Prothrombin activation in the presence of C6PS. Initial rates of human prothrombin activation were determined in reaction mixtures containing 50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM CaCl₂, 1 nM human FXa, 5 nM human rFVa₂, 1 μM prothrombin, and different concentrations of C6PS as described in “Methods.” The rate of prothrombin activation was determined in the presence of 0 to 400 μM C6PS and rFVa₂ having either an intact (A, circles) or a mutant ²³C1 domain (A,B, triangles). Data obtained with rFVa₂ having an intact C2 domain are shown in red (wild-type, circles in A; C1 mutant, triangles in panel B), whereas data with a mutant C2 domain¹⁸  are shown in green (C2 mutants, circles in A; and C1-C2 mutant, triangles in panel B). The lines drawn through the data in panel A are hyperbola, corresponding to a simple, single-site binding model, with parameters K_d^app = 3.8 (± 0.5) μM and saturating activity 179 (± 7) nM/sec (wild-type) and K_d^app = 3.7 (± 0.8) μM and saturating activity 177 (± 12) nM/sec (C2 mutant). Hyperbolic fits to the data up to 50 μM of C6PS are shown as dashed lines in the inset to panel A, with one of these fits (wild-type) also plotted as a dashed line in panel A. The data for rFVa₂ with a mutated C1 domain were also well described by hyperbola (B), but with K_d^app's = 60 (± 4) μM and 56 (± 3) μM and saturating activities of 0.0079 (± 0.0003) and 0.0078 (± 0.0002) nM IIa/sec for the C1 and C1-C2 mutants, respectively.