Evidence for an α-Granular Pool of the Cytoskeletal Protein α-Actinin in Human Platelets That Redistributes With the Adhesive Glycoprotein Thrombospondin-1 During the Exocytotic Process

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Abstract
Abstract In a previous study, we have demonstrated that the platelet adhesive glycoprotein thrombospondin-1 (TSP-1) interacts specifically with the cytoskeletal protein α-actinin in a solid-phase binding assay. Stored in the α-granules of platelets, TSP-1 is secreted during cell activation and binds to the plasma membrane promoting the platelet macroaggregate formation. However, the molecular mechanism by which TSP-1 reaches and binds to the platelet surface is to date unelucidated. α-Actinin is an actin-binding and actinin–cross-linking protein that is present in most cells and may act as a link between the bundles of F-actin and the plasma membrane. In this study, we have investigated a possible interaction of α-actinin with TSP-1 in platelets by examining their respective subcellular location during the platelet activation process. By indirect immunofluorescence, α-actinin was found to display a granular staining in resting platelets similar to that of TSP-1. Performing postembedding immunogold labeling for electron microscopy, we detected the presence of α-actinin throughout the cytoplasm, but the strongest gold staining was found in organelles identified as α-granules on the basis of their ultrastructure and TSP-1 content. With the use of double immunogold labeling on platelets at different stages of activation by thrombin, both α-actinin and TSP-1 were seen redistributing from the α-granules to the platelet surface via the open canalicular system (OCS). At the same time, the cytoplasmic α-actinin concentrated toward the plasma membrane, but no colocalization with the F-actin bundles was evidenced. Finally, preembedding immunogold labeling and immunoprecipitation of 125 I-surface–labeled, thrombin-activated platelets further demonstrated that α-actinin was expressed on the plasma membrane in the absence of any detectable expression of actin and that it could form molecular complexes with TSP-1 on activated platelets. These results suggest that α-actinin found to be present on the platelet surface together with TSP-1 originates in the α-granules by fusion of the α-granules with the plasma membrane during platelet exocytosis.