Characterization of PG assay. (A) Platelet-poor plasma was mixed with TF, phospholipids, and rtPA. Calibrator wells contained plasma and calibrator (α₂-macroglobulin/plasmin complex). Reactions were initiated by automatically dispensing fluorogenic substrate and CaCl₂ to each well. These reactions produced 2 fluorescence curves (raw fluorescence from plasmin generation in plasma [solid line], calibrator curve [dashed line]) from which a PG curve was derived and associated parameters were calculated: time to measurable plasmin generation (lag time), TtPeak, velocity, maximum plasmin produced (peak), and time integral of PG (EPP). (B) PG was measured in Plg^+/+ and Plg^−/− plasmas diluted 1:6 (N = 3 mice/group); averaged curves are shown. (C-D) PG was measured in normal, pooled plasma (diluted 1:6) in the presence of (C) 1.25 μg/mL or (D) 0.31 μg/mL rtPA, with increasing concentrations of α₂-antiplasmin (α₂-AP, note longer time scale in panel D). (E) PG was measured in normal, pooled plasma diluted in HEPES-buffered saline. Dilutions are indicated as plasma-to-buffer ratio. Normal, pooled plasma was diluted (F) 1:3 or (G) 1:6 and PG was measured in the presence of the indicated concentrations of rtPA. (H-P) Thrombin generation, fibrin formation, and PG were measured in 1:6 diluted plasmas from littermate-matched wild-type mice and (H-J) fibrinogen-deficient mice (Fga^+/−, Fga^−/−), (K-M) mice expressing mutated fibrinogen that cannot polymerize (Fgn^AEK), and (N-P) factor XIII-deficient mice (F13a1^+/−, F13a1^−/−), or (Q-S) different TF concentrations (0.05-6 pM). All reactions included 1 pM TF and 1.25 µg/mL rtPA unless otherwise indicated. Panels C-S show representative curves.