Inhibition of FXIIa amplification of PK and FXI activation on HUVECs.

HUVECs were grown in endothelial cell culture media containing human FXII-deficient serum. When confluent, the cells were washed 3 times with HCB containing 10 μM ZnCl_{2 .} (A) Amplification of PK activation. HUVECs were incubated with 10 nM HK, 10 nM PK, and 20 nM FXII in HCB in the absence or presence of 100 μM of peptide YHK9 or FPF9 for 1 hour at 37°C. At the end of the incubation, the cells were washed, 1 mM S2302 was added in HCB, and hydrolysis of the substrate was monitored for an additional hour. The extent of kallikrein/FXIIa hydrolytic activity was determined by measuring the absorbance of the reaction mixture at 405 nm using a microplate autoreader EL 311. (B) Initiation of FXI activation. HUVECs were incubated with 10 nM HK, 5 nM FXI, and 20 nM FXII in HCB in the absence or presence of 100 μM of peptide YHK9, FPF9, or IPP19 for 1 hour at 37°C. At the end of the incubation, the cells were washed, 0.8 mM S2366 was added in HCB, and hydrolysis of the substrate was monitored for an additional hour. The generated FXIa was determined by measuring the absorbance of the reaction mixture at 405 nm using a microplate autoreader EL 311. The results presented are the means ± SEM of 3 independent experiments.