Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Aug 31.
Published in final edited form as: Blood. 2025 Dec 4;146(23):2833–2842. doi: 10.1182/blood.2025029430

Cryo-EM structure of the tissue factor/factor VIIa complex with a factor X mimetic reveals a novel allosteric mechanism

Josepha C Sedzro 1,*, Amanda L Photenhauer 2,*, Fabienne Birkle 1, Katarina Meze 2, Alex Mortenson 3, Cade Duckworth 3, Po-Chao Wen 3, Sarah Kearns 2, Michael A Cianfrocco 1,2, Emad Tajkhorshid 3, Melanie D Ohi 2,4,#, James H Morrissey 1,#
PMCID: PMC12397995  NIHMSID: NIHMS2105441  PMID: 40811856

Abstract

Blood clotting is triggered in hemostasis and thrombosis when the membrane-bound tissue factor (TF)/factor VIIa (FVIIa) complex activates factor X (FX). There are no structures of TF/FVIIa on membranes, with or without FX. Using cryo-EM to address this gap, we assembled TF/FVIIa complexes on nanoscale membrane bilayers (nanodiscs), bound to XK1 and an antibody fragment. XK1 is a FX mimetic whose protease domain is replaced by the first Kunitz-type (K1) domain of tissue factor pathway inhibitor, while 10H10 is a non-inhibitory, anti-TF antibody. We determined a cryo-EM structure of a TF/FVIIa/XK1/10H10/nanodisc complex with a resolution of 3.7 Å, allowing us to model all the protein backbones. TF/FVIIa extends perpendicularly from the membrane, interacting with a “handle shaped” XK1 at two locations: the K1 domain docks into FVIIa’s active site, while the γ-carboxyglutamate-rich (GLA) domain binds to TF’s substrate-binding exosite. The FX and FVIIa GLA domains also contact each other and the membrane surface. Except for a minor contact between the first epidermal growth factor (EGF)-like domain of XK1 and TF, the rest of the FX light chain does not interact with TF/FVIIa. The structure reveals a previously unrecognized, membrane-dependent allosteric activation mechanism between FVIIa and TF where a serine-rich loop in TF that partially obscures the TF exosite must undergo a shift to allow access of the FX GLA domain to its final binding location on the membrane-bound TF/FVIIa complex. This mechanism also provides a novel explanation for the otherwise puzzling phenomenon of TF encryption/decryption on cell surfaces.

Keywords: Cryo-EM, Factor VII, Factor X, Membrane, Tissue factor

Introduction

Tissue factor (TF), an integral membrane protein, initiates blood coagulation in normal hemostasis and many thrombotic disorders.1,2 TF binds factor VII (FVII), facilitating its conversion into the active protease, FVIIa. The membrane-bound TF/FVIIa complex then proteolytically activates membrane-bound factors X (FX) and IX (FIX), with FX being preferred under most conditions. Aberrant TF expression can trigger arterial and venous thrombosis, and TF also has non-hemostatic, signaling roles.2

A combination of approaches has shown that TF/FVIIa recognizes FIX and FX as substrates via interactions with not only the FVIIa catalytic cleft,3,4 but also the TF exosite, a cluster of surface-exposed residues near the membrane surface located ~60 Å away from the catalytic site.5–14 Encompassing TF residues Y157, K159, S163, G164, K165, K166, and Y185, this exosite is defined by mutations that diminish FIX or FX activation while not altering TF/FVIIa binding affinity or allosteric activation of FVIIa’s catalytic domain.11 Mutating K165 and K166 caused the largest decreases in FIX9–11,15 and FX5,6,8–11 activation. The γ-carboxyglutamate (GLA)-rich domains of FIX, FX, and FVIIa are implicated as major sites of interaction with the TF exosite region.9,13,16–18

Experimentally determined structures of membrane-bound TF/FVIIa are lacking, but crystal structures of the soluble TF ectodomain (sTF),19 and the sTF/FVIIa complex,20 show that the normally flexible FVIIa is stabilized upon binding the rigid sTF.20,21 While these structures have facilitated models for how TF allosterically activates the FVIIa catalytic domain,22 understanding TF/FVIIa assembly on membranes and binding to protein substrates has been limited to computational modeling.21,23 Docking studies using models of FX(a) or FIX bound to sTF/FVIIa (usually without membranes) predicted extensive protein-protein contacts between TF/FVIIa and all the domains of FIX or FX.24–27 In contrast, our molecular dynamics (MD) simulation on membranes predicted that FX interacts with TF/FVIIa chiefly via its GLA and protease domains, with little to no contact with its epidermal growth factor (EGF)-like domains.28 Thus, competing models have substantially different predictions for how TF/FVIIa engages FX.

To address how TF/FVIIa recognizes its substrates in the presence of a lipid bilayer, we used single particle cryo-electron microscopy (cryo-EM) to determine a structure of TF/FVIIa bound to both a FX mimetic, XK1,29 and the Fab fragment of the non-inhibitory anti-TF antibody, 10H10,30,31 assembled on phospholipid nanodiscs (Figure 1A). XK1 is a FX substrate mimetic in which the FX light chain (GLA-EGF1-EGF2) is fused to the first Kunitz-type (K1) domain of tissue factor pathway inhibitor (TFPI) to create a slow, tight-binding inhibitor of TF/FVIIa,29 which we confirm interacts with TF/FVIIa in a substrate-like manner.

Figure 1. Biochemical characterization of TF/FVIIa/XK1/10H10 complexes.

Figure 1.

Open in a new tab

(A) Schematic of the TF/FVIIa/XK1/10H10/nanodisc complex. FVIIa, light orange; TF, purple; MBP, gray; XK1, light sea green; and 10H10 Fab, blue; GLA, gamma (γ)-carboxyglutamate-rich domain; EGF, epidermal growth factor-like domain; K1, first Kunitz-type domain of TFPI. (B) TF exosite mutations K165A and K166A reduce the rates of FX activation by TF/FVIIa, with higher PS content moderating this effect. TF was incorporated into small unilamellar liposomes after which FVIIa and FX were added, and initial rates of FX activation were quantified. Bar graphs show FX activation rates (normalized to wild-type TF) observed when TF/liposomes contained 5% PS (left) or 20% PS (right). Fold-reductions are listed in supplemental Table S1. (C) Elevated XK1 concentrations are required to inhibit TF/FVIIa when TF exosite residues are mutated, with higher PS content moderating this effect. TF/FVIIa was assembled on TF/liposomes with 5% PS (left) or 20% PS (right), then pre-incubated with varying XK1 concentrations. The residual TF/FVIIa enzymatic activity was quantified (normalized to the FX activation rate without XK1) and plotted versus XK1 concentration, from which IC50 values were derived (listed in supplemental Table S1). Individual data points are graphed in panels B and C (n ≥ 3). ***, p<0.001.

This structure shows that the TF/FVIIa complex extends as a “mushroom shaped” rod perpendicular to the nanodisc, with XK1 forming a “handle” contacting TF/FVIIa in two regions: the K1 domain of XK1 docked into the FVIIa active site, and extensive interactions of the FVIIa and XK1 GLA domains (and a portion of EGF1 domain of XK1) with the TF exosite region. The structure also reveals a previously unrecognized allosteric activation mechanism in which a loop on TF obscures access of the FX GLA domain until being displaced during formation of the TF/FVIIa/XK1/membrane complex. This work provides crucial structural evidence for understanding how TF/FVIIa interacts with its primary substrate on the membrane surface.

Methods

A more detailed methods section is provided in the supplemental Methods, available on the Blood website.

Recombinant proteins

Amino acid sequences and residue numbering of the recombinant proteins employed in this study are given in supplemental Methods, as are details of their production. Recombinant, membrane-anchored TF consisted of TF residues 3–244, followed by a short linker and the Escherichia coli maltose-binding protein (MBP) sequence. For simplicity, this TF-MBP fusion protein is referred to as “TF” throughout the paper, except in supplemental Figure 1A.

Assembly and evaluation of TF/FVIIa/XK1 complexes on nanodiscs

Nanodiscs with TF embedded in the bilayers (TF/nanodiscs) were assembled largely as described,32 with changes detailed in the supplemental Methods. Briefly, we employed the truncated membrane scaffold protein, MSP1D1ΔH5,33 and the phospholipid composition was 50% phosphatidylserine (PS)/50% phosphatidylcholine (PC). TF/FVIIa/XK1 complexes for cryo-EM analyses were assembled by mixing TF/nanodiscs, FVIIa, and XK1 (with or without 10H10 Fab) in 20 mM Tris-HCl pH 7.4, 100 mM NaCl, 1.25 mM CaCl2, 0.5 mM MgCl2, 0.02% NaN3.

Cryo-EM data analysis

CryoSPARC was used for data processing with detailed steps outlined in the supplemental Methods.34

Movies were dose-weighted, motion corrected, and CTF corrected to generate micrographs. Particles were picked in an unbiased approach using blob picker on a subset of 100 micrographs. All classes with nanodiscs were selected as templates. Selection of 2D classes with discernible signal protruding from the nanodiscs resulted in 479,810 particles. These were subjected to ab initio reconstruction with three distinct classes and heterogeneous refinement. The class containing well-resolved signal for the TF/FVIIa/XK1/10H10 complex was further refined using homogeneous refinement. A mask was used to subtract the nanodiscs from the particles. Two more rounds of 3D classification were used, including one with a filter mask around XK1 to classify based on the presence of XK1. The final 20,107 particles were refined with non-uniform refinement.

Refinement was also performed with the nanodisc included. The initial 2,140,190 particles were subjected to 2 rounds of ab initio reconstruction and heterogeneous refinement to eliminate junk particles. The class of particles belonging to a class with nanodisc and protein were subjected to homogeneous refinement followed by local refinement with a mask around the TF/FVIIa/XK1/10H10 complex. The resulting particles were subjected to 3D classification with a filter resolution of 12 Å and a focus mask around the protein complex. The best class of 91,001 particles was refined with non-uniform refinement resulting in a 3.2 Å map. The distinct regions of the complex were segmented in ChimeraX and used for 3D Flexible Refinement in cryoSPARC with k=2 latent dimensions with the segments corresponding to FVIIa/TF/10H10 kept rigid for mesh generation. The results confirm significant variability in the nanodisc and movement of the XK1 handle in the sagittal and frontal planes.

Results

Assembly and biochemical analysis of membrane-bound TF/FVIIa/XK1 complexes

To investigate how the membrane-bound TF/FVIIa complex interacts with FX, we assembled TF/FVIIa complexes on membrane nanodiscs for single particle cryo-EM analysis. Since FX only transiently interacts with TF/FVIIa,35 we employed the FX mimetic, XK1, to build a stable membrane complex. XK1 is a hybrid protein wherein the FX protease domain is replaced by the K1 domain of TFPI, which tightly binds into the FVIIa active site29 (Figure 1A). To further stabilize TF within nanodiscs during cryo-EM analysis, we created a TF fusion protein with MBP. We also added the Fab fragment of antibody 10H10 as an alignment fiducial to the complex. 10H10 binds TF but does not interfere with FVIIa binding or FX activation.30 We confirmed that TF/FVIIa complexes on nanodiscs robustly activated FX, irrespective of the presence of MBP or the 10H10 Fab (supplemental Figure 1A). 10H10 Fab had no effect on the ability of XK1 to inhibit TF/FVIIa enzymatic activity (supplemental Figure 1B).

As a FX mimetic, XK1 should interact with TF/FVIIa in a substrate-like manner.29 If so, then TF exosite mutations K165A and K166A5,6,9,11 should comparably reduce both the rate of FX activation by TF/FVIIa and the affinity of XK1 for TF/FVIIa. Indeed, the TF K165A and K166A mutations diminished FX activation by 16- to 59-fold, respectively, when TF/FVIIa was assembled on liposomes with 5% PS (Figure 1B, supplemental Table S1). Defects in FX activation rates were less pronounced when liposomes had 20% PS, consistent with reports that higher PS partially masks the severity of TF exosite mutations (Figure 1B, supplemental Table S1).5,15 Next, we assessed the ability of XK1 to inhibit TF/FVIIa complexes assembled on liposomes with 5% or 20% PS. As shown in Figure 1C and supplemental Table S1, the TF K165A and K166A mutations increased the IC50 of XK1 (i.e., diminished its efficacy) for inhibiting TF/FVIIa enzyme activity by 4.1- to 23-fold relative to wild-type TF, depending on mutation and PS content. This confirms our previous finding with TF exosite mutations and XK1,36 and is consistent with TF exosite mutations reducing the rate of inhibition of TF/FVIIa by TFPI in the presence of FXa.8 Taken together, this demonstrates that XK1’s interaction with TF/FVIIa relies on an intact TF exosite, paralleling the effects on FX activation.

Cryo-EM structure of membrane-bound TF/FVIIa/XK1 with and without 10H10

TF/FVIIa/XK1 complexes were then assembled on membrane nanodiscs, in the absence or presence of 10H10 Fab (supplemental Figure 2). The assemblies formed ordered protein complexes as evidenced by negative stain EM (supplemental Figure 2C,D,F). Using single-particle cryo-EM analysis, we determined the structure of the TF/FVIIa/XK1 complex assembled on a nanodisc and bound to a 10H10 Fab (Figure 2, supplemental Figure 3, supplemental Figure 4, and supplemental Video 1). The structure has a resolution of 3.7 Å (loose mask); however, the local resolution across the EM density is not uniform (Figure 2A, supplemental Figure 3C,E, and supplemental Table S2). The quality of the EM density map allowed us to confidently identify the location of TF, FVIIa, XK1, and 10H10 Fab. The structure shows a mushroom-shaped density matching the available sTF/FVIIa crystal structures, with a thinner “handle-shaped” density, corresponding to XK1, interacting with the “stem” and “cap” of the mushroom (Figure 2A). There is clear density for the 10H10 Fab bound to the “stem” of the of the mushroom, while the weaker density of the “handle” in both the 2D averages and 3D EM map suggests that this region of XK1 is structurally flexible, which was confirmed by 3D Flexible Refinement, indicating there are continuous conformations in this region of XK1 (Figure 2A, supplemental Figure 3, supplemental Figure 5, and supplemental Video 2).

Figure 2. Cryo-EM structure of the TF/FVIIa/XK1/10H10 complex.

Figure 2.

Open in a new tab

(A) Four views of the cryo-EM density map (at contour level 0.1) together with the model of the TF/FVIIa/XK1/10H10 complex. The less structured portions of the XK1 density map (contour level 0.04) are shown in mesh, and the density for the nanodisc and MBP was subtracted for clarity. Views are rotated 90° around the y-axis. FVIIa is orange, TF is purple, XK1 is light sea green and 10H10 Fab is blue. (B) Views of the model of TF/FVIIa/XK1 complex, rotated 90° around the y-axis, with the same color scheme as in panel A. For clarity, the 10H10 Fab is not included in these views.

The local resolution of most of the protein regions of the EM map was sufficient to model the peptide backbones and/or fit crystal structures into the density map to create an atomic model of the TF/FVIIa/XK1/10H10 complex associated with membrane (Figure 2, supplemental Figure 6A,B, and supplemental Video 3). The structure of the 10H10 Fab fits easily into the EM density map (Figure 2A). Notably, the hinge between the Fab domains is at a different angle than was captured in the sTF/10H10 crystal structure (4M7L) (supplemental Figure 6C). The best resolved region of the EM density map is the TF/FVIIa complex (supplemental Figure 3E,H), which closely matches the 1DAN crystal structure of sTF/FVIIa in the absence of membranes, except for the orientation of FVIIa GLA domain (supplemental Figure 6D). In the EM density map this domain has shifted by ~16° (supplemental Figure 6D). This structural change propagates through the FVIIa α-helix linking the GLA domain to the FVIIa EGF1 domain, resulting in a shift of the EGF1 domain relative to FVIIa GLA domain when compared with the crystal structures, as discussed in detail below in the section titled “TF exosite interactions reveal a novel allosteric mechanism” (supplemental Figure 6D). While there are no structures of full-length FX or XK1, crystal structures and homology models of their individual domains are available. The K1 domain of XK1 is also clearly resolved, allowing us to build a complete homology model in this region of the cryo-EM density (Figure 3). Placement of the crystal structure of the bovine FX GLA domain (1IOD) into the cryo-EM map was done using the clearly resolved FX α-helix (residues 34–43) that connects the GLA domain with the EGF1 domain. This α-helix allowed us to confidently orient a homology model of the human FX GLA domain into the map (Figure 2A, Figure 3A, supplemental Figure 6E). Overall, the cryo-EM structure resembles the recent MD simulation of the TF/FVIIa/FX/membrane complex,28 although in that model the FX GLA and EGF1 domains are rotated ~120° relative to the present structure (supplemental Figure 6E).

Figure 3. Protein–protein interactions between the FVIIa protease domain and the K1 domain of XK1.

Figure 3.

Open in a new tab

(A) Cryo-EM density map (shown as a mesh) and model of the TF/FVIIa/XK1 complex, shown with density for the 10H10 Fab, nanodisc and MBP subtracted for clarity. Color scheme: FVIIa, orange; TF, purple; XK1, light sea green. Residues 31–41 of the XK1 K1 domain, which form a hairpin-like conformation, are in dark red. The cryo-EM density map is shown at contour level 0.1, while the density for EGF2 is visible at higher counter levels. (B) View of the interaction between the protease domain of FVIIa and the K1 domain of XK1 with the density map at contour level 0.1. Boxes indicate areas of the structure magnified in panels C and D. (C,D) Magnified views of specific regions of the FVIIa–XK1 interface boxed in panel B. Critical residues are shown as sticks and labeled, with carbon atoms colored according to the respective protein, oxygens in red, and nitrogens in blue. Selected atomic distances are indicated with gray dotted lines. The backbones of G365 and G375 are shown in gray. The cryo-EM density in panels C and D is shown at contour level 0.13.

To ensure that the 10H10 Fab did not alter the overall structure of the TF/FVIIa/XK1/nanodisc complex, we also determined a lower resolution cryo-EM 3D structure without 10H10 (supplemental Figures 7, 8, and 9, supplemental Video 4, and supplemental Table S2). The mushroom-shaped TF/FVIIa density in the 2D averages contains detailed secondary structural elements, while the XK1 “handle” has a weaker signal, suggesting the same structural flexibility in this region of XK1 as seen in the presence of the 10H10 Fab (Figure 2A, Figure 3A, and supplemental Figure 9A). The resolution of the 3D map without 10H10 (supplemental Figure 7C–E and supplemental Figure 9B) was not as high as the complex bound to the Fab, but the maps match well (supplemental Figure 9C) and the 3D model of TF/FVIIa/XK1 derived from the 10H10 Fab-bound map fits well into the non-Fab map with no significant domain shifts (supplemental Figure 9D). This confirms that 10H10 does not alter the orientation of the TF/FVIIa/XK1/nanodisc complex.

Key protein-protein interactions between XK1 and TF/FVIIa

The 3D EM structure and resulting atomic model of the TF/FVIIa/XK1/10H10 complex provide new insights into how TF/FVIIa interacts with substrate in the context of a membrane. The EM density map reveals that the substrate mimetic, XK1, interacts with TF/FVIIa through two main contact points and one minor one (Figure 3A). One major interaction occurs between the K1 domain of XK1 and the FVIIa protease domain (Figure 2B and Figure 3C,D), which is explored in more detail below in the section titled, “Molecular basis of XK1 binding to the FVIIa active site.” In the minor contact, R200 at the upper end of the TF exosite is positioned to form a salt bridge with E51 in the EGF1 domain of XK1 (Figure 4B), consistent with reports for a role for FX’s EGF1 domain in substrate recognition by TF/FVIIa and formation of the TF/FVIIa/FXa/TFPI complex.13,37,38 We note that this residue is also a Glu at the equivalent position in FIX (the alternate substrate for TF/FVIIa), but is not conserved in other vitamin K-dependent clotting proteins (supplemental Figure 10). Near the membrane, the other major area of interaction occurs between the GLA domain of XK1, the TF exosite, and the FVIIa GLA domain (Figure 2B, Figure 3A, and Figure 4C,D,E). The rest of XK1’s two EGF domains make no discernable contact with TF/FVIIa, and much of this density is at lower resolution, consistent with flexibility. Such flexibility of the FX light chain is not inconsistent with the recent cryo-EM structure of the prothrombinase complex and of single-molecule fluorescence measurements of FXa,39,40 especially taken together with prior fluorescence resonance energy transfer studies estimating the distance of the FXa active site from the membrane surface.41

Figure 4. Key protein–protein interactions between the TF/FVIIa complex and the FX light chain portion of XK1.

Figure 4.

Open in a new tab

(A) The TF/FVIIa/XK1 cryo-EM density map, together with the atomic model, is repeated here from Figure 3A for orientation purposes. Color scheme: FVIIa, orange; TF, purple; XK1, light sea green. (B) Close-up view of the interaction between TF residue R200 and FX residue E51 (in the EGF1 domain of XK1), shown with (left panel) and without (right panel) the cryo-EM density map. (C) Close-up view of the GLA domains of FVIIa and XK1 in proximity to the TF 4×Ser loop (indicated by purple arrow), shown with (left panel) and without (right panel) the cryo-EM density map. (D) Another close-up view of GLA domain–GLA domain and GLA domain–TF interactions, with and without the density map. Interactions between TF residue K165, FVIIa residues γ35 and R36, and XK1 residue K45 are highlighted. (E) A close-up view (from a different angle relative to panels C and D), revealing interactions between the XK1 GLA domain and the TF exosite, shown with (left panel) and without (right panel) the cryo-EM density map. Interactions between XK1 residues γ14 and γ19 with TF residues K159, K166, and Y185 are highlighted.

Molecular basis of XK1 binding to the FVIIa active site

Figure 3C,D shows that K1 residues 31–41 (10–20 in the numbering of bovine pancreatic trypsin inhibitor, BPTI) adopt a hairpin-like conformation that inserts into the FVIIa active site, positioning itself near the active site triad of FVIIa (H193, D242, and S344; corresponding to H57c, D102c, and S195c in chymotrypsinogen numbering), as previously described for homologous Kunitz-type inhibitor/protease interactions.42,43 The P1 residue, K36 in K1, docks into the S1 pocket of FVIIa (Figure 3C).42,43 Kunitz domain residues D11, R20, and E46 (BPTI numbering) are thought to play key roles in determining TFPI domain specificity.42–45 In our model, K1 residue D32 (D11 in BPTI numbering) is in a position that would support interaction with FVIIa residues R290 (R147c) and K341 (K192c) (Figure 3C), consistent with K341 being reported to play a significant role in TFPI-mediated inhibition of the TF/FVIIa complex.8,46 K1 residue E67 (E46 in BPTI numbering) is positioned so that it could interact with FVIIa residues K197 and K199 (Figure 3D).42–44

TF exosite interactions reveal a novel allosteric mechanism

Previous biochemical data identified residues that comprise the TF exosite for FX activation (Y157, K159, S163, G164, K165, K166, and Y185), whose roles can now be examined in the context of our EM structure. The TF exosite clearly forms a connecting interface with the GLA domains of both FVIIa and XK1 (Figure 2B, Figure 3A, and Figure 4C–E). This binding interface includes TF residue K166, which is well positioned for electrostatic interactions with γ14 and γ19 in the XK1 GLA domain (Figure 4E). Furthermore, XK1 residue γ19 is positioned to form a salt bridge to TF residue K159 and a hydrogen bond with TF residue Y185 (Figure 4E). Despite its known importance for FX activation, TF residue Y157 does not interact directly with any XK1 or FVIIa residues but instead is positioned behind TF K166, possibly playing a structural role (Figure 4E). The region spanning the TF 4×Ser loop (S160-S163) and G164 lies at the interface between FVIIa and XK1 in the cryo-EM structure (Figure 4C). While individual interactions between the side chains of residues in this region of the map cannot be fully resolved at the current resolution, the structure clearly shows this loop intercalating between the FVIIa and XK1 GLA domains (Figure 4C–E).

Aligning the cryo-EM structure of membrane-bound TF/FVIIa/XK1 to a crystal structure of sTF/FVIIa obtained without membranes (3TH2) reveals a few notable structural differences (Figure 5). In 3TH2, sTF residues S162, S163, and G164 sterically clash with the location of the XK1 GLA domain in the EM structure (particularly, with XK1 residues γ19 and γ20; Figure 5B,C,D). However, when looking just within the EM structure, there is no steric clash, and in fact the TF residues in the 4×Ser loop (and the backbone at G164) comprise part of the binding interface with the XK1 GLA domain, as described in the previous paragraph and shown in Figure 4C–E and Figure 5E. Relative to the positions of sTF residues in the 3TH2 crystal structure, the TF 4×Ser loop and adjacent G164 residue in the cryo-EM structure are shifted about 4 Å toward the FVIIa GLA domain (shown with curved arrows in Figure 5A). Intriguingly, the backbone of FVIIa residue R36 (located in the α-helix that comprises the “aromatic stack” portion of the FVIIa GLA domain) is shifted in the same direction by ~4–5 Å (Figure 5C). This movement positions the R36 side chain closer to TF residue K165 and primes γ35 of the FVIIa GLA domain to form a salt bridge with K45 of the XK1 GLA domain (Figure 4D and Figure 5C). Additionally, this movement creates potential hydrogen bonding interactions involving TF residues S162 and S163, including: S162 hydroxyl with a γ20 carboxyl of XK1; S162 and S163 backbone carbonyls with the T21 hydroxyl in the XK1 GLA domain; and S163 hydroxyl with the backbone carbonyl of F31 in the FVIIa GLA domain (Figure 5E).

Figure 5. Novel allosteric regulation of the TF exosite revealed by comparing the cryo-EM structure of TF/FVIIa/XK1/10H10 with the sTF/FVIIa crystal structure (3TH2).

Figure 5.

Open in a new tab

(A) Superposition of 3TH2 crystal structure of sTF/FVIIa with the cryo-EM map and model of TF/FVIIa/XK1, aligned via their TF components. Insets highlight conformational differences in the FVIIa GLA domain (left box) and the TF 4×Ser loop (right box). The color scheme for the cryo-EM map (mesh) and model is: FVIIa in orange, TF in purple, and XK1 in light sea green. For the 3TH2 crystal structure, it is: FVIIa in light gray and sTF in black/dark gray. The map is contoured at 0.1. For clarity, the 10H10 density was subtracted from the cryo-EM map and omitted from the model. In the following close-up views (panels B-E), the map is contoured at 0.145 and key interacting residues are shown as sticks and labeled, with carbon atoms colored by molecule, nitrogen in blue, and oxygen in red. (B) Close-up view focusing on the FVIIa and XK1 GLA domains within the alignment of TF/FVIIa/XK1 (cryo-EM structure) with the 3TH2 crystal structure. Arrows indicate the 4×Ser loop. (C) Close-up comparison of the positions of residues R36 of FVIIa and K165 of TF in the cryo-EM structure versus the sTF/FVIIa crystal structure, with the 4×Ser loop indicated by arrows. (D) Close-up view of the TF 4×Ser loop (arrows) and the XK1 GLA domain in the alignment between the cryo-EM structure and the sTF/FVIIa crystal structure. Note that the 4×Ser loop in the sTF/FVIIa crystal structure (but not the cryo-EM structure) sterically clashes with the cryo-EM density map of the XK1 GLA domain. FVIIa was omitted in this view for clarity. (E) Another close-up view of the region including the TF 4×Ser loop (arrows), highlighting interactions between TF residues S162 and S163 with FVIIa residue F31 and XK1 residues γ20 and T21.

The significant rearrangement of the 4×Ser loop in this complex suggests an important role in facilitating the interaction between TF, FVIIa and FX. We propose that the FVIIa GLA domain exerts an allosteric effect which includes FVIIa residue R36 repelling TF residue K165, causing the adjacent loop (TF residues S160 to G164) to move aside in order to expose the binding interface for interacting with the XK1 GLA domain (Figure 5 and supplemental Video 5).

Discussion

Our cryo-EM density maps of the TF/FVIIa/membrane complex show that this elongated complex is arranged perpendicular to the membrane surface, confirming prior fluorescence resonance energy transfer measurements.47,48 The high-affinity FX mimetic, XK1, was also bound to the complex, providing new insights into the structural basis of FX recognition by TF/FVIIa. While XK1 is a surrogate for the natural substrate, FX, which could represent a potential limitation of this study, we demonstrated that its affinity for the membrane-bound TF/FVIIa complex depends on a functional TF exosite in the same way that FX requires this exosite for efficient activation by membrane-bound TF/FVIIa. These data add confidence that the interaction of XK1’s FX light chain with TF/FVIIa reflects the enzyme/substrate interactions within the TF/FVIIa/FX Michaelis complex.

We found two main contact points between XK1 and TF/FVIIa: (1) the K1 domain of XK1 docks into the FVIIa active site (designed to mimic FX bound to FVIIa within the Michaelis complex); and (2) the TF exosite near the membrane surface engages the GLA domains of both FVIIa and XK1. It was technically challenging to obtain a high-resolution structure of what are two elongated peripheral membrane proteins (FVIIa and XK1) with very small membrane footprints, bound to a small, elongated, single-pass membrane protein (TF), all projecting away from a supported nanoscale membrane. Incorporating the Fab fragment of 10H10 facilitated a higher-resolution structure, which in turn allowed identification of protein-protein and protein-membrane interactions in greater detail. 10H10 has been shown to have no effect on the rate FX activation by TF/FVIIa, which we confirmed. We also demonstrated that it had no effect of the affinity of XK1 for TF/FVIIa.

Much of the FX light chain (i.e., most of EGF1 and all of EGF2) makes no discernable contact with TF/FVIIa, making this part of the complex flexible. This fits with the idea that FX must interact selectively with the TF/FVIIa complex, while at the same time, not bind so tightly that egress of the product, FXa, would be too slow. Our structure contrasts with most computational models of the TF/FVIIa/FX complex (typically in the absence of the membrane), which predicted extensive contact surfaces between TF/FVIIa and all FX domains.24–27 We previously reported that aligning these prior models with the sTF/10H10 crystal structure reveals extensive steric clash between FX and the 10H10 Fab,28,31 indicating that their trajectories for FX cannot be correct. In our more recent MD simulation of the TF/FVIIa/FX/membrane complex, FX interacts with TF/FVIIa in a manner that resembles that of XK1 in the membrane associated cryo-EM complex, in that both EGF domains of FX are in the solvent, projecting away from TF/FVIIa (and also avoiding the steric clash with 10H10).28 However, in that MD simulation, the FX GLA domain is rotated ~120° compared to its arrangement in the EM structure, so it has a different face of the FX GLA domain interacting with TF/FVIIa, and also has a different trajectory for the neighboring EGF1 domain.

A surprise in the EM structure was the location and movement of TF’s 4×Ser loop and adjacent G164 and K165 residues. Residues S163, G164, and K165 are among those whose mutation to alanine defined the TF exosite.11 While residues S160, S161 and S162 were not mutated in the original exosite studies, more recently we showed that any change in the length of TF’s 4×Ser loop—including deleting or inserting even one Ser—drastically reduces the rate of FX activation.36 This is in spite of the lack of electron density for this loop in almost all crystal structures (supplemental Figure 11), indicating it is surface-exposed and likely disordered. The cryo-EM structure now reveals that residue R36 in the FVIIa GLA domain, previously shown to be important in FX activation,16 is positioned near TF residue K165 in what appears to be a charge-charge repulsion. This is a consequence of the FVIIa GLA domain moving relative to its position in the sTF/FVIIa crystal structures, accompanied by upward movement of TF residue K165 shown in the view in Figure 5C, and movement of adjacent TF residue G164 and the entire 4×Ser loop towards FVIIa by some 4 to 5 Å (Figure 5C,D,E and supplemental Video 5). As a result, the EM structure shows that the FX GLA domain is now abutted up against TF residues S162, S163 and G164 in a location that clashes with these same TF residues in sTF/FVIIa crystal structures (Figure 5). The fact that this TF loop undergoes substantial movement relative to the crystal structures helps explain why none of the prior computational or docking models (including our own) predicted the correct placement of the FX GLA domain on the TF/FVIIa complex.

We now propose a novel allosteric activation mechanism for the TF exosite. When TF/FVIIa assembles on a membrane bilayer, the FVIIa GLA domain engages PS and becomes repositioned, resulting in FVIIa residue R36 repelling TF residue K165. This charge-charge repulsion in turn forces TF residues S160-G164 to move aside to uncover a cryptic FX GLA-domain binding site. This mechanism explains why reversing the charge of TF residue K165 (i.e., the K165E mutation) tightens the FVIIa-TF binding affinity threefold while greatly decreasing FX activation.9,15 It also comports with the finding that mutating FVIIa residue R36 substantially decreases the rate of FX activation by TF/FVIIa,16 and agrees with a series of domain deletion and swapping studies that implicated the FVIIa and FX GLA domains as the likeliest interaction points with the TF exosite.9,13,16–18 It explains why changing the length of the 4×Ser loop by even one residue is so deleterious for FX activation, given that this loop must move aside and become part of the binding interface between the FVIIa and FX GLA domains. We propose that it is the engagement of the FVIIa and FX GLA domains with PS in the bilayer that drives these structural rearrangements. The Kd for binding of FVIIa to membrane-embedded TF drops from 2.7 nM when TF is embedded in pure PC membranes down to 41 pM when TF is in PC/PS membranes.17 Thus, engagement of PS with the FVIIa GLA domain contributes −10.4 kJ/mol to the binding energy (ΔΔG),17 which we propose drives allosteric activation of the TF exosite. This may also help explain why an elevated PS content can partially compensate for TF exosite mutations.

An extensive literature exists for the phenomenon of TF encryption/decryption.49 TF on the surface of unactivated, undamaged cells binds FVIIa with high affinity but poorly supports FX activation, while agents that induce the appearance of PS on the outer leaflet of the plasma membrane are known to “decrypt” TF, resulting in full FX activation. While other mechanisms have been proposed,50 the allostery uncovered in the present study provides a mechanistic explanation for understanding TF encryption/decryption on cell surfaces, in which the appearance of PS on the outer membrane leaflet repositions the FVIIa GLA domain to force the S160-K165 loop in TF to move aside. This then uncovers the previously cryptic binding site on TF for the FX GLA domain, permitting efficient recognition of FX as a substrate.

In conclusion, the cryo-EM structure of the TF/FVIIa/membrane complex bound to a high-affinity substrate mimetic provides crucial new structural insights into how the enzyme that triggers blood clotting in health and disease interacts with its primary substrate on the membrane surface. Unlike previous models—most of which lack a membrane context, a resolved TF 4×Ser loop, or a stably bound substrate—our structure captures key protein-membrane and protein-substrate interactions within a fully assembled complex (supplemental Figure 11). And finally, this 3D structure now allows us to propose a novel allosteric regulatory mechanism for the TF exosite region that explains a host of biochemical and mutational findings from the past 30 years, and the puzzling phenomenon of TF encryption/decryption.

Supplementary Material

Supplemental_Video_1
Download video file (4.8MB, mp4)
Supplemental_Video_2
Download video file (5MB, mp4)
Supplemental_Video_3
Download video file (4.8MB, mp4)
Supplemental_Video_4
Download video file (4.9MB, mp4)
Supplemental_Video_5
Download video file (4.1MB, mp4)
Supplemental_Material

Key points.

  • XK1 interacts with TF/FVIIa via the FVIIa protease domain and TF exosite, with much of the FX light chain making no contact with TF/FVIIa.

  • A novel, membrane-dependent allosteric mechanism controls FX GLA domain binding to TF/FVIIa and can explain decryption of cell-surface TF.

Acknowledgements

We would like to thank Melanie P. Muller for her early MD modeling work, Xueyuan Peng for help with initial cryo-EM data collection, and the UM cryo-EM facility scientists for technical support. The UM cryo-EM facility is supported by the UM Biosciences Initiative, the Beckman Foundation, and the Life Sciences Institute. This study was supported in part by the National Institutes of Health (NIH) as follows: National Heart, Lung, and Blood Institute grants R35 HL135823 and R35 HL171334 (J.H.M.); NIH Director’s Office grant S10OD030275 (M.D.O.); and National Institute for General Medical Sciences grant R24 GM145965 (E.T.). F.B. was supported by predoctoral fellowship N028347 from the American Heart Association. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Footnotes

Conflict-of-interest disclosure: All the authors declare no competing financial interests.

Agreement to Share Publication-Related Data and Data Sharing Statement:

Atomic coordinates and cryo-EM density maps have been deposited in the Protein Data Bank and the Electron Microscopy Data Bank with the following accession codes: 9P0X and EMD-71090 for the TF/FVIIa/XK1/10H10 complex with density for the nanodisc subtracted during refinement; EMD-71093 for the TF/FVIIa/XK1/10H10 complex with nanodisc density included; EMD-71095 for the TF/FVIIa/XK1/10H10 complex with nanodisc density analyzed by 3D Flexible Refinement; and EMD-71094 for the TF/FVIIa/XK1 complex (no 10H10) with nanodisc density included.

Additional data are available on request from the corresponding authors, James H. Morrissey (jhmorris@umich.edu) and Melanie D. Ohi (mohi@umich.edu).

References

  • 1.Smith SA, Travers RJ, Morrissey JH. How it all starts: Initiation of the clotting cascade. Crit Rev Biochem Mol Biol. 2015;50(4):326–336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Grover SP, Mackman N. Tissue factor: an essential mediator of hemostasis and trigger of thrombosis. Arteriosclerosis, Thrombosis and Vascular Biology. 2018;38(4):709–725. [DOI] [PubMed] [Google Scholar]
  • 3.Baugh RJ, Dickinson CD, Ruf W, Krishnaswamy S. Exosite interactions determine the affinity of factor X for the extrinsic Xase complex. J Biol Chem. 2000;275(37):28826–28833. [DOI] [PubMed] [Google Scholar]
  • 4.Shobe J, Dickinson CD, Edgington TS, Ruf W. Macromolecular substrate affinity for the tissue factor-factor VIIa complex is independent of scissile bond docking. J Biol Chem. 1999;274:24171–24175. [DOI] [PubMed] [Google Scholar]
  • 5.Roy S, Hass PE, Bourell JH, Henzel WJ, Vehar GA. Lysine residues 165 and 166 are essential for the cofactor function of tissue factor. J Biol Chem. 1991;266(32):22063–22066. [PubMed] [Google Scholar]
  • 6.Ruf W, Miles DJ, Rehemtulla A, Edgington TS. Cofactor residues lysine 165 and 166 are critical for protein substrate recognition by the tissue factor-factor VIIa protease complex. J Biol Chem. 1992;267(9):6375–6381. [PubMed] [Google Scholar]
  • 7.Ruf W, Miles DJ, Rehemtulla A, Edgington TS. Tissue factor residues 157–167 are required for efficient proteolytic activation of factor X and factor VII. J Biol Chem. 1992;267(31):22206–22210. [PubMed] [Google Scholar]
  • 8.Rao LV, Ruf W. Tissue factor residues Lys165 and Lys166 are essential for rapid formation of the quaternary complex of tissue factor.VIIa with Xa.tissue factor pathway inhibitor. Biochemistry. 1995;34(34):10867–10871. [DOI] [PubMed] [Google Scholar]
  • 9.Huang Q, Neuenschwander PF, Rezaie AR, Morrissey JH. Substrate recognition by tissue factor-factor VIIa. Evidence for interaction of residues Lys165 and Lys166 of tissue factor with the 4-carboxyglutamate-rich domain of factor X. J Biol Chem. 1996;271(36):21752–21757. [DOI] [PubMed] [Google Scholar]
  • 10.Dittmar S, Ruf W, Edgington TS. Influence of mutations in tissue factor on the fine specificity of macromolecular substrate activation. Biochem J. 1997;321(3):787–793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Kirchhofer D, Lipari MT, Moran P, Eigenbrot C, Kelley RF. The tissue factor region that interacts with substrates factor IX and factor X. Biochemistry. 2000;39(25):7380–7387. [DOI] [PubMed] [Google Scholar]
  • 12.Kirchhofer D, Eigenbrot C, Lipari MT, Moran P, Peek M, Kelley RF. The tissue factor region that interacts with factor Xa in the activation of factor VII. Biochemistry. 2001;40(3):675–682. [DOI] [PubMed] [Google Scholar]
  • 13.Carlsson K, Freskgård PO, Persson E, Carlsson U, Svensson M. Probing the interface between factor Xa and tissue factor in the quaternary complex tissue factor-factor VIIa-factor Xa-tissue factor pathway inhibitor. Eur J Biochem. 2003;270(12):2576–2582. [DOI] [PubMed] [Google Scholar]
  • 14.Gajsiewicz JM, Nuzzio KM, Rienstra CM, Morrissey JH. Tissue factor residues that modulate magnesium-dependent rate enhancements of the tissue factor/factor VIIa complex. Biochemistry. 2015;54(30):4665–4671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Ke K, Yuan J, Morrissey JH. Tissue factor residues that putatively interact with membrane phospholipids. PLoS ONE. 2014;9(2):e88675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Ruf W, Shobe J, Rao SM, Dickinson CD, Olson A, Edgington TS. Importance of factor VIIa Gla-domain residue Arg-36 for recognition of the macromolecular substrate factor X Gla-domain. Biochemistry. 1999;38(7):1957–1966. [DOI] [PubMed] [Google Scholar]
  • 17.Neuenschwander PF, Morrissey JH. Roles of the membrane-interactive regions of factor VIIa and tissue factor. The factor VIIa Gla domain is dispensable for binding to tissue factor but important for activation of factor X. J Biol Chem. 1994;269(11):8007–8013. [PubMed] [Google Scholar]
  • 18.Ndonwi M, Broze GJ Jr., Agah S, Schmidt AE, Bajaj SP. Substitution of the Gla domain in factor X with that of protein C impairs its interaction with factor VIIa/tissue factor: lack of comparable effect by similar substitution in factor IX. J Biol Chem. 2007;282(21):15632–15644. [DOI] [PubMed] [Google Scholar]
  • 19.Harlos K, Martin DMA, O’Brien DP, et al. Crystal structure of the extracellular region of human tissue factor. Nature. 1994;370:662–666. [DOI] [PubMed] [Google Scholar]
  • 20.Banner DW, D’Arcy A, Chène C, et al. The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature. 1996;380(6569):41–46. [DOI] [PubMed] [Google Scholar]
  • 21.Ohkubo YZ, Morrissey JH, Tajkhorshid E. Dynamical view of membrane binding and complex formation of human factor VIIa and tissue factor. J Thromb Haemost. 2010;8(5):1044–1053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Madsen JJ, Persson E, Olsen OH. The intricate allostery in factor VIIa: triggering the trigger. J Thromb Haemost. 2025;23(1):1–10. [DOI] [PubMed] [Google Scholar]
  • 23.Lee CJ, Wu S, Bartolotti LJ, Pedersen LG. Molecular dynamic simulations of the binary complex of human tissue factor (TF(1–242)) and factor VIIa (TF(1–242) /FVIIa) on a 4:1 POPC/POPS lipid bilayer. J Thromb Haemost. 2012;10(11):2402–2405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Norledge BV, Petrovan RJ, Ruf W, Olson AJ. The tissue factor/factor VIIa/factor Xa complex: a model built by docking and site-directed mutagenesis. Proteins. 2003;53(3):640–648. [DOI] [PubMed] [Google Scholar]
  • 25.Venkateswarlu D, Duke RE, Perera L, Darden TA, Pedersen LG. An all-atom solution-equilibrated model for human extrinsic blood coagulation complex (sTF-VIIa-Xa): a protein-protein docking and molecular dynamics refinement study. J Thromb Haemost. 2003;1(12):2577–2588. [DOI] [PubMed] [Google Scholar]
  • 26.Lee CJ, Chandrasekaran V, Wu S, Duke RE, Pedersen LG. Recent estimates of the structure of the factor VIIa (FVIIa)/tissue factor (TF) and factor Xa (FXa) ternary complex. Thromb Res. 2010;125 Suppl 1:S7–S10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Banerjee S, Sen P. A molecular dynamics simulation study to understand the effect of cholesterol and tissue factor palmitoylation on tissue factor-factor VIIa-factor Xa ternary complex in different lipid environments. J Thromb Haemost. 2023;21(4):917–932. [DOI] [PubMed] [Google Scholar]
  • 28.Muller MP, Mortenson A, Sedzro JC, Wen PC, Morrissey JH, Tajkhorshid E. Membrane-bound model of the ternary complex between factor VIIa/tissue factor and factor X. Blood Adv. 2025;9(4):729–740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Girard TJ, MacPhail LA, Likert KM, Novotny WF, Miletich JP, Broze GJ Jr. Inhibition of factor VIIa-tissue factor coagulation activity by a hybrid protein. Science. 1990;248(4961):1421–1424. [DOI] [PubMed] [Google Scholar]
  • 30.Morrissey JH, Fair DS, Edgington TS. Monoclonal antibody analysis of purified and cell-associated tissue factor. Thromb Res. 1988;52(3):247–261. [DOI] [PubMed] [Google Scholar]
  • 31.Teplyakov A, Obmolova G, Malia TJ, et al. Crystal structure of tissue factor in complex with antibody 10H10 reveals the signaling epitope. Cell Signal. 2017;36:139–144. [DOI] [PubMed] [Google Scholar]
  • 32.Shaw AW, Pureza VS, Sligar SG, Morrissey JH. The local phospholipid environment modulates the activation of blood clotting. J Biol Chem. 2007;282(9):6556–6563. [DOI] [PubMed] [Google Scholar]
  • 33.Hagn F, Nasr ML, Wagner G. Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR. Nat Protoc. 2018;13(1):79–98. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Punjani A, Rubinstein JL, Fleet DJ, Brubaker MA. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods. 2017;14(3):290–296. [DOI] [PubMed] [Google Scholar]
  • 35.Ruf W, Rehemtulla A, Morrissey JH, Edgington TS. Phospholipid-independent and -dependent interactions required for tissue factor receptor and cofactor function. J Biol Chem. 1991;266(4):2158–2166. [PubMed] [Google Scholar]
  • 36.Birkle F, Morrissey JH. A serine loop in tissue factor mediates substrate selectivity by the tissue factor-factor VIIa complex. J Thromb Haemost. 2021;19(1):75–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Ndonwi M, Broze G Jr., Bajaj SP. The first epidermal growth factor-like domains of factor Xa and factor IXa are important for the activation of the factor VII--tissue factor complex. J Thromb Haemost. 2005;3(1):112–118. [DOI] [PubMed] [Google Scholar]
  • 38.Thiec F, Cherel G, Christophe OD. Role of the Gla and first epidermal growth factor-like domains of factor X in the prothrombinase and tissue factor-factor VIIa complexes. J Biol Chem. 2003;278(12):10393–10399. [DOI] [PubMed] [Google Scholar]
  • 39.Stojanovski BM, Di Cera E. Conformation of factor Xa in solution revealed by single-molecule spectroscopy. J Thromb Haemost. 2024;22(10):2767–2772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Ruben EA, Summers B, Rau MJ, Fitzpatrick JAJ, Di Cera E. Cryo-EM structure of the prothrombin-prothrombinase complex. Blood. 2022;139(24):3463–3473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Husten EJ, Esmon CT, Johnson AE. The active site of blood coagulation factor Xa. Its distance from the phospholipid surface and its conformational sensitivity to components of the prothrombinase complex. J Biol Chem. 1987;262(27):12953–12961. [PubMed] [Google Scholar]
  • 42.Bajaj MS, Birktoft JJ, Steer SA, Bajaj SP. Structure and biology of tissue factor pathway inhibitor. Thromb Haemost. 2001;86(4):959–972. [PubMed] [Google Scholar]
  • 43.Schmidt AE, Chand HS, Cascio D, Kisiel W, Bajaj SP. Crystal structure of Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in complex with trypsin. Implications for KD1 specificity of inhibition. J Biol Chem. 2005;280(30):27832–27838. [DOI] [PubMed] [Google Scholar]
  • 44.Zhang E, St Charles R, Tulinsky A. Structure of extracellular tissue factor complexed with factor VIIa inhibited with a BPTI mutant. J Mol Biol. 1999;285(5):2089–2104. [DOI] [PubMed] [Google Scholar]
  • 45.Vadivel K, Bajaj SP. Structural biology of factor VIIa/tissue factor initiated coagulation. Front Biosci. 2012;17(7):2476–2494. [Google Scholar]
  • 46.Neuenschwander PF, Morrissey JH. Alteration of the substrate and inhibitor specificities of blood coagulation factor VIIa: importance of amino acid residue K192. Biochemistry. 1995;34(27):8701–8707. [DOI] [PubMed] [Google Scholar]
  • 47.McCallum CD, Hapak RC, Neuenschwander PF, Morrissey JH, Johnson AE. The location of the active site of blood coagulation factor VIIa above the membrane surface and its reorientation upon association with tissue factor. A fluorescence energy transfer study. J Biol Chem. 1996;271(45):28168–28175. [DOI] [PubMed] [Google Scholar]
  • 48.McCallum CD, Su B, Neuenschwander PF, Morrissey JH, Johnson AE. Tissue factor positions and maintains the factor VIIa active site far above the membrane surface even in the absence of the factor VIIa Gla domain. A fluorescence resonance energy transfer study. J Biol Chem. 1997;272(48):30160–30166. [DOI] [PubMed] [Google Scholar]
  • 49.Rao LV, Kothari H, Pendurthi UR. Tissue factor encryption and decryption: facts and controversies. Thromb Res. 2012;129 Suppl 2(Suppl 2):S13–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Ansari SA, Pendurthi UR, Rao LVM. Role of cell surface lipids and thiol-disulphide exchange pathways in regulating the encryption and decryption of tissue factor. Thromb Haemost. 2019;119(6):860–870. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental_Video_1
Download video file (4.8MB, mp4)
Supplemental_Video_2
Download video file (5MB, mp4)
Supplemental_Video_3
Download video file (4.8MB, mp4)
Supplemental_Video_4
Download video file (4.9MB, mp4)
Supplemental_Video_5
Download video file (4.1MB, mp4)
Supplemental_Material
NIHMS2105441-supplement-Supplemental_Material.pdf (5.9MB, pdf)

Data Availability Statement

Atomic coordinates and cryo-EM density maps have been deposited in the Protein Data Bank and the Electron Microscopy Data Bank with the following accession codes: 9P0X and EMD-71090 for the TF/FVIIa/XK1/10H10 complex with density for the nanodisc subtracted during refinement; EMD-71093 for the TF/FVIIa/XK1/10H10 complex with nanodisc density included; EMD-71095 for the TF/FVIIa/XK1/10H10 complex with nanodisc density analyzed by 3D Flexible Refinement; and EMD-71094 for the TF/FVIIa/XK1 complex (no 10H10) with nanodisc density included.

Additional data are available on request from the corresponding authors, James H. Morrissey (jhmorris@umich.edu) and Melanie D. Ohi (mohi@umich.edu).

RESOURCES