PRT318对胶原诱导人血小板Syk磷酸化的影响
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摘要
目的观察脾酪氨酸激酶(Syk)抑制剂PRT060318(PRT318)在体外对人血小板聚集率及Syk磷酸化的影响。方法采用比浊法测定PRT318对胶原、二磷酸腺苷(ADP)及花生四烯酸(AA)诱导的人血小板聚集率的影响;运用Western blot检测Syk525/6磷酸化水平。结果 Syk特异性抑制剂PRT318可抑制胶原诱导的人血小板聚集率,但对ADP或AA诱导的人血小板聚集率无影响。在蛋白水平上PRT318可促进人血小板在胶原诱导下Syk525/6磷酸化水平升高。酪氨酸蛋白激酶抑制剂PP2可抑制胶原诱导的人血小板聚集,降低人血小板在胶原诱导下Syk525/6磷酸化水平,并可阻断胶原诱导下PRT318促Syk525/6磷酸化作用。结论 PRT318对胶原诱导的血小板聚集具有抑制作用,并能促进胶原诱导的血小板膜受体糖蛋白VI信号通路上Syk525/6磷酸化,提示PRT318可能通过作用于Syk525/6磷酸化而抑制血小板聚集。
Objective To observe the effect of PRT060318(PRT318), an inhibitor of spleen tyrosine ki-nase(Syk), on human platelet aggregation and Syk phosphorylation in vitro. Methods The effect of PRT318 on platelet aggregation induced by collagen, adenosine diphosphate(ADP) and arachidonic acid(AA) was measured by turbidimetry. The phosphorylation of Syk525/6 was detected by Western blot. Results The Sykspecific inhibitor PRT318 suppressed collagen-induced platelet aggregation, while it did not have significantly inhibitory effect on ADP-or AA-induced platelet aggregation. The present data showed that PRT318 promoted the phosphorylation of Syk525/6 in collagen-induced platelets. PP2, tyrosine protein kinase(Src) inhibitor,inhibited collagen-induced platelet aggregation and decreased the level of Syk525/6 phosphorylation induced by collagen. PP2 also blocked PRT318-enhanced Syk525/6 phosphorylation in collagen-treated platelets.Conclusions PRT318 significantly inhibits collagen-induced platelet aggregation and promotes collagen-induced phosphorylation of Syk525/6 locating in the downstream of glycoprotein VI(GPVI) signal pathway, suggesting that PRT318 may inhibit platelet activation through regulating Syk525/6 phosphorylation.
Objective To observe the effect of PRT060318(PRT318), an inhibitor of spleen tyrosine ki-nase(Syk), on human platelet aggregation and Syk phosphorylation in vitro. Methods The effect of PRT318 on platelet aggregation induced by collagen, adenosine diphosphate(ADP) and arachidonic acid(AA) was measured by turbidimetry. The phosphorylation of Syk525/6 was detected by Western blot. Results The Sykspecific inhibitor PRT318 suppressed collagen-induced platelet aggregation, while it did not have significantly inhibitory effect on ADP-or AA-induced platelet aggregation. The present data showed that PRT318 promoted the phosphorylation of Syk525/6 in collagen-induced platelets. PP2, tyrosine protein kinase(Src) inhibitor,inhibited collagen-induced platelet aggregation and decreased the level of Syk525/6 phosphorylation induced by collagen. PP2 also blocked PRT318-enhanced Syk525/6 phosphorylation in collagen-treated platelets.Conclusions PRT318 significantly inhibits collagen-induced platelet aggregation and promotes collagen-induced phosphorylation of Syk525/6 locating in the downstream of glycoprotein VI(GPVI) signal pathway, suggesting that PRT318 may inhibit platelet activation through regulating Syk525/6 phosphorylation.
引文
[1]陈一竹,张俊峰.抗血小板治疗新靶点:糖蛋白Ⅵ[J].上海交通大学学报(医学版),2015,35(7):1078-1081.
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[4]M譫CSAI A,RULAND J,TYBULEWICZ V L J.The syk tyrosine kinase:A crucial player in diverse biological functions[J].Nat Rev Immunol,2010,10(6):387-402.
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[6]MARJORAM R J,LI Z,HE L,et al.α2β1 integrin,GPVI receptor,and common Fc Rγchain on mouse platelets mediate distinct responses to collagen in models of thrombosis[J].Plos One,2014,9(11):DOI:10.1371/journal.pone.0114035.
[7]SHIDA Y,RYDZ N,STEGNER D,et al.Analysis of the role of von Willebrand factor,platelet glycoproteinⅥ-,andα2β1-mediated collagen binding in thrombus formation[J].Blood,2014,124(11):1799-1807.
[8]THOMAS D H,GETZ TMNEWMAN T N,DANGELMAIER C A,et al.A novel histidine tyrosine phosphatase,tula-2,associates with syk and negatively regulates gpvi signaling in platelets[J].Blood,2010,116(14):2570-2578.
[9]CHEN X,REN L,KIM S,et al.Determination of the substrate specificity of protein-tyrosine phosphatase tula-2 and identification of syk as a tula-2 substrate[J].J Biol Chem,2010,285(41):31268-31276.
[10]NEGRESCU E V,SIESS W.Dissociation of the alphaiib beta3-integrin by egta stimulates the tyrosine kinase pp72(syk)without inducing platelet activation[J].J Biol Chem,1996,271(43):26547-26553.
[11]TOURDOT B E,BRENNER M K,KEOUGH K C,et al.Immunoreceptor tyrosine-based inhibitory motif(ITIM)-mediated inhibitory signaling is regulated by sequential phosphorylation mediated by distinct nonreceptor tyrosine kinases:a case study involving PECAM-1[J].Biochemistry,2013,52(15):2597-2608.
[12]BOULAFTALI Y,HESS P R,KAHN M L,et al.Platelet ITAM signaling and vascular integrity[J].Circ Res,2014,114(7):1174-1184.
[2]ANDREWS R K,ARTHUR J F,GARDINER E E.Targeting GPVI as a novel antithrombotic strategy[J].J Blood Med,2014(5):59-68.
[3]BERNDT M C,METHAROM P,ANDREWS R K.Primary haemostasis:Newer insights[J].Haemophilia,2014,20(s4):15-22.
[4]M譫CSAI A,RULAND J,TYBULEWICZ V L J.The syk tyrosine kinase:A crucial player in diverse biological functions[J].Nat Rev Immunol,2010,10(6):387-402.
[5]ANDRE P,MOROOKA T,SIM D,et al.Critical role for syk in responses to vascular injury[J].Blood,2011,118(18):5000-5010.
[6]MARJORAM R J,LI Z,HE L,et al.α2β1 integrin,GPVI receptor,and common Fc Rγchain on mouse platelets mediate distinct responses to collagen in models of thrombosis[J].Plos One,2014,9(11):DOI:10.1371/journal.pone.0114035.
[7]SHIDA Y,RYDZ N,STEGNER D,et al.Analysis of the role of von Willebrand factor,platelet glycoproteinⅥ-,andα2β1-mediated collagen binding in thrombus formation[J].Blood,2014,124(11):1799-1807.
[8]THOMAS D H,GETZ TMNEWMAN T N,DANGELMAIER C A,et al.A novel histidine tyrosine phosphatase,tula-2,associates with syk and negatively regulates gpvi signaling in platelets[J].Blood,2010,116(14):2570-2578.
[9]CHEN X,REN L,KIM S,et al.Determination of the substrate specificity of protein-tyrosine phosphatase tula-2 and identification of syk as a tula-2 substrate[J].J Biol Chem,2010,285(41):31268-31276.
[10]NEGRESCU E V,SIESS W.Dissociation of the alphaiib beta3-integrin by egta stimulates the tyrosine kinase pp72(syk)without inducing platelet activation[J].J Biol Chem,1996,271(43):26547-26553.
[11]TOURDOT B E,BRENNER M K,KEOUGH K C,et al.Immunoreceptor tyrosine-based inhibitory motif(ITIM)-mediated inhibitory signaling is regulated by sequential phosphorylation mediated by distinct nonreceptor tyrosine kinases:a case study involving PECAM-1[J].Biochemistry,2013,52(15):2597-2608.
[12]BOULAFTALI Y,HESS P R,KAHN M L,et al.Platelet ITAM signaling and vascular integrity[J].Circ Res,2014,114(7):1174-1184.