Binding of human rFVa₂ to DEGR-FXa in the presence of C6PS. Binding of factor rFVa₂ to DEGR-FXa was detected by the change in fluorescence intensity of DEGR, which is covalently bound to the active site of FXa. Small aliquots of wild-type rFVa₂, C2 mutant, C1 mutant, and C1-C2 mutant were added to DEGR-FXa (1 nM in 5 mM Ca²⁺, 50 mM Tris, 150 mM NaCl, 0.6% polyethylene glycol, 400 μM C6PS, pH 7.5). The lines through the data were obtained by least-squares regression to a simple single-site binding model with the best fit K_d values for wild-type rFVa₂, C2 mutant, C1 mutant, and C1-C2 mutant being 3.1 (± 0.4) nM, 4.0 (± 0.6) nM, 564 (± 35) nM, and 624 (± 40) nM, respectively.