Activities of antibody 14E11. (A) Nonreducing Western blots showing fIX (100nM) activation by fXIa (1nM) in the presence of control vehicle (top), 200nM 14E11 (middle), or 200nM O1A6 (bottom). Blots were developed with a polyclonal anti-human fIX IgG. Arrows to the right of each blot indicate zymogen fIX (IX) and fIXaβ (IXa). Time of incubation in minutes is shown at the bottom, and positions of molecular mass standards are at the left of the figure. (B) Competition binding assay in which biotinylated fXI is allowed to bind to immobilized HK in the presence of 14E11 (10⁻⁵ to 10⁰μM). Bound fXI was detected with strepavidin-HRP, as described in “Methods.” Each symbol is the average of results for duplicate experiments. (C) Factor XI (85nM) was activated by fXIIa (17nM) in the presence of HK (70nM and kaolin (0.5 mg/mL) in the presence (●) or absence (○) of 300nM 14E11. At various time points, samples were removed from the reaction into CTI, and fXIa generated was determined with a chromogenic substrate, as described in “Methods.” Each symbol is the average of results for duplicate experiments. (D) Thrombin generation in fXII deficient plasma initiated by addition of Ca²⁺ and fXIIa (10nM curves 1 and 3, or 1nM curves 2 and 4) in the absence (curves 1 and 2) or presence (curves 3 and 4) of 300nM 14E11. (E) Thrombin generation in fXII deficient plasma initiated by addition of Ca²⁺ in the absence (curve 1) or presence (curvess 2, 3, and 4) of tissue factor (0.23pM). Reaction for curves 2, 3, and 4 included control vehicle, 300nM 14E11, or 300nM O1A6, respectively.