Reducing agents induce thrombomodulin shedding in human endothelial cells

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Medicine

Background:

• Soluble thrombomodulin (sTM) levels reflect cell surface thrombomodulin (TM) dynamics, including shedding.
• Understanding sTM release mechanisms is crucial for inflammatory diseases where protein C activation is diminished.

Purpose of the Study:

• To investigate the mechanisms regulating the release of soluble thrombomodulin (sTM) from human umbilical endothelial cells (HUVECs).
• To identify factors that modulate TM shedding and its potential impact on protein C activation.

Main Methods:

• Treatment of HUVECs with various compounds including cytokines, reducing agents, and protease activators.
• Assessment of sTM release and protein C activation.
• Pharmacological inhibition studies targeting metalloproteases and serine proteases.

Main Results:

• Pro-inflammatory cytokines did not affect sTM release, unlike EPCR shedding.
• Natural and synthetic reducing compounds, but not their oxidized forms, significantly upregulated sTM release.
• 4-aminophenylmercuric acetate (APMA), a metalloprotease activator, effectively induced TM shedding and inhibited protein C activation.
• Both metalloproteases and serine proteases contribute to basal sTM release, with serine proteases playing a key role in thiol-induced shedding.
• Disulfide bond modification by thiols likely increases TM susceptibility to serine protease cleavage.

Conclusions:

• The extracellular redox state critically regulates TM shedding in HUVECs.
• Thiol-mediated reduction of disulfide bonds enhances TM susceptibility to protease cleavage.
• These findings offer novel strategies to modulate sTM shedding and potentially improve protein C activation in inflammatory conditions.