Thrombin-independent FVIII activation by nascent TF-FVIIa-FXa. (A) Representative immunoblots showing FVIIIa A1 activation fragment generation induced by 200 pM FVIIa and 50 pM rTF (0.37 μM phospholipids) in reactions containing 700 pM FVIII, 3 nM FV, 135 nM FX, 1 µM prothrombin (FII) (WT without or with 4 μM DAPA [n = 4] or inactive S195A mutant [n = 3]), and 2.5 mM CaCl₂ incubated at 37°C for 120 s (top). Quantification of FVIIIa-A1 calibrated with known quantities of the fragment (bottom). (B) FVIIIa activity calculated from FXa generation in reactions as in panel A but with 10 nM FIXa added and incubated for 180 s at 37°C (n = 4-12). FXa generation dependent on FVIIIa-FIXa activity was calculated by subtracting FXa generated by FVIIa and FIXa added individually from that by FVIIa/FIXa added together. (C) FVIIIa activity generated and calculated in reaction as in panels A and B but with the addition of 10 nM TFPIα. Results in panel A (bottom) and panels B and C (shown as 25th-75th percentile bars, min-to-max whiskers, line at the median; or min-to-max floating bars with line at the mean when n ≤ 3) were analyzed by ANOVA/Tukey tests. (D) Effect of different hirugen concentrations on TG initiated by 10 pM FIXa in normal PRP (180 ⋅ 10³ platelets/μL) containing 30 μg/mL CTI and 20 µg/mL anti-FXIa blocking MoAb O1A6 (n = 3). (E) Representative thrombograms initiated in normal PRP, containing CTI and anti-FXIa MoAb as in D, by 0.15 pM rTF, 10 pM FIXa, or both without (left) or with (right) 2 µM hirugen (n = 3). ***P < .001. NS, not significant.