THROMBIN MODULATES VECTORIAL SECRETION OF EXTRACELLULAR-MATRIX PROTEINS IN CULTURED ENDOTHELIAL-CELLS

We have identified a novel cellular action of thrombin on cultured rat adrenal medullary endothelial cells (RAMEC). Five-minute incubation o f RAMEC with physiological concentrations of thrombin (<1 U/ml) caused within 3 h an increase in the basolateral deposition of the extracell ular 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 E CM proteins, quantitated with enzyme-linked immunoassays, was time dep endent. Maximal stimulation of ECM protein deposition was observed aft er 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 depend ence, peaking for all proteins at 0.25 U/ml of thrombin, and was indep endent of de novo mRNA or protein synthesis. Maximal amounts of deposi ted proteins increased between 2.5-fold (fibronectin) and 4-fold (coll agen I) over baseline values. Similar results were obtained with throm bin receptor agonist peptide (TRAP), proteolytically active gamma-thro mbin, 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, th ese results suggest that the thrombin effects are mediated by proteoly tic activation of the thrombin receptor. Possible involvement of the p hospholipase C-signaling pathway in thrombin-mediated ECM protein depo sition was also investigated. Inhibition or downregulation of protein kinase C (PKC) and chelation of intracellular or extracellular Ca2+ di d not suppress, but rather enhanced, basal and thrombin-stimulated ECM protein deposition. Quantitative differences in augmentation of basol ateral deposition by these treatments suggest differential regulatory pathways for individual ECM proteins. Our data indicate that, in cultu red RAMEC, short-term activation of the thrombin receptor causes an in crease in amounts of deposited ECM protein by a cellular signaling pat hway that is independent of PKC activation and/or elevation of intrace llular Ca2+.