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

J. L. Aschner, J. M. Lennon, J. W. Fenton, M. Aschner, A. B. Malik

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)L270-L275
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume259
Issue number4 3-2
DOIs
StatePublished - 1990

Keywords

  • endothelial monolayer permeability

ASJC Scopus subject areas

  • Physiology
  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology

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