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.
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.
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