Enzymatic activity is necessary for thrombin-mediated increase in endothelial permeability.
alpha-Thrombin causes a dose-dependent increase in endothelial permeability as measured by the clearance rate of 125I-albumin across a monolayer of bovine pulmonary artery endothelial cells. We determined if an active catalytic site is necessary for the thrombin-mediated increase in endothelial permeability. alpha-Thrombin was reacted with 10-fold excess D-phenylalanyl-prolyl-arginine chloromethyl ketone (PPACK), an irreversible inhibitor that forms a covalent bond with thrombin's active site, producing an enzymatically inactive thrombin. PPACK completely inhibited the alpha-thrombin-mediated increase in 125I-albumin permeability. Similar results were obtained with gamma-thrombin, an enzymatically active alpha-thrombin form with an altered fibrinogen recognition domain. PPACK alone and the active site-inhibited PPACK-alpha-thrombin had no effect on permeability. Diisopropylphospho (DIP)-alpha-thrombin was effective only in very high concentrations (10(-6)M), and this effect was abolished by the addition of PPACK. These studies demonstrate that binding alone is insufficient for the thrombin-mediated increase in endothelial monolayer permeability. Thrombin's active catalytic site is a requirement for the increase in transendothelial albumin permeability.
Aschner
J L
JL
Department of Pediatrics, Albany Medical Center, New York 12208.
Lennon
J M
JM
Fenton
J W
JW
Aschner
M
M
Malik
A B
AB
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000494:pharmacology*
D000590:Amino Acid Chloromethyl Ketones; D013917:Thrombin; C050815:phenylalanyl-prolyl-arginine chloromethyl ketone-thrombin; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone