Surface recruitment but not activation of integrin α_IIbβ₃ (GPIIb-IIIa) requires a functional actin cytoskeleton

Binding of integrin α_IIbβ₃ (glycoprotein [GP] IIb-IIIa) to soluble fibrinogen requires that the receptor undergo a conformational change (receptor activation), which occurs rapidly in agonist-stimulated platelets. Agonist stimulation of platelets also results in α_IIbβ₃ recruitment from intracellular membranes (α-granules and open canalicular system) to the platelet surface. Once activated and accessible, the receptor can engage, a process that corresponds to the binding of the receptor to its soluble fibrinogen ligand, leading to intracellular signaling reactions and centripetal migration of bound receptor molecules. Because these processes occur concurrently with a marked reorganization of the actin cytoskeleton, we investigated the role of actin in fibrinogen receptor activation and surface recruitment. We used a flow cytometric assay to directly quantitate the binding of α_IIbβ₃ to fluorescently labeled fibrinogen on the platelet surface. Cytochalasin D, which inhibits elongation of actin filaments, was used to prevent the actin response to platelet agonists. Despite its ability to inhibit the actin response and α_IIbβ₃ binding to the actin cytoskeleton, cytochalasin D did not alter the agonist-induced intramolecular changes resulting in increased affinity of α_IIbβ₃ for soluble fibrinogen and therefore did not inhibit ADP-induced aggregation. Thus, disruption of the actin network with cytochalasin D had no effect on the dissociation constant of the complex between activated α_IIbβ₃ and fibrinogen (K_d=0.26 to 0.28 μmol/L). However, cytochalasin D suppressed the recruitment of cryptic α_IIbβ₃ molecules to the platelet surface. While the physiological consequence of exposing additional α_IIbβ₃ molecules on the surface of platelets is unclear, it is tempting to speculate that this process plays an important role in consolidating intra-arterial platelet thrombi, despite the shear strain generated by the arterial blood flow.