EVIDENCE FOR AN ALPHA-GRANULAR POOL OF THE CYTOSKELETAL PROTEIN ALPHA-ACTININ IN HUMAN PLATELETS THAT REDISTRIBUTES WITH THE ADHESIVE GLYCOPROTEIN THROMBOSPONDIN-1 DURING THE EXOCYTOTIC PROCESS

In a previous study, we have demonstrated that the platelet adhesive g lycoprotein thrombospondin-1 (TSP-1) interacts specifically with the c ytoskeletal protein alpha-actinin in a solid-phase binding assay. Stor ed in the alpha-granules of platelets, TSP-1 is secreted during cell a ctivation and binds to the plasma membrane promoting the platelet macr oaggregate formation. However, the molecular mechanism by which TSP-1 reaches and binds to the platelet surface is to date unelucidated. alp ha-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 alpha-actinin with TSP-1 in platelets by exam ining their respective subcellular location during the platelet activa tion process. By indirect immunofluorescence, alpha-actinin was found to display a granular staining in resting platelets similar to that of TSP-1. Performing postembedding immunogold labeling for electron micr oscopy, we detected the presence of alpha-actinin throughout the cytop lasm, but the strongest gold staining was found in organelles identifi ed as alpha-granules on the basis of their ultrastructure and TSP-1 co ntent. With the use of double immunogold labeling on platelets at diff erent stages of activation by thrombin, both alpha-actinin and TSP-1 w ere seen redistributing from the alpha-granules to the platelet surfac e via the open canalicular system (OCS). At the same time, the cytopla smic alpha-actinin concentrated toward the plasma membrane, but no col ocalization with the F-actin bundles was evidenced. Finally, preembedd ing immunogold labeling and immunoprecipitation of I-125-surface-label ed, thrombin-activated platelets further demonstrated that alpha-actin in was expressed on the plasma membrane in the absence of any detectab le expression of actin and that it could form molecular complexes with TSP-1 on activated platelets. These results suggest that alpha-actini n found to be present on the platelet surface together with TSP-1 orig inates in the alpha-granules by fusion of the alpha-granules with the plasma membrane during platelet exocytosis.