Activation appeared to be thrombin-specific, since it was prevented by the catalytic thrombin inhibitor PPACK (Figure 4(a)).
This effect appeared to be thrombin-specific, prevented by PPACK (Figure 5(a)).
Epifluorescence analysis in Figure 6(b) shows that stimulation with 10 nM thrombin for 5 min significantly decreased PAR1 expression at the plasma membrane; this effect was prevented by the thrombin inhibitor PPACK. To determine if thrombin effect on PAR1 membrane expression is mediated by the activation of PAR1, we analyzed the effect of PAR1-AP on PAR1 membrane expression.
We demonstrated that thrombin induces calpain activation through a specific process prevented by PPACK and identified to PAR1 as the receptor responsible for this effect (Figure 4(a)).
Specificity of thrombin effect was assessed by inclusion of the thrombin inhibitor PPACK (25 [micro]M).
ARPE-19 cells were serum-deprived for 24 hours prior to stimulation for 1 min with 10 nM thrombin or 2.5 [micro]M PAR1-AP PPACK, BAPTA-AM, and calpain inhibitors were included 30 minutes prior to stimulation.
Serum-deprived ARPE-19 cells were stimulated for 1 minute with 10 nM thrombin or 2.5 [micro]M PAR1-AP PPACK, calcium chelators, and calpain inhibitors were included 30 minutes ahead of stimulation.
 Nonstandard abbreviations: tPA, tissue plasminogen activator; PAI-1, plasminogen activator inhibitor 1; uPA, urokinase plasminogen activator; EIA, enzyme immunoassay; PPACK
, d-Phe-Pro-Arg chloromethyl ketone; and Ab, antibody.
The activation of proCPU in plasma was stopped by the addition of 10 [micro]L of 20 [micro]mol/L PPACK, and the activity was measured with Hip-Arg and analyzed by HPLC.
After incubation for 10 min at room temperature, the activation was stopped with 10 [micro]L of 20 [micro]mol/L PPACK. Activity was measured with the substrate Hip-Arg, and the released hippuric acid was determined by HPLC.
The activation was stopped after a 10-min incubation at room temperature by the addition of 5 [micro]L of 20 [micro]mol/L PPACK. Subsequently, 5 [micro]L of inactivated serum was added and the carboxypeptidase activity was measured.
 Nonstandard abbreviations: CPN, carboxypeptidase N; CPU, carboxypepfidase U; Hip-Arg, hippuryl-L-arginine; PPACK, D-phenylalanyl-L-prolylarginyl chloromethyl ketone; and SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.