Effect of TPP-26870 amino acid substitutions within each CDR on APC-mediated histone H3 cleavage. (A) Histone H3 cleavage by APC in presence of TPP-26870 parental antibody was monitored by measuring the decrease in the intact H3 band over time. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed TPP-26870 did not affect H3 cleavage. (B) Time course of H3 cleavage by APC in the presence of TPP-26870 V_H variants. The amount of H3 cleaved by APC after 120 minutes is defined as 100%. (C) Time course of H3 cleavage by APC in the presence of TPP-26870 V_L variants. (D) SDS-PAGE analysis of the enhancement of H3 cleavage by the V_L Q27E substitution. (E) Identification of V_H amino acid substitutions with improved inhibition of APC anticoagulant activity and enhanced H3 cleavage compared with the parental antibody TPP-26870. (F) Identification of V_L substitutions with improved inhibition of APC anticoagulant activity and enhanced H3 cleavage. Histone H3 cleavage assay (n = 3-6 independent experiments). BSA, bovine serum albumin.