The two-receptor system PAR-1/PAR-4 mediates alpha-thrombin-induced [Ca(2+)](i) mobilization in human astrocytoma cells.

The proteinase-activated receptor 1 (PAR-1) was characterized as a functional receptor for thrombin in cells from different brain tumor entities. Whether PAR-1 alone accounts for thrombin-induced effects in human cancer cells, or whether other PAR contribute is unknown. We established primary cultures from two neurosurgically removed human astrocytomas and investigated intracellular signaling roles of PAR-1 and PAR-4 by estimating the effect of alpha-thrombin and PAR-activating peptides on [Ca(2+)](i) mobilization in single astrocytoma cells. alpha-Thrombin or the PAR-1-activating peptide SFLLRN induced a transient calcium mobilization. This suggests the involvement of PAR-1 in alpha-thrombin-induced calcium signaling in human astrocytoma cells. In addition, a second, PAR-4-dependent, mechanism exists. This was deduced from the findings that a further calcium signal could be observed in human astrocytoma cells stimulated with alpha-thrombin after SFLLRN and the PAR-4-activating peptide GYPGQV also induced a calcium response. In addition, the observation that trypsin, known to activate both PAR-2 and PAR-4, but not the specifically PAR-2-activating peptide SLIGRL induced calcium signaling is a further indication of functional PAR-4-type thrombin receptors in human astrocytoma cells. This is the first report demonstrating a signaling role for a dual thrombin receptor system in human tumor cells.