Effect of cellular and receptor activation on the extent of integrin α IIb β 3 internalization

Summary.  Previous studies by our laboratory demonstrated that internalization of fibrinogen‐bound α IIb β 3 correlated with both a loss of aggregation and a loss of bound fibrinogen from the platelet surface. However, these studies do not address whether cellular activation, receptor activation and/or receptor occupancy are responsible for the observed internalization of α IIb β 3 . The present studies were designed to evaluate the roles of cellular and receptor activation states on the α IIb β 3 internalization process. In these studies, washed platelets were allowed to bind FITC‐D57, an antiα IIb monoclonal antibody, and were subsequently treated with ADP, thrombin receptor activation peptide (TRAP) or antiLIBS6 monoclonal antibody. Following flow cytometric analyses for log green fluorescence, rabbit antifluorescein was added, and the samples were re‐analyzed for residual/unquenched fluorescence. Because access of the quenching antibody is limited to extracellular/surface‐associated fluorescein, protection from quenching by antifluorescein is taken as evidence of internalization. Stimulation of platelets with ADP or TRAP resulted in a significant increase in the percent internalization of α IIb β 3 compared to control (8.7% and 12.8% vs. 2.9%). Addition of cytochalasin E prior to stimulation resulted in a greater than 90% inhibition of both TRAP and ADP‐induced internalization, suggesting that activation‐dependent internalization is mediated by the actin cytoskeleton. To investigate whether receptor activation increases the extent of α IIb β 3 internalization, platelets were treated with anti‐LIBS6, which directly activates α IIb β 3 . Stimulation with anti‐LIBS6 caused an approximate 8‐fold increase in the extent of α IIb β 3 internalization. To evaluate whether the activated pool of α IIb β 3 is preferentially internalized, platelets were incubated with PAC‐1, an antibody specific for activated α IIb β 3 . Platelets stimulated with TRAP, demonstrated a dose‐dependent internalization of PAC‐1. However, approximately 29% of total PAC‐1 binding was internalized, irrespective of TRAP concentration, suggesting that a constant proportion of activated α IIb β 3 is selectively internalized in platelets. Collectively, these data suggest that α IIb β 3 is internalized to a greater extent in activated platelets in a cytoskeleton‐dependent manner. Furthermore, the active conformer of α IIb β 3 is preferentially internalized which may act as a mechanism for downregulating adhesiveness of activated platelets in the circulation.