Factor Xa Binding Sites on Factor Va in the Prothrombinase Complex.

Factor Va (FVa), a non-enzymatic cofactor for proteolytic activation of prothrombin by factor Xa (FXa), interacts with FXa via both its heavy and light chains. Phospholipids can bind both FVa and FXa and enhance prothrombin activation. The action of the prothrombinase complex (FXa:FVa) on its substrate, prothrombin, involves stepwise binding and enzymatic interactions, including FVa interactions with exosites on FXa that are topologically distinct from its active site. Synthetic peptides used as inhibitors of the prothrombinase complex implicated FVa residues 311–335 and 493–506 as probable FXa binding sites. Analysis of sequence alignments of FV with factor FVIII and of our 3-dimensional FVa model lead us to hypothesize that residues 312–315, 319–323, and 499–505, which delineate a remarkable compact triangular surface on the A2 domain are involved in FXa binding. Hence, we used homologous loop mutagenesis and substituted these three FV sequences with FVIII sequences, replacing 312-TREQ-315 with RSVA, 319-MKRWE-323 with PKTWV, and 499-KSRSLDR-505 with YKESVDQ. Each recombinant FV mutant was purified, characterized and compared to control wild type (wt)-FVa using ELISA and functional assays. Prothrombinase assays using thrombin-activated FVa and purified components were used to determine functional apparent Kd values of FVa for FXa by varying FXa concentrations in the absence or presence of saturating phospholipid (PL) vesicles (20%PS/80%PC). In the absence of PL vesicles, the kinetically determined apparent Kd values of each FVa mutant were greatly increased, such that mutation of either 312–315 or 319–323 in FVa decreased the apparent affinity for FXa by greater than 80-fold while mutation of 499–505 resulted in a 20-fold lower apparent affinity for FXa. However, in the presence of PL vesicles, these three FVa mutants each showed only a moderately reduced affinity for FXa (2.0–2.5 fold increased apparent Kd). None of the three FVa mutations significantly altered the Km for prothrombin. Therefore, each of the three mutated sequences in FVa is critical for normal functional interactions with FXa. Furthermore, these results illustrate the importance of each different kind of interactions, namely protein-protein and protein-PL, for the overall formation of the prothrombinase complex. Binding of both FXa and FVa to PL surfaces can help optimize the relative orientation of FVa to FXa to facilitate enzyme:cofactor exosite interactions and/or can promote conformational changes in one or both of FXa and FVa that affects exosite interactions. In the absence of PL, the effects of the A2 domain mutations on protein-protein interactions are magnified whereas when FXa and FVa are PL-bound, the observable effects of these mutations are greatly reduced. In summary, these mutagenesis studies support the hypothesis that three, closely located sequences on the surface of the FVa A2 domain comprising residues 312–315, 319–323, and 499–505 contribute significantly to FXa binding.