Previous studies have suggested that qualitative changes in platelet bound
fibrinogen modulate platelet aggregation. The present study used confocal scanning
laser microscopy to further evaluate post-
ligand binding events over a 60-minute time course. When
fluorescein isothiocyanate (
FITC)-
streptavidin was added to
ADP-stimulated platelets 1 minute after biotinylated
fibrinogen binding at 22 degrees C, bound
fibrinogen was found in variously sized patches on the cell surface. When
streptavidin was added 60 minutes later, bound
fibrinogen had been cleared from the platelet surface and was observed in clusters penetrating into platelets to various extents.
ADP-activated platelets did not
stain with a
monoclonal antibody against CD62 suggesting that platelets were not permeabilized during the experiment and had not released alpha-granules. Additional studies using either biotinylated
fibrinogen that had been prelabeled with
FITC-
streptavidin or
FITC-labeled
fibrinogen revealed similar patterns of platelet-associated
fibrinogen clearance and redistribution. Pretreatment of platelets with
cytochalasin D prevented this redistribution. Dual labeling experiments using biotinylated
fibrinogen and
FITC-
streptavidin as well as a monoclonal anti-
GPIIIa antibody labeled with
rhodamine-conjugated anti-mouse
IgG demonstrated the co-localization of
fibrinogen and
GPIIIa. Similar observations were made with
fibrinogen bound to
thrombin-stimulated platelets. In contrast,
fibronectin bound to
thrombin-activated platelets retained a predominantly surface membrane distribution under identical experimental conditions. Since surface-cleared
fibrinogen was accessible to exogenous
FITC-
streptavidin under conditions that did not lead to platelet permeabilization, the data suggest
fibrinogen deposition in compartments that are accessible to the extracellular milieu. This is consistent with the ability of exogenous
plasmin to completely remove cleared
fibrinogen pools without detectable
fibrinogen reexpression on the platelet surface or alpha-granule secretion. The data provide morphological evidence for the selective, GPIIb-IIIa mediated, actin-dependent clearance of bound
fibrinogen from the activated platelet surface, suggesting a mechanism for preventing and limiting
thrombus development.