Thrombin modulates vectorial secretion of extracellular matrix
proteins in cultured endothelial cells.
Papadimitriou, Evangelia, Vangelis G. Manolopoulos, G. Thomas Hayman,
Michael E. Maragoudakis, Brian R. Unsworth, John W. Fenton Ii, and
Peter I. Lelkes.
Marquette University, Dept. of Biology, Milwaukee, WI, USA; Dept.
of Pharmacology, University of Patras, Patras, Greece; New York State
Department of Health, Albany, N.Y., Wadworth Center for Laboratories
and Research, and University of Wisconsin Medical School, Milwaukee
Clinical Campus, Milwaukee, WI, USA
APStracts 3:0331C, 1996.
We have identified a novel cellular action of thrombin on cultured rat
adrenal medullary endothelial cells (RAMEC). Incubation of RAMEC for
five minutes with physiological concentrations of thrombin (<
1 U/ml) caused, within 3 hours, an increase in the basolateral
deposition of the extracellular matrix (ECM) proteins fibronectin,
laminin, collagen-IV and collagen-I concomitant with a corresponding
decrease in the apical release of these proteins into the medium.
This shift in the vectorial secretion of ECM proteins, quantitated
with enzyme-linked immunoassays, was time-dependent. Maximal
stimulation of ECM protein deposition was observed after incubation
of the cells with thrombin for 5 to 15 min. Prolonged exposure
(> 1 hour) to thrombin resulted in loss of proteins from the
ECM. Thrombin-stimulated ECM protein deposition exhibited a bell
-shaped dose-dependence, peaking for all for proteins at 0.25 U/ml of
thrombin, and was independent of de novo mRNA or protein synthesis.
The maximal amounts of deposited proteins increased between 2.5 fold
(fibronectin) and 4 fold (collagen I) over base line values. Similar
results were obtained with a peptide agonist of the thrombin receptor
(TRAP), with proteolytically active [delta]-thrombin, and, to a
lesser extent, with other serine proteases, such as trypsin and
plasmin. By contrast, a scrambled TRAP, proteolytically inactive
PPACK-thrombin and DIP-thrombin, as well as type IV collagenase were
ineffective. Taken together, these results suggest that the observed
thrombin effects are mediated by proteolytic activation of the
thrombin receptor. The possible involvement of the phospholipase C
signaling pathway in thrombin-mediated ECM protein deposition was
also investigated. Inhibition or down-regulation of protein kinase C
as well as chelation of intracellular or extracellular Ca2+ did not
suppress, but rather enhanced, basal and thrombin-stimulated ECM
protein deposition. Quantitative differences in the augmentation of
the basolateral deposition by either of the above treatments suggest
differential regulatory pathways for individual ECM proteins. Our
data indicate that in cultured RAMEC, short-term activation of the
thrombin receptor causes an increase in the amounts of deposited ECM
proteins by a cellular signaling pathway which is independent of
protein kinase C activation and/or elevation of intracellular Ca2+.
Received 12 August 1996; accepted in final form 8 October 1996.
APS Manuscript Number C452-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 5 November 1996