Effects Of Thymic Stromal Lymphopoietin On The Function Of Platelet And Its Mechanism

Part I Expression of thymic stromal lymphopoietin receptor in human and murine plateletsOjective:To investigate expression of thymic stromal lymphopoietin receptor protein in human and murine plateletsMethods:Washed platelets from human and10-week-old wild-type C57BL/6mice, western blot and flow cytometry (FCM) were used to detect the expressions of TSLPR, DCs served as positive control.Rresults:Western blotting and flow cytometry of human and murine platelets revealed that TSLPR is expressed on plateletsConclusion:TSLPR protein expression in human and murine platelets.Thymic stromal lymphopoietin is a newly founded inflammatory cytokine,it is important to further examine the effects of TSLP/TSLPR on platelet function. Objective:To investigate the effects of thymic Stromal Lymphopoietin on the Function of Platelet.Methods:Washed platelets were isolated and collected from human. Then, washed platelets were cultured with PBS or stimulated with TSLP. The platelet fluorescent-gathered analyzer was used to test the effects of TSLP stimulation on platelet aggregation and platelet ATP release. Flow cytometry was used to determine the expression of P-selectin and activated integrin αⅢbβ3in human platelets after treatment with TSLP.Results:Upon stimulated with TSLP, TSLP alone was induced minimal aggregation of resting platelets, TSLP alonewas sufficient to induced the release of ATP, but theaggregation and amount of ATP release induced by TSLP stimulation is much lower than that induced by ADP or thrombin. TSLP increased the expression of P-selectin and activated integrin αⅢbβ3, whereas quantity of expression was minimal. Meanwhile, we found that TSLP strongly amplified platelet aggregation, P-selectin expression and integrin αⅢbβ3activation with low concentration of platelet agonists.Conclusion:Results collectively suggest that via binding to TSLPR, TSLP trigger activation of platelet,we propose that TSLP promote platelet activation and make a magnified effect via co-stimulation with platelet agonists. Objective:To investigate the effects of thymic stromal lymphopoietin receptor deficiency on the function of platelet and thrombus formationMethods:Washed platelets were isolated and collected from wild-type (WT) C57BL/6mice or TSLPR knockout (TSLPR KO)mice.Then, washed platelets were cultured with PBS or stimulated with TSLP. Effects of TSLPR deficiency on platelet aggregation and secretion were detected viathe platelet fluorescent-gathered analyzer. Flow cytometry was used to determine the expression of P-selectin and activated integrin αⅢbβ3in mice or human platelets after treatment with TSLP. Perfusion flow chamber was assessed theeffects of TSLPR deficiency on platelet thrombus formation under flow conditions. Effect of TSLPR deficiency on FeCl3-induced carotid artery thrombosis model was detected by a miniature Dopplerflow probe. Western blot was used to determine the involvement effect of PI3K/Akt pathway in platelet activation after stimulated with TSLP,the effect of TSLPR deficiency on Akt phosphorlytion of platelet after treatmented with TSLP.Results:1. Compared with wild-type control, TSLPR KO platelets showed a markedly defective aggregation following stimulation with low dose thrombin (47.0±8.2vs.64.2±8.1, p<0.01),25%reduction in TSLPR KO relative to WT platelets. Platelet serotonin release consistent with platelet aggregation was markedly reduced in TSLPR KO platelets in response to low dose thrombin (41.2±8.0vs.28.9±7.1, p<0.05).2. Following stimulation with TSLP, compared to WT mice, TSLP stimulated shorten vessel occlusion time of WT mice, and vessel occlusion time was significantly delayed in TSLPR KO mice (P<0.01); we observed that significantly defective thrombus formation of TSLPR KO mice stimulation with TSLP at both low (600s-1) and high shear rates (1800s-1) compared with WT mice.3. TSLP significantly increased phosphorylation of Akt which could be prevented by preincubation with phosphatidylinositide3-kinase (PI3K) inhibitors wortmannin and LY294002. Consistent with these findings the effect of TSLP on platelet activation (degranulation, integrin αⅢbβ3activation) was abolished after preincubation with the PI3K inhibitors wortmannin (P-selectin:18.1±2.9vs.8.6±2.4, p<0.01; PAC-1:17.8±5.1vs.8.9±3.0, p<0.01) and LY294002(P-selectin:18.1±2.9vs.10.2±2.2, p<0.01; PAC-1:17.8±5.1vs.10.0±2.8, p<0.01). TSLP-dependent Akt phosphorylation was abrogated in TSLPR KO platelets compared to WT platelets. TSLP-induced platelet degranulation (22.2±7.1vs.14.5±6.5, p<0.05) and integrin αⅢbβ3activation (42.3±11.0vs.25.6±10.6, p<0.05) was significantly reduced in TSLPR KO platelets compared to WT platelets.Conclusion:Our study here identified that TSLPR deficiency caused defective platelet aggregation and defective platelet secretion, and markedly attenuated thrombus growth in perfusion chambers at both low and high shear rates. TSLPR deficiency exhibited defective carotid artery thrombus formation. TSLP triggers platelet TSLPR-dependent PI3K/Akt signaling leading to degranulation and integrin αⅢbβ3activation.