Bound fibrinogen distribution on stimulated platelets: Examination by confocal scanning laser microscopy

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°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 α-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 α-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.