Intracellular trafficking, localization, and mobilization of platelet-borne thiol isomerases

Alastair Poole, Marilena Crescente, Fred G. Pluthero, Tony Walsh, Jonathan M. Gibbins, Walter H Kahr

Research output: Contribution to journalArticle (Academic Journal)peer-review

34 Citations (Scopus)
284 Downloads (Pure)

Abstract

Objective - Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including PDI 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 for the first time 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 upon activation.

Approach and Results – Immunofluorescence microscopy (IFM) imaging was used to localize PDI and ERp57 in murine and human megakaryocytes at various developmental stages. IFM 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 SERCA3. IFM 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 Munc 13-4 (absent in platelets from Unc13dJinx mice).

Conclusions – 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 SNARE/Munc 13-4-dependent vesicular-plasma membrane fusion.
Original languageEnglish
Pages (from-to)1164-1173
Number of pages10
JournalArteriosclerosis, Thrombosis, and Vascular Biology
Volume36
Issue number6
Early online date14 Apr 2016
DOIs
Publication statusPublished - Jun 2016

Keywords

  • platelets
  • megakaryocytes
  • thiol isomerases
  • trafficking
  • granules

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