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

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 DESCRIPTION (provided by applicant): The platelet adhesion receptor, integrin αIIbβ3, plays a critical physiological role in hemostasis and also a critical pathological role in thrombosis and in the development of thrombotic diseases such as heart attack and stroke. The integrin antagonists are effective anti-thrombotics but also have significant adverse effect of hemorrhage, which can be life-threatening. Thus, it is important to develop a new generation of anti-thrombotics that minimally cause adverse effect of bleeding. Integrin αIIbβ3 not only mediates platelet adhesion and aggregation but also transmits signals bidirectionally: Agonist-induced intracellular signals from within platelets activate the extracellular ligand binding function of integrin αIIbβ3 ("inside-out" signaling). The binding of extracellular ligand to αIIβ3 then induces "outside-in" signals, which elicit cellular responses such as platelet spreading, granule secretion, and platelet-dependent clot retraction. The "outside-in signaling" response is critical in amplifying and stabilizing thrombi, which is critically important in occlusive thromboss. During the current funding period, we have shown that the G protein subunit, Gα13, directly interacts with a highly conserved ExE motif in the cytoplasmic domain of several integrin β subunits between talin binding sites, and "time-share" the binding region with talin in opposing waves. Talin binding occurs during inside-out signaling and late phase outside-in signaling, whereas the Gα13 binding occurs during early phase outside-in signaling. We further show that Gα13 is selectively important in the early phase outside-in signaling leading to platelet spreading and amplification of platelet thrombus formation. Importantly, we have developed selective inhibitors of Gα13-integrin interaction that potently inhibited integrin outside-in signaling and arterial thrombosis without causing adverse effect of bleeding. Based on these data, we propose the overall hypothesis that the opposing waves of talin and Gα13 binding to integrin cytoplasmic domain switch the direction of integrin signaling and also control outcomes of integrin outside-in signaling. To test this hypothesis, we propose the following specific aims: (1) To investigate the switch between talin and Gα13 binding to β3 during integrin signaling and its regulatory mechanisms. (2)To further investigate the roles of Gα13-integrin interaction in mediating integrin outside-in signaling, amplification of platelet activation, and thrombosis. These studies will facilitate the development of new generations of anti-thrombotic drugs for treating thrombosis without causing excessive bleeding.