Figure 8. Thrombin does not directly cleave Cyr61 but activates a leupeptin-sensitive Cyr61 cleaving enzyme. A: The culture medium (cell naïve) was incubated with recombinant Cyr61 (rCyr61, 31.6 nM) in the presence (+) or absence (-) of thrombin (1.0 U/ml) for 2 h at 37 °C; n = 2 independent experiments. B and C: The conditioned media from 3 h untreated or 1.0 U/ml thrombin-treated fibroblast (B) and myofibroblast (C) cultures were incubated with rCyr61 (31.6 nM) in the presence of additional medium, additional thrombin (1.0 U/ml), hirudin (4.4 AT-U/ml), or additional thrombin plus hirudin for 2 h at 37 °C; n = 3 independent experiments. D and E: The conditioned media from 3 h thrombin-treated fibroblast (D) and myofibroblast (E) cultures were incubated with rCyr61 (31.6 nM) in the presence of additional medium, additional thrombin (1.0 U/ml), leupeptin (100 µM), or additional thrombin plus leupeptin for 2 h at 37 °C; n = 2 independent experiments. The conditioned media from 3 h thrombin-treated fibroblast (F) and myofibroblast (G) cultures were incubated with rCyr61 (31.6 nM) in the presence of additional medium, additional thrombin (1.0 U/ml), hirudin (4.4 AT-U/ml), hirudin plus thrombin, aprotinin (0.3 µM), aprotinin plus thrombin, E-64 (10 µM), or E-64 plus thrombin for 2 h at 37 °C; n = 2 independent experiments. A-G: The aliquots, without concentration, on western blots were probed for Cyr61 using a specific antibody against the central linker region of Cyr61 (Figure 1D). All conditioned media plus inhibitor experiments were repeated at least twice using the conditioned media from cells of independent donors.