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: 8186790
• 负责人: Xiaoping Du
• 金额: $ 39.75万
• 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/8186790
• 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. PUBLIC HEALTH RELEVANCE: Thrombotic diseases such as heart attack and stroke are a leading cause of death in United States. Integrins are a family of adhesion receptors expressed on the surface of blood platelets that are critical to thrombosis ("clotting" in blood vessels). Following integrin mediated platelet adhesion to the site of vascular injury, there is a signal sent by integrins from the outside to the inside of platelets (outside-in signaling) that induces platelet responses including platelet spreading, clot retraction and platelet release of pro-thrombotic factors that greatly amplify thrombus formation. In the previous funding period, we discovered that integrin outside-in signaling requires the binding of G protein, Ga13 to the inner side of integrin ¿3 subunit (published in Science, 2010). The binding of Ga13 to ¿3 sends a signal inhibiting another G protein named RhoA. The function of RhoA is to transmit a signal causing platelet contraction. By inhibiting contraction, integrins signaling allows platelets to spread out helping sealing the wound. This competitive renewal application is aimed at further investigating how the binding of Ga13 to integrins transmits signals that induce platelet responses and thrombosis. This study will help in the development of new concepts and new drugs in fighting thrombotic diseases.

描述（由申请人提供）：血小板整合素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，整合蛋白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也可以双向传递信号：整合素aIIb¿3的配体结合功能被血小板内部的信号激活（“内向外”信号）。与aIIb¿3结合的配体诱导“外向内”信号，这些信号传递到血小板并引发细胞反应，这对扩大和稳定血栓、伤口愈合和血管再通至关重要。本应用程序的目标是了解整合素的内外信号传导机制。众所周知，整合素的外向内信号需要激活Src家族激酶和调节小的gtpase，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最初介导血小板扩散，但随后诱导血小板介导的凝块缩回，这需要血小板膜向相反方向运动。目前还不清楚整合蛋白aIIb¿3如何改变膜运动的方向。在目前的资助期内，我们取得了以下发现：(1)G蛋白亚基Ga13直接与整合素¿3亚基的细胞质结构域相互作用，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)钙蛋白酶对¿3的裂解破坏了c- src依赖性RhoA抑制，诱导RhoA依赖性凝块缩回。(3)小G蛋白RhoA和Rac1在整合素依赖的收缩信号和凝块收缩中都很重要。基于这些研究数据，我们提出以下假设：(1)整合素aIIb¿3的外向内信号通路是由Ga13直接结合到¿3的细胞质结构域介导的。(2) Ga13通过传递整合素信号激活c-Src，诱导c-Src依赖性的RhoA抑制，从而抑制血小板收缩，促进血小板扩散。(3) Calpain切割¿3可能通过使抑制RhoA的Ga13/c- src依赖性信号通路失活而下调Ga13信号通路。这促进了整合素依赖性RhoA激活和刺激凝块缩回。为了验证这些假设，我们提出了以下具体目标：目的1，确定Ga13与¿3结合在介导整合素外向内信号传导中的作用。为此，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，以及这种相互作用是如何激活c- Src的。目的2：探讨Ga13和整合素依赖的RhoA动态调控血小板扩张和收缩的机制。在这个目的中，我们将研究Ga13-¿3相互作用在抑制RhoA和刺激p190RhoGAP中的作用，¿3对Ga13- p115rhogef的竞争性抑制，以及calpain诱导的晚期RhoA激活和收缩信号传导的机制。