Figure 5.

FVIIa mutants with impaired FXa product turnover support FVIII activation by nascent FXa when thrombin feedback is blocked. (A) Time course (mean ± SEM) of 1 µM FX activation by 2 µM phospholipid-free soluble rTF with 10 nM FVIIa WT (n = 4-7), T99Y (n = 2-3), or E154A mutants (n = 3-4); incubation at 37°C (left). FXa generation in reactions with 50 pM phospholipid-reconstituted rTF, 200 pM FVIIa WT or mutants, 135 nM FX, and 700 pM FVIII incubated for 2 (n = 2-3), 4 (n = 4-5), or 6 (n = 6-9) min (right). (B) Representative immunoblots (n = 2) of FVIII activation by 50 pM rTF, 200 pM FVIIa WT or mutants, 135 nM FX, 700 pM FVIII, 3 nM FV, and 200 nM lepirudin, without or with 10 nM TFPIα incubated for 180 s. (C) Representative immunoblots (n = 2) of FV activation in reactions as in panel B incubated 420 s. (D) FVIIIa activity (25th-75th percentile bars, min-to-max whiskers, line at the median) generated as in panel B but without TFPIα, measured as FXa produced by 10 nM FIXa (n = 9 for FVIIa WT and T99Y; n = 5 for E154A) by ANOVA/Tukey tests (left). FVIIIa-FIXa activity generated with 90 nM FIX replacing FIXa; incubation 360 s (n = 5-6) (right). (E) Representative TG (n = 3) initiated by 2.5 pM rTF and 400 pM FVIIa WT or mutants in FVII-deficient reconstituted PRP containing 30 µg/mL CTI, 8 µg/mL anti-FVIII MoAb 8D4-blocking FVIIIa cofactor activity. (F) Representative TG (n = 3) as in panel E but without anti-FVIII MoAb and without (left) or with (right) 20 µg/mL anti-FXI MoAb O1A6-blocking FXIa activity in FIX activation. (G) Representative TG (n = 3) initiated by 20 pM FIXa in FVII-deficient reconstituted PRP containing 4 µM hirugen, 30 µg/mL CTI, 20 µg/mL anti-FXI MoAb. (H) Representative TG as in panel G but initiated by 2.5 pM rTF and 400 pM FVIIa WT or E154A with (left; n = 3) or without (right; n = 5) 8 µg/mL anti-FVIII MoAb. (I) Representative TG as in panel H but with added 3 nM FVa with (left; n = 4) or without (right; n = 5) anti-FVIII MoAb. **P < .01. ***P < .001.