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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1466-1473.)
© 1995 American Heart Association, Inc.

Articles

Surface Recruitment but Not Activation of Integrin _IIbß₃ (GPIIb-IIIa) Requires a Functional Actin Cytoskeleton

John B. Addo; Paul F. Bray; Dmitriy Grigoryev; Nauder Faraday; Pascal J. Goldschmidt-Clermont

From the Divisions of Cardiology and Hematology, Department of Medicine, and Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Md.

Correspondence to Pascal J. Goldschmidt-Clermont, Bernard Laboratory, Ross 1023, Johns Hopkins University, 720 Rutland Ave, Baltimore, MD 21287.

Abstract Binding of integrin _IIbß₃ (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 _IIbß₃ recruitment from intracellular membranes (-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 _IIbß₃ 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 _IIbß₃ binding to the actin cytoskeleton, cytochalasin D did not alter the agonist-induced intramolecular changes resulting in increased affinity of _IIbß₃ 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 _IIbß₃ and fibrinogen (K_d=0.26 to 0.28 µmol/L). However, cytochalasin D suppressed the recruitment of cryptic _IIbß₃ molecules to the platelet surface. While the physiological consequence of exposing additional _IIbß₃ 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.

Key Words: cytoskeleton • integrin • actin • fibrinogen receptor • platelets

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