ASSOCIATION OF VINCULIN TO THE PLATELET CYTOSKELET0N DURING RELEASE REACTION

Abstract

Vinculin is an Mr 130 kD protein, which in various cell types is located at the membrane attachment site of microfilaments and has been implicated in membrane-cytoskeleton interaction. Though we have previously verified its presence in platelets and showed that in resting platelets it is localized submembra-neously and around the ±-granules, its relation to the cytos-keleton is still to be elucidated. It has been also revealed by biochemical studies that in resting bovine gel filtered platelets vinculin is not cytoskeletal component, however about 50% of the total vinculin content became incorporated into the cytoskeleton during thrombin activation. To establish if its association to the cytoskeleton occured during pseudopode formation, aggregation or release reaction, cytoskeletons were prepared from platelets activated in various conditions and the time course of vinculin incorporation was analyzed by immuno-blotting. When pseudopode formation was inhibited by cytochalasin B pretreatment but neither aggregation nor release reaction induced by thrombin was prevented, the amount of vinculin in the Triton insoluble residue even increased. Phorbol myristate acetate (PMA), which induces pseudopode but not contractile gel formation, is a strong stimulus for aggregation, but induces only a slight release of the granule content. In parallel, only a low amount of vinculin was recovered in the cytoskeletal fraction following PMA induced aggregation. In contrast, when release reaction elicited by thrombin occured in the absence of aggregation the association of vinculin did not diminished.The results clearly demonstrate, that platelet shape change and pseudopode formation are not prerequisites of the incorporation of vinculin into the cytoskeleton, which occurs parallel to the release reaction.