| ObjectiveTo investigate the antiplatelet effect of the novel STAT3 inhibitor SC99 and its possible mechanism,and to evaluate the synergetic effect and safety of antiplatelet with aspirin and SC99 combination in vitro.MethodsThe current study uses the vitro wash platelets of C57BL/6 mice as research object.The effects of different concentrations of collagen or at different phases on the phosphorylation of JAK2/STAT3 signaling pathway were observed by collagen-induced platelet aggregation.The alteration of platelet maximum aggregation rate and the effect on the phosphorylation of related proteins were observed after the selective inhibitors AG490 and SC99 were used respectively.The relationship of Syk,STAT3 and PLCγ2 in the STAT3 signaling pathway was verified by platelet aggregation rate assay,immunoblotting and immunoprecipitation in the use of the specific protein molecular inhibitors SykI and SC99.The effect of SC99 on activation of STAT3 signaling pathway was detected by the inflammatory stimuli factor IL-6.Combining aspirin with SC99 the efficacy and safety of antiplatelet effects was evaluated with the following method: platelet aggregation;blood clot retraction observed by dynamic imaging;the release,activation and apoptosis of platelets detected by flow cytometry;and the time of the mice tail vein bleeding calculated.Results1.Administration with different concentrations of collagen stimulated mice platelet aggregation in vitro,it showed that the expression of p-STAT3 and p-JAK2 was the highest at 2μg / ml(P<0.05).2μg / ml of collagen stimulated platelets for 2min,the expression of p-JAK2 was significantly higher than that of other time points(P<0.05),and the expression of p-STAT3 reached a peak at 5min(P<0.05).The platelets were pretreated with AG490(0,25,50,100μM),the maximal aggregation in AG490 group lowered by 13%,25% and 66% respectively compared with control group(P<0.05).The expression of p-JAK2,p-STAT3,p-P65 and p-AKT decreased gradually with the increase of AG490 concentration(P<0.05),There was no significant change in p-c-Src expression(P>0.05);The platelets were pretreated with 50μM AG490 and different concentrations of collagen(1,2,5μg/ml)induced platelet aggregation.The maximal aggregation in AG490 group lowered by 65%,27%,16% respectively compared with control group(P<0.05).The platelets were pretreated with SC99(0,1.25,2.5,5.0μM),Compared with the control group,the maximum aggregation decreased by 13%,34% and 63% in SC99 groups respectively(P<0.05).The expression of p-JAK2 and p-STAT3 decreased gradually with the increase of SC99 concentration(P<0.05),while the expression of p-P65,p-AKT and p-c-Src had no significant change(P>0.05).The platelets were pretreated with 2.5μM SC99 and different concentrations of collagen(1,2,5μg/ml)induced platelet aggregation.The maximal aggregation of SC99 groups were decreased by 67%,37% and 18% respectively compared with control group(P<0.05).2.The platelets were pretreated with Syk I(0,0.05,0.5,5μM),respectively.The maximal aggregation in SykI groups were decreased by 5%,23% and 47% respectively compared with control group(P<0.05).The expression of p-STAT3 and p-PLCγ2 was also decreased with the increase of SykI concentration(P<0.05).The platelets were pretreated with 0.5μM Syk I and different concentrations of collagen(1,2,5μg/ml)induced platelet aggregation.Compared with the control group,the maximum aggregation of the SykI group decreased by 56%,36% and 23% respectively(P<0.05).The platelets were pretreated with SC99(0,1.25,2.5,5μM)respectively,The expression of p-PLCγ2 decreased gradually with the increase of SC99 concentration(P<0.05),but the expression of p-Syk did not change significantly(P>0.05).STAT3 antibody immunoprecipitation showed that STAT3,Syk and PLCγ2 have a significant combination,PLCγ2 antibody immunoprecipitation showed that PLCγ2,STAT3 and Syk have a combination as well.STAT3 and PLCγ2 were capable to bind to p-PLCγ2 and p-STAT3 antibodies,respectively(P<0.05).The p-STAT3 binding to PLCγ2 antibody decreased with the increase of SC99 concentration(P<0.05)when the platelets were pretreated with SC99(0,1.25,2.5,5μM).Compared with IL-6,IL-6+sIL-6R complex can significantly induce the expression of p-STAT3(P<0.05).The expression of platelet p-STAT3 increased with the dose of IL-6+s IL-6R complex(P<0.05),which could be inhibited by SC99(P<0.05).3.The platelets were pretreated with ASA 2mM,ASA(2mM)+ SC99(2.5μM),ASA 5mM,respectively,the maximum aggregation decreased by 15%,61%,64% compared with the control group.There was no significant difference between the last two groups(P>0.05),The difference in the rest groups was statistically significant(P<0.05);Thrombosis retraction experiment showed that the difference of time-points in the intra-group,ASA(5mM)and SC99(5μM)was statistically significant(P<0.05),the difference of time-points in the ASA(5mM)+ SC99(5μM)group had no significant difference(P>0.05).There was no significant difference between ASA(5m M)and SC99(5μM)group at 10 min,20min and 30min(P>0.05).The difference in the rest groups was statistically significant(P<0.05).ASA + SC99 group inhibited 22.6% expression of P-selectin and 36.3% expression of integrinαⅡbβ3 on platelet surface,the difference was statistically significant(P<0.05)compared with ASA group.There was no significant difference in platelet apoptosis gate number and the expression of the apoptotic related protein in ASA + SC99 group(P>0.05)compared with ASA(5mM)group.There was no significant difference in the mice tail vein bleeding time(326.6 ± 42.9 s)between ASA + SC99 group and ASA group(307.0 ± 36.9 s)(P>0.05).Conclusion1.The platelet aggregation of collagen-induced in C57BL/6 mice was associated with the activation of JAK2/STAT3 signaling pathway.AG490 and SC99,which were the inhibitors of JAK2/STAT3 signaling pathway,inhibited the platelet aggregation,while the molecular signal specificity of SC99 was higher than that of AG490.2.As a protein scaffold,STAT3 promotes Syk to activate PLCγ2;Collagen and IL-6 have established a common signaling pathway related to inflammatory factors with STAT3 as the intermediate molecule,which can be inhibited by SC99.3.SC99 combined with ASA can synergistically inhibit the platelet aggregation in vitro,and also which can effectively inhibit the plaltelet release,activation and thrombosis retraction in mice,however not significantly affect the platelet apoptosis and coagulation function compared with the control group. |
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