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Distribution of ligand-occupied alpha IIb beta 3 in resting and activated
human platelets determined by expression of a novel class of ligand-induced
binding site recognized by monoclonal antibody AP6
P Nurden, M Humbert, RS Piotrowicz, C Bihour, C Poujol, AT Nurden and TJ Kunicki
URA 1464 CNRS, Hopital Cardiologique, Pessac, France.
The sequence beta (3)203-228 is involved, in a yet undetermined manner, in
alpha IIb beta 3 function. We now show that murine monoclonal antibody
(MoAb) AP6, specific for beta (3)211-221, binds to alpha IIb beta 3 on
adenosine diphosphate (ADP)-activated platelets only when the receptor is
occupied by intact fibrinogen. The ligand-induced binding- site reported by
AP6 is unique in that it is not expressed following occupancy by either RGD
peptides or the gamma-chain carboxy-terminal dodecapeptide. Binding of AP6
to platelets coincides temporally with the binding of the MoAb 9F9,
specific for a receptor-induced binding site on fibrinogen. Thus, AP6
reports the binding of fibrinogen to the recognition pocket of alpha IIb
beta 3. Its binding to thrombin- stimulated washed platelets correlates
with secretion as determined using an MoAb to P-selectin. When ultrathin
sections of nonactivated platelets were examined by immunogold staining and
electron microscopy, AP6 identified a pool of alpha IIb beta 3 colocalizing
with P-selectin and suggesting the presence of alpha IIb beta 3-ligand
complexes in the alpha-granule membrane. There was little binding of AP6 to
surface alpha IIb beta 3 of unstimulated platelets. After ADP-induced
activation, AP6 was abundantly distributed over the entire platelet
surface, including pseudopods, but only when fibrinogen was present in the
medium. ADP had little effect on AP6 reactivity within platelets. This
contrasted with washed platelets and thrombin, where extensive AP6 binding
was observed within internal membrane pools as early as 10 to 15 seconds
after stimulation. Surface labeling with AP6 followed slower kinetics. Flow
cytometry on Triton X-100 permeabilized fixed platelets confirmed AP6
binding to alpha IIb beta 3 within the platelet. Thus, our results provide
evidence of (1) a pool of alpha-granule alpha IIb beta 3 occupied by ligand
in nonactivated platelets; (2) thrombin- induced activation of alpha IIb
beta 3 within the platelet, and (3) thrombin-induced mobilization of
ligand-bound alpha IIb beta 3 to the surface.
Volume 88,
Issue 3,
pp. 887-899,
08/01/1996
Copyright © 1996 by The American Society of Hematology
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