A critical role for the regulation of Syk from agglutination to aggregation in human platelets.
A critical role for the regulation of Syk from agglutination to aggregation in human platelets.
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DOI：
10.1016/j.bbrc.2013.12.001
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发表时间：
2014-01
影响因子：
3.1
通讯作者：
Chun-Ho Shih;Tin-Bin Chiang;Wen‐Jeng Wang
中科院分区：
文献类型：
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作者：
Chun-Ho Shih;Tin-Bin Chiang;Wen‐Jeng Wang
凝集素是一种来自福尔摩沙Agkistrodon acutus毒液的四聚体糖蛋白 (GP) Ibα 激动剂，已被定性为人洗涤血小板 (WP) 中的凝集诱导剂。在富含血小板的血浆（PRP）中，凝集素显着引发凝集和随后聚集的双相反应。为了阐明人血小板中从凝集到聚集的细胞内信号传导事件，我们通过检测蛋白质酪氨酸磷酸化 (PTP) 来检查所涉及的重要信号传导分子。在 WP 中，抗 GPIbα 单克隆抗体 (mAb) AP1（而非 Src 激酶抑制剂 PP1）完全抑制了凝集素诱导的凝集。然而，PP1（而非 AP1）通过 GPVI 激活剂 convulxin 对血小板聚集具有有效抑制作用。 PTP 分析显示，单独的凝集素可引起涉及 Lyn/Fyn、Syk、SLP-76 和磷脂酶 Cγ2 (PLCγ2) 顺序磷酸化的弱模式。此外，Syk 选择性激酶抑制剂白皮杉醇显着抑制凝集素激活的 PRP 中的聚集反应。通过流式细胞术分析，荧光团缀合的 PAC-1（一种识别激活的整合素 αIIbβ3 的单克隆抗体）在凝集素刺激的血小板中显示出结合能力增加。同样，白皮杉醇（而非 PP1）对凝集素诱导的 αIIbβ3 暴露具有浓度依赖性抑制。此外，信号体的形成，包括 Syk、SLP-76、VAV、粘附和脱颗粒促进接头蛋白 (ADAP) 和 PLCγ2，是凝集素/纤维蛋白原激活血小板中血小板聚集所必需的。此外，通过凝集素的 GPIbα 连接可以显着促进 αIIbβ3 和纤维蛋白原之间的相互作用。因此，Lyn/Fyn/Syk/SLP-76/ADAP/VAV/PLCγ2/PKC信号通路足以触发凝集素/纤维蛋白原预处理的血小板中的血小板聚集。重要的是，Syk 可能是人血小板中从 GPIbα 引发的凝集到整合素 αIIbβ3 依赖性聚集反应的主要调节因子。
Agglucetin, a tetrameric glycoprotein (GP) Ibα agonist from FormosanAgkistrodon acutusvenom, has been characterized as an agglutination inducer in human washed platelets (WPs). In platelet-rich plasma (PRP), agglucetin dramatically elicits a biphasic response of agglutination and subsequent aggregation. For clarifying the intracellular signaling events from agglutination to aggregation in human platelets, we examined the essential signaling molecules involved through the detection of protein tyrosine phosphorylation (PTP). In WPs, an anti-GPIbα monoclonal antibody (mAb) AP1, but not a Src kinase inhibitor PP1, completely inhibited agglucetin-induced agglutination. However, PP1 but not AP1 had a potent suppression on platelet aggregation by a GPVI activator convulxin. The PTP analyses showed agglucetin alone can cause a weak pattern involving sequential phosphorylation of Lyn/Fyn, Syk, SLP-76 and phospholipase Cγ2 (PLCγ2). Furthermore, a Syk-selective kinase inhibitor, piceatannol, significantly suppressed the aggregating response in agglucetin-activated PRP. Analyzed by flow cytometry, the binding capacity of fluorophore-conjugated PAC-1, a mAb recognizing activated integrin αIIbβ3, was shown to increase in agglucetin-stimulated platelets. Again, piceatannol but not PP1 had a concentration-dependent suppression on agglucetin-induced αIIbβ3 exposure. Moreover, the formation of signalosome, including Syk, SLP-76, VAV, adhesion and degranulation promoting adapter protein (ADAP) and PLCγ2, are required for platelet aggregation in agglucetin/fibrinogen-activated platelets. In addition, GPIbα-ligation via agglucetin can substantially promote the interactions between αIIbβ3 and fibrinogen. Therefore, the signal pathway of Lyn/Fyn/Syk/SLP-76/ADAP/VAV/PLCγ2/PKC is sufficient to trigger platelet aggregation in agglucetin/fibrinogen-pretreated platelets. Importantly, Syk may function as a major regulator for the response from GPIbα-initiated agglutination to integrin αIIbβ3-dependent aggregation in human platelets.