![Figure 3. The TF initiation complex activates FVIII. (A) TG was induced by 20 pM FIXa; or 400 pM FVIIa WT or inactive iFVIIa; or FIXa combined with FVIIa WT or iFVIIa added into FVII-deficient reconstituted PRP (180 ⋅ 103 platelets/μl) containing 30 μg/mL CTI, 0.15 pM rTF, and 18 mM CaCl2, followed by incubation for 5 (n = 2-5), 8 (n = 2-5), or 11 (n = 3) min at 37°C. (B) FVIII (1.4 nM) activation by 400 pM rTF and 500 pM FVIIa with or without 135 nM FX in reactions containing 40 nM TFPIα and 200 nM lepirudin incubated for 30 s at 37°C. After blocking residual FXa and TF-FVIIa with 70 nM of TAP and 20 µg/mL each of MoAbs 5G9 and 12C7, respectively, FVIIIa clotting activity (n = 2-4) was measured by adding the reaction mixture into FVIII-deficient plasma with 10 nM FIXa, 20 µM phospholipids, and 8 mM CaCl2. (C) TG as in panel A but induced by a preformed complex of 10 nM rTF, 10 nM iFVIIa, 5 nM FXa, and 40 nM TFPIα or NAPc2 added at the indicated TF/FXa concentrations without (left) or with (right) 10 pM FIXa. Incubation for 8 min at 37°C (n = 3-5). Results in panels A-C are shown as 25th-75th percentile bars (minimum-to-maximum [min-to-max] whiskers, line at the median) or, when n ≤ 3, min-to-max floating bars (line at the mean); analysis by ANOVA/Tukey tests (after y = log10 y transformation in panel C). (D) Representative immunoblots (n = 2) of FVIIIa and FVa generation after 30 or 60 s in reactions with the indicated combinations of 200 pM FXa, 500 pM iFVIIa, 40 nM NAPc2, and 1 µM TAP added to 400 pM rTF, 700 pM FVIII, 3 nM FV, and 200 nM lepirudin. (E) Representative immunoblots showing the effect of TAP (1 µM) on FVIII activation by TF-FVIIa (400 and 500 pM, respectively) and 135 nM FX (top). Reactions, containing 700 pM FVIII, 3 nM FV, and 200 nM lepirudin also, were incubated for 30 s at 37°C. Quantification of experiments as shown in the top panel (n = 4), presented as 25th-75th percentile bars and analyzed by 2-tailed t test (bottom). (F) Dose response of FVa effect on 1 µM prothrombin conversion to thrombin in reactions containing 10 pM FXa, 50 pM rTF, and 700 pM FVIII incubated for 180 s at 37°C; mean ± 95% confidence interval (CI) (n = 4). (G) Dose response of FVa effect on FVIII activation by 10 pM FXa in reactions with 50 pM rTF, 700 pM FVIII, and 200 nM lepirudin incubated for 180 s at 37°C. FVIIIa activity (mean ± 95% CI, n = 4) was measured as generated FXa in the presence of 2 nM FIXa and expressed as percentage of that measured in the absence of FVa. (H) Representative immunoblots (n = 3) of FVIIIa generation in reactions as in panel G (left). Quantification of generated FVIIIa (n = 3) expressed as in panel G and shown by min-to-max floating bars (right); analysis by 1-sample t test. *P < .05. **P < .01. ***P < .001.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/130/14/10.1182_blood-2017-02-767079/4/blood767079f3.jpeg?Expires=1724851416&Signature=TM0mZGGIyW60h~e5KDYDZPFsslthIn3HzdQ-hY1BXEkdtqhr9biQ1HodYAyii-cLtL2jdJBnYHnIyRjvsGf~D~hBzjkCy~VMXCAU1VyWugpGkysKcQVU8Q1r2uiObOY75QKWykTRD2yWKYxEw8D5n1LgxY9~~1vvpkoWqTH9n7nG~lScuL5EEXFAgdseIYVYdrxNmHWGg7cRlh2L23JUDyR2II043WBcRDhul2Mj-cmHDvVMUjQ-09FVfudZ20PcO0xvh2OkJ49DzqAU7iMAqLVxza8DTTir6SiIKHpyf60M3bPdyU8GWvX8GRKV6Z-P5MUVeqsbvI66aSZ6pye-~w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The TF initiation complex activates FVIII. (A) TG was induced by 20 pM FIXa; or 400 pM FVIIa WT or inactive iFVIIa; or FIXa combined with FVIIa WT or iFVIIa added into FVII-deficient reconstituted PRP (180 ⋅ 103 platelets/μl) containing 30 μg/mL CTI, 0.15 pM rTF, and 18 mM CaCl2, followed by incubation for 5 (n = 2-5), 8 (n = 2-5), or 11 (n = 3) min at 37°C. (B) FVIII (1.4 nM) activation by 400 pM rTF and 500 pM FVIIa with or without 135 nM FX in reactions containing 40 nM TFPIα and 200 nM lepirudin incubated for 30 s at 37°C. After blocking residual FXa and TF-FVIIa with 70 nM of TAP and 20 µg/mL each of MoAbs 5G9 and 12C7, respectively, FVIIIa clotting activity (n = 2-4) was measured by adding the reaction mixture into FVIII-deficient plasma with 10 nM FIXa, 20 µM phospholipids, and 8 mM CaCl2. (C) TG as in panel A but induced by a preformed complex of 10 nM rTF, 10 nM iFVIIa, 5 nM FXa, and 40 nM TFPIα or NAPc2 added at the indicated TF/FXa concentrations without (left) or with (right) 10 pM FIXa. Incubation for 8 min at 37°C (n = 3-5). Results in panels A-C are shown as 25th-75th percentile bars (minimum-to-maximum [min-to-max] whiskers, line at the median) or, when n ≤ 3, min-to-max floating bars (line at the mean); analysis by ANOVA/Tukey tests (after y = log10y transformation in panel C). (D) Representative immunoblots (n = 2) of FVIIIa and FVa generation after 30 or 60 s in reactions with the indicated combinations of 200 pM FXa, 500 pM iFVIIa, 40 nM NAPc2, and 1 µM TAP added to 400 pM rTF, 700 pM FVIII, 3 nM FV, and 200 nM lepirudin. (E) Representative immunoblots showing the effect of TAP (1 µM) on FVIII activation by TF-FVIIa (400 and 500 pM, respectively) and 135 nM FX (top). Reactions, containing 700 pM FVIII, 3 nM FV, and 200 nM lepirudin also, were incubated for 30 s at 37°C. Quantification of experiments as shown in the top panel (n = 4), presented as 25th-75th percentile bars and analyzed by 2-tailed t test (bottom). (F) Dose response of FVa effect on 1 µM prothrombin conversion to thrombin in reactions containing 10 pM FXa, 50 pM rTF, and 700 pM FVIII incubated for 180 s at 37°C; mean ± 95% confidence interval (CI) (n = 4). (G) Dose response of FVa effect on FVIII activation by 10 pM FXa in reactions with 50 pM rTF, 700 pM FVIII, and 200 nM lepirudin incubated for 180 s at 37°C. FVIIIa activity (mean ± 95% CI, n = 4) was measured as generated FXa in the presence of 2 nM FIXa and expressed as percentage of that measured in the absence of FVa. (H) Representative immunoblots (n = 3) of FVIIIa generation in reactions as in panel G (left). Quantification of generated FVIIIa (n = 3) expressed as in panel G and shown by min-to-max floating bars (right); analysis by 1-sample t test. *P < .05. **P < .01. ***P < .001.
