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
| Autor: | Véronique Dubernard, Monique Lemesle, Brigitte Arbeille, Chantal Legrand |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
Blood Platelets
Blotting Western Thrombin macromolecular substances Immunogold labelling Biology Cytoplasmic Granules Precipitin Tests Exocytosis Cell biology Cytoplasm Humans Actinin Platelet Platelet activation Fluorescent Antibody Technique Indirect Microscopy Immunoelectron Thrombospondins Cardiology and Cardiovascular Medicine Cytoskeleton Cell activation Actin |
| Zdroj: | Arteriosclerosis, Thrombosis, and Vascular Biology. 17:2293-2305 |
| ISSN: | 1524-4636 1079-5642 |
| DOI: | 10.1161/01.atv.17.10.2293 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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