Effect of TPP-26870 amino acid substitutions on healthy human plasma APC anticoagulant activity. (A) Clotting time standard curve of protein C–depleted plasma in the Protac-APTT assay. Healthy human plasma was serially diluted in protein C–depleted plasma (o) to achieve a range of protein C levels from 1.56% to 100%. Protac is a fast-acting snake venom that rapidly converts available protein C into APC. Protac was added into these serially diluted samples with different amounts of protein C zymogen to establish a Protac-APTT clotting time standard curve in the presence of different percent of APC in plasma. The APTT clotting time of healthy human plasma (△) and protein C–depleted plasma (▽) in the absence of Protac are also shown (n = 3-6 independent experiments; most error bars are smaller symbols). (B) Protac-APTT clotting time (left; y-axis) and the percentage of APC activity remaining in plasma (right; y-axis) in the presence of TPP-26870 variants with amino acid substitutions on the V_H. The percent APC activity in plasma of the TPP-26870 variants–treated samples was interpolated based on their clotting time against the standard curve in panel A (n = 6 independent experiments; some error bars are too small to appear). M34F provided the most reduction in the plasma clotting time and percent of APC in plasma. (C) Protac-APTT clotting time and the percent of APC anticoagulant activity in plasma in the presence of TPP-26870 V_L variants (n = 6 independent experiments; some error bars are too small to appear). N57R provided the most inhibition. (D) Effects of V_H amino acid substitutions on K_D and inhibition of APC anticoagulant activity compared with the parental antibody TPP-26870 (K_D, n = 2 independent measurements; APC activity, n = 6 independent measurements). (E) Effects of V_L amino acid substitutions on K_D and inhibition of APC anticoagulant activity compared with the parental antibody TPP-26870 (K_D, n = 2 independent measurements; APC activity, n = 6 independent measurements).