Endothelial cell prekallikrein activation. (A) CN represents biotin-PK directly added to the SDS-PAGE. XIIa represents factor XIIa-cleaved soluble biotin-PK. PK represents the form of 20 nmol/L biotin-PK bound to HK on HUVECs. In this experiment, HUVECs were incubated with 20 nmol/L HK for 1 hour at 37°C. After washing the cells, 20 nmol/L biotin-PK was added in the absence (PK) or presence of two neutralizing antibodies to FXII (AFXII₁ and AFXII₂) or normal goat IgG (IgG). The biotin-PK was detected by chemilluminesence. The figure is a photograph of a 10% SDS-PAGE after the proteins were reduced with 5% β-mercaptoethanol and boiling. (B) CN represents biotin-PK directly added to the SDS-PAGE. XIIa represents factor XIIa-cleaved soluble biotin-PK. In this experiment, HUVECs were treated with 20 nmol/L HK for 1 hour at 37°C. After washing the cells, 20 nmol/L biotin-PK was added in the absence (PK₁) or presence of PMSF (1 mmol/L), SBTI (2 μg/mL), Pro-Phe-Arg-chloromethylketone (PFRCK; 100 μmol/L), or benzamidine (BZ; 1 mmol/L). PK₂ was 40 nmol/L biotin-PK bound to HUVECs in the absence of HK. The biotin-PK was detected by chemilluminesence. The figure is a photograph of a 7% SDS-PAGE after the proteins were reduced with 5% β-mercaptoethanol and boiling.