A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase complex by Rinku Majumder, Mary Ann Quinn-Allen, William H. Kane, and Barry R. Lentz Blood Volume 112(7):2795-2802 October 1, 2008 ©2008 by American Society of Hematology

Amino acid residues in human FVa that contribute to PS binding. Amino acid residues in human FVa that contribute to PS binding. The structure of bovine FVai solved by Adams et al38 is shown as rendered by the Cn3D software package.51 Selected amino acids are labeled using the corresponding residues in human FVa. Amino acid residues previously implicated in FVa binding to PS membranes are highlighted in yellow. The indole side chains of Trp2063 and Trp2064, located in the FVa C2 domain, penetrate the lipid bilayer and contribute a majority of the free energy associated with FVa binding to PS membranes.21 The hydrophobic side chains of Tyr1956 and Leu1957 are located in a structurally analogous position within the FVa C1 domain and provide a modest contribution to the free energy associated with FVa binding to PS membranes.23 Glycosylation of Asn2181 (green) results in the FVa1 glycoform. FVa2 is not glycosylated at Asn2181. Rinku Majumder et al. Blood 2008;112:2795-2802 ©2008 by American Society of Hematology

Prothrombin activation in the presence of C6PS. 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 Kdapp = 3.8 (± 0.5) μM and saturating activity 179 (± 7) nM/sec (wild-type) and Kdapp = 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 Kdapp'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. Rinku Majumder et al. Blood 2008;112:2795-2802 ©2008 by American Society of Hematology

Binding of C1 mutant human rFVa2 to soluble C6PS. Binding of C1 mutant human rFVa2 to soluble C6PS. The intrinsic fluorescence intensities of 0.2 μM of C1 mutant rFVa2 in 50 mM Tris, 150 mM NaCl, 5 mM CaCl2, 0.6% polyethylene glycol, pH 7.5, was measured as a function of C6PS concentration at 24°C to follow C6PS binding. The data were analyzed according to a simple binding model as described in “Binding of C6PS to human rFVa2,” with results shown in Table 1. Error bars represent SEM. Rinku Majumder et al. Blood 2008;112:2795-2802 ©2008 by American Society of Hematology

Binding of human rFVa2 to DEGR-FXa in the presence of C6PS. 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 Kd 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. Rinku Majumder et al. Blood 2008;112:2795-2802 ©2008 by American Society of Hematology

Activity of human rFVa C1 and C2 domain mutants in the presence of C6PS. Activity of human rFVa C1 and C2 domain mutants in the presence of C6PS. The cofactor activity of rFVa mutants was determined as described in the equation 1 in “Lipid-dependent cofactor activity.” Reaction mixtures contained 1 μM prothrombin, 1 nM FXa, 5 nM rFVa, 5 μM DAPA in 50 mM Tris, 150 mM NaCl, 5 mM Ca2+, pH 7.5. Shown are means (± SD) of 4 measurements. *P < .002 compared with wild-type rFVa. Rinku Majumder et al. Blood 2008;112:2795-2802 ©2008 by American Society of Hematology

Proposed model of prothrombinase assembly for wild-type human FVa, and for C1- and C2-mutated FVa. This model is based on the well-known fact that the free energy of binding of extrinsic membrane proteins to membranes often reflects multiple interactions, a... Proposed model of prothrombinase assembly for wild-type human FVa, and for C1- and C2-mutated FVa. This model is based on the well-known fact that the free energy of binding of extrinsic membrane proteins to membranes often reflects multiple interactions, and on the results presented in this paper. Rinku Majumder et al. Blood 2008;112:2795-2802 ©2008 by American Society of Hematology