Effect of cellular and receptor activation on the extent of integrin alphaIIbbeta3 internalization

Abstract

Previous studies by our laboratory demonstrated that internalization of fibrinogen-bound alphaIIbbeta3 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 alphaIIbbeta3. The present studies were designed to evaluate the roles of cellular and receptor activation states on the alphaIIbbeta3 internalization process. In these studies, washed platelets were allowed to bind FITC-D57, an antialphaIIb 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 alphaIIbbeta3 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 alphaIIbbeta3 internalization, platelets were treated with anti-LIBS6, which directly activates alphaIIbbeta3. Stimulation with anti-LIBS6 caused an approximate 8-fold increase in the extent of alphaIIbbeta3 internalization. To evaluate whether the activated pool of alphaIIbbeta3 is preferentially internalized, platelets were incubated with PAC-1, an antibody specific for activated alphaIIbbeta3. 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 alphaIIbbeta3 is selectively internalized in platelets. Collectively, these data suggest that alphaIIbbeta3 is internalized to a greater extent in activated platelets in a cytoskeleton-dependent manner. Furthermore, the active conformer of alphaIIbbeta3 is preferentially internalized which may act as a mechanism for downregulating adhesiveness of activated platelets in the circulation.