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

描述（由申请人提供）：血小板整联蛋白aIIb <$3与粘附配体（如纤维蛋白原和纤维蛋白）相互作用，并介导血小板粘附和聚集。因此，整合素aIIb <$3在血栓性疾病如心脏病发作和中风的发展中起关键作用。整合素aIIb？3也双向传递信号：整合素aIIb <$3的配体结合功能被血小板内的信号激活（“由内而外”信号传导）。配体与aIIb结合？3诱导“由外向内”信号，其传递到血小板中并引发在扩大和稳定血栓、伤口愈合和血管再通中至关重要的细胞应答。本申请的目的是了解整合素由外向内信号传导的机制。已知整联蛋白由外向内信号传导需要Src激酶家族的激活和小GTP酶如RhoA的调节。然而，启动整合素介导的Src激活的上游机制一直是未知的。此外，整合素aIIb 3最初介导血小板扩散，但后来诱导血小板介导的凝块收缩，这需要血小板膜沿相反方向移动。目前还不清楚整合素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Ga 13直接与整合素3亚基的胞质结构域相互作用，并且这种相互作用是整合素依赖性c-Src激活、RhoA抑制和血小板扩散所必需的（Gong et al，Science，2010）。(2)钙蛋白酶裂解Δ 3破坏c-Src依赖性RhoA抑制，诱导RhoA依赖性凝块收缩。(3)小G蛋白RhoA和Rac 1在整联蛋白依赖性回缩信号和血块回缩中都是重要的。基于这些研究的数据，我们提出了以下假设：（1）整合素aIIb <$3由外向内的信号传导是由Ga 13直接结合到<$3的胞质结构域介导的。(2)Ga 13通过传递整合素信号激活c-Src，诱导c-Src依赖性抑制RhoA，从而抑制血小板收缩，促进血小板铺展。(3)Calpain卵裂3作为一种机制，通过失活抑制RhoA的Ga 13/c-Src依赖性信号传导途径下调Ga 13信号传导。这有助于整合素依赖性RhoA活化并刺激凝块收缩。为了验证这些假设，我们提出了以下具体目标：目标1，确定Ga 13结合到3在介导整合素由外向内信号传导中的作用。在这个目标中，我们将确定Ga 13的重要性，特别是Ga 13-<$3相互作用在各种整合素信号通路中的作用，并确定Ga 13-<$3相互作用如何调节以及这种相互作用如何激活c-Src。目的2、探讨Ga 13和整合素依赖性RhoA对血小板伸展和收缩的动态调节机制。为此，我们将研究Ga 13-<$3相互作用在抑制RhoA和刺激p190 RhoGAP中的作用，<$3对Ga 13-p115 RhoGEF的竞争性抑制，以及钙蛋白酶诱导的晚期RhoA激活和回缩信号的机制。

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.