Physical Proximity and Functional Interplay of the Glycoprotein Ib-IX-V Complex and the Fc Receptor FcγRIIA on the Platelet Plasma Membrane

Abstract

Although the glycoprotein (GP) Ib-IX-V complex andFc␥RIIA are distinct platelet membrane receptors, previous studies have suggested that these structures may be co-localized. To determine more directly the proximity of GP Ib-IX-V and Fc␥RIIA, we assessed the effects of anti-GP Ib␣ monoclonal antibodies on Fc␥RIIA-mediated platelet aggregation and on the direct binding of polymeric IgG to human platelets. In addition, we directly examined the proximity of Fc␥RII and GP Ib-IX-V using flow cytometric fluorescence energy transfer and immunoprecipitation studies. Preincubation of platelets with either of two monoclonal antibodies (AN51 or SZ2) directed against GP Ib␣ completely blocked platelet aggregation by polymeric IgG. Similarly, these antibodies totally inhibited platelet aggregation by two strains of viridans group streptococci known to induce aggregation via Fc␥RIIA. In addition, AN51 and SZ2 significantly reduced the binding of polymeric IgG to washed fixed platelets. When assessed by flow cytometry, significant levels of bidirectional energy transfer were detected between Fc␥RIIA and GP Ib␣, indicating a physical proximity of less than 10 nm between these receptors. This energy transfer was not due to high receptor density, because no homoassociative energy transfer was seen. Moreover, immunoprecipitation of Fc␥RIIA from platelet lysates also co-precipitated GP Ib␣. These results indicate that GP Ib␣ and Fc␥RIIA are co-localized on the platelet membrane and that this association is not random. The glycoprotein (GP)1 Ib-IX-V complex and Fc␥RIIA are distinct platelet membrane receptors for von Willebrand factor and polymeric IgG, respectively. The GP Ib-IX-V complex contains four polypeptides (GP Ib␣, GP Ib␤, GP IX, and GP V), which are present in a stoichiometry of 2:2:2:1 on the platelet plasma membrane (1). The precise configuration by which these peptides form a functional receptor is unknown, although more than one copy of each polypeptide per receptor complex is probable (1). This receptor mediates platelet adhesion to sites of blood vessel injury by binding von Willebrand factor in the subendothelium and participates in platelet activation by thrombin by providing a high affinity binding site for this agonist. In contrast to the GP Ib-IX-V complex, the platelet receptor for the Fc portion of IgG, Fc␥RIIA, is much simpler in structure, consisting of a single transmembrane polypeptide. This receptor is responsible for the aggregation and activation of platelets induced by immune complexes, opsonized bacteria, and certain antibodies that bind other platelet surface proteins (2).Although GP Ib-IX-V and Fc␥RIIA are structurally unrelated, a functional interaction between these receptors has been suspected for some time. Moore et al. reported that both polymeric IgG and IgG Fc fragments could inhibit platelet aggregation induced by von Willebrand factor and ristocetin (3). The reciprocal phenomenon was also observed, i.e. prior incubation of platelets with von Willebrand factor and ristocetin inhibited plate...

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