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

α-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. α-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 α-thrombin-mediated increase in ¹²⁵I-albumin permeability. Similar results were obtained with γ-thrombin, an enzymatically active α-thrombin form with an altered fibrinogen recognition domain. PPACK alone and the active site-inhibited PPACK-α-thrombin had no effect on permeability. Diisopropylphospho (DIP)-α-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.