Phosphatidylserine-induced factor Xa dimerization and binding to factor Va are competing processes in solution

Abstract

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 (K(d) ~ 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 Ca(2+) to show that the apparent K(d) of FVa-FXa interaction increased with an increase in FXa concentration at 5 mM Ca(2+), but the K(d) was only slightly affected at 3 mM Ca(2+). 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 weakened dimerization (K(d) ~ 147 nM) and weakened FVa binding (apparent K(d) values of 58, 92, and 128 nM for 5, 20, and 50 nM R165A FXa, respectively). Native gel electrophoresis showed that the GLA-EGF(NC) 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 Ca(2+). 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.

Figure 1. Initial Rate of Prothrombin Activation by FXa-FVa at increasing Concentrations of FVa under Conditions Favoring Dimers

The FXa-FVa interaction was monitored as an increase in the ability of FXa at 5 (squares), 20 (triangles) and 50 (circles) nM concentrations to activate 1 µM prothrombin while titrating with FVa in the presence of 400 µM C6PS at 5 mM Ca²⁺. These conditions favor formation of both the FXa dimer and the FXa-FVa complex. A) the deviation of experimental data from a value predicted by Model 1 (open symbols) and Model 5a (closed symbols) relative to the average standard deviation (R_exp – R_fit)/σ_mean. B) The black curves drawn through the symbols indicate the global fit of the data to a simple competition model as described in the Supplement (dashed black line) or to the (XaVa)₂ model (Model 5a in Supplement; solid black line. Both models assume that the FXa dimer and XaVa dimer are inactive. Solid red lines represent a fit with hyperbola. Inset: The apparent K_ds of FXa-FVa dissociation were 2.2 ±0.6, 20±4.2, and 43 ± 13.3 nM for 5, 20 and 50 nM FXa, respectively. The inset shows a plot of apparent K_d versus % of monomer of FXa obtained using Equation 2 in Methods.

Figure 2. Initial Rate of Prothrombin Activation by FXa-FVa at increasing concentrations of FVa under Conditions Favoring Monomers

Conditions and symbols are as in Figure 1 except that Ca²⁺ concentration was 3 mM. A) the deviation of experimental data from a value predicted by the Model 1 (open symbols) and Model 5a (closed symbols) relative to the average standard deviation (R_exp–R_fit)/σ_mean. B) The black curves drawn through the symbols indicate the global fit of the data to a simple dimer competition model as described in Appendix A (dashed black line) or to an aggregate model as described in Appendix B (solid black line), both models predict that a dimer or aggregates are inactive. Solid red lines represent a fit with hyperbola. B inset) The apparent equilibrium constants (K_d) for FXa-FVa dissociation were 1.7 ± 0.4, 5.8 ± 0.8, and 8.6 ± 1.2 nM for 5, 20 and 50 nM FXa, respectively. The inset shows a plot of apparent K_d versus % of monomer obtained using Equation 2 in Methods.

Figure 3

3A. Native Gel Electrophoresis of a FXa and FVa Mixture in the Presence and Absence of C6PS. A 5% polyacrylamide gel of a mixture 50 nM FXa and 50 nM FVa in the presence of 400 µM C6PS and 5 mM Ca²⁺ (lane 1) shows coexistence of homodimers (FXa ·FXa; 90 KD), heterodimers (FXa · FVa; 224 KD), and FVa (178 KD), showing that FXa monomer interacts with both FVa and with FXa, thus consistent with the hypothesis that FXa and FVa compete to bind FXa. Lane 2 shows that the same reaction mixture in the absence of C6PS contains only monomers of both FVa and FXa, showing that this competition is regulated by phosphatidylserine (in this case C6PS). Lane 3 shows molecular mass markers. 3B: Native Gel Electrophoresis of FXa and its GLA-EGFNC Fragment. 8% native polyacrylamide gel electrophoresis of FXa and GLA-EGF_NC fragment of FXa in the presence and absence of µM 400 C6PS and 5 mM Ca²⁺ shows that the GLA-EGF^NC fragment does not dimerize in the presence of C6PS and 5 mM Ca²⁺. Figure 3C: 5% native polyacrylamide gel electrophoresis of 50 nM FVa with and without 50 nM GLA-αEGF_NC domain in the presence of 400 µM C6PS, 5 mM Ca²⁺. The results show that the GLA-EGF_NC fragment of FXa does not form a complex with FVa.

Figure 4. Prothrombin activation by increasing concentration of wild type and mutant (R165A) FXa in the presence of C6PS and 5 mM Ca²⁺

The initial rates of prothrombin activation by FXa in the presence of FVa and 400 µM C6PS at different of FXa concentrations were plotted as a function of FXa. The appearance of thrombin was determined by the rate of hydrolysis of DAPA (as described in Methods) at 37°C. The reaction was carried out at increasing concentrations of wild type FXa (closed circles) and mutant R165A FXa (open circles). The reaction mixture contained 1 µM prothrombin, FXa in 50 mM Tris, 175 nM NaCl, 0.6% poly(ethylene glycol), 5 mM Ca⁺² and 400 µM C6PS. The dimerization K_ds obtained in the presence of wild type and mutant FXa are 16 and 147 nM respectively.