SURFACE RECRUITMENT BUT NOT ACTIVATION OF INTEGRIN ALPHA(IIB)BETA(3) (GPIIB-IIIA) REQUIRES A FUNCTIONAL ACTIN CYTOSKELETON

Binding of integrin alpha(IIb)beta(3) (glycoprotein [GP] IIb-IIIa) to soluble fibrinogen requires that the receptor undergo a conformational change (receptor activation), which occurs rapidly in agonist-stimula ted platelets. Agonist stimulation of platelets also results in alpha( IIb)beta(3) recruitment from intracellular membranes (alpha-granules a nd open canalicular system) to the platelet surface. Once activated an d accessible, the receptor can engage, a process that corresponds to t he binding of the receptor to its soluble fibrinogen ligand, leading t o 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 recruitmen t. We used a flow cytometric assay to directly quantitate the binding of alpha(IIb)beta(3) to fluorescently labeled fibrinogen on the platel et surface. Cytochalasin D, which inhibits elongation of actin filamen ts, was used to prevent the actin response to platelet agonists. Despi te its ability to inhibit the actin response and alpha(IIb)beta(3) bin ding to the actin cytoskeleton, cytochalasin D did not alter the agoni st-induced intramolecular changes resulting in increased affinity of a lpha(IIb)beta(3), for soluble fibrinogen and therefore did not inhibit ADP-induced aggregation. Thus, disruption of the actin network with c ytochalasin D had no effect on the dissociation constant of the comple x between activated alpha(IIb)beta(3) and fibrinogen (K-d=0.26 to 0.28 mu mol/L). However, cytochalasin D suppressed the recruitment of cryp tic alpha(IIb)beta(3) molecules to the platelet surface. While the phy siological consequence of exposing additional alpha(IIb)beta(3) molecu les on the surface of platelets is unclear, it is tempting to speculat e that this process plays an important role in consolidating intra-art erial platelet thrombi, despite the shear strain generated by the arte rial blood flow.