Ero1α Is Expressed on Blood Platelets in Association with Protein-disulfide Isomerase and Contributes to Redox-controlled Remodeling of αIIbβ3

Maria Swiatkowska,Gianluca Padula,Lidia Michalec,Marta Stasiak,Szymon Skurzynski,Czeslaw S Cierniewski

doi:10.1074/jbc.m109.092486

Maria Swiatkowska, Gianluca Padula + Show 4 more

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https://doi.org/10.1074/jbc.m109.092486

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Abstract

Recent evidence supports a role of protein-disulfide isomerase (PDI) in redox-controlled remodeling of the exofacial domains of α(IIb)β(3) in blood platelets. The aim of this study was to explain whether Ero1α can be responsible for extracellular reoxidation of the PDI active site. We showed that Ero1α can be found on platelets and is rapidly recruited to the cell surface in response to platelet agonists. It is physically associated with PDI and α(IIb)β(3), as suggested by colocalization analysis in confocal microscopy and confirmed by immunoprecipitation experiments. Apart from monomeric oxidized Ero1α, anti-α(IIb)β(3) immunoprecipitates showed the presence of several Ero1α-positive bands that corresponded to the complexes α(IIb)β(3)-PDI-Ero1α, PDI-Ero1α, and Ero1α-Ero1α dimers. It binds more efficiently to the activated α(IIb)β(3) conformer, and its interaction is inhibited by RGD peptides. Ero1α appears to be involved in the regulation of α(IIb)β(3) receptor activity because of the following: (a) blocking the cell surface Ero1α by antibodies leads to a decrease in platelet aggregation in response to agonists and a decrease in fibrinogen and PAC-1 binding, and (b) transfection of MEG01 with Ero1α increases α(IIb)β(3) receptor activity, as indicated by increased binding of fibrinogen.

Highlights

2008/0 from Polish Ministry of Scientific Research and Higher Education. 1 Both authors contributed to this work. 2 To whom correspondence should be addressed: Dept. of Molecular and oxygen to liberate hydrogen peroxide [5, 6]
Recent studies showed that substantial amounts of protein-disulfide isomerase (PDI), a major Ero1␣-binding protein, are present on the surface of blood platelets [9]
We raised polyclonal antiserum against recombinant Ero1␣ in rabwere fixed in 3% paraformaldehyde, incubated with anti-Ero1␣ antibodies conjugated with rhodamine, and subjected to confocal microscopy analysis

Summary

Introduction

2008/0 from Polish Ministry of Scientific Research and Higher Education. 1 Both authors contributed to this work. 2 To whom correspondence should be addressed: Dept. of Molecular and oxygen to liberate hydrogen peroxide [5, 6]. PDI was identified on the platelet surface, where it appears to play an important role in platelet reactions such as adhesion, aggregation, and secretion [9]. Both ␣IIb␤3 and ␣2␤1 were reported to be substrates of PDI, and their thiol groups seem to be implicated in platelet adhesion and aggregation. PDI was found to directly interact with ␣V␤3 in endothelial cells, and the exofacial disulfide exchange appears to be involved in the conformational changes that follow ␣V␤3 activation [10]. Ero1␣ can support PDI activity toward ␣IIb␤3 providing oxidative equivalents to PDI, which in turn reduces or rearranges disulfide bonds in the cargo protein

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