CYTOSKELETAL DOMAINS IN THE ACTIVATED PLATELET

Platelets circulate in the blood as discoid cells which, when activate d, change shape by polymerizing actin into various structures, such as filopodia and stress fibers. In order to understand this process, it is necessary to determine how many other proteins are involved. As a f irst step in defining the full complement of actin-binding proteins in platelets, filamentous (F)-actin affinity chromatography was used. Th is approach identified >30 different proteins from ADP-activated human blood platelets which represented 4% of soluble protein. Although a n umber of these proteins are previously identified platelet actin-bindi ng proteins, many others appeared to be novel. Fourteen different poly clonal antibodies were raised against these apparently novel proteins and used to sort them into nine categories based on their molecular we ights and on their location in the sarcomere of striated muscle, in fi broblasts and in spreading platelets. Ninety-three percent of these pr oteins (13 of 14 proteins tested) were found to be associated with act in-rich structures in vivo. Four distinct actin filament structures we re found to form during the initial 15 min of activation on glass: fil opodia, lamellipodia, a contractile ring encircling degranulating gran ules, and thick bundles of filaments resembling stress fibers. Actin-b inding proteins not localized in the discoid cell became highly concen trated in one or another of these actin-based structures during spread ing, such that each structure contains a different complement of prote ins. These results present crucial information about the complexity of the platelet cytoskeleton, demonstrating that four different actin-ba sed structures form during the first 15 min of surface activation, and that there remain many as yet uncharacterized proteins awaiting furth er investigation that are differentially involved in this process. (C) 1995 Wiley-Liss, Inc.