A soluble, short chain phosphatidylserine, 1,2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), binds to discrete sites on FXa, FVa, and prothrombin to alter their conformations, to promote FXa dimerization (Kd ~ 14 nM), and to enhance both the catalytic activity of FXa and the cofactor activity of FVa. In the presence of calcium, C6PS binds to two sites on FXa, one in the epidermal growth factor like (EGF) domain and one in the catalytic domain; the latter interaction is sensitive to Na+ binding and probably represents a protein recognition site. Here we ask whether dimerization of FXa and its binding to FVa in the presence of C6PS are competitive processes. We monitored FXa activity at 5, 20 and 50 nM FXa while titrating with FVa in the presence of 400 µM C6PS and 3 or 5 mM Ca2+ to show that the apparent Kd of FVa-FXa interaction increased with increasing FXa concentration at 5 mM Ca2+, but the Kd was only slightly affected at 3 mM Ca2+. A mixture of 50 nM FXa and 50 nM FVa in the presence of 400 µM C6PS yielded both Xa homodimers and Xa ·Va heterodimers but no FXa dimers bound to FVa. A mutant FXa (R165A) that has reduced prothrombinase activity showed both reduced dimerization (Kd~147 nM) and reduced FVa binding (apparent Kd, = 58, 92 and 128 nM, respectively for 5, 20 and 50 nM R165A FXa). Native gel electrophoresis showed that the GLA-EGFNC fragment of FXa (lacking the catalytic domain) neither dimerized nor formed a complex with FVa in the presence of 400 µM C6PS and 5 mM Ca2+. Our results demonstrate that the dimerization site and FVa binding site are both located in the catalytic domain of FXa and that these sites are linked thermodynamically
