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Enzymatic activity is necessary for thrombin-mediated increase in endothelial permeability
J L Aschner1, J M Lennon, J W Fenton
1Department of Pediatrics, Albany Medical Center, New York 12208.
The American Journal of Physiology
|October 1, 1990
Summary
The active catalytic site of alpha-thrombin is essential for increasing endothelial permeability. Inhibiting this site with PPACK prevents thrombin from affecting albumin permeability across cell monolayers.
Area of Science:
- Biochemistry
- Cell Biology
- Physiology
Background:
- Endothelial permeability is crucial for regulating substance exchange.
- alpha-Thrombin is known to increase endothelial permeability.
- The role of thrombin's active site in this process requires clarification.
Purpose of the Study:
- To investigate whether an active catalytic site is necessary for thrombin-induced endothelial permeability.
- To determine if thrombin binding alone is sufficient to increase permeability.
Main Methods:
- Utilized bovine pulmonary artery endothelial cells in a monolayer model.
- Measured 125I-albumin clearance to quantify endothelial permeability.
- Employed D-phenylalanyl-prolyl-arginine chloromethyl ketone (PPACK) to irreversibly inhibit thrombin's active site.
- Tested gamma-thrombin and Diisopropylphospho (DIP)-alpha-thrombin to assess the role of enzymatic activity.
Main Results:
- PPACK completely blocked the alpha-thrombin-induced increase in 125I-albumin permeability.
- Active site-inhibited thrombin (PPACK-alpha-thrombin) did not affect permeability.
- Gamma-thrombin also showed inhibited permeability increase when pre-treated with PPACK.
- Diisopropylphospho (DIP)-alpha-thrombin was only effective at very high concentrations and its effect was abolished by PPACK.
Conclusions:
- Thrombin's active catalytic site is required for increasing endothelial monolayer permeability.
- Enzymatic activity, not just binding, is necessary for thrombin to mediate changes in transendothelial albumin permeability.