Thromb Haemost 2022; 122(09): 1469-1478
DOI: 10.1055/s-0042-1744378
Coagulation and Fibrinolysis

N-Glycosylation Deficiency Reduces the Activation of Protein C and Disrupts Endothelial Barrier Integrity

Tiffany Pascreau
1   HITh, UMR_S 1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Saclay, Le Kremlin-Bicêtre, France
2   Laboratoire d'Hématologie, AP-HP, Hôpital Necker-Enfants malades, Paris, France
,
François Saller
1   HITh, UMR_S 1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Saclay, Le Kremlin-Bicêtre, France
,
1   HITh, UMR_S 1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Saclay, Le Kremlin-Bicêtre, France
,
Dominique Lasne
1   HITh, UMR_S 1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Saclay, Le Kremlin-Bicêtre, France
2   Laboratoire d'Hématologie, AP-HP, Hôpital Necker-Enfants malades, Paris, France
,
Arnaud Bruneel
3   Biochimie Métabolique et Cellulaire, AP-HP, Hôpital Bichat-Claude Bernard, Paris, France
4   INSERM UMR1193, Université Paris-Saclay, Châtenay-Malabry, France
,
Christelle Reperant
1   HITh, UMR_S 1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Saclay, Le Kremlin-Bicêtre, France
,
François Foulquier
5   Université de Lille, CNRS, UMR 8576–UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France
,
Cécile V. Denis
1   HITh, UMR_S 1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Saclay, Le Kremlin-Bicêtre, France
,
Pascale De Lonlay
6   Centre de référence des maladies héréditaires du métabolisme de l'enfant et de l'adulte - MAMEA, Filière G2M, MetabERN, Imagine Institute, AP-HP, Hôpital Necker-Enfants Maladies, University Paris-Descartes, Paris, France
,
Delphine Borgel
1   HITh, UMR_S 1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Saclay, Le Kremlin-Bicêtre, France
2   Laboratoire d'Hématologie, AP-HP, Hôpital Necker-Enfants malades, Paris, France
› Author Affiliations
Funding The study was funded by ERA-NET cofounding (Reference 643578) and Fondation pour la Recherche Médicale (DEA20140731350).

Abstract

Phosphomannomutase 2 (PMM2) deficiency is the most prevalent congenital disorder of glycosylation. It is associated with coagulopathy, including protein C deficiency. Since all components of the anticoagulant and cytoprotective protein C system are glycosylated, we sought to investigate the impact of an N-glycosylation deficiency on this system as a whole. To this end, we developed a PMM2 knockdown model in the brain endothelial cell line hCMEC/D3. The resulting PMM2low cells were less able to generate activated protein C (APC), due to lower surface expression of thrombomodulin and endothelial protein C receptor. The low protein levels were due to downregulated transcription of the corresponding genes (THBD and PROCR, respectively), which itself was related to downregulation of transcription regulators Krüppel-like factors 2 and 4 and forkhead box C2. PMM2 knockdown was also associated with impaired integrity of the endothelial cell monolayer—partly due to an alteration in the structure of VE-cadherin in adherens junctions. The expression of protease-activated receptor 1 (involved in the cytoprotective effects of APC on the endothelium) was not affected by PMM2 knockdown. Thrombin stimulation induced hyperpermeability in PMM2low cells. However, pretreatment of cells with APC before thrombin simulation was still associated with a barrier-protecting effect. Taken as a whole, our results show that the partial loss of PMM2 in hCMEC/D3 cells is associated with impaired activation of protein C and a relative increase in barrier permeability.

Author Contributions

T.P. performed the research, analyzed data, and wrote the manuscript; F.S. analyzed data and wrote the manuscript; E.P.B., D.L., A.B., C.V.D., and P.D.L. wrote the manuscript; F.F. contributed for reagents; C.R. performed research and analyzed data; D.B. designed the research, analyzed data, and wrote the manuscript; all authors read and approved the final version of the manuscript.


Supplementary Material



Publication History

Received: 05 July 2021

Accepted: 02 February 2022

Article published online:
19 June 2022

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