Physical proximity and functional interplay of the glycoprotein Ib-IX-V complex and the Fc receptor FcγRIIA on the platelet plasma membrane

Although the glycoprotein (GP) Ib-IX-V complex and Fc gamma 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 gamma RIIA, we assessed the effects of anti-GP Ib alpha monoclonal antibodies on Fc gamma RIIA-mediated platelet aggregation and on the direct binding of polymeric IgG to human platelets. In addition, we directly examined the proximity of Fc gamma 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 alpha 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 gamma 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 gamma RIIA and GP Ib alpha, 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 gamma RIIA from platelet lysates also co-precipitated GP Ib alpha. These results indicate that GP Ib alpha and Fc gamma RIIA are co-localized on the platelet membrane and that this association is not random.