Abstract 380: PDI and ERp57 Localization in Platelets Is Regulated by Actin Polymerization
Introduction: PDI and ERp57 are members of the thiol isomerase family of enzymes that are released to the platelet surface where they contribute to a range of platelet responses. PDI has been reported to be present in subcellular structures in resting platelets, although it is excluded from α - and δ -granules, and lysosomes. It was, however, recently shown to co-localise with TLR9 in T-granules, intracellular bodies underlying the plasma membrane. The subcellular localisation of other platelet thiol isomerases or their mechanisms of translocation to the cell surface have not been established.
Hypothesis and Methods: Using spinning disk confocal microscopy we sought to explore whether platelet thiol isomerases ERp57 and PDI are co-localised in resting and activated platelets and to test the hypothesis that translocation of thiol isomerases to the platelet surface is dependent on actin polymerization.
Results and Conclusions: In resting platelets, PDI and ERp57 were organized in punctate structures both on the platelet surface and in the cytoplasm. They did not colocalise with P-selectin consistent with them residing outside α -granules. In contrast to expectations, TLR9 did not colocalize with PDI and ERp57, suggesting their exclusion from T-granules. Partial colocalization was observed in resting platelets between PDI and ERp57 in granule-like structures. Upon platelet activation, this degree of co-distribution persisted with PDI and ERp57 migrating to the platelet surface. Latrunculin A, an inhibitor of actin polymerization, decreased P-selectin exposure on the platelet surface, as measured by flow cytometry, and agonist-stimulated platelets treated with this agent retained a rounded shape, as shown by tubulin staining. Importantly, latrunculin A also blocked the translocation of PDI and ERp57 from internal granule-like structures to the platelet surface. We conclude that in resting platelets PDI and ERp57 are organized, and partially co-localised, in punctuate structures unlikely to represent α- or T-granules. The polymerization of actin during platelet activation exerts a fundamental role in the relocalization of PDI and ERp57 from these cytoplasmic structures to the platelet surface.
Author Disclosures: M. Crescente: None. F.G. Pluthero: None. M.P. Schenk: None. W.H.A. Kahr: None. J.M. Gibbins: None.
- © 2014 by American Heart Association, Inc.