Studies on mitogenic signal transduction by thrombin in vascular cells

The serine protease a-thrombin may be an important mediator of vascular disease states such as atherosclerosis, restenosis and pulmonary hypertension that are characterized by the hyperactive division of cell types of the blood vessel wall. Fundamental to an understanding of the role of thrombin in these conditions will be the identification of intracellular signal transduction processes activated by thrombin in vascular cells that are involved in proliferation. The aim of this study was to define protein kinase signalling pathways implicated in the mitogenic effects of thrombin on bovine pulmonary arterial (BPA) fibroblasts. In BPA fibroblasts, thrombin was found to stimulate the time and concentration dependent activation of 1) components of the mitogen-activated protein (MAP) kinase cascade including p42/p44 MAP kinases, MEK-1/2, and c-Raf-1, by a protein kinase C (PKC)- independent and pertussis toxin-insensitive mechanism; 2) the 70 kDa ribosomal S6 kinase (p70s6k) in a manner consistent with an involvement of PKC, a heterotrimeric G protein of the Gi/o family, and a phosphoinositide 3-kinase different from p85/pl 10; 3) a MAP kinase- activated protein kinase 2 (MAPKAP kinase-2) -associated kinase activity, that may be attributable to the MAP kinase homologue p38. All three signalling mechanisms contributed to a strong mitogenic effect of thrombin in BPA fibroblasts as thrombin-stimulated DNA synthesis was inhibited to varying extents when components of these pathways were specifically blocked by pharmacological intervention. The signalling events evoked by thrombin in BPA fibroblasts did not appear to be a cellular consequence of activation of the cloned tethered ligand thrombin receptor, nor the recently described proteinase-activated receptor-2 (PAR-2), and occurred by an undefined mechanism, possibly involving a novel receptor species. In contrast, both of the currently defined proteolytically-activated receptors coupled to activation of the MAP kinase cascade in rat aortic smooth muscle cells. The results presented demonstrate that multiple signal transduction mechanisms are employed by thrombin to control cell cycle re-entry in a single cell type. In addition, they highlight roles for different protease-sensitive cell surface proteins in initiating signalling events in vascular cells.

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