抗氧剂2246对血小板功能的作用及机制OA

OBJECTIVE To investigate the effects of the 2,2'-Methylenebis(6-tert-butyl-4-methyl-phenol)(AO2246)on platelet function in vitro and the underlying mechanism.METHODS Venous blood was collected from healthy volunteers before washed platelets were prepared that were divided into the vehicle group,AO2246 1,2 and 4 μmol·L-1 groups,and positive drug group(0.1%Trinton X-100 group).The lactate dehydrogenase(LDH)content in platelet suspension was detected using a LDH kit.Platelets were divided into vehicle group,AO2246 1,2,and 4 μmol·L-1 groups.The platelet aggrega-tion rate and adenosine triphosphate(ATP)secretion at 5 min induced by thrombin,collagen,or ade-nosine diphosphate(ADP)were detected using a platelet aggregation instrument.Platelets were divided into a blank group,vehicle group,AO2246 1,2 and 4 μmol·L-1 groups,and positive drug group[100 μmol·L-1 aspirin group,P-selectin(CD62P)expression detection or 1 μmol·L-1 tirofiban group,platelet activator combined-1(PAC-1)binding detection].Flow cytometry was used to detect the effects of AO2246 1,2,and 4 μmol·L-1 on the number of platelet surface receptors(CD41b,CD42b,CD61,and GPⅥ),as well as on the expression of CD62P and the binding rate of PAC-1 induced by convulxin and thrombin at 5 min.Platelets were divided into a blank group,vehicle group,AO2246 1,2,and 4 μmol·L-1 groups.The spreading area of platelets on fibrinogen and the number of adhesions on the collagen surface were observed by fluorescence microscopy.The platelet clot retraction was also observed.The binding ability of AO2246 to phosphatidylinositol 3-kinase/protein kinase B(PI3K/AKT1)protein was verified by molecular docking.In the rescue experiment,platelets were divided into a vehicle group,AKT agonist SC-79 10 μmol·L-1 group,SC-79 10 μmol·L-1+AO2246 4 μmol·L-1 group,and AO2246 4 μmol·L-1 group.The aggregation rate of each group was detected using a platelet aggregation instrument.Western blotting was used to detect the protein phosphorylation levels of PI3K,AKT,and p38 mitogen-activated protein kinase(p38 MAPK)in platelets.RESULTS Compared with the vehicle group,AO2246 1,2 and 4 μmol·L-1 groups did not affect the number of platelet surface receptors,but significantly inhibited platelet aggregation and ATP secretion induced by thrombin,collagens or ADP,significantly lowered CD62P expressions and PAC-1 binding rate induced by convulxin and thrombin,reduced the spreading area of platelets on fibrinogen and the number of adhesions on collagen surface,and inhibited platelet clot retraction.Western blotting results showed that compared with the vehicle group,the phosphoryla-tion of PI3K,AKT,and p38 MAPK induced by thrombin,collagens or ADP was significantly inhibited in AO2246 1,2 and 4 μmol·L-1 groups.Molecular docking results showed that AO2246 had a high binding affinity with PI3K and AKT1(binding energies were-6.7 and-7.1 kcal·mol-1,respectively).The SC-79 10 μmol·L-1+AO2246 4 μmol·L-1 group could partially reverse the inhibitory effect of AO2246 4 μmol·L-1 group on platelet aggregation rates and AKT phosphorylation induced by thrombin.CONCLUSION AO2246 exerts anti-platelet effects by inhibiting platelet aggregation,secretion,adhesion and spread.The mechanism may be related to the inhibition of the PI3K/AKT/p38 MAPK signaling pathway.