Lipid raft localization regulates the cleavage specificity of protease activated receptor 1 in endothelial cells

J Thromb Haemost. 2008 Jun;6(6):954-61. doi: 10.1111/j.1538-7836.2008.02924.x. Epub 2008 Feb 12.


Background: The endothelial protein C receptor (EPCR)-dependent cleavage of protease activated receptor 1 (PAR-1) by either activated protein C (APC) or thrombin in lipid rafts initiates protective signaling responses in endothelial cells.

Objectives: To investigate the mechanism by which APC and thrombin interact with and cleave PAR-1 in lipid rafts.

Methods: We constructed two types of PAR-1 cleavage reporter constructs in which a secreted alkaline phosphatase (ALP) was fused to the extracellular domain of PAR-1. The first construct has a transmembrane domain capable of uniformly anchoring the fusion protein to the membrane surface, while the second construct has the recognition sequence for targeting the fusion protein to lipid rafts/caveolae in transfected cells.

Results: Both APC and the Gla-domainless (GD)-APC cleaved the PAR-1 exodomain with similar efficiency in HUVECs transfected with the first construct. Unlike APC, GD-APC did not cleave PAR-1 in cells transfected with the second construct; however, prior treatment of cells with S195A mutants of either protein C or thrombin led to the GD-APC cleavage of PAR-1 with a comparable or higher catalytic efficiency. The same results were obtained if the cellular signaling properties of APC and GD-APC were monitored in the TNF-alpha-induced endothelial cell apoptosis and permeability assays.

Conclusions: The lipid raft localization renders the scissile bond of the PAR-1 exodomain unavailable for interaction with coagulation proteases. The binding of either the Gla-domain of protein C to EPCR or exosite-1 of thrombin to the C-terminal hirudin-like sequence of PAR-1 changes the membrane localization and/or the conformation of the PAR-1 exodomain to facilitate its recognition and subsequent cleavage by these proteases.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alkaline Phosphatase / metabolism
  • Apoptosis
  • Blood Coagulation Factors / metabolism
  • Endothelial Cells / metabolism*
  • Humans
  • Lipids / chemistry
  • Membrane Microdomains / chemistry*
  • Models, Biological
  • Permeability
  • Protein C / metabolism
  • Protein Structure, Tertiary
  • Receptor, PAR-1 / metabolism*
  • Receptors, Cell Surface / metabolism
  • Signal Transduction
  • Thrombin / chemistry
  • Thrombin / metabolism*
  • Transfection


  • Blood Coagulation Factors
  • Lipids
  • Protein C
  • Receptor, PAR-1
  • Receptors, Cell Surface
  • activated protein C receptor
  • Alkaline Phosphatase
  • Thrombin