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Arg-Gly-Asp-dependent occupancy of GPIIb/IIIa by applaggin: evidence for
internalization and cycling of a platelet integrin
JD Wencel-Drake, AL Frelinger , MG Dieter and SC Lam
Department of Medical Laboratory Sciences, University of Illinois, Chicago
60612.
Using indirect immunofluorescence microscopy we examined the distribution
and cycling of GPIIb/IIIa after binding to applaggin, a high-affinity
Arg-Gly-Asp (RGD)--containing ligand. Resting, unfixed platelets were
incubated with applaggin for 30 minutes at 37 degrees C, and bound
applaggin was detected by an affinity-purified rabbit anti- applaggin
antibody. Examination of intact cells showed a rim pattern for applaggin,
consistent with its binding to the platelet surface. Staining of Triton
X-100--permeabilized cells showed an intracellular pool of applaggin.
Competition of applaggin binding by either AP-2, an anti-GPIIb/IIIa
monoclonal antibody (MoAb) that blocks fibrinogen binding, or the synthetic
peptide RGDW eliminated both surface and intracellular staining, indicating
that applaggin is binding to GPIIb/IIIa in an RGD-dependent manner.
Inhibition of platelet activation by PGE1 and theophylline had no effect on
the observed staining patterns, indicating that cellular activation is not
required for surface binding and subsequent internalization. To evaluate
whether occupancy of functional binding sites on GPIIb/IIIa is required for
internalization, we used mAb15, an anti-GPIIIa antibody that neither blocks
fibrinogen binding nor induces the expression of ligand-induced binding
sites on GPIIb/IIIa. In these studies mAb15 was internalized in a manner
analogous to both AP-2 and applaggin, showing that occupancy of the RGD
binding site is not required to initiate receptor internalization. To
estimate the size of the newly internalized pool of applaggin,
125I-applaggin--binding studies were performed. Displacement of bound
125I-applaggin by excess unlabeled applaggin or EDTA showed that at least
17% of bound applaggin was nondisplaceable when binding was performed under
conditions permitting membrane flow and internalization. These data
indicate that GPIIb/IIIa is internalized in unstimulated platelets
independent of cellular activation or occupancy of the functional binding
site(s) of GPIIb/IIIa by RGD-containing ligands. Thus, internalization of
GPIIb/IIIa may represent a mechanism by which the surface expression of
this adhesion receptor is regulated.
Volume 81,
Issue 1,
pp. 62-69,
01/01/1993
Copyright © 1993 by The American Society of Hematology
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