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

• Published in: Journal of thrombosis and haemostasis 2007-12, Vol.5 (12), p.2437-2444
• Main Authors: BANFI, C., BRIOSCHI, M., BARCELLA, S., PIGNIERI, A., PAROLARI, A., BIGLIOLI, P., TREMOLI, E., MUSSONI, L.
• Format: Article
• Language: English
• Subjects: Caveolin 1 - genetics; Caveolin 1 - metabolism; Cells, Cultured; Cholesterol - deficiency; Cholesterol - metabolism; Endothelial Cells - drug effects; Endothelial Cells - enzymology; Endothelial Cells - metabolism; endothelium; Humans; membrane microdomains; Membrane Microdomains - drug effects; Membrane Microdomains - metabolism; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3 - metabolism; Nystatin - pharmacology; Oligopeptides - pharmacology; Phosphorylation; protease‐activated receptors; Protein Transport; Receptor, PAR-1 - agonists; Receptor, PAR-1 - metabolism; Receptor, PAR-2 - metabolism; RNA Interference; RNA, Messenger - biosynthesis; RNA, Small Interfering - metabolism; Signal Transduction - drug effects; signaling pathways; Thromboplastin - biosynthesis; Thromboplastin - genetics; tissue factor; Transfection
• ISSN: 1538-7933; 1538-7836; 1538-7836
• DOI: 10.1111/j.1538-7836.2007.02759.x

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