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

描述(申请人提供)：血小板整合素，AIIB？3，与纤维蛋白原和纤维蛋白等黏附配体相互作用，并介导血小板黏附和聚集。因此，整合素AIIB？3在心脏病发作和中风等血栓性疾病的发展中起着关键作用。整合素AIIB？3还双向传递信号：整合素AIIB？3的配体结合功能被来自血小板内的信号激活(“内向外”信号)。与AIIB？3的配体结合可诱导“由外而内”的信号，这些信号传递到血小板中，并在伤口愈合和血管再通畅过程中引发对放大和稳定血栓至关重要的细胞反应。本应用的目的是了解整合素自外向内信号转导的机制。已知整合素Outside-In信号转导需要激活Src家族的激酶和调节小的GTP酶，如RhoA。然而，启动整合素介导的Src激活的上游机制尚不清楚。此外，整合素AIIB？3最初介导了血小板的扩散，但后来诱导了血小板介导的凝块回缩，这需要血小板膜向相反的方向移动。目前尚不清楚整合素AIIB？3如何改变膜运动的方向。在当前的资助期间，我们取得了以下发现：(1)G蛋白亚基Ga13直接与整合素β3亚单位的细胞质结构域相互作用，这种相互作用是整合素依赖的c-Src激活、RhoA抑制和血小板扩散所必需的(Gong等人，《科学》，2010)。(2)c-Src依赖的RhoA抑制作用被calain切割破坏，导致依赖于RhoA的凝块收缩。(3)小G蛋白RhoA和rac1在整合素依赖的回缩信号和凝块回缩中都起重要作用。根据这些研究的数据，我们提出了以下假设：(1)整合素AIIB？3由外向内信号是通过Ga13与β3的细胞质结构域直接结合而介导的。(2)Ga13传递整合素信号来激活c-Src，并诱导c-Src依赖的抑制RhoA，从而抑制血小板回缩和促进血小板扩散。(3)通过抑制RhoA的依赖于Ga13/c-Src的信号通路，CaP3的裂解作用是下调Ga13信号的一种机制。这有助于整合素依赖的RhoA的激活，并刺激凝块回缩。为了验证这些假说，我们提出了以下具体目标：目的1，确定Ga13与？3结合在介导整合素由外向内信号转导中的作用。在这一目标中，我们将确定Ga13的重要性，特别是Ga13-3相互作用在各种整合素信号通路中的作用，并确定Ga13-？3相互作用是如何调节的，以及这种相互作用如何激活c-Src。目的2，探讨Ga13和RhoA整合素依赖的动态调节在控制血小板伸展和回缩中的作用机制。为此，我们将研究Ga13-β3相互作用在抑制RhoA和刺激p190RhoGAP中的作用，竞争抑制Ga13-p115RhoGlobal的作用，以及Calain诱导的晚期RhoA激活和收缩信号的机制。

DESCRIPTION (provided by applicant): The platelet integrin, aIIb¿3, interacts with adhesive ligands such as fibrinogen and fibrin, and mediates platelet adhesion and aggregation. Integrin aIIb¿3 thus plays a critical role in the development of thrombotic diseases such as heart attack and stroke. Integrin aIIb¿3 also transmits signals bidirectionally: The ligand binding function of integrin aIIb¿3 is activated by signals from within platelets ("inside-out" signaling). Ligand binding to aIIb¿3 induces "outside-in" signals, which transmit into platelets and elicit cellular response critical in amplifying and stabilizing thrombi, in wound healing and the re-canalization of blood vessels. The goal of this application is to understand the mechanisms of integrin outside-in signaling. It is known that integrin outside-in signaling requires the activation of the Src family of kinases and regulation of small GTPases such as RhoA. However, the upstream mechanisms that initiate integrin-mediated Src activation have been unknown. Furthermore, integrin aIIb¿3 initially mediates platelet spreading, but later induces platelet-mediated clot retraction, which requires the movement of platelet membranes in the opposite direction. It has been unclear how integrin aIIb¿3 switches the direction of membrane movements. During the current funding period, we have made the following discoveries: (1) The G protein subunit, Ga13, directly interacts with the cytoplasmic domain of the integrin ¿3 subunit, and this interaction is required for the integrin-dependent c-Src activation, RhoA inhibition and platelet spreading (Gong et al, Science, 2010). (2) a calpain cleavage of ¿3 disrupts the c- Src-dependent RhoA inhibition, inducing RhoA-dependent clot retraction. (3) the small G proteins, RhoA and Rac1, are both important in integrin-dependent retractile signaling and clot retraction. Based on data from these studies, we propose following hypotheses: (1) integrin aIIb¿3 outside-in signaling is mediated by the direct binding of Ga13 to the cytoplasmic domain of ¿3. (2) Ga13 transmits integrin signal to activate c-Src and induce c-Src-dependent inhibition of RhoA, resulting in inhibition of platelet retraction and promotion of platelet spreading. (3) Calpain cleavage of ¿3 serves as a mechanism that down regulates Ga13 signaling by inactivating the Ga13/c-Src-dependent signaling pathway that inhibits RhoA. This facilitates integrin-dependent RhoA activation and stimulates clot retraction. To test these hypotheses, we propose the following specific aims: Aim 1, to determine the role of Ga13 binding to ¿3 in mediating integrin outside-in signaling. In this aim, we will determine the importance of Ga13, particularly the role of Ga13-¿3 interaction, in various integrin signaling pathways, and determine how Ga13-¿3 interaction is regulated and how this interaction activates c- Src. Aim 2, to investigate the mechanism of Ga13 and integrin-dependent dynamic regulation of RhoA in controlling platelet spreading and retraction. In this aim, we will investigate the role of Ga13-¿3 interaction in inhibiting RhoA and stimulating p190RhoGAP, competitive inhibition of Ga13-p115RhoGEF by ¿3, and the mechanisms of calpain-induced late RhoA activation and retractile signaling.