FIGURE 3.
Lysine residues 1967 and 1968 have a differential effect on activity of FVIIIa over time. A–D, 0.3 nm FVIII WT and the variants thereof activated by thrombin as described under “Experimental Procedures.” FXa generation was allowed for 1 min in the presence of increasing concentrations of FIXa (0–16 nm), phospholipids, and Ca2+ ions 2 min (A) 5 min (B) 20 min (C), or 40 min (D) after thrombin activation. FVIII WT is displayed in black circles, K1967I in blue triangles, K1967A in green squares, and K1968A in red diamonds. Data represent mean ± S.D. (error bars) of at least three independent experiments. E, normalized decrease in residual FVIIIa activity over time for FVIII WT and FVIII variants. The inactivation rate constants, kinact, were assessed as described under “Experimental Procedures.” The S.D. values were within 15% of the obtain values for kinact.