Human coagulation factor Xa (FXa) plays a key role in blood coagulation by activating prothrombin to thrombin on “stimulated” platelet membranes in the presence of its cofactor factor Va (FVa). Phosphatidylserine (PS) exposure on activated platelet membranes promotes prothrombin activation by FXa by allosterically regulating FXa. To identify the structural basis of this allosteric regulation, we used fluorescence resonance energy transfer (FRET) to monitor changes in FXa length in response 1] to soluble PS (dicaproyl-phosphatidylserine; C6PS), 2] to PS membranes, and 3] to FVa in the presence of C6PS and membranes. We incorporated a FRET pair with donor (fluorescein) at the active site and acceptor (Alexa fluor 555) at FXa N-terminus near the membrane. The results demonstrated that FXa structure changes upon binding of C6PS to two sites, a regulatory site (Reg site) at the N-terminus (previously identified as involving the Gla and EGFN domains) and a presumptive protein-recognition site in the catalytic domain (Prot site). Binding of C6PS to the regulatory site increased the inter-probe distance by ~ 3 Å, while saturation of both sites further increased the distance by ~ 6.4 Å. FXa binding to a membrane produced a smaller length increase (~1.4 Å), indicating that FXa has a somewhat different structure on a membrane than when bound to C6PS in solution. However, when both FVa2 (a FVa glycoform) and either C6PS or PS-containing membranes bound to FXa, the overall change in length was comparable (~ 5.6–5.8 Å), indicating that C6PS and PS-containing membranes in conjunction with FVa2 have comparable regulatory effects on FXa. We conclude that the similar functional regulation of FXa by C6PS or membranes in conjunction with FVa2 correlates with similar structural regulation. The results demonstrate the usefulness of FRET in analyzing structure-function relationships in FXa and in the FXa.FVa2 complex
