Effect of TPP-26870 amino acid substitutions within each CDR on APC-mediated cleavage of SEAP-PAR1 on HEK293 cells. (A) SEAP-PAR1 cleavage by APC in the presence of TPP-26870 V_H CDR1 variants. The amount of SEAP released into the medium by APC without antibodies is defined as 100%. (B) SEAP-PAR1 cleavage in the presence of V_H CDR2 variants. (C) SEAP-PAR1 cleavage in the presence of a V_H CDR3 variant. (D) Identification of V_H amino acid substitutions with improved APC anticoagulant activity inhibition and enhanced PAR1 cleavage compared with the parental antibody TPP-26870. (E) SEAP-PAR1 cleavage in the presence of V_L CDR1 variants. (F) SEAP-PAR1 cleavage in the presence of V_L CDR2 variants. (G) SEAP-PAR1 cleavage in the presence of V_L CDR3 variants. (H) Identification of V_L amino acid substitutions with improved inhibition of APC anticoagulant activity and enhanced PAR1 cleavage compared with the parental antibody TPP-26870. SEAP-PAR1 cleavage assay (n = 4-6 independent experiments).