribution characteristics of SEPTIN in the process of platelet spreading and the effect of FCF on the rearrangement of platelet skeleton proteins.The effect of FCF on platelet activity was detected by Alma blue staining.The effect of FCF on platelet aggregation induced by thrombin,collagen and ADP was detected by platelet aggregometer.The effect of FCF on thrombin-induced expression of CD62 P and JON/A on platelets was detected by flow cytometry.The effect of FCF on thrombin-induced platelet activated ATP release and reactive oxygen species(ROS)production were detected by kit and probe.Immunofluorescence technique was used to observe the effect of FCF on the morphological changes of platelets during the process of fibrinogen matrix spreading.The effect of FCF on thrombin-induced thrombus retraction was evaluated.The effect of FCF on the signaling pathways related to platelet activation was detected by Western blot.An inferior vena cava stenosis model was used to explore the effect of FCF on thrombosis in mice.The bleeding risk of FCF was evaluated by rat tail bleeding test.Results:During the process of platelet spreading,SEPTINS rearranged along with the cytoskeleton movement and highly co-localized with actin and tubulin.The platelet cytoskeleton rearrangement caused by thrombin was destroyed by SEPTINS inhibitor FCF.FCF inhibited the aggregation of washed platelets induced by different agonists in a dose-dependent manner,and had no toxic effect on platelets within the range of doses used.FCF blocked thrombin-induced platelet plasma membrane CD62 P and JON/A expression.FCF inhibited the release of ATP and the generation of ROS after thrombin-induced platelet activation.FCF dose-dependently inhibited thrombininduced fibrinogen binding and spreading area of platelets.FCF significantly inhibited thrombin-induced platelet retraction.FCF dose-dependently inhibited the phosphorylation of AKT and ERK1/2 during platelet activation and selectively blocked the agonist-induced dephosphorylation of VASP at Ser239,but had no significant effect on the phosphorylation of VASP at Ser157.Inhibition of GSK3β activity by SB216763 partially reversed the inhibitory effect of FCF on platelet function.A mouse model of inferior vena cava stenosis was used to confirm that tail vein injection of FCF inhibited thrombosis in mice.The tail bleeding experiment showed that FCF had no obvious bleeding risk in mice.Conclusion:Our study shows that SEPTIN dynamic organization is essential for the morphological transition of platelet activation by coordinating with actin and microtubule organization during platelet activation deformation.Moreover,FCF inhibited agonise-induced platelet aggregation,degranulation,reactive oxygen species(ROS)production,integrin αIIbβ3 activation,as well as corresponding internal and external signal transduction,accompanied by attenuated phosphorylation of AKT and ERK1/2 and maintenance of VASP(Ser529)phosphorylation.In addition,we found that FCF delayed IVC thrombosis in mice without significant bleeding risk.Therefore,we suggest that SEPTINS inhibitor FCF has the potential to become a new antiplatelet agent for the prevention and treatment of thrombotic diseases,and targeting SEPTIN activity or its dynamic organization may provide a new direction for the treatment of thrombotic diseases. |
