fXI autoactivation. (A) Plasma fXI (120 nM subunits) was incubated with 1 μg/mL DS (●) or 4 μM poly-P (○). At various times, fXIa generation was determined by chromogenic substrate assay. (B) Plasma fXI (120 nM subunits) was incubated with 1 μg/mL DS. Samples were collected into nonreducing sample buffer at the indicated time points and size fractionated on 6% polyacrylamide gels, then stained with GelCode Blue. (C) 120 nM subunits of plasma fXI (○) or 1/2-fXIai (●) were incubated with 1 μg/mL DS. At various times, fXIa generation was determined by chromogenic substrate assay. The fXI preparations in this panel were treated with DIP to inactivate contaminating fXIa, accounting for the longer lag phase compared with progress curves in panel A. (D) fXI^WT (○), fXI^C321S (●), fXI^C321S,L284A (□), or fXI^C321S,I290A (△), all 120 nM subunits, was incubated with 1 μg/mL DS. At various times, fXIa generation was determined by chromogenic substrate assay. (E) fXI^C321S,L284A (321/284), fXI^C321S,I290A (321/290), or fXI^C321S (321), 120 nM subunits, was incubated with 1 μg/mL DS. Samples were collected into nonreducing sample buffer at the indicated time points and size fractionated on 7.5% polyacrylamide gels, then stained with GelCode Blue. (F) fXI^WT (○), fXI^C321S,L284A (□), or fXI^C321S,I290A (△), each at a concentration of 120 nM subunits, was incubated with 4 μM poly-P. At various times, fXIa generation was determined. For all panels, error bars represent 1 standard deviation.