Kinetics of FXII cleavage by PK and PKa. (A) Varying concentrations of FXII-S544A were incubated with 1 nM PKa (left panel) or 200 nM PK-R371A (right panel) in HEPES buffer at 37°C in the presence of 70 μM poly-P. After 120 minutes, reactions were stopped by adding polybrene (0.1 mg/mL) and the antikallikrein IgG HO3 (5 nM). Conversion of FXII-S544A to the FXIIa conformation was measured by ELISA. (B) Kinetic parameters of FXII-S544A cleavage by PKa or PK-R371A in the presence of poly-P determined from the curves in panel A. (C) FXII-WT (200 nM) was incubated in the absence (−) or presence (+) of 70 μM poly-P. Reactions were run in the absence of PK (None) or in the presence of 200 nM PK-R371A or PK-S559A. Blue columns are reactions run in the absence of HK; red columns indicate reactions run in the presence of 200 nM HK. Reactions were stopped after 60 minutes with polybrene (0.1 mg/mL), and FXIIa cleavage of S-2302 (500 μM) was measured at OD 405 nm. *Compares FXII activation by PK-R371A in the presence or absence of HK (P = .0001); †compares FXII activation in the presence or absence of PK-R371A (P = .01).