Beating tissue factor at its own game: Design and properties of a soluble tissue factor-independent coagulation factor VIIa

Abstract

Two decades of research have uncovered the mechanism by which the complex of tissue factor (TF) and the plasma serine protease factor VIIa (FVIIa) mediates the initiation of blood coagulation. Membrane-anchored TF directly interacts with substrates and induces allosteric effects in the protease domain of FVIIa. These properties are also recapitulated by the soluble ectodomain of TF (sTF). At least two interdependent allosteric activation pathways originate at the FVIIa:sTF interface are proposed to enhance FVIIa activity upon sTF binding. Here, we sought to engineer an sTF-independent FVIIa variant by stabilizing both proposed pathways, with one pathway terminating at segment 215-217 in the activation domain and the other pathway terminating at the N terminus insertion site. To stabilize segment 215-217, we replaced the flexible 170 loop of FVIIa with the more rigid 170 loop from trypsin and combined it with an L163V substitution (FVIIa-VY_T). The FVIIa-VYT variant exhibited 60-fold higher amidolytic activity than FVIIa, and displayed similar FX activation and antithrombin inhibition kinetics to the FVIIa.sTF complex. The sTF-independent activity of FVIIa-VY_T was partly mediated by an increase in the N terminus insertion and, as shown by X-ray crystallography, partly by Tyr-172 inserting into a cavity in the activation domain stabilizing the S1 substrate-binding pocket. The combination with L163V likely drove additional changes in a delicate hydrogen-bonding network that further stabilized S1-S3 sites. In summary, we report the first FVIIa variant that is catalytically independent of sTF and provide evidence supporting the existence of two TF-mediated allosteric activation pathways.

Keywords: X-ray crystallography; allosteric regulation; blood clotting; coagulation factor; cofactor; inflammation; protein engineering; serine protease; tissue factor; trypsin.

Figure 1.

Overview of FVIIa-WT and FVIIa-Y_T structural features and variant nomenclature. A, overview of the FVIIa-WT:sTF structure (PDB code 1DAN (42)) with the protease domain in gray and the active site in magenta. The light chain is shown in purple, containing the phospholipid-interacting γ-carboxyglutamic acid (Gla) domain and two epidermal growth factor (EGF)-like domains. sTF is shown in wheat. B, the protease domain of FVIIa-WT is shown in gray, and the domain in the FVIIa-Y_T variant is shown in blue (C), with the 170 loop from trypsin (PDB code 4Z6A (21)). Residues found to be involved in the TF-mediated allosteric increase in FVIIa activity are shown in blue for pathway I and green for pathway II. Residues manipulated in this study are shown in red, including the entire 170 loop. D, sequence alignment of generated variants with nomenclature, showing both full-length FVIIa and chymotrypsin numbering.

Figure 2.

Functional characterization of FVIIa variants. A, binding affinities for sTF measured using either a functional amidolytic activity–based assay or SPR. B, amidolytic activity (using S-2288) shown as k_cat/K_m. C, inhibition constants (K_i) for a small molecule S1 pocket inhibitor (pABA) measured in the presence of 1 mm S-2288. D, the effects of KNCO on FVIIa variant activity on 1 mm S-2288 reported as activity half-life (t_½). All functional experiments were conducted at 25 °C in the presence or absence of 3 μm sTF, with data shown as mean with range bars (n = 2 on the same day). The SPR data were also collected at 25 °C and shown as the mean ± S.D. (n = 3, three separate runs over 2 days). All data were collected in conjunction with FVIIa-WT and FVIIa-Y_T, for which the data were previously published (21).

Figure 3.

Physiological inhibition and substrate activation. A, AT inhibition was measured at different time points after the addition of 2.5 μm AT with 12 μm low-molecular-weight heparin to samples containing 200 nm FVIIa-WT (○), FVIIa-V (▴), FVIIa-Q (▵), FVIIa-Y_T (♢), FVIIa-QY_T (□), FVIIa-VY_T (▾), or FVIIa-WT with 5 μm sTF (○). Residual activity is shown as a percentage of activity corresponding to time 0, which was fitted to a one-phase decay model represented by the solid line from which an inhibition rate (K_inh) was estimated (mean ± S.D., n = 3 as separate runs on different days). B and C, FX activation was measured in solution by 100 nm FVIIa-WT (○), 50 nm FVIIa-V (▴), 50 nm FVIIa-Q (▵), 5 nm FVIIa-Y_T (♢), 5 nm FVIIa-QY_T (□), or 5 nm FVIIa-VY_T (▾) in the absence of sTF (B) or in the presence of sTF and 5 nm of FVIIa-WT or variants (C). The initial activation rate was fitted to linear regression model from which a k_cat/K_m could be derived (mean ± S.D., n = 3 as separate runs on different days).

Figure 4.

Probing conformational changes by exosite inhibitor binding. A and B, crystal structures of FVIIa-WT in complex with the exosite inhibitor E-76 (PDB code 1DVA (38)) or A-183 (PDB code 1JBU (49)), respectively. C and D, residual amidolytic activity measurements in the presence of either 600 nm E-76 or A-183 for FVIIa-WT (black), FVIIa-WT:sTF (red), FVIIa-Q (dark blue), FVIIa-V (gray), FVIIa-Y_T (light blue), FVIIa-QY_T (green), and FVIIa-VY_T (orange). Results are mean ± S.D. (error bars) with n = 3 from separate runs, and significance relative to FVIIa-WT was evaluated with one-way analysis of variance with Dunnett's multiple-comparison test in GraphPad Prism.

Figure 5.

Calorimetric evaluations of FVIIa variants. A, thermal stability of FVIIa variants obtained by monitoring the fluorescence ratio at 330/350 nm while heating from 20 to 90 °C (top). Each variant is shown with the respective transition temperature (T_m) in ºC, and the FVIIa-WT data are included in all graphs for comparison. Data are shown as mean ± S.D. (dotted lines), n = 8. The transition temperature (T_m) was determined by analyzing the first derivative and is reported for the largest change (bottom). Vertical lines mark the corresponding T_m. B, ITC data collected at 20 °C for binding of sTF to FVIIa-WT, FVIIa-Y_T and FVIIa-VY_T. Top, single thermographs as representative of a triplicate run, with single replicates prepared and measured individually on different days. Bottom, integrated enthalpies shown as mean ± S.D. (error bars) (n = 3) with obtained thermodynamic values from nonlinear regression fit with a 1:1 binding model shown for each variant (K_d (nm), ΔH (kcal/mol), TΔS (kcal/mol)).

Figure 6.

Structural features of a TF-independent FVIIa variant. A, structural alignment of the FVIIa-FFR:sTF (PDB code 1DAN) and FVIIa-VY_T-FFR:sTF protease domains. B, close-up of the 170 loop and AL2 and -3 area, with 2mF_o − dF_c density for mutated residues contoured at σ = 1.0. Water molecules in similar locations for FVIIa-WT and FVIIa-VY_T are numbered in pairs 1–6. The inset shows the leucine-to-valine mutation and resulting move of Phe-225 compared with both FVIIa-Y_T and WT. The green arrow indicates the viewing angle used in C–E. C–E, comparison of changes in hydrogen-bonding networks in the region of the S1 pocket (AL2-3) for FVIIa-WT (gray), FVIIa-Y_T (PDB code 4Z6A; blue), and FVIIa-VY_T (orange). Residues and water molecules colored in green have changed hydrogen bonding pattern and likely effect interactions with both P1-Arg and the backbone of P3-Phe of FFR. 2mF_o − dF_c density for the shown water molecules is contoured at σ = 1.0.