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

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 ant ibody (MoAb) AP6, specific for beta(3)211-221, binds to alpha(IIb)beta (3) on adenosine diphosphate (ADP)-activated platelets only when the r eceptor is occupied by intact fibrinogen. The ligand-induced binding-s ite reported by AP6 is unique in that it is not expressed following oc cupancy by either RGD peptides or the gamma-chain carboxy-terminal dod ecapeptide. 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 reco gnition pocket of alpha(IIb)beta(3). Its binding to thrombin-stimulate d washed platelets correlates with secretion as determined using an Mo Ab to P-selectin. When ultrathin sections of nonactivated platelets we re examined by immunogold staining and electron microscopy, AP6 identi fied a pool of alpha(IIb)beta(3) colocalizing with P-selectin and sugg esting the presence of alpha(IIb)beta(3)-ligand complexes in the alpha -granule membrane. There was little binding of AP6 to surface alpha(II b)beta(3) of unstimulated platelets. After ADP-induced activation, Ape was abundantly distributed over the entire platelet surface, includin g 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 ob served within internal membrane pools as early as 10 to 15 seconds aft er stimulation. Surface labeling with Ape followed slower kinetics. Fl ow cytometry on Triton X-100 permeabilized fixed platelets confirmed A pe 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) occ upied by ligand in nonactivated platelets, (2) thrombin-induced activa tion of alpha(IIb)beta(3) within the platelet, and (3) thrombin-induce d mobilization of ligand-bound alpha(IIb)beta(3) to the surface. (C) 1 996 by The American Society of Hematology.