Elevations of PAIgG are observed in platelets from patients with ITP and nonimmune thrombocytopenic (NIT) disorders. Most of the PAIgG within these platelets is thought to be non-specific plasma IgG trafficked into the platelet by platelet and/or megakaryocyte endocytosis. The trafficking of murine anti-GPHb/IIIa antibodies into platelet alpha granules has been described, however a comparison of the trafficking of immune and non-immune human IgG in platelets has not been reported. In this study we investigated pathways of IgG trafficking in platelets following incubation with a human anti-GPIIb/IIIa antibody (from a transfused Glanzmann's patient) or normal human IgG, by determining the immunolocalization of PAIgG over time (10-mins, 3-hrs & 20-hrs). Radioimmunoprecipitation and an antigen capture assay demonstrated that isolated immune IgG reacted with human GPIIb/IIIa, whereas isolated non-immune IgG did not react with normal platelet antigens. Confocal microscopy analysis showed that platelets incubated with non-immune IgG demonstrated IgG staining within platelets and an absence of IgG staining on the platelet surface. These results were similar to that observed for platelets from patients with non-immune thrombocytopenia. In comparison, platelets incubated with immune IgG demonstrated IgG staining both within platelets and on the platelet surface. This pattern of surface IgG staining was similar to that observed in ITP platelets. In platelets incubated with non-immune IgG, immunogold labeling of IgG was observed primarily within alpha granules with little to no labeling of their platelet surface membrane or OCS. Enumeration of gold particles within these structures (n=65 analyzed pits) did not differ from control platelets incubated in buffer alone (n=65 analyzed pits). Additionally, the number of gold particles per platelet did not increase over time. In comparison, platelets incubated with immune IgG demonstrated gold particles primarily within alpha granules and their OCS, with less labeling of their platelet surface membrane. Enumeration of gold particles in these platelets (n=65 analyzed pits) demonstrated significantly more labeling within their OCS and platelet surface membrane than control platelets or platelets incubated with non-immune IgG. These observations were consistent over time. Additionally, the number of gold particles within alpha granules increased over time indicating an accumulation of IgG within storage granules. These results suggest that platelet internalization of immune versus non-immune human IgG may differ.
Authors
Hughes M; Hayward CPM; Horsewood P; Warkentin TE; Kelton JG