title: Intracellular Trafficking, Localization, and Mobilization of Platelet-Borne Thiol Isomerases.
abstract: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation.
  
  
  
  Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13d(Jinx) mice).
  
  
  
  Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.
authors.affiliation: From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.).
authors.forename: Marilena
authors.initials: M
authors.lastname: Crescente
authors.affiliation: From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.).
authors.forename: Fred G
authors.initials: FG
authors.lastname: Pluthero
authors.affiliation: From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.).
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.affiliation: From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.).
authors.forename: Richard W
authors.initials: RW
authors.lastname: Lo
authors.affiliation: From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.).
authors.forename: Tony G
authors.initials: TG
authors.lastname: Walsh
authors.affiliation: From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.).
authors.forename: Michael P
authors.initials: MP
authors.lastname: Schenk
authors.affiliation: From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.).
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Holbrook
authors.affiliation: From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.).
authors.forename: Silvia
authors.initials: S
authors.lastname: Louriero
authors.affiliation: From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.).
authors.forename: Marfoua S
authors.initials: MS
authors.lastname: Ali
authors.affiliation: From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.).
authors.forename: Sakthivel
authors.initials: S
authors.lastname: Vaiyapuri
authors.affiliation: From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.).
authors.forename: Hervé
authors.initials: H
authors.lastname: Falet
authors.affiliation: From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.).
authors.forename: Ian M
authors.initials: IM
authors.lastname: Jones
authors.affiliation: From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.).
authors.forename: Alastair W
authors.initials: AW
authors.lastname: Poole
authors.affiliation: From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.). j.m.gibbins@reading.ac.uk walter.kahr@sickkids.ca.
authors.forename: Walter H A
authors.initials: WH
authors.lastname: Kahr
authors.affiliation: From the School of Biological Sciences, University of Reading, Reading, United Kingdom (M.C., M.P.S., L.M.H., S.L., M.S.A., S.V., I.M.J., J.M.G.); Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada (F.G.P., L.L., R.W.L., W.H.A.K.); Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada (R.W.L., W.H.A.K.); School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (T.G.W., A.W.P.); and Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA (H.F.). j.m.gibbins@reading.ac.uk walter.kahr@sickkids.ca.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Gibbins
mesh_terms: D000199:Actins / Q000097:blood; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D001798:Blood Proteins / Q000172:deficiency / Q000235:genetics; D037281:Calnexin / Q000097:blood; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D005838:Genotype; D006801:Humans; D008533:Megakaryocytes / Q000187:drug effects / Q000201:enzymology*; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000097:blood / Q000172:deficiency / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010641:Phenotype; D015539:Platelet Activation / Q000187:drug effects; D019704:Protein Disulfide-Isomerases / Q000096:biosynthesis / Q000097:blood*; D021381:Protein Transport; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000097:blood
chemical_list: D000199:Actins; D001798:Blood Proteins; D008565:Membrane Proteins; C585423:Nbeal2 protein, mouse; C507969:UNC13D protein, human; C520225:Unc13d protein, mouse; D037281:Calnexin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C513195:Pdia3 protein, mouse; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human
keywords: blood platelets; megakaryocytes; membrane fusion; protein disulfide isomerases; secretory vesicles