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Factor Xa activates endothelial cells by a receptor cascade between EPR-1 and PAR-2
F Bono1, P Schaeffer, J P Hérault
1Sanofi-Synthélabo Recherche, Toulouse, France.
Arteriosclerosis, Thrombosis, and Vascular Biology
|November 14, 2000
Summary
Factor Xa activates endothelial cells by binding to EPR-1 and cleaving protease-activated receptor 2 (PAR-2), initiating intracellular signaling. This novel pathway involves both receptor binding and proteolysis for cell activation.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Signaling
Background:
- Factor Xa plays a key role in hemostasis and interacts with endothelial cells via effector cell protease receptor 1 (EPR-1).
- This interaction triggers signal transduction, second messenger generation, and cytokine gene modulation, requiring local proteolysis for cell activation.
- The mechanism by which factor Xa induces cell activation, particularly the role of protease-activated receptors (PARs), remained unclear as EPR-1 lacks proteolysis-sensitive sites.
Purpose of the Study:
- To investigate whether factor Xa-mediated signal transduction in endothelial cells requires the proteolytic activation of a protease-activated receptor (PAR) family member.
- To elucidate the specific PAR involved in factor Xa-induced endothelial cell activation and the interaction with EPR-1.
Main Methods:
- Utilized DX9065, a catalytic inhibitor of factor Xa, to assess its effect on factor Xa-induced calcium (Ca2+) signaling and ligand binding to EPR-1.
- Employed desensitization experiments using trypsin or a PAR-2-specific activator peptide (SLIGKV) to probe the role of PAR-2.
- Demonstrated direct cleavage of PAR-2 by factor Xa on endothelial cells using synthetic peptide cleavage assays and immunofluorescence.
Main Results:
- Catalytic inactivation of factor Xa suppressed endothelial cell Ca2+ signaling but did not affect ligand binding to EPR-1.
- Factor Xa-induced Ca2+ signaling was ablated by trypsin or PAR-2 activation, and factor Xa pretreatment blocked PAR-2-dependent signaling.
- Direct cleavage of PAR-2 by factor Xa was confirmed through peptide cleavage assays and immunofluorescence, indicating PAR-2 activation.
Conclusions:
- Factor Xa induces endothelial cell activation through a novel signaling cascade involving initial docking to EPR-1.
- This activation requires the subsequent local proteolytic cleavage and activation of protease-activated receptor 2 (PAR-2) by factor Xa.
- The findings reveal a new mechanism of endothelial cell activation mediated by factor Xa and PAR-2.