Journal of Applied Physiology, Vol 69, Issue 5 1599-1606, Copyright © 1990 by American Physiological Society

ARTICLES

Thrombin's enzymatic activity increases permeability of endothelial cell monolayers

M. A. DeMichele, D. G. Moon, J. W. Fenton 2nd and F. L. Minnear
Department of Physiology, Albany Medical College 12208.

Human alpha-thrombin increases the permeability of bovine pulmonary artery endothelial cell (CCL-209) monolayers. To determine if this increase is via an enzymatic or receptor-mediated mechanism, enzymatically active forms of alpha-thrombin and enzymatically inactive forms with cell binding activity were incubated with the monolayers. Enzymatic forms included alpha-thrombin and two digestion products, zeta-thrombin (chymotryptic product with 89% clotting activity) and gamma-thrombin (tryptic product). Enzymatically inactive forms included D-Phe-Pro-Arg-chloromethylketone-(PPACK) alpha-thrombin and diisopropylphosphorofluoridate-(DIP) alpha-thrombin. Cell binding activity of alpha- and PPACK-alpha-thrombin was demonstrated to be similar to each other and comparable to that cited in the literature for DIP-alpha-thrombin. gamma-Thrombin, on the other hand, did not compete for binding of 125I-labeled alpha-thrombin. All enzymatic forms of alpha-thrombin increased endothelial permeability as assessed by the clearance of 125I-albumin across the monolayers. Coincubation of PPACK, an enzymatic site inhibitor, with alpha- or gamma-thrombin prevented the increase in permeability, further indicating that alpha-thrombin increased permeability by its enzymatic activity. Both enzymatically inactive forms of alpha-thrombin with high-affinity binding activity had no effect on permeability. To further examine whether cell binding activity of alpha-thrombin contributed to the increased permeability, a sulfated COOH-terminal fragment of hirudin (hirugen) that binds to the anion-binding site of alpha-thrombin but, unlike hirudin, does not interact with the catalytic site was coincubated with alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

This article has been cited by other articles:

				A. Siflinger-Birnboim and A. Johnson Protein kinase C modulates pulmonary endothelial permeability: a paradigm for acute lung injury Am J Physiol Lung Cell Mol Physiol, March 1, 2003; 284(3): L435 - L451. [Abstract] [Full Text] [PDF]

				T. Imamura, K. Matsushita, J. Travis, and J. Potempa Inhibition of Trypsin-Like Cysteine Proteinases (Gingipains) from Porphyromonas gingivalis by Tetracycline and Its Analogues Antimicrob. Agents Chemother., October 1, 2001; 45(10): 2871 - 2876. [Abstract] [Full Text]

				M. Cugno, M. Cicardi, B. Bottasso, R. Coppola, R. Paonessa, P. M. Mannucci, and A. Agostoni Activation of the Coagulation Cascade in C1-Inhibitor Deficiencies Blood, May 1, 1997; 89(9): 3213 - 3218. [Abstract] [Full Text] [PDF]

				T. Imamura, A. Banbula, P. J. B. Pereira, J. Travis, and J. Potempa Activation of Human Prothrombin by Arginine-specific Cysteine Proteinases (Gingipains R) from Porphyromonas gingivalis* J. Biol. Chem., May 25, 2001; 276(22): 18984 - 18991. [Abstract] [Full Text] [PDF]