Activation of the fibrinogen binding site on platelets isolated from a patient with the Strasbourg I variant of Glanzmann's thrombasthenia

Abstract

One proposed ligand binding site on platelet integrin α(IIb)β3 is the region of the β3 subunit encompassing amino acids 211-221. However, we recently showed that synthetic peptides corresponding to amino acids 211-221 inhibit fibrinogen binding to α(IIb)β3 by binding to α(IIb)β3 and not to fibrinogen. In this study, we show that AP6, a monoclonal antibody (MoAb) directed against amino acids 214-221 of β3, bound to immobilized active α(IIb)β3 but did not inhibit fibrinogen binding to the complex. We then determined whether non-functional α(IIb)β3 on platelets with a β3 Arg- 214 → Trp mutation (Strasbourg I variant of Glanzmann's thrombasthenia or GTV) could be induced to aggregate after treatment with dithiothreitol (DTT). DTT has been shown to expose the fibrinogen receptor on normal platelets. DTT treatment of GTV platelets did result in the formation of the fibrinogen binding site as indicated by the binding of pl-55, an MoAb that only binds to the activated form of α(IIb)β3. Furthermore, DTT-treated GTV platelets aggregated in the presence of fibrinogen and divalent cations. This aggregation was inhibited by EDTA, RGDS, and the selective α(IIb)β3 antagonist, Ro 43-5054. These data show that Arg-214 of β3 is not required for fibrinogen binding or for platelet aggregation. However, this amino acid appears to be critical for the formation and for the maintenance of the correct tertiary structure of the fibrinogen binding site on α(IIb)β3.