Protein disulfide isomerase‐A1 regulates intraplatelet reactive oxygen species–thromboxane A <sub>2</sub>‐dependent pathway in human platelets

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Abstract

Background

Platelet‐derived protein disulfide isomerase 1 (PDIA1) regulates thrombus formation, but its role in the regulation of platelet function is not fully understood.

Aims

The aim of this study was to characterize the role of PDIA1 in human platelets.

Methods

Proteomic analysis of PDI isoforms in platelets was performed using liquid chromatography tandem mass spectometry, and the expression of PDIs on platelets in response to collagen, TRAP‐14, or ADP was measured with flow cytometry. The effects of bepristat, a selective PDIA1 inhibitor, on platelet aggregation, expression of platelet surface activation markers, thromboxane A ₂ (TxA ₂), and reactive oxygen species (ROS) generation were evaluated by optical aggregometry, flow cytometry, ELISA, and dihydrodichlorofluorescein diacetate‐based fluorescent assay, respectively.

Results

PDIA1 was less abundant compared with PDIA3 in resting platelets and platelets stimulated with TRAP‐14, collagen, or ADP. Collagen, but not ADP, induced a significant increase in PDIA1 expression. Bepristat potently inhibited the aggregation of washed platelets induced by collagen or convulxin, but only weakly inhibited platelet aggregation induced by TRAP‐14 or thrombin, and had the negligible effect on platelet aggregation induced by arachidonic acid. Inhibition of PDIA1 by bepristat resulted in the reduction of TxA ₂ and ROS production in collagen‐ or thrombin‐stimulated platelets. Furthermore, bepristat reduced the activation of αIIbβ3 integrin and expression of P‐selectin.

Conclusions

PDIA1 acts as an intraplatelet regulator of the ROS‐TxA ₂ pathway in collagen‐GP VI receptor‐mediated platelet activation that is a mechanistically distinct pathway from extracellular regulation of αIIbβ3 integrin by PDIA3.

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Most cited references 55

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Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant.

Rudi Busse, Sven Wind, Sven Wind … (2008)

A large body of literature suggest that vascular reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases are important sources of reactive oxygen species. Many studies, however, relied on data obtained with the inhibitor apocynin (4'-hydroxy-3'methoxyacetophenone). Because the mode of action of apocynin, however, is elusive, we determined its mechanism of inhibition on vascular NADPH oxidases. In HEK293 cells overexpressing NADPH oxidase isoforms (Nox1, Nox2, or Nox4), apocynin failed to inhibit superoxide anion generation detected by lucigenin chemiluminescence. In contrast, apocynin interfered with the detection of reactive oxygen species in assay systems selective for hydrogen peroxide or hydroxyl radicals. Importantly, apocynin interfered directly with the detection of peroxides but not superoxide, if generated by xanthine/xanthine oxidase or nonenzymatic systems. In leukocytes, apocynin is a prodrug that is activated by myeloperoxidase, a process that results in the formation of apocynin dimers. Endothelial cells and smooth muscle cells failed to form these dimers and, therefore, are not able to activate apocynin. Dimer formation was, however, observed in Nox-overexpressing HEK293 cells when myeloperoxidase was supplemented. As a consequence, apocynin should only inhibit NADPH oxidase in leukocytes, whereas in vascular cells, the compound could act as an antioxidant. Indeed, in vascular smooth muscle cells, the activation of the redox-sensitive kinases p38-mitogen-activate protein kinase, Akt, and extracellular signal-regulated kinase 1/2 by hydrogen peroxide and by the intracellular radical generator menadione was prevented in the presence of apocynin. These observations indicate that apocynin predominantly acts as an antioxidant in endothelial cells and vascular smooth muscle cells and should not be used as an NADPH oxidase inhibitor in vascular systems.

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Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein.

Yasushi Ishihama, Yoshiya Oda, Tsuyoshi Tabata … (2005)

To estimate absolute protein contents in complex mixtures, we previously defined a protein abundance index (PAI) as the number of observed peptides divided by the number of observable peptides per protein (Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome. Res. 12, 1231-1245). Here we report that PAI values obtained at different concentrations of serum albumin show a linear relationship with the logarithm of protein concentration in LC-MS/MS experiments. This was also the case for 46 proteins in a mouse whole cell lysate. For absolute quantitation, PAI was converted to exponentially modified PAI (emPAI), equal to 10PAI minus one, which is proportional to protein content in a protein mixture. For the 46 proteins in the whole lysate, the deviation percentages of the emPAI-based abundances from the actual values were within 63% on average, similar or better than determination of abundance by protein staining. emPAI was applied to comprehensive protein expression analysis and to a comparison study between gene and protein expression in a human cancer cell line, HCT116. The values of emPAI are easily calculated and add important quantitation information to proteomic experiments; therefore we suggest that they should be reported in large scale proteomic identification projects.

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Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents.

Reema Jasuja, Freda H Passam, Daniel Kennedy … (2012)

Thrombosis, or blood clot formation, and its sequelae remain a leading cause of morbidity and mortality, and recurrent thrombosis is common despite current optimal therapy. Protein disulfide isomerase (PDI) is an oxidoreductase that has recently been shown to participate in thrombus formation. While currently available antithrombotic agents inhibit either platelet aggregation or fibrin generation, inhibition of secreted PDI blocks the earliest stages of thrombus formation, suppressing both pathways. Here, we explored extracellular PDI as an alternative target of antithrombotic therapy. A high-throughput screen identified quercetin-3-rutinoside as an inhibitor of PDI reductase activity in vitro. Inhibition of PDI was selective, as quercetin-3-rutinoside failed to inhibit the reductase activity of several other thiol isomerases found in the vasculature. Cellular assays showed that quercetin-3-rutinoside inhibited aggregation of human and mouse platelets and endothelial cell-mediated fibrin generation in human endothelial cells. Using intravital microscopy in mice, we demonstrated that quercetin-3-rutinoside blocks thrombus formation in vivo by inhibiting PDI. Infusion of recombinant PDI reversed the antithrombotic effect of quercetin-3-rutinoside. Thus, PDI is a viable target for small molecule inhibition of thrombus formation, and its inhibition may prove to be a useful adjunct in refractory thrombotic diseases that are not controlled with conventional antithrombotic agents.

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Author and article information

Contributors

Kamil Przyborowski:

ORCID: https://orcid.org/0000-0002-8526-945X

kamil.przyborowski@jcet.eu

Stefan Chlopicki:

ORCID: https://orcid.org/0000-0002-2878-3858

stefan.chlopicki@jcet.eu

Journal

Journal ID (nlm-ta): J Thromb Haemost

Journal ID (iso-abbrev): J Thromb Haemost

Journal ID (doi): 10.1111/(ISSN)1538-7836

Journal ID (publisher-id): JTH

Title: Journal of Thrombosis and Haemostasis

Publisher: John Wiley and Sons Inc. (Hoboken )

ISSN (Print): 1538-7933

ISSN (Electronic): 1538-7836

Publication date (Electronic): 14 October 2021

Publication date (Print): January 2022

Volume: 20

Issue: 1 ( doiID: 10.1111/jth.v20.1 )

Pages: 157-169

Affiliations

[ ¹ ] Jagiellonian Centre for Experimental Therapeutics (JCET) Jagiellonian University Krakow Poland

[ ² ] Department of Haemostasis and Haemostatic Disorders Medical University of Lodz Lodz Poland

[ ³ ] Mass Spectrometry Laboratory Institute of Biochemistry and Biophysics Polish Academy of Sciences Warszawa Poland

[ ⁴ ] Laboratory of Carbocyclic Compounds Latvian Institute of Organic Synthesis Riga Latvia

[ ⁵ ] Chair of Biomedical Sciences Medical University of Lodz Lodz Poland

[ ⁶ ] Chair of Pharmacology Jagiellonian University Medical College Krakow Poland

Author notes

[*] [* ] Correspondence

Stefan Chlopicki and Kamil Przyborowski, Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14. 30‐348 Krakow, Poland.

Email: stefan.chlopicki@ 123456jcet.eu and kamil.przyborowski@ 123456jcet.eu

Author information

Kamil Przyborowski https://orcid.org/0000-0002-8526-945X

Anna Kurpinska https://orcid.org/0000-0003-2130-0099

Dagmara Wojkowska https://orcid.org/0000-0002-9317-9817

Patrycja Kaczara https://orcid.org/0000-0003-0106-4172

Joanna Suraj‐Prazmowska https://orcid.org/0000-0002-0190-2474

Kamil Karolczak https://orcid.org/0000-0001-5866-7244

Agata Malinowska https://orcid.org/0000-0001-6599-901X

Agnieszka Pelesz https://orcid.org/0000-0002-7907-7750

Agnieszka Kij https://orcid.org/0000-0003-2233-0564

Ivars Kalvins https://orcid.org/0000-0001-6076-7818

Cezary Watala https://orcid.org/0000-0002-5627-7872

Stefan Chlopicki https://orcid.org/0000-0002-2878-3858

Article

Publisher ID: JTH15539

DOI: 10.1111/jth.15539

PMC ID: 9292974

PubMed ID: 34592041

SO-VID: 318cedc2-cda8-41ab-9615-647c4cc903b6

License:

This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

History

Date revision received : 17 September 2021

Date received : 03 April 2021

Date accepted : 27 September 2021

Page count

Figures: 6, Tables: 2, Pages: 13, Words: 8648

Funding

Funded by: The National Centre for Research and Development, Warsaw, Poland

Award ID: STRATEGMED1/233226/11/NCBR/2015

Custom metadata

source-schema-version-number 2.0

cover-date January 2022

details-of-publishers-convertor Converter:WILEY_ML3GV2_TO_JATSPMC version:6.1.7 mode:remove_FC converted:18.07.2022

ScienceOpen disciplines: Hematology

Keywords: bepristat,flow cytometry,pdis expression,platelet activation,proteomics

Data availability:

ScienceOpen disciplines: Hematology

Keywords: bepristat, flow cytometry, pdis expression, platelet activation, proteomics