Role of G protein signaling in the formation of the fibrin(ogen)-integrin IIb3-actin cytoskeleton complex in platelets

Effective platelet function requires formation of a physical link between fibrin(ogen), integrin IIb3, and cytoplasmic actin filaments. We investigated the role of the Gq, Gi, and G12/13 families of heterotrimeric GTP-binding proteins (G proteins) in the assembly of a ligand-IIb3-actin cytoskeleton complex. Selective and combined activation of the G proteins was achieved by using combinations of various platelet agonists and inhibitors. Formation and stability of fibrinogen-IIb3 interaction were evaluated by the extent of platelet aggregation and the rate of eptifibatide-induced platelet disaggregation; association of IIb3 with the cytoskeleton was analyzed by western blot. Formation of the fibrin-IIb3-actin cytoskeleton complex was evaluated by rotational thromboelastometry assay in which clot formation was induced by the mixture of reptilase and factor XIIIa. We demonstrated that involvement of heterotrimeric G proteins in the formation of the ligand-IIb3-cytoskeleton complex depends on whether fibrinogen or fibrin serves as the integrin ligand. Formation of the fibrinogen-IIb3-cytoskeleton complex requires combined activation of at least two G protein pathways while the maximal IIb3-cytoskeleton association and the strongest IIb3-fibrinogen binding supporting irreversible platelet aggregation require combined activation of all threeGq, Gi, and G12/13G protein families. In contrast, formation of the fibrin-IIb3-cytoskeleton complex mediating clot retraction is critically dependent on the activation of the Gi family, especially on the activation of Gz.