Download PDF
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
,
Elsa P. Bianchini 
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).
› Further Information
    Permissions and Reprints

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.

Keywords

protein C - phosphomannomutase 2 - endothelial - N-glycosylation

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

© 2022. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

  • References

  • 1 Péanne R, de Lonlay P, Foulquier F. et al. Congenital disorders of glycosylation (CDG): quo vadis?. Eur J Med Genet 2018; 61 (11) 643-663
    CrossrefPubMedGoogle Scholar
  • 2 Jaeken J, Péanne R. What is new in CDG?. J Inherit Metab Dis 2017; 40 (04) 569-586
    CrossrefPubMedGoogle Scholar
  • 3 Schiff M, Roda C, Monin M-L. et al. Clinical, laboratory and molecular findings and long-term follow-up data in 96 French patients with PMM2-CDG (phosphomannomutase 2-congenital disorder of glycosylation) and review of the literature. J Med Genet 2017; 54 (12) 843-851
    CrossrefPubMedGoogle Scholar
  • 4 Arnoux JB, Boddaert N, Valayannopoulos V. et al. Risk assessment of acute vascular events in congenital disorder of glycosylation type Ia. Mol Genet Metab 2008; 93 (04) 444-449
    CrossrefPubMedGoogle Scholar
  • 5 Linssen M, Mohamed M, Wevers RA, Lefeber DJ, Morava E. Thrombotic complications in patients with PMM2-CDG. Mol Genet Metab 2013; 109 (01) 107-111
    CrossrefPubMedGoogle Scholar
  • 6 Tamminga RYJ, Lefeber DJ, Kamps WA, van Spronsen FJ. Recurrent thrombo-embolism in a child with a congenital disorder of glycosylation (CDG) type Ib and treatment with mannose. Pediatr Hematol Oncol 2008; 25 (08) 762-768
    CrossrefPubMedGoogle Scholar
  • 7 Pascreau T, de la Morena-Barrio ME, Lasne D. et al. Elevated thrombin generation in patients with congenital disorder of glycosylation and combined coagulation factor deficiencies. J Thromb Haemost 2019; 17 (11) 1798-1807
    CrossrefPubMedGoogle Scholar
  • 8 Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood 2007; 109 (08) 3161-3172
    CrossrefPubMedGoogle Scholar
  • 9 Griffin JH, Fernández JA, Liu D, Cheng T, Guo H, Zlokovic BV. Activated protein C and ischemic stroke. Crit Care Med 2004; 32 (5, Suppl) S247-S253
    CrossrefPubMedGoogle Scholar
  • 10 Lopez-Ramirez MA, Pham A, Girard R. et al. Cerebral cavernous malformations form an anticoagulant vascular domain in humans and mice. Blood 2019; 133 (03) 193-204
    CrossrefPubMedGoogle Scholar
  • 11 Parkinson JF, Vlahos CJ, Yan SCB, Bang NU. Recombinant human thrombomodulin. Regulation of cofactor activity and anticoagulant function by a glycosaminoglycan side chain. Biochem J 1992; 283 (Pt 1): 151-157
    CrossrefPubMedGoogle Scholar
  • 12 Liaw PCY, Mather T, Oganesyan N, Ferrell GL, Esmon CT. Identification of the protein C/activated protein C binding sites on the endothelial cell protein C receptor. Implications for a novel mode of ligand recognition by a major histocompatibility complex class 1-type receptor. J Biol Chem 2001; 276 (11) 8364-8370
    CrossrefPubMedGoogle Scholar
  • 13 Soto AG, Smith TH, Chen B. et al. N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias. Proc Natl Acad Sci U S A 2015; 112 (27) E3600-E3608
    PubMedGoogle Scholar
  • 14 Soto AG, Trejo J. N-linked glycosylation of protease-activated receptor-1 second extracellular loop: a critical determinant for ligand-induced receptor activation and internalization. J Biol Chem 2010; 285 (24) 18781-18793
    CrossrefPubMedGoogle Scholar
  • 15 Grinnell BW, Walls JD, Gerlitz B. Glycosylation of human protein C affects its secretion, processing, functional activities, and activation by thrombin. J Biol Chem 1991; 266 (15) 9778-9785
    CrossrefPubMedGoogle Scholar
  • 16 Stearns-Kurosawa DJ, Kurosawa S, Mollica JS, Ferrell GL, Esmon CT. The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex. Proc Natl Acad Sci U S A 1996; 93 (19) 10212-10216
    CrossrefPubMedGoogle Scholar
  • 17 Hwang J, Qi L. Quality control in the endoplasmic reticulum: crosstalk between ERAD and UPR pathways. Trends Biochem Sci 2018; 43 (08) 593-605
    CrossrefPubMedGoogle Scholar
  • 18 Crone C, Christensen O. Electrical resistance of a capillary endothelium. J Gen Physiol 1981; 77 (04) 349-371
    CrossrefPubMedGoogle Scholar
  • 19 Srinivasan B, Kolli AR, Esch MB, Abaci HE, Shuler ML, Hickman JJ. TEER measurement techniques for in vitro barrier model systems. J Lab Autom 2015; 20 (02) 107-126
    CrossrefPubMedGoogle Scholar
  • 20 Giannotta M, Trani M, Dejana E. VE-cadherin and endothelial adherens junctions: active guardians of vascular integrity. Dev Cell 2013; 26 (05) 441-454
    CrossrefPubMedGoogle Scholar
  • 21 Thiel C, Lübke T, Matthijs G, von Figura K, Körner C. Targeted disruption of the mouse phosphomannomutase 2 gene causes early embryonic lethality. Mol Cell Biol 2006; 26 (15) 5615-5620
    CrossrefPubMedGoogle Scholar
  • 22 He P, Srikrishna G, Freeze HH. N-glycosylation deficiency reduces ICAM-1 induction and impairs inflammatory response. Glycobiology 2014; 24 (04) 392-398
    CrossrefPubMedGoogle Scholar
  • 23 Isermann B, Hendrickson SB, Zogg M. et al. Endothelium-specific loss of murine thrombomodulin disrupts the protein C anticoagulant pathway and causes juvenile-onset thrombosis. J Clin Invest 2001; 108 (04) 537-546
    CrossrefPubMedGoogle Scholar
  • 24 Stibler H, Holzbach U, Tengborn L, Kristiansson B. Complex functional and structural coagulation abnormalities in the carbohydrate-deficient glycoprotein syndrome type I. Blood Coagul Fibrinolysis 1996; 7 (02) 118-126
    CrossrefPubMedGoogle Scholar
  • 25 Pathak R, Shao L, Chafekar SM. et al. IKKβ regulates endothelial thrombomodulin in a Klf2-dependent manner. J Thromb Haemost 2014; 12 (09) 1533-1544
    CrossrefPubMedGoogle Scholar
  • 26 Villarreal Jr G, Zhang Y, Larman HB, Gracia-Sancho J, Koo A, García-Cardeña G. Defining the regulation of KLF4 expression and its downstream transcriptional targets in vascular endothelial cells. Biochem Biophys Res Commun 2010; 391 (01) 984-989
    CrossrefPubMedGoogle Scholar
  • 27 Lin Z, Kumar A, SenBanerjee S. et al. Kruppel-like factor 2 (KLF2) regulates endothelial thrombotic function. Circ Res 2005; 96 (05) e48-e57
    PubMedGoogle Scholar
  • 28 Sangwung P, Zhou G, Nayak L. et al. KLF2 and KLF4 control endothelial identity and vascular integrity. JCI Insight 2017; 2 (04) e91700
    CrossrefPubMedGoogle Scholar
  • 29 Welsh JD, Hoofnagle MH, Bamezai S. et al. Hemodynamic regulation of perivalvular endothelial gene expression prevents deep venous thrombosis. J Clin Invest 2019; 129 (12) 5489-5500
    CrossrefPubMedGoogle Scholar
  • 30 Griffin JH, Zlokovic BV, Mosnier LO. Activated protein C: biased for translation. Blood 2015; 125 (19) 2898-2907
    CrossrefPubMedGoogle Scholar
  • 31 Alabanza LM, Bynoe MS. Thrombin induces an inflammatory phenotype in a human brain endothelial cell line. J Neuroimmunol 2012; 245 (1–2): 48-55
    CrossrefPubMedGoogle Scholar
  • 32 Komarova YA, Mehta D, Malik AB. Dual regulation of endothelial junctional permeability. Sci STKE 2007; 2007 (412) re8
    CrossrefPubMedGoogle Scholar
  • 33 Weksler BB, Subileau EA, Perrière N. et al. Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 2005; 19 (13) 1872-1874
    CrossrefPubMedGoogle Scholar
  • 34 Scott DW, Tolbert CE, Graham DM, Wittchen E, Bear JE, Burridge K. N-glycosylation controls the function of junctional adhesion molecule-A. Mol Biol Cell 2015; 26 (18) 3205-3214
    CrossrefPubMedGoogle Scholar
  • 35 Lertkiatmongkol P, Paddock C, Newman DK, Zhu J, Thomas MJ, Newman PJ. The role of sialylated glycans in human platelet endothelial cell adhesion molecule 1 (PECAM-1)-mediated trans homophilic interactions and endothelial cell barrier function. J Biol Chem 2016; 291 (50) 26216-26225
    CrossrefPubMedGoogle Scholar
  • 36 Geyer H, Geyer R, Odenthal-Schnittler M, Schnittler H-J. Characterization of human vascular endothelial cadherin glycans. Glycobiology 1999; 9 (09) 915-925
    CrossrefPubMedGoogle Scholar
  • 37 Schneider A, Thiel C, Rindermann J. et al. Successful prenatal mannose treatment for congenital disorder of glycosylation-Ia in mice. Nat Med 2011; 18 (01) 71-73
    CrossrefPubMedGoogle Scholar
  • 38 Truin G, Guillard M, Lefeber DJ. et al. Pericardial and abdominal fluid accumulation in congenital disorder of glycosylation type Ia. Mol Genet Metab 2008; 94 (04) 481-484
    CrossrefPubMedGoogle Scholar
  • 39 Makhamreh MM, Cottingham N, Ferreira CR, Berger S, Al-Kouatly HB. Nonimmune hydrops fetalis and congenital disorders of glycosylation: a systematic literature review. J Inherit Metab Dis 2020; 43 (02) 223-233
    CrossrefPubMedGoogle Scholar
  • 40 Léticée N, Bessières-Grattagliano B, Dupré T. et al. Should PMM2-deficiency (CDG Ia) be searched in every case of unexplained hydrops fetalis?. Mol Genet Metab 2010; 101 (2-3): 253-257
    CrossrefPubMedGoogle Scholar