Protease-activated receptor (PAR)-independent growth and pro-inflammatory actions of thrombin on human cultured airway smooth muscle

1 Thrombin, a mitogen for human cultured airway smooth muscle (HASM), has many actions that have been attributed to activation of protease-activated receptor (PARs). However, the role of PARs in the proliferative action has not been clearly identified. Moreover, thrombin elicits cytokine production in a. number of cell types, but these effects have not been characterized in human ASM. 2 Thrombin (0.03-3 U ml(-1))-stimulated increases in the levels of the pro-inflammatory and fibrogenic cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF) were observed over the same concentration range observed for thrombin-stimulated mitogenesis. 3 Inhibition of thrombin proteolytic activity, with either D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK)- or hirudin-treated thrombin (0.3 U ml(-1)) or in the presence of the thrombin serine protease-selective inhibitor, SDZ 217-766 (0.15 muM), reduced the thrombin-stimulated GM-CSF levels by 91 +/- 3, 65 +/- 12 and 83 +/- 9% (n = 8, P < 0.05), respectively. PPACK treatment, hirudin and SDZ 217-766 inhibited thrombin-stimulated increase in cell number by 70 +/- 8, 63 +/- 11 and 69 +/- 8%, respectively. 4 PAR-selective peptides SFLLRN (PARI; 10,muM), SLIGKV (PAR2; 10 muM), GYPGQV (PAR4; 100 muM) or the combination of SFLLRN and GYPGQV elicited mitogenic responses of only 15% of that to thrombin and surprisingly, had no effect on GM-CSF levels (n = 8). Nevertheless, inhibition of thrombin responses by pertussis toxin (50 ng ml(-1)) suggests that the PAR-independent actions also involve a G-protein-coupled receptor. 5 PARI receptor expression was evident by immunohistochemistry and these receptors were coupled to increases in intracellular calcium, but not to the phosphorylation of ERK or the increases in cyclin D1 protein levels that are essential for cell proliferation. Cross-desensitization of intracellular calcium increases by thrombin and the PARI-selective peptide provides evidence that the PARI receptor responds to both ligands. 6 The failure of PAR-selective peptides to mimic thrombin responses together with the inhibition of thrombin responses by serine protease inhibitors suggest the involvement of novel proteolytic receptor targets for thrombin-induced mitogenesis and cytokine production.