Regulation of Outside-In Integrin Signaling in Platelets

描述（由申请人提供）：血管损伤后，纤维蛋白原和血管性血友病因子等黏附配体与整合素allbbeta3结合，在止血和血栓形成过程中影响血小板聚集和扩散。这些反应是由配体介导的allbbeta3聚集引发的，它启动了“由外而内”的信号来重组肌动蛋白细胞骨架。该实验室最近的研究表明，血小板中的外向内信号需要Src家族酪氨酸激酶（SFKs），特别是c-Src，它与β 3结合，并以依赖于PTP-1B（一种蛋白酪氨酸激酶）的方式被所有β 3聚集激活。在这里，三个主要的未解决的问题将被问及血小板内外信号的分子基础及其生物学后果。首先，整合素和sfk之间的直接相互作用是否代表血小板中内外信号时空启动的一般机制？由于血小板含有五种不同的整合素和至少六种不同的sfk，因此这种可能性将通过共免疫沉淀技术、活细胞中的双分子荧光互补成像以及使用基于fret的报告细胞定位活细胞中的Src激活来评估。此外，将在果蝇细胞中评估整合素/SFK的相互作用，以确定整合素直接激活SFK在多大程度上是一个进化保守过程。第二，PTP-1B如何激活整合素下游的c-Src ？PTP-1B被募集到allbbeta3/c-Src复合体以及其他整合素/SFK复合体的机制将在血小板和模型细胞系统中进行评估，重点关注适配蛋白的可能作用。此外，还将确定整合素聚类对PTP-1B催化活性的影响。第三，体外信号的选择性中断是否会影响体内血栓的形成？本研究将在新的基因靶向小鼠中研究动脉血栓形成，这些小鼠被预测在allbbeta与c-Src或其他sfk的相互作用中存在选择性缺陷，或在肌动蛋白重组所需的下游事件中存在缺陷。总之，这些研究将提供关于内外整合素信号如何启动的分子见解，并确定该过程在体内调节血小板功能的程度。因此，这条研究路线可能导致新的抗血栓药物靶点的鉴定，并作为其他血细胞中整合素信号传导的范例。

DESCRIPTION (provided by applicant): Following vascular injury, adhesive ligands such as fibrinogen and von Willebrand factor engage integrin allbbeta3 to effect platelet aggregation and spreading during hemostasis and thrombosis. These responses are triggered by ligand-mediated allbbeta3 clustering, which initiates "outside-in" signals to reorganize the actin cytoskeleton. Recent work from this laboratory has established that outside-in signaling in platelets requires Src family tyrosine kinases (SFKs), c-Src in particular, which bind to beta3 and are activated by allbbeta3 clustering in a manner dependent on PTP-1B, a protein tyrosine kinase. Here, three major unresolved questions will be asked concerning the molecular basis of outside-in signaling in platelets and its biological consequences. First, do direct interactions between integrins and SFKs represent a general mechanism for spatio-temporal initiation of outside-in signaling in platelets? Since platelets contain five different integrins and at least six different SFKs, this possibility will be evaluated by co-immunoprecipitation techniques, by bimolecular fluorescence complementation imaging in live cells, and by localization of Src activation in live cells using a FRET-based reporter. In addition, integrin/SFK interactions will be assessed in Drosophila cells to determine the extent to which direct activation of SFKs by integrins is an evolutionary conserved process. Second, how does PTP-1B activate c-Src downstream of integrins? The mechanism by which PTP-1B is recruited to the allbbeta3/c-Src complex, and possibly to other integrin/SFK complexes, will be evaluated in platelets and model cell systems, focusing on the possible role of adapter proteins. In addition, the effect of integrin clustering on PTP-1B catalytic activity will be determined. Third, does selective disruption of outside- in signaling affect thrombus formation in vivo? Here arterial thrombosis will be studied in novel gene-targeted mice predicted to have selective defects in the interaction of allbbeta with c-Src or other SFKs, or defects in downstream events required for actin reorganization. Altogether, these studies will provide molecular insights into how outside-in integrin signaling is initiated and establish the extent to which this process regulates platelet function in vivo. Thus, this line of investigation may lead to identification of new anti-thrombotic drug targets and serve as a paradigm for integrin signaling in other blood cells.