Thrombin-induced gap formation in confluent endothelial cell monolayers in vitro

Area of Science:

• Endothelial cell biology
• Vascular physiology
• Thrombosis research

Background:

• Endothelial cells form a barrier crucial for vascular health.
• Disruption of this barrier can lead to various vascular diseases.
• Thrombin is a key enzyme in blood coagulation.

Purpose of the Study:

• To investigate the effect of thrombin on human umbilical vein endothelial cell (HUVEC) monolayer integrity.
• To quantify thrombin-induced changes in endothelial cell shape and barrier function.
• To explore potential inhibitors of thrombin-induced endothelial gap formation.

Main Methods:

• Incubation of HUVEC monolayers with thrombin in vitro.
• Utilizing a grid assay to measure surface area changes.
• Testing inhibitors like hirudin, antithrombin III, heparin, and dibutyryl cyclic adenosine monophosphate (dbcAMP).
• Comparing thrombin's effects with histamine, bradykinin, serotonin, C5a, and C3a.

Main Results:

• Thrombin induced rapid (within 2 min) and significant gap formation in HUVEC monolayers, uncovering up to 80% of the surface area.
• These gaps were transient, disappearing completely within 2 hours.
• Thrombin-induced gap formation was inhibited by hirudin, antithrombin III/heparin, and dbcAMP.
• Histamine also caused gap formation, but thrombin's effect was not blocked by histamine antagonists (pyrilamine, cimetidine).
• Bradykinin, serotonin, C5a, and C3a did not disrupt intact monolayers.

Conclusions:

• Low concentrations of thrombin can transiently disrupt endothelial integrity, exposing the subendothelium.
• This repeated exposure may play a role in the development of atherosclerosis and thrombosis.
• The findings highlight thrombin as a significant modulator of endothelial barrier function.