title: Formation and contraction of a microfilamentous shell in saponin-permeabilized platelets.
abstract: To study the mechanism of granule centralization in platelets, we permeabilized with saponin in either EGTA (5 mM) or calcium (1 or 10 microM). Under all conditions, platelets retained 40-50% of their total actin and greater than 70% of their actin-binding protein (ABP) but lost greater than 80% of talin and myosin to the supernatant. Thin sections of platelets permeabilized in EGTA showed a microfilament network under the residual plasma membrane and throughout the cytoplasm. Platelets permeabilized in calcium contained a microfilament shell partly separated from the residual membrane. The shell stained brightly for F-actin. A less dense microfilament shell was also seen in sections of ADP-stimulated intact platelets subsequently permeabilized in EGTA. In the presence of 1 mM ATP gamma S and calcium, myosin was retained (70%) and was localized by indirect immunofluorescence in bright central spots that also stained intensely for F-actin. Electron micrographs showed centralized granules surrounded by a closely packed mass of microfilaments much like the structures seen in thrombin-stimulated intact platelets subsequently permeabilized in EGTA. Permeabilization in calcium, ATP, and okadaic acid, produced the same configuration of centralized granules and packed microfilaments; myosin was retained and the myosin regulatory light chain became phosphorylated. Microtubule coil disassembly before permeabilization did not inhibit granule centralization. These results suggest a possible mechanism for granule centralization in these models. The cytoskeletal network first separates from some of its connections to the plasma membrane by a calcium-dependent mechanism not involving ABP proteolysis. Phosphorylated myosin interacts with the microfilaments to contract the shell moving the granules to the platelet's center.
authors.affiliation: Department of Anatomy, University of Pennsylvania School of Medicine, Philadelphia 19104-6058.
authors.forename: F
authors.initials: F
authors.lastname: Stark
authors.forename: R
authors.initials: R
authors.lastname: Golla
authors.forename: V T
authors.initials: VT
authors.lastname: Nachmias
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry / Q000648:ultrastructure*; D000199:Actins / Q000032:analysis; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D001792:Blood Platelets / Q000737:chemistry / Q000648:ultrastructure*; D001798:Blood Proteins / Q000032:analysis; D002118:Calcium / Q000032:analysis; D002458:Cell Fractionation; D003594:Cytoplasmic Granules / Q000648:ultrastructure*; D003598:Cytoskeletal Proteins / Q000032:analysis; D004533:Egtazic Acid; D004988:Ethers, Cyclic / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D008840:Microfilament Proteins / Q000032:analysis / Q000378:metabolism; D008854:Microscopy, Electron; D009218:Myosins / Q000032:analysis / Q000378:metabolism; D019319:Okadaic Acid; D010539:Permeability; D012503:Saponins / Q000494:pharmacology; D016608:Talin
chemical_list: D000199:Actins; D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D004988:Ethers, Cyclic; D008840:Microfilament Proteins; D012503:Saponins; D016608:Talin; D019319:Okadaic Acid; C022571:adenosine 5'-O-(3-thiotriphosphate); D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D009218:Myosins; D002118:Calcium