Editors' ChoicePlatelet Biology

Thrombosis Prevention Without Bleeding Extension

Science Signaling  12 Nov 2013:
Vol. 6, Issue 301, pp. ec276
DOI: 10.1126/scisignal.2004896

Platelets are key mediators of blood clotting, but excessive aggregation can contribute to vessel occlusion (thrombosis). As mediators of cell adhesion, integrins, which are heteromeric dimers consisting of α and β subunits, are important participants of platelet activation and clotting. Integrins exhibit inside-out signaling, by which agonists of other receptors can stimulate the binding of talin to the cytoplasmic portion of the β subunit to alter integrin conformation and enable interaction with soluble matrix components, such as fibrinogen. Integrins also exhibit outside-in signaling, by which ligand binding to the extracellular portion of integrin stimulates intracellular signals. By coimmunoprecipitation of wild-type and various β subunit mutants from CHO (Chinese hamster ovary) cells, Shen et al. identified a Gα13 binding site within the region where talin bound and found that these two proteins exhibited mutually exclusive binding to the β3 subunit. Coimmunoprecipitation of Gα13 and talin with the β3 subunit from platelets activated with a G protein–coupled receptor (GPCR) ligand thrombin showed that talin bound rapidly (within 1 minute) and transiently (undetectable by 2 minutes), then Gα13 bound, and by 30 minutes talin binding was increased and Gα13 binding was decreased. Inhibition of ligand binding to the integrin extracellular domain prevented the decrease in talin coimmunoprecipitation and the increase in Gα13 coimmunoprecipitation. Talin-deficient platelets did not aggregate or spread in response to stimuli that required the inside-out signaling-mediated conformational change in integrin, but aggregation and spreading were induced by stimuli that directly stimulated outside-in signaling. Mutation of the EXE motif, to which Gα13 bound, to AAA disrupted the interaction between Gα13 and β3 but did not prevent the interaction between β3 and talin. Platelets were isolated from irradiated mice with bone marrow transplantation of cells from β3-knockout mice reconstituted with β3-AAA. These platelets stimulated with a GPCR ligand bound soluble fibrinogen, indicating inside-out signaling, but failed to spread on immobilized fibrinogen, indicating that outside-in signaling was compromised. Myristoylated peptides that selectively blocked Gα13 binding to β3 prevented platelet spreading, but not binding to fibrinogen, and increased clot retraction in vitro, indicating specific inhibition of the Gα13-mediated phase of outside-in signaling. When injected into mice, the peptide reduced thrombosis in two different assays in vivo without triggering enhanced tail bleeding, which occurred with the currently available integrin inhibitor Integrilin. Thus, selective targeting of the Gα13-mediated outside-in integrin signaling in platelets may improve available therapies for thrombotic disease.

B. Shen, X. Zhao, K. A. O’Brien, A. Stojanovic-Terpo, M. K. Delaney, K. Kim, J. Cho, S. C.-T. Lam, X. Du, A directional switch of integrin signalling and a new anti-thrombotic strategy. Nature 503, 131–135 (2013). [PubMed]