Enzymatic activity is necessary for thrombin-mediated increase in endothelial permeability

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Abstract
.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.