Fig. 3

Effect of PAR-1 antagonist (SCH 79797), PAR-4 antagonist (transcinnamoyl- YPGKF-NH2), and PAR-3 antibody on cat K-induced platelet aggregation and intracellular calcium measurements. (a) Human platelets were preincubated with the specific PAR-1 SCH 79797 antagonist (140 nM) for 30 min at 37 °C and evaluated in the aggregometer. Representative traces show the interference of SCH 79797 on cat K-induced platelet aggregation. AP-PAR1 (0.2 µM) was completely inhibited. (b) The specific PAR-4 trans-cinnamoyl-YPGKF-NH2 antagonist (30 µM) inhibits human platelet aggregation induced by cat K (20 nM). AP-PAR-4 (60 µM) was used as control. (c) Inhibitory effect of the PAR-3 antibody (0.02 µM) on cat Kinduced human platelet aggregation. Platelets were preincubated with the PAR-3 antibody for 30 min at 37 °C and aggregation was measured. Representative traces show the inhibition of the cat K effect (red and green lines). Papain-induced human platelet aggregation inhibition, black line. The bar graph shows percentages of aggregation. Data are expressed as mean ± SEM from 3 independent experiments (**p<0.001, *** p<0.0001). (d) Reverse transcriptase-PCR using total platelet RNA. Error bars indicate S.D. from triplicate samples. **p <0.01. (e). PAR-1, -3, and -4 were detected in washed platelets treated with cat K (20 nM), papain (1.6 µM), and α-thrombin (0.001 UNIH/500 µL) for 10 min at 37 °C; lysate proteins were separated by 10% SDS-PAGE and electrotransferred to nitrocellulose membrane. Membranes were blocked and incubated with anti-PAR1, anti-PAR3, anti-PAR4 rabbit primary antibodies, and anti-ß-actin (control). Antibody binding was visualized by chemiluminescence and the relative levels of these proteins were determined by densitometric analysis. Data are expressed as mean ± SEM from 3 independent experiments (**p<0.001). (f) Intracellular calcium measurements in ThrR -/- fibroblasts (absence in PAR-1). Increases in cytosolic calcium in response to cat K (20 nM) and α-thrombin (0.001 UNIH/500 µL). (g) ThrR -/- fibroblasts (absence in PAR-3). Increases in cytosolic calcium in response to cat K (20 nM) and α-thrombin (0.001 UNIH/500 µL). Inhibition of human cat K-activity on human platelet aggregation by E-64 (5 µM). (h) ThrR -/- fibroblasts (absence in PAR-4). Increases in cytosolic calcium in response to cat K (20 nM), papain (1.6 µM), and α-thrombin (0.001 UNIH/500 µL)