Activation of the KKS in human plasma. (A) Human FXII-deficient plasma was supplemented with 400 nM FXII-WT and incubated in the presence (top row) or absence (bottom row) of kaolin (2.5 mg/mL). Kaolin will induce contact activation in plasma. (B) Human FXII-deficient plasma was supplemented with 400 nM FXII-Ala³⁶ (top row) or FXII-Ala³⁴⁶ (bottom row). For all experiments, FXII-supplemented plasma was incubated at 37°C. Samples were removed at the indicated times into nonreducing sample buffer, size fractionated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were developed with antibodies to FXII (left), PK (middle), or FXI (right). Positions of molecular mass standards in kilodaltons are shown to the left of each panel. Positions for free FXII and FXIIa (XII[a]), free PK and PKa (PK[a]), and FXI and FXIa; and FXIIa, PKa, and FXIa in complex with plasma protease inhibitors (XIIa + INH, PKa + INH, and XIa + INH) are shown to the right of each panel. Note that free FXIa migrates more slowly than free FXI in panel A (right column). Shown are representative blots for experiments that were run in duplicate.