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

血小板整合素α-IIb-β3由外而内的信号传导机制

• 批准号: 8528688
• 负责人: Xiaoping Du
• 金额: $ 37.96万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-15 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8528688
• 关键词: Adhesions; Adhesives; Agonist; Binding; Blood; Blood Platelets; Blood Vessels; Bone Marrow Transplantation; Calpain; Cause of Death; Chinese Hamster Ovary Cell; Clot retraction; Coagulation Process; Cytoplasmic Granules; Cytoplasmic Tail; Cytoskeleton; Data; Development; Disease; Extracellular Matrix; Family; Fibrin; Fibrinogen; Funding; GTP-Binding Proteins; Goals; In Vitro; Injury; Integrin Signaling Pathway; Integrin alpha Chains; Integrins; Ligand Binding; Ligands; Ligation; Mediating; Membrane; Modeling; Monomeric GTP-Binding Proteins; Movement; Myocardial Infarction; Names; Peptides; Pharmaceutical Preparations; Platelet Activation; Play; Protein Subunits; Publishing; Regulation; Role; SRC gene; Science; Side; Signal Pathway; Signal Transduction; Site; Stroke; Surface; Techniques; Testing; Thrombosis; Thrombus; Transgenic Mice; Tyrosine Phosphorylation; United States; Wound Healing; adhesion receptor; base; extracellular; fighting; in vivo; inhibitor/antagonist; mutant; response; rho guanine nucleotide exchange factor p115; seal; src-Family Kinases; wound

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.

描述（由申请人提供）：血小板整合素，aIIb¿ 3，与纤维蛋白原和纤维蛋白等粘附配体相互作用，介导血小板粘附和聚集。因此，整合素 aIIb 3 在心脏病和中风等血栓性疾病的发展中起着至关重要的作用。 3 还双向传输信号：整合素 aIIb 的配体结合功能¿ 3 由血小板内的信号激活（“由内而外”的配体与 aIIb 结合）。 3 诱导“由外向内”信号，该信号传输到血小板中并引发对放大和稳定血栓、伤口愈合和血管再通至关重要的细胞反应。该应用的目的是了解整合素外部的机制。众所周知，整合素从外到内的信号传导需要激活 Src 激酶家族并调节 RhoA 等小 GTP 酶。此外，整合素 aIIb 介导的 Src 激活尚不清楚。 3 最初介导血小板扩散，但随后诱导血小板介导的凝块回缩，这需要血小板膜向相反方向移动目前尚不清楚整合素 aIIb 如何发生。 3 改变膜运动方向 在当前资助期间，我们取得了以下发现：（1）G 蛋白亚基 Ga13 直接与整合素 ¿ 3 亚基，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 等人，Science，2010）。 3 破坏 c-Src 依赖性 RhoA 抑制，诱导 RhoA 依赖性血栓收缩 (3) 小 G 蛋白 RhoA 和 Rac1 在整合素依赖性收缩信号传导和血栓收缩中都很重要。我们提出以下假设：(1) 整合素 aIIb¿ 3 由外向内信号传导是由 Ga13 与 ¿ 的细胞质结构域直接结合介导的3. (2) Ga13 传递整合素信号激活 c-Src 并诱导 RhoA 的 c-Src 依赖性抑制，从而抑制血小板回缩并促进血小板扩散 (3) Calpain 裂解 ¿ 3 作为一种通过失活抑制 RhoA 的 Ga13/c-Src 依赖性信号通路来下调 Ga13 信号传导的机制，这促进了整合素依赖性 RhoA 激活并刺激血栓收缩。为了检验这些假设，我们提出了以下具体目标：目标 1，确定 Ga13 与 ¿ 结合的作用3 在介导整合素外向内信号传导方面，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，并确定 Ga13-¿ 3 相互作用的调节以及这种相互作用如何激活 c-Src 目的 2，研究 Ga13 和整合素依赖性 RhoA 控制血小板扩散和收缩的动态调节机制。 3 抑制 RhoA 和刺激 p190RhoGAP 的相互作用，通过 ¿ 竞争性抑制 Ga13-p115RhoGEF 3、钙蛋白酶诱导的晚期RhoA激活和回缩信号传导的机制。