Activation of PK on HUVECs. (A) Endothelial cell monolayers (HUVECs) were preincubated with 200 μL of a solution containing 2% bovine serum albumin. HK (20 nmol/L) or buffer was then added for 1 hour at 37°C, the unbound protein was removed by washing, and 20 nmol/L PK or 20 nmol/L plasma kallikrein (Kal) was incubated for an additional 1 hour. The wells were then washed and 0.4 mmol/L S2302 was added in the absence or presence of 20 nmol/L FXII (XII), 3.4 nmol/L αFXIIa (XIIa), or 3.4 nmol/L βFXIIa (XIIf), as indicated. The data presented are the mean ± SEM of three experiments. The absence of standard error bars in some columns indicates that the variation was too little to portray visually. (B) Endothelial cell monolayers (HUVECs) were preincubated with 200 μL of a solution containing 2% bovine serum albumin. HK (20 nmol/L) or buffer were then added for 1 hour at 37°C, the unbound protein was removed by washing, and 20 nmol/L PK was incubated for 1 additional hour in the absence or presence of 0.4 mg/mL of an anti-FXII antibody (Anti-FXII). In other experiments, HUVECs saturated with HK (20 nmol/L) were incubated with 50 μL of pooled normal plasma (NHP), FXII-deficient plasma, or PK-deficient plasma for 1 hour at 37°C. After washing, 0.4 mmol/L S2302 was added and hydrolysis was monitored for 1 hour. The data presented are the mean ± SEM of three experiments. The absence of standard error bars in some columns indicates that the variation was too little to portray visually.