Thrombin activates nuclear factor-kappaB and potentiates endothelial cell activation by TNF

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• Thrombin is a central enzyme in hemostasis.
• Inflammatory responses occur in vascular beds where thrombin is generated.
• Endothelial cells (EC) play a crucial role in regulating vascular inflammation.

Purpose of the Study:

• To investigate the effect of thrombin on gene expression in porcine aortic endothelial cells.
• To determine if thrombin activates the nuclear factor-kappaB (NF-kappaB) transcription factor.
• To examine the synergistic effect of thrombin and TNF on endothelial cell activation.

Main Methods:

• Gene expression analysis using mRNA levels.
• Electrophoretic mobility shift assay (EMSA) to detect NF-kappaB binding activity.
• Luciferase reporter assays to measure transcriptional activity.
• Flow cytometry to assess E-selectin surface expression.

Main Results:

• Thrombin upregulated mRNA levels of E-selectin, monocyte chemoattractant protein-1, IL-8, plasminogen activator inhibitor-1, and IkappaB-alpha.
• Thrombin activated NF-kappaB binding activity, primarily involving RelA (p65) and NF-kappaB1 (p50).
• Thrombin potentiated TNF-induced E-selectin surface expression and NF-kappaB activity.

Conclusions:

• Thrombin induces inflammatory gene expression in endothelial cells via NF-kappaB activation.
• Thrombin synergizes with TNF to enhance endothelial cell activation and inflammatory responses.
• This study reveals a mechanism by which coagulation factors amplify cytokine-mediated inflammation.