Plasma clotting assays. (A) fXI^WT (WT), fXI^C321S (321), fXI^C321S,L284A (284/321), or fXI^C321S,I290A was added to fXI-deficient human plasma anticoagulated with 0.32% sodium citrate to a final concentration of 30 nM. –XI indicates vehicle-treated control plasma. Plasma containing fXI (30 μL) was mixed with an equal volume of PTT reagent, followed by incubation for 5 minutes at 37°C. Thirty microliters of 25 mM CaCl₂ was added and time to fibrin clot formation was determined. Proteins were tested in triplicate. (B-C) fXI-deficient plasma-containing vehicle or 30 nM fXI^WT, fXI^C321S,L284A, or fXI^C321S,I290A supplemented with 60 μM PC–PS vesicles was incubated with (B) 30 μL 1:256 dilution of PTT-reagent or (C) 30 μL poly-P (final concentration 4 μM). Mixtures were incubated for 1 minute at 37°C before addition of CaCl₂ and measuring time to clot formation, as above. (D) fXI-deficient plasma containing 8 μM corn trypsin inhibitor (to inhibit fXIIa), 60 μM PC–PS vesicles, and 16 μM poly-P (final concentration 4 μM) was mixed with an equal volume of 30 nM fXI^WT, fXI^C321S,L284A, or fXI^C321S,I290A. β-thrombin was added to a final concentration of 1.5 nM, followed by incubation for 1 minute at 37°C. CaCl₂ was added and the time to clot formation was measured. For all panels, error bars represent 1 standard deviation.