Interactions of E149A-APC with EPCR and PAR-1. (A,B) Binding of wt-hAPC (A) or E149A-hAPC (B) to immobilized sEPCR was determined by SPR analysis at various APC concentrations (480, 240, 140, and 65 nM). Derived kinetic-binding parameters for association rate constants (k_on), dissociation rate constants (k_off), and equilibrium-binding constants (K_D) were indistinguishable (k_on, 2.3 × 10⁵ vs 2.0 × 10⁵ M⁻¹s⁻¹; k_off, 4.3 × 10⁻⁴ vs 3.6 × 10⁻⁴s⁻¹; K_D, 183 vs 180 nM for wt-hAPC vs E149A-hAPC, respectively). No binding of wt-hAPC to E86A-sEPCR was observed (data not shown). (C) Binding of wt-hAPC (□), E149A-hAPC (◇), and 5A-hAPC (○) to K293 cells transfected with wt-EPCR. (D) Cleavage of the TR33-62 PAR-1 peptide by E149A-hAPC was monitored over time as disappearance of the TR33-62 substrate peptide peak (open symbols) and as appearance of the TR42-62 product peptide peak (solid symbols). Symbols denote effects of wt-hAPC (■, □) and E149A-hAPC (♦, ◇). (E,F) Cleavage of a SEAP-PAR1 reporter construct by wt-hAPC (□) and E149A-hAPC (◇) on transfected K293 cells that lack (E) or contain EPCR (F). (G,H) Endogenous PAR-1 cleavage by APC on EA.hy 926 endothelial cells was monitored using cleavage-sensitive PAR-1 antibodies ATAP-2 (G) or H111 (data not shown). Cell surface PAR-1 antigen was detected using cleavage-insensitive PAR-1 antibodies WEDE15 (H) or S19 (data not shown). Antibody-staining results were corrected for residual nonspecific staining and normalized to non–agonist-treated cells (100%). Controls include plasma-derived APC (pl-APC) and DEGR-inhibited APC (DEGR-pl-APC) that is unable to cleave PAR-1. Each point represents the mean plus or minus SEM (n ≥ 3).