Mapping the binding domains of the αIIb subunit

Abstract

α_IIbβ₃, a member of the integrin family of adhesive protein receptors, is the most abundant glycoprotein on platelet plasma-membranes and binds to adhesive proteins via the recognition of short amino acid sequences, for example the ubiquitous RGD motif. However, elucidation of the ligand-binding domains of the receptor remains controversial, mainly owing to the fact that integrins are conformationally labile during purification and storage. In this study, a detailed mapping of the extracellular region of the α_IIb subunit is presented, using overlapping 20-peptides, in order to identify the binding sites of α_IIb potentially involved in the platelet-aggregation event. Regions α_IIb 313–332, α_IIb 265–284 and α_IIb 57–64 of α_IIbβ₃ were identified as putative fibrinogen-binding domains because the corresponding peptides inhibited platelet aggregation and antagonized fibrinogen association, possibly by interacting with this ligand. The latter is further supported by the finding that the above peptides did not interfere with the binding of PAC-1 to the activated form of α_IIbβ₃. Furthermore, α_IIb 313–332 was found to bind to fibrinogen in a solid-phase binding assay. It should be emphasized that all the experiments in this study were carried out on activated platelets and consequently on the activated form of this integrin receptor. We hypothesize that RAD and RAE adhesive motifs, encompassed in α_IIb 313–332, 265–284 and 57–64, are capable of recognizing complementary domains of fibrinogen, thus inhibiting the binding of this ligand to platelets.