Elucidation of the mechanisms whereby integrin mediates platelet activation.

与整合素结合的特定配体启动“自外向内”信号，该信号与通过生长因子、细胞因子和G蛋白偶联受体启动的信号级联相协调，以调节肌动蛋白的重组、细胞存活和增殖。在血小板中，纤维蛋白原或血管性血友病因子与整合素αIIbβ3的结合触发信号，促进细胞骨架的改变，扩散和形成稳定的血小板血栓。αIIbβ3与非受体蛋白激酶如sRc和syk之间的物理相互作用已被证实，纤维蛋白原与整合素结合导致这些酪氨酸激酶的激活。对人血小板、基因靶向小鼠血小板和异源表达系统的研究表明，整合素β3胞浆结构域与c-Src的SH3结构域直接和结构性地相互作用。在整合素连接和聚集后，有人认为整合素相关的c-src被迅速激活，导致…更多的NG到c-Src底物的酪氨酸磷酸化，以及迄今已知的基于αIIbβ3的新生信号复合体的组装，该信号复合体包括Syk、SLP-76、Vav和ADAP(促进黏附和脱颗粒的适配器蛋白；以前称为Fyn结合蛋白，Fyb或SLAP-130)。LNK是一种含有SH2结构域的接头蛋白，可抑制细胞因子信号转导。LNK^&lt；-/-&gt；小鼠表现出明显的血小板增多症，这是由于前体细胞在细胞因子的作用下增殖和成熟所致，血小板计数增加了5倍。我们发现，LnK在人和小鼠的血小板中都有表达，并且在血小板整合素αIIbβ3的外向内信号转导中起着意想不到的作用。在流动状态下，血小板在纤维蛋白原表面铺展、β3亚单位酪氨酸磷酸化、凝块回缩以及在胶原上形成血栓等方面存在缺陷。相反，内向外信号在LNK^-lt；-/-&gt；血小板中是正常的，激动剂诱导的纤维蛋白原结合证明了这一点。在血小板中，LNK以依赖于αIIbβ3连接和Src激活的方式，与c-Src和Fyn结合接头ADAP形成复合体，可能是Fyn重新聚集到αIIbβ3以及β3亚单位的酪氨酸磷酸化所必需的。这些结果首次证明，LNKA通过调节外源整合素αIIbβ3信号，在血小板的黏附反应和血栓形成中发挥关键作用。此外，我们还讨论了WASP/WAVE家族在巨核细胞和血小板肌动蛋白细胞骨架变化中的作用。在项目的这一部分，我们证明了WAVE2/Abil信号复合体介导了整合素依赖的板脂蛋白的形成和巨核细胞生成的成熟。作为最终的结果，我们建立了一种在体外产生人ES细胞来源的血小板的新方法。较少

Specific ligand binding to integrins initiates 'outside-in' signaling that coordinates with signaling cascades initiated through growth factor-, cytokine-, and G protein-coupled receptors to regulate actin reorganization, cell survival, and proliferation. In platelets, the binding of fibrinogen or von Willebrand factor(VWF) to integrin αIIbβ3 triggers signals that promote cytoskeletal changes, spreading and formation of stable platelet thrombi. Physical interactions between αIIbβ3 and non-receptor protein kinases, such as Src and Syk, have been demonstrated, and fibrinogen binding to the integrin results in activation of these tyrosine kinases. Studies with human platelets, platelets from gene-targeted mice and heterologous expression systems have suggested a model in which the integrin β3 cytoplasmic domain interacts directly and constitutively with the SH3 domain of c-Src. Upon integrin ligation and clustering, it is proposed that integrin-associated c-Src is activated rapidly, leadi … More ng to tyrosine phoshorylation of c-Src substrates and assembly of a nascent αIIbβ3-based signaling complex known to date that includes Syk, SLP-76, Vav, and ADAP(adhesion and degranulation promoting adaptor protein ; previously called Fyn-binding protein, Fyb or SLAP-130). Lnk is an SH2 domain-containing adapter protein that inhibits cytokine signaling. Lnk^<-/-> mice exhibit a marked thrombocytosis, as evidenced by 5-fold increase in platelet count, due to proliferation and maturation of precursor cells in response to cytokines. We show that Lnk is expressed in human and mouse platelets, and that Lnk plays an unanticipated role in platelet integrin αIIbβ3 outside-in signaling. Lnk^<-/-> platelets exhibit defects in spreading on fibrinogen, β3 subunit tyrosine phosphorylation, clot retraction and formation of thrombi on collagen under flow conditions. In contrast, inside-out signaling is normal in Lnk^<-/-> platelets, as evidenced by agonist-induced fibrinogen binding. In platelets, Lnk forms a complex with c-Src and ADAP, Fyn-binding adaptor, in a manner dependent on αIIbβ3 ligation and Src activation, and may be required for Fyn recruitment to αIIbβ3 together with tyrosine phosphorylation of the β3 subunit. These results demonstrate for the first time that Lnk adaptor plays a pivotal role in the adhesion responses of platelets and thrombus formation through regulation of outside-in integrin αIIbβ3 signaling. In addition, we addressed the role of WASP/WAVE family on actin cytoskeletal changes in megakaryocytes and platelets. In this part of the projects, we demonstrated that the WAVE2/Abil signaling complex mediates integrin-dependent lamellipodia formation and maturation of megakaryocytopoiesis. As a final result, we established a novel method to generate human ES cell-derived platelets in vitro. Less