Outside-in signaling mechanisms of platelet integrin alpha-IIb-beta3

 描述(申请人提供)：血小板黏附受体，整合素αIIbβ3，在止血中起关键的生理作用，也在血栓形成和血栓形成中起关键的病理作用。 在心脏病发作和中风等血栓性疾病的发展过程中。整合素拮抗剂是有效的抗血栓药物，但也有明显的出血副作用，可能危及生命。因此，开发新一代抗血栓药物，将出血的不良反应降至最低，是非常重要的。整合素αIIbβ3不仅介导血小板的黏附和聚集，而且还能双向传递信号：激动剂诱导的细胞内信号激活整合素αIIbβ3的细胞外配体结合功能(“内向外”信号)。细胞外配体与αIIβ3结合后会诱导“由外而内”的信号，从而引发细胞反应，如血小板铺展、颗粒分泌和血小板依赖的凝块回缩。“由外而内”的信号反应是放大和稳定血栓的关键，而血栓的放大和稳定对闭塞性血栓形成至关重要。在目前的资助期间，我们已经证明G蛋白亚基Gα13直接与Talin结合位点之间的几个整合素β亚基的细胞质区域中高度保守的EXE基序相互作用，并在相反的波中与Talin“分时共享”结合区域。α结合发生在自内向外信号转导和晚期自外向内信号转导过程中，而G Talin 13结合发生在早期自外向内信号转导过程中。我们进一步证明，G-α13在早期阶段选择性地重要，外向内信号导致血小板扩散和放大血小板血栓形成。重要的是，我们已经开发了G-α13-整合素相互作用的选择性抑制剂，有效地抑制了整合素由外向内的信号转导和动脉血栓形成，而不会引起出血的不良反应。基于这些数据，我们提出了总体假设，即Talin和Gα13与整合素胞质结构域结合的相反波改变了整合素信号的方向，并控制了整合素由外向内的信号转导结果。为了验证这一假设，我们提出了以下具体目标：(1)研究整合素信号转导过程中Talin和Gα13与β3结合的转换及其调控机制。(2)进一步研究G-α13-整合素相互作用在介导整合素由外向内信号转导、血小板活化放大和血栓形成中的作用。这些研究将促进新一代抗血栓药物的开发，以治疗血栓形成，而不会导致大量出血。

 DESCRIPTION (provided by applicant): The platelet adhesion receptor, integrin αIIbβ3, plays a critical physiological role in hemostasis and also a critical pathological role in thrombosis and in the development of thrombotic diseases such as heart attack and stroke. The integrin antagonists are effective anti-thrombotics but also have significant adverse effect of hemorrhage, which can be life-threatening. Thus, it is important to develop a new generation of anti-thrombotics that minimally cause adverse effect of bleeding. Integrin αIIbβ3 not only mediates platelet adhesion and aggregation but also transmits signals bidirectionally: Agonist-induced intracellular signals from within platelets activate the extracellular ligand binding function of integrin αIIbβ3 ("inside-out" signaling). The binding of extracellular ligand to αIIβ3 then induces "outside-in" signals, which elicit cellular responses such as platelet spreading, granule secretion, and platelet-dependent clot retraction. The "outside-in signaling" response is critical in amplifying and stabilizing thrombi, which is critically important in occlusive thromboss. During the current funding period, we have shown that the G protein subunit, Gα13, directly interacts with a highly conserved ExE motif in the cytoplasmic domain of several integrin β subunits between talin binding sites, and "time-share" the binding region with talin in opposing waves. Talin binding occurs during inside-out signaling and late phase outside-in signaling, whereas the Gα13 binding occurs during early phase outside-in signaling. We further show that Gα13 is selectively important in the early phase outside-in signaling leading to platelet spreading and amplification of platelet thrombus formation. Importantly, we have developed selective inhibitors of Gα13-integrin interaction that potently inhibited integrin outside-in signaling and arterial thrombosis without causing adverse effect of bleeding. Based on these data, we propose the overall hypothesis that the opposing waves of talin and Gα13 binding to integrin cytoplasmic domain switch the direction of integrin signaling and also control outcomes of integrin outside-in signaling. To test this hypothesis, we propose the following specific aims: (1) To investigate the switch between talin and Gα13 binding to β3 during integrin signaling and its regulatory mechanisms. (2)To further investigate the roles of Gα13-integrin interaction in mediating integrin outside-in signaling, amplification of platelet activation, and thrombosis. These studies will facilitate the development of new generations of anti-thrombotic drugs for treating thrombosis without causing excessive bleeding.