Thrombin modulates vectorial secretion of extracellular matrix proteins in cultured endothelial cells

Am J Physiol. 1997 Apr;272(4 Pt 1):C1112-22. doi: 10.1152/ajpcell.1997.272.4.C1112.


We have identified a novel cellular action of thrombin on cultured rat adrenal medullary endothelial cells (RAMEC). Five-minute incubation of RAMEC with physiological concentrations of thrombin (<1 U/ml) caused within 3 h an increase in the basolateral deposition of the extracellular matrix (ECM) proteins fibronectin, laminin, and collagens IV and I, concomitant with a corresponding decrease in the apical release of these proteins into the medium. This shift in vectorial secretion of ECM proteins, quantitated with enzyme-linked immunoassays, was time dependent. Maximal stimulation of ECM protein deposition was observed after incubation of cells with thrombin for 5-15 min. Prolonged exposure (>1 h) to thrombin resulted in loss of proteins from the ECM. Thrombin-stimulated ECM protein deposition exhibited a bell-shaped dose dependence, peaking for all proteins at 0.25 U/ml of thrombin, and was independent of de novo mRNA or protein synthesis. Maximal amounts of deposited proteins increased between 2.5-fold (fibronectin) and 4-fold (collagen I) over baseline values. Similar results were obtained with thrombin receptor agonist peptide (TRAP), proteolytically active gamma-thrombin, and, to a lesser extent, other serine proteases such as trypsin and plasmin. A scrambled TRAP, proteolytically inactive PPACK-thrombin, DIP-thrombin, and type IV collagenase were ineffective. Together, these results suggest that the thrombin effects are mediated by proteolytic activation of the thrombin receptor. Possible involvement of the phospholipase C-signaling pathway in thrombin-mediated ECM protein deposition was also investigated. Inhibition or downregulation of protein kinase C (PKC) and chelation of intracellular or extracellular Ca2+ did not suppress, but rather enhanced, basal and thrombin-stimulated ECM protein deposition. Quantitative differences in augmentation of basolateral deposition by these 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 amounts of deposited ECM protein by a cellular signaling pathway that is independent of PKC activation and/or elevation of intracellular Ca2+.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adrenal Medulla / cytology
  • Adrenal Medulla / drug effects
  • Adrenal Medulla / metabolism*
  • Animals
  • Calcium / physiology
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Dose-Response Relationship, Drug
  • Endothelium / cytology
  • Endothelium / drug effects
  • Endothelium / metabolism
  • Extracellular Matrix Proteins / antagonists & inhibitors
  • Extracellular Matrix Proteins / genetics
  • Extracellular Matrix Proteins / metabolism*
  • Peptide Fragments / pharmacology
  • Peptide Hydrolases / pharmacology
  • Protein Kinase C / physiology
  • RNA, Messenger / biosynthesis
  • Rats
  • Receptors, Thrombin / agonists
  • Serine Endopeptidases / pharmacology
  • Thrombin / pharmacology*
  • Time Factors


  • Extracellular Matrix Proteins
  • Peptide Fragments
  • RNA, Messenger
  • Receptors, Thrombin
  • Protein Kinase C
  • Peptide Hydrolases
  • Serine Endopeptidases
  • Thrombin
  • Calcium