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

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

Author information

1
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St Louis, MO 63104, USA.

Abstract

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.

[Indexed for MEDLINE]
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