Tissue factor induction by protease-activated receptor 1 requires intact caveolin-enriched membrane microdomains in human endothelial cells

Cristina Banfi, M. Brioschi, S. Barcella, A. Pignieri, A. Parolari, P. Biglioli, E. Tremoli, L. Mussoni

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Background: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique mechanism of proteolytic activation. PARs regulate a broad range of cellular functions and are active in the pathogenesis of disorders characterized by chronic inflammation or activation of the coagulation cascade. Signaling through PAR1 and PAR2 shifts the endothelium towards a prothrombotic phenotype, thereby exacerbating the initial pathophysiologic condition. Objectives: This study aimed to analyze the localization of PARs in the cell membrane and how their compartmentalization affects tissue factor (TF) in human endothelial cells. Methods: TF expression was determined by quantitative real-time polymerase chain reaction analysis and by activity assays. The interaction of PARs with caveolin was investigated through: (i) caveolin-1 gene knockdown performed by transfection with specific small interfering RNA (siRNA); (ii) caveolin-enriched membrane microdomain disruption; and (iii) coimmunoprecipitation assay. Results: We have shown that PAR1, but not PAR2, is present in endothelial caveolin-enriched membrane microdomains, where it is bound to caveolin-1, and that these structures must be intact if PAR1-induced signaling is to increase TF activity. Cholesterol depletion of endothelial cells by cholesterol-sequestering agents caused the PAR1 to relocate to high-density membranes, and impaired the induction of TF (P <0.01) without affecting the PAR2-mediated procoagulant effect. In addition, siRNA directed against caveolin-1 inhibited TF activation by PAR1 (P <0.01 and P <0.01, respectively). Conclusions: PAR1 localization in the caveolin-enriched membrane microdomain, bound to caveolin-1, represents a crucial requirement for TF induction in endothelial cells.

Original languageEnglish
Pages (from-to)2437-2444
Number of pages8
JournalJournal of Thrombosis and Haemostasis
Volume5
Issue number12
DOIs
Publication statusPublished - Dec 2007

Fingerprint

PAR-1 Receptor
Membrane Microdomains
Caveolins
Thromboplastin
Proteinase-Activated Receptors
Caveolin 1
Endothelial Cells
Small Interfering RNA
Sequestering Agents
Cholesterol
Gene Knockdown Techniques
G-Protein-Coupled Receptors
Endothelium
Transfection
Real-Time Polymerase Chain Reaction
Cell Membrane
Inflammation
Phenotype
Membranes

Keywords

  • Endothelium
  • Membrane microdomains
  • Proteaseactivated receptors
  • Signaling pathways
  • Tissue factor

ASJC Scopus subject areas

  • Medicine(all)

Cite this

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title = "Tissue factor induction by protease-activated receptor 1 requires intact caveolin-enriched membrane microdomains in human endothelial cells",
abstract = "Background: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique mechanism of proteolytic activation. PARs regulate a broad range of cellular functions and are active in the pathogenesis of disorders characterized by chronic inflammation or activation of the coagulation cascade. Signaling through PAR1 and PAR2 shifts the endothelium towards a prothrombotic phenotype, thereby exacerbating the initial pathophysiologic condition. Objectives: This study aimed to analyze the localization of PARs in the cell membrane and how their compartmentalization affects tissue factor (TF) in human endothelial cells. Methods: TF expression was determined by quantitative real-time polymerase chain reaction analysis and by activity assays. The interaction of PARs with caveolin was investigated through: (i) caveolin-1 gene knockdown performed by transfection with specific small interfering RNA (siRNA); (ii) caveolin-enriched membrane microdomain disruption; and (iii) coimmunoprecipitation assay. Results: We have shown that PAR1, but not PAR2, is present in endothelial caveolin-enriched membrane microdomains, where it is bound to caveolin-1, and that these structures must be intact if PAR1-induced signaling is to increase TF activity. Cholesterol depletion of endothelial cells by cholesterol-sequestering agents caused the PAR1 to relocate to high-density membranes, and impaired the induction of TF (P <0.01) without affecting the PAR2-mediated procoagulant effect. In addition, siRNA directed against caveolin-1 inhibited TF activation by PAR1 (P <0.01 and P <0.01, respectively). Conclusions: PAR1 localization in the caveolin-enriched membrane microdomain, bound to caveolin-1, represents a crucial requirement for TF induction in endothelial cells.",
keywords = "Endothelium, Membrane microdomains, Proteaseactivated receptors, Signaling pathways, Tissue factor",
author = "Cristina Banfi and M. Brioschi and S. Barcella and A. Pignieri and A. Parolari and P. Biglioli and E. Tremoli and L. Mussoni",
year = "2007",
month = "12",
doi = "10.1111/j.1538-7836.2007.02759.x",
language = "English",
volume = "5",
pages = "2437--2444",
journal = "Journal of Thrombosis and Haemostasis",
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TY - JOUR

T1 - Tissue factor induction by protease-activated receptor 1 requires intact caveolin-enriched membrane microdomains in human endothelial cells

AU - Banfi, Cristina

AU - Brioschi, M.

AU - Barcella, S.

AU - Pignieri, A.

AU - Parolari, A.

AU - Biglioli, P.

AU - Tremoli, E.

AU - Mussoni, L.

PY - 2007/12

Y1 - 2007/12

N2 - Background: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique mechanism of proteolytic activation. PARs regulate a broad range of cellular functions and are active in the pathogenesis of disorders characterized by chronic inflammation or activation of the coagulation cascade. Signaling through PAR1 and PAR2 shifts the endothelium towards a prothrombotic phenotype, thereby exacerbating the initial pathophysiologic condition. Objectives: This study aimed to analyze the localization of PARs in the cell membrane and how their compartmentalization affects tissue factor (TF) in human endothelial cells. Methods: TF expression was determined by quantitative real-time polymerase chain reaction analysis and by activity assays. The interaction of PARs with caveolin was investigated through: (i) caveolin-1 gene knockdown performed by transfection with specific small interfering RNA (siRNA); (ii) caveolin-enriched membrane microdomain disruption; and (iii) coimmunoprecipitation assay. Results: We have shown that PAR1, but not PAR2, is present in endothelial caveolin-enriched membrane microdomains, where it is bound to caveolin-1, and that these structures must be intact if PAR1-induced signaling is to increase TF activity. Cholesterol depletion of endothelial cells by cholesterol-sequestering agents caused the PAR1 to relocate to high-density membranes, and impaired the induction of TF (P <0.01) without affecting the PAR2-mediated procoagulant effect. In addition, siRNA directed against caveolin-1 inhibited TF activation by PAR1 (P <0.01 and P <0.01, respectively). Conclusions: PAR1 localization in the caveolin-enriched membrane microdomain, bound to caveolin-1, represents a crucial requirement for TF induction in endothelial cells.

AB - Background: Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique mechanism of proteolytic activation. PARs regulate a broad range of cellular functions and are active in the pathogenesis of disorders characterized by chronic inflammation or activation of the coagulation cascade. Signaling through PAR1 and PAR2 shifts the endothelium towards a prothrombotic phenotype, thereby exacerbating the initial pathophysiologic condition. Objectives: This study aimed to analyze the localization of PARs in the cell membrane and how their compartmentalization affects tissue factor (TF) in human endothelial cells. Methods: TF expression was determined by quantitative real-time polymerase chain reaction analysis and by activity assays. The interaction of PARs with caveolin was investigated through: (i) caveolin-1 gene knockdown performed by transfection with specific small interfering RNA (siRNA); (ii) caveolin-enriched membrane microdomain disruption; and (iii) coimmunoprecipitation assay. Results: We have shown that PAR1, but not PAR2, is present in endothelial caveolin-enriched membrane microdomains, where it is bound to caveolin-1, and that these structures must be intact if PAR1-induced signaling is to increase TF activity. Cholesterol depletion of endothelial cells by cholesterol-sequestering agents caused the PAR1 to relocate to high-density membranes, and impaired the induction of TF (P <0.01) without affecting the PAR2-mediated procoagulant effect. In addition, siRNA directed against caveolin-1 inhibited TF activation by PAR1 (P <0.01 and P <0.01, respectively). Conclusions: PAR1 localization in the caveolin-enriched membrane microdomain, bound to caveolin-1, represents a crucial requirement for TF induction in endothelial cells.

KW - Endothelium

KW - Membrane microdomains

KW - Proteaseactivated receptors

KW - Signaling pathways

KW - Tissue factor

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U2 - 10.1111/j.1538-7836.2007.02759.x

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M3 - Article

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JO - Journal of Thrombosis and Haemostasis

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SN - 1538-7933

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