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. Author manuscript; available in PMC: 2019 Apr 1.
Published in final edited form as: J Thromb Haemost. 2018 Mar 12;16(4):734–748. doi: 10.1111/jth.13968

Evolutionary conservation of the allosteric activation of factor VIIa by tissue factor in lamprey

DL Beeler *, WC Aird *,†, MA Grant *,†
PMCID: PMC5893411  NIHMSID: NIHMS940042  PMID: 29418058

Summary

Background

Previous studies have provided insight into the molecular basis of human tissue factor (TF) activation of activated factor VII (FVIIa). TF-induced allosteric networks of FVIIa activation have been rationalized through analysis of the dynamic changes and residue connectivities in the human soluble TF (sTF)/FVIIa complex structure during molecular dynamics (MD) simulation. Evolutionary conservation of the molecular mechanisms for TF-induced allosteric FVIIa activation between human and extant vertebrate jawless fish (lamprey), where blood coagulation emerged more than 500 million years ago, is unknown and of considerable interest.

Objective

To model sTf/FVIIa complex from cloned Petromyzon marinus lamprey sequences, and with comparisons to human sTF/FVlla investigate conservation of allosteric mechanisms of FVIIa activity enhancement by soluble TF using MD simulations.

Methods

Full-length cDNAs of lamprey tf and f7 were cloned and characterized. Comparative models of lamprey sTf/FVIIa complex and free FVIIa were determined based on constructed human sTF/FVIIa complex and free FVIIa models, used in full-atomic MD simulations, and characterized using dynamic network analysis approaches.

Results

Allosteric paths of correlated motion from Tf contact points in lamprey sTf/FVIIa to the FVIIa active site were determined and quantified, and were found to encompass residue-residue interactions along significantly similar paths compared with human.

Conclusions

Despite low conservation of residues between lamprey and human proteins, 30% TF and 39% FVII, the structural and protein dynamic effects of TF activation of FVIIa appear conserved and moreover, present in extant vertebrate proteins from 500 million years ago when TF/FVIIa initiated extrinsic pathway blood coagulation emerged.

Keywords: allosteric regulation, blood coagulation factors, factor VIIa, thromboplastin, tissue factor

Introduction

The initiation of blood coagulation and subsequent wound repair is a fundamental defense mechanism conserved in vertebrates [1]. High blood pressure generated in the vertebrate circulation requires a strong, immediate, and strictly localized procoagulant response to minimize blood loss from sites of vascular injury without compromising blood flow. Initiating the network of interacting pathways of blood coagulation in vertebrates, from jawless fish to mammals, is the catalytic protease factor VIIa (FVIIa) and its cofactor tissue factor (TF) (reviewed, [2, 3]).

Human FVII is a 50-kDa glycoprotein synthesized in the liver and secreted into the blood as a zymogen (reviewed, [4]). Ten N-terminal γ-carboxyglutamic acid (Gla) residues facilitate calcium binding and phosphatidylserine-containing membrane association. Human TF is a 44-kDa transmembrane glycoprotein (reviewed, [5]) synthesized in many cell types, including adventitial fibroblasts, smooth muscle cells, pericytes, and several organ capsule epithelial layers [6–8]. When blood comes into contact with the sub-endothelium following vascular injury, circulating FVII/FVIIa binds TF to form a bimolecular complex on phosphatidylserine-containing membranes, and with activation of FVIIa triggers blood coagulation (reviewed, [9]). In mammals, the TF/FVIIa complex initiates coagulation by activating factors X (FX) and IX (FIX), leading to thrombin generation.

Previous studies provided details of TF-binding regions and insight for activating allosteric changes in FVIIa upon TF cofactor binding [10–13]. Conversion of FVII to FVIIa involves a two-step process where in the first step, proteolytic cleavage at R152-I153 produces a disulfide-linked (C135-C262) two-chain molecule with a light chain (Gla and two EGF-like domains) and heavy chain (trypsin-like protease domain). Unlike in trypsin, the newly formed FVIIa N-terminus does not completely insert [14] into the activation pocket to form a salt bridge with aspartate and optimally align the oxyanion hole and primary specificity pocket (S1) for catalytic efficiency [15], leading to “zymogen-like” FVIIa with sub-optimal catalytic efficiency. In the second step, TF binding to FVIIa allosterically affects the catalytic domain by stabilizing specific sub-regions and N-terminus insertion [16, 17]. These allosteric TF-mediated effects transform FVIIa into an optimal catalytically active form with ~106-fold enhanced proteolytic activity (summarized, [18]).

All vertebrates studied to date, including the extant vertebrate lamprey (a jawless fish), are capable of generating fibrin clots [19], possess functionally similar extrinsic arm clotting [20], and demonstrate TF-like activity in tissue extracts [21]. However lamprey which diverged from the vertebrate lineage >500 mya entirely lack the intrinsic arm of the clotting cascade [20, 22, 23], defining a critical evolutionary precedent for TF/FVIIa initiated blood coagulation across vertebrates from a common ancestor. Here, we report full-length tf and f7 cDNAs from the sea lamprey, Petromyzon marinus, and characterization of structural features in the complete proteins. Using models of lamprey Tf soluble extracellular-domain (sTf) and factor VIIa (sTf/FVIIa) complex, we assess the evolutionary conservation of Tf-binding regions in FVIIa and allosteric mechanisms of Tf-mediated FVIIa stabilization. Novel molecular dynamics (MD) simulations of lamprey sTf/FVIIa complex show conservation of Tf-mediated allosteric and dynamic changes in FVIIa. Together, these findings reveal molecular mechanisms of allosteric activation shared by human and lamprey sequences and conserved since the Gla domain-linked FVIIa trypsin-like serine protease emerged functionally in TF-mediated extrinsic blood coagulation more than 500 mya in a common ancestral vertebrate.

Materials and methods

Experimental animals

Adult lampreys (Petromyzon marinus), maintained at 11°C for 20–40 days before sacrifice, were anesthetized in 0.1g/L tricaine methanesulfonate (MS-222, Sigma) prior to blood sampling, tissue collection, and euthanasia. Experimental procedures were approved by the Animal Care and Use Committee, Mount Desert Island Biological Laboratory.

Isolation of lamprey genes

Total lamprey liver RNA was prepared using RNeasy RNA extraction (Qiagen) and used to prepare cDNA using oligo dT primer and SuperscriptIII (Invitrogen). Sense and antisense degenerate primer pools (Table S1) were used in PCR. Amplified cDNA fragments were purified, subcloned and sequenced. In RACE, initial and nested sense primers for 3’-RACE or antisense primers for 5′-RACE (Table S1) were used with KOD polymerase (EMD Millipore) and lamprey liver RACE cDNA pools made using SMARTer RACE 5′/3′ (TaKaRa).

Quantitative real-time polymerase chain reaction (qPCR) assays

Total lamprey tissue RNA prepared as above or total RNA from lamprey blood prepared using RiboPure Blood kit (Ambion) was converted into cDNA using random primers and SuperscriptIII (Invitrogen). The SYBR Green I assay was used for real-time PCR (ABI Prism 7700). Target gene expression data is presented as mRNA copies per 106 18S copies in each sample. PCR was performed in duplicate on samples from three animals (N=3).

Clotting assays

Lamprey blood was collected and citrated (1:6 v/v citrate buffer to whole blood) with 3.2% sodium citrate in 20mM HEPES, pH 7.4, 150mM NaCl, and 0.1% PEG8000. Cell-free plasma was prepared by centrifuging at 4000 × g for 4 min. For prothrombin time (PT), calcium-depleted lamprey thromboplastin, a phospholipid extract of tissue factor prepared from lamprey skin and muscle similarly as described previously [24], was mixed with plasma and CaCl2. Plasma mixtures were incubated (25°C) and the time for clot formation observed. For recalcification time, citrated plasma mixed only with CaCl2 was incubated (25°C) and clotting observed.

Homology modeling of Pm-sTf and Pm-FVIIa

Coordinates of human TF extracellular-domain (soluble sTF) and FVIIa complex (Hu-sTF/FVIIa) were built in YASARA [25] from PDB 1DAN [10] similarly as described [18], with missing loops modeled from corresponding residues of PDBs 1BOY [26] and 2HFT [27] of unbound sTF, and 1QFK [17] of human FVIIa and 2H9E [28] of human FXa. The template complex models residues 1-213 of human TF and residues 1-144 (light chain) and 153-406 (heavy chain) of human FVIIa. Co-crystallized inhibitor and chloride ion were removed, all calcium ions were retained, and hydrogens and termini groups were added. Hu-sTF/FVIIa was refined by energy minimization in a solvent shell in YASARA [29] and used as template for homology modeling lamprey sTf (residues 1-214) and FVIIa (light chain residues 1-153 and heavy chain residues 199-447) complex (Pm-sTf/FVIIa) with manually refined sequence alignments and model-packing assessment (see supplement footnote 1, regarding EGF2-SP intervening residues). Topologies were built using AutoSMILES [30]. Coordinates for free human FVIIa (Hu-FVIIa) based on PDB 1KLI [31], the heavy chain and light chain EGF2 domain, were used as a template for homology modeling corresponding regions of lamprey FVIIa (Pm-FVIIa). The calcium ion was retained, and hydrogens and termini groups were added. Both Pm-sTf/FVIIa and free Pm-FVIIa models were refined by energy minimization as above and iterative manual alignment adjustments.

Molecular dynamics simulations

MD simulations were performed using YASARA [25] and AMBER14 [32] force field parameters with additional parameterizations by AutoSMILES [30]. Gla residue topology and parameterizations included improper dihedrals to maintain near planarities of side-chain carboxylates. Proteins were solvated with TIP3P [33] water molecules in a cubic cell for periodic boundary conditions (10 Å larger along each axis), at pH 7.4 and physiological ionic strength (0.9%) with added sodium and chloride ions [34]. The Particle Mesh Ewald (PME) method [35] was used for long-range electrostatic forces (without cut off). Simulations were performed without distance constraints, except dative bonds to coordinated calcium and sodium ions were translated into distance constraints [25]. Equations of motion were integrated with a 1.25 fs time step, and van der Waals (cut off 8 Å) and electrostatics forces were evaluated every second step (2.5 fs) and added with a scaling factor of 2 [36]. Rescaling the atom velocities using a Berendsen thermostat [37] was applied to enforce constant temperature (298 K) and solvent probe pressure control [25] was used to isotropically resize the cell to maintain solvent density (0.997 g/mL) and pressure (1 bar). System equilibrium MD simulations following initial energy minimization using 500 conjugated gradient steps comprised 75 ns of experimental trajectory with coordinate snapshots taken every 25 ps.

Results

Clotting time assays

In prothrombin time (PT) assays using rabbit thromboplastin (membrane phospholipid and TF) at RT, clotting times were fast with human plasma, while lamprey clotted eventually at times greater than 350 sec (Fig. 1A). In contrast, using lamprey thromboplastin resulted in a significantly more rapid PT with lamprey plasma compared with human. Human plasma recalcification time at RT was 260 ±26 sec (data not shown). We observed no clotting of recalcified lamprey plasma (up to 75 min), indicating the intrinsic pathway is not conserved in lamprey and observed PT times are Tf/phospholipid dependent. Taken together, these PT results and previously published results [38], indicate that the extrinsic pathway is conserved between lamprey and humans and suggest that blood coagulation is fundamentally similar in all vertebrates with initiation by TF cofactor, albeit with species specificity.

Fig. 1. Comparative functional clotting assays and domain structure of human and lamprey FVII and TF(Tf).

Fig. 1

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(A) PT assay of plasma from human and lamprey (Petromyzon marinus) using thromboplastin from rabbit (mammalian) or lamprey at room temperature. Data are presented as mean ± SD (n=3). (B) Lamprey f7 cDNA encodes a putative protein of 484 amino acids. Processing of a putative N-terminal 38-amino acid signal-prosequence (PRO) would result in a predicted protein of 446 amino acids with putative homologous post-translational modifications, including γ-carboxylation of glutamic acid residues in the N-terminal domain. Annotated with yellow circles are human Gla-residue modifications and conserved Glu residues in lamprey FVII that are putative Gla-residue modification sites. Identified domains are the γ-carboxyglutamic acid rich Gla domain (GLA), two EGF-like domains (EGF), and the trypsin-family serine protease (SP) domain. Lamprey tf cDNA encodes a putative protein of 295 amino acids. Processing of a putative N-terminal 24-amino acid signal sequence (SS) would result in a mature protein of 271 amino acids. Identified protein domains of lamprey Tf are the extracellular domain fibronectin type III N- and C-modules (N-module, C-module), transmembrane region (TM) and cytoplasmic region (C).

Cloning and characterization of lamprey tf and f7 cDNA

To clone full-length tf and f7 cDNAs from P. marinus lamprey and portions of the UTRs, we used PCR with degenerate primers (Table S1) designed for conserved regions within multiple sequence alignments of other vertebrate TF and FVII proteins (Table S2), followed by rapid amplification of cDNA ends (RACE) using gene-specific and nested primers (Table S1). Lamprey tf cDNA (888-bp) encodes a protein (pro-Tf) of 295 amino acids (Fig. 1B). Processing the putative N-terminal 24-amino acid signal sequence would result in a protein (Tf) of 271 amino acids (30.0 kDa). Lamprey Tf (Pm-Tf) has 29.9% sequence identity to human TF (Table S3). Sequence alignment of Pm-Tf to TF sequences from multiple vertebrates, ranging from human to zebrafish (Fig. 2A and Fig. S1), and sequence analysis (InterProScan [39]) identifies extracellular N- and C-modules of fibronectin type III (FN3) domains and tissue factor-specific motifs that are conserved in higher vertebrates (Fig. 1B), and predicts transmembrane and signal sequences. After cloning lamprey tf cDNA, a report of partial Tf sequence was published [22] based on P. marinus draft-genome BLAST searching. However, our full-length Pm-Tf sequence identifies five novel sequence regions (from 2-10 amino acids), four novel residue substitutions, and contributes 14 additional C-terminal residues (Fig. S2) and 5′ and 3′ UTR sequences. A single tf cDNA was obtained after exhaustive sequencing of cDNA fragments, and to our knowledge, no tf paralog has been reported in jawed fish, higher vertebrates, or in the prior reporting of partial P. marinus sequence [22].

Fig. 2. Human and lamprey TF(Tf) and FVII sequence alignment.

Fig. 2

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(A) Sequence alignment of human and lamprey TF. Boxed regions in aligned sequences, which are colored according to domain structure as in Fig. 1B. (magenta, N- and C-module; yellow-orange, TM; cyan, C), identify sequence conservation (color-shaded background) and sequence similarity (white background). (B) Sequence alignment of human and lamprey FVII. Boxed regions in aligned sequences, which are colored according to domain structure as in Fig. 1B. (red, GLA; green,EGF; blue, SP), identify sequence conservation (color-shaded background) and sequence similarity (white background). Important functional sub-regions of FVII SP domain are annotated. Gaps created in aligning sequences are indicated by (●). Crosses (black) denote residues in human TF extracellular domain or FVIIa serine protease which are in close contact in the X-ray structure of sTF/FVIIa (inhibited) complex.[10]

Lamprey f7 cDNA (1455-bp) encodes a protein (pro-FVII) of 484 amino acids (Fig. 1B). Processing the putative N-terminal 38-amino acid signal-prosequence would result in a protein (FVII) of 446 amino acids (48.7 kDa). Mature lamprey FVII (Pm-FVII) has 39.3% overall sequence identity to human FVII (Table S3). Alignment of Pm-FVII to vertebrate FVII sequences, ranging from human to zebrafish (Fig. 2B, Fig. S3 and Table S2), and sequence analysis identifies specific domains that are conserved in higher vertebrates: the γ-carboxyglutamic acid-rich Gla domain, two EGF-like domains, and trypsin-family serine protease domain (Fig. 1B). Of ten N-terminal Gla residues in human FVII, nine Glu residues are found at homologous sites in Pm-FVII suggesting significant conservation of post-translational modifications for calcium-dependent membrane binding. After cloning lamprey f7 cDNA, a report of partial lamprey FVII sequence was published [22] based on P. marinus draft-genome BLAST searching. However, our full-length Pm-FVII sequence identifies 21 additional N-terminal amino acids, contributes a large 62 amino acid sequence within the serine protease domain, reveals nine residue substitutions, shows a SP domain residue deletion, and contributes 5′ and 3′ UTR sequences (Fig. S4).

A single f7 cDNA was identified after exhaustive sequencing of gene fragments from PCR similar to previously reported f7 from a Lethenteron japonicum lamprey liver cDNA library [23]. The SP domains of these lamprey FVII sequences share 98.8% identity. In previous P. marinus draft-genome searches [22], 3 reconstituted partial sequences were identified as putative FVII gene fragments (FVIIA/B/C), with the first (FVIIA) being nearly identical to the Pm-FVII sequence (Fig. S4). In alignments of candidate lamprey FVII sequences to jawed fish Danio rerio FVII, where a single zebrafish f7 gene was identified and expressed FVII protein functionally characterized [40], the Pm-FVII SP domain showed the greatest sequence identity to zebrafish FVII SP (50.9%), compared to FVIIB (46.1%) and FVIIC (46.0%). Additionally, BLASTp [41] analysis of the SP domains, matched FVIIB (60% of top 40 alignment hits) and FVIIC (80% of top 40 alignment hits) to FX SP sequences of other species including fish and birds (Table S4). In contrast, 92.5% of the top alignment hits for Pm-FVII SP were vertebrate FVII SP domains including fish and birds.

Expression of lamprey tf and f7 transcripts

To demonstrate expression of cloned tf and f7, qPCR analyses using designed primers (Table S1) were carried out for lamprey tf and f7 mRNAs in P. marinus lamprey liver, heart, muscle and blood (Fig. 3). qPCR analysis confirmed specific expression of tf and f7 mRNAs in various lamprey tissues. We found the expression of lamprey tf to be comparatively low, with the highest transcript levels in liver, followed by muscle; while f7 was expressed at higher levels in liver, followed by heart. These results are the first to identify expressed tf and f7 transcripts in lamprey.

Fig. 3. Lamprey tf and f7 expression in lamprey tissues.

Fig. 3

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Quantitative PCR (qPCR) analysis of tf (A) and f7 (B) in lamprey organs and whole blood. mRNA expression is represented as copy number per 106 18S copies. Data are presented as mean ± SD (n=3 fish).

Structure of the lamprey sTf/FVIIa complex

To understand the molecular basis of lamprey Tf-FVII interaction, we determined structural models of Pm-sTf and FVII protein domains. Views of the human sTF/FVIIa complex (Hu-sTF/FVIIa), derived from modeled experimental structures, and the computationally determined lamprey sTf/FVIIa complex (Pm-sTf/FVIIa) are shown in Fig. 4A–B. Superimposition of protein backbone Cα atoms of Hu-sTF/FVIIa onto Pm-sTf/FVIIa yields a RMSD of 0.774 Å over 563 aligned residues with 41.4% sequence identity. Side-chains of sTF important for binding FVIIa along the extensive regions of contact are shown in Fig. 4A–B. A listing of interacting residues between sTF and all FVIIa domains in human and lamprey complexes is given in Table S5. Contact maps for Hu-sTF/FVIIa or Pm-sTf/FVIIa show similar intermolecular profiles between the TF N-module and FVIIa heavy chain (4.4 Å distance cutoff) (Fig. S5). This Pm-sTF/FVIIa interface region comprises 24 contacts, compared to 27 contacts in Hu-sTF/FVIIa. Detailed inspection shows a conserved binding mechanism (Fig. 4C–D). In both, two neighboring ‘key-and-lock’ interactions involving FVIIa residues Met306-Asp309 (TF-binding loop), Arg271 and Phe275-Phe278 in human FVIIa and Asp351-Gln354 and Arg319-Val322 in lamprey FVIIa, fit into and over the combined surface of TF N-module subregions defined by residues within Gln37-Trp45, Leu72-Phe78, and Pro92-Asn96 in Hu-TF, and within Glu37, Thr42-Gln48, Arg76,Arg78 and Val90-Trp92 in Pm-Tf. As this interface in Hu-sTF/FVIIa was identified as the molecular basis of TF-induced allosteric activation of FVIIa heavy chain [11–13, 42–45] conservation of such ‘key-and-lock’ interaction suggests conservation of Tf-induced allosteric activation in Pm-sTf/FVIIa.

Fig. 4. Structural comparisons of human and lamprey sTF/FVIIa complexes.

Fig. 4

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Structures of human (A) and lamprey (B) sTF/FVIIa complexes visualized using PyMol [67]. TF N- and C-modules (magenta), FVIIa Gla domain (red), EGFs (green), and heavy chain (blue) are shown in backbone cartoon representation. Catalytic triad residues of FVIIa serine protease (SP) domain are shown as sticks (orange). Bound and modeled calcium ions are shown as spheres (yellow). Residues involved in the sTF-FVIIa interaction interface are shown in sticks. Detailed inspection of the interface sub-region between TF N-module and FVIIa heavy chain (4.4 Å distance cutoff) in human (C) and lamprey (D) complexes. In both, two neighboring ‘key-and-lock’ interactions involving FVIIa catalytic domain residues Met306-Asp309 (TF-binding loop), Arg271 and Phe275-Phe278 in human FVIIa and Asp351-Gln354 and Arg319-Val322 in lamprey FVIIa, fit into and over the combined surface of TF N-module subregions within Gln37-Trp45, Leu72-Phe78, and Pro92-Asn96 in human TF, and Glu37, Thr42-Gln48, Arg76,Arg78 and Val90-Trp92 in lamprey Tf.

Molecular dynamics simulation of Hu-sTF/FVIIa and Pm-sTf/FVIIa

TF-mediated allosteric activation of Hu-FVIIa was shown to comprise significant stabilization and reduced flexibility of the protease domain, in particular the TF-binding helix (Fig. 2B), 170-loop, 94-shunt, and activation loop3 and loop1 [11–13, 16]. Additionally, previously identified effects of sTF on FVIIa included stabilized, reduced exposure of Trp364 [45] and reductions in Cβ distances between the TF-binding helix/170-loop region and activation loop3 or loop2 residues, and between loop1 and loop3 residues [11, 13, 16]. To identify evolutionary conservation of TF-induced allosteric FVIIa activation, we determined such conformational differences between free and sTF-bound human or lamprey FVIIa heavy chain during MD simulations. MD simulations were performed using initial structures derived from the free Hu-FVIIa crystal structure or our homology-based free Pm-FVIIa structure, and compared to simulations of modeled Hu-sTF/FVIIa or Pm-sTf/FVIIa complexes. During MD simulations of human and lamprey complex, sTF binds to all FVIIa domains through salt bridges, hydrogen bonds, ionic contacts and hydrophobic interactions, and domains of sTF and FVIIa remain stably folded. Stability of the FVIIa heavy chain N-terminus insertion into the activation pocket to form a salt bridge with Asp is shown in N-O salt bridge distances in starting and ending Hu-sTF/FVIIa and Pm-sTf/FVIIa simulation structures (Table S6). The N-terminal Gla domain residue Ala1 remains ‘tucked’ in the Ca2+-binding region sustaining hydrogen bonds with Gla20/Gln21/Gla26 (human) or Gla22/Gly23/Gla28 (lamprey) and a closed ω-loop-like conformation. Distances between Ala1-N and Gla side-chain and main-chain oxygens in starting and ending simulation structures are listed in Table S7. The time-evolved backbone Cα position RMSD (Fig. 5A–B) from initial equilibrated structures across the MD trajectories of free verses sTF-bound FVIIa shows significant reduction of FVIIa heavy chain structural deviations. Similar to human, the entire lamprey FVIIa heavy chain is substantially ‘rigidified’ as Cα RMSDs are notably reduced in sTf-bound lamprey FVIIa. sTF-bound FVIIa active site geometry shows shortening of Cβ distances between Asp-Ser and Ser-His triad pairs in both human and lamprey, indicating stabilization and narrowing of active site geometries (Fig. 5C–D). The FVIIa residues Trp364 (human) and Trp405 (lamprey), which are both highly flexible and undergo structural exchange in the absence of sTF, become stabilized in less solvent exposed conformations in sTF-bound complexes (Fig. 5E). Additionally, sTF-induced reductions and stabilizations in Cβ distances between FVIIa TF-binding helix and activation loop3 or loop2 residues, and between loop1 and loop3 residues (Fig. 5F) are comparable in lamprey.

Fig. 5. Structural dynamics in human and lamprey sTF/FVIIa complexes during MD simulation.

Fig. 5

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Cα atom RMSDs verses simulation time (relative to the initial structure) for human (A) and lamprey (B) free (black) or sTF-bound (red) FVIIa heavy chain. Distances between catalytic site triad residues for human (C) and lamprey (D) free (black) or sTF-bound (red) FVIIa heavy chain. (E) Structural dynamics of the 170-loop and activation loops monitored by solvent accessible surface area (SASA) of FVIIa Trp364 (human) or Trp405 (lamprey), and (F) distances between side-chain atoms in FVIIa heavy chain verses time for human or lamprey free and sTF-bound FVIIa. Human distances from (green) Arg315N-Gly372C, (blue) Ser333Cβ-Gln313Cβ, (purple) Asp289Cβ-Thr370Cβ, and (black) Ile153N-Asp343CG. Lamprey distances from (green) Met359Cβ -Lys415Cβ, (blue) Glu374Cβ-Ala358Cβ, (purple) Glu334Cβ-Arg411Cβ, and (black) Val199N-Asp384CG. The latter distances (black) are to monitor the N-termini inserted positions as controls for human and lamprey FVIIa domain stability during MD simulation.

Qualitative binding energy determinations of Hu-sTF/FVIIa and Pm-sTf/FVIIa complexes in MD simulations carried out using molecular mechanics (MM)/Poisson-Boltzmann surface area (PBSA) methods [25] show stably overlapped profiles (Fig. S6A). The time averaged binding energy across the final 25 nsec of simulation shows only a small relative 1.15-fold increase in binding energy for human compared to lamprey (Fig. S6B). For comparison, qualitative interaction energies [46] and energies of binding [47] also determined indicate very similar, small relative fold increases for human compared to lamprey (Fig. S6B). The similar binding energies are consistent with observed extensive contact profiles along both sTF/FVIIa interfaces (Table S5), noting a few more contacts between human TF N-module and FVIIa heavy chain than lamprey (Fig. 4C–D).

Measurements of average per residue Cα RMSDs and root-mean-square fluctuations (RMSF) in residue backbone atom positions across the MD trajectory structures characterize the structural change and conformational flexibility of FVIIa heavy chain residues. Plots of average RMSD (structural change) and RMSF (conformational flexibility) verses residue number in free and sTF-complexed human or lamprey FVIIa heavy chain demonstrate that nearly all sTF-bound FVIIa subregions have reduced conformational deviations and diminished fluctuations (Fig. 6A–B). Projection of B-factor values (the per residue fluctuation of atoms from their average positions across trajectories) on the average FVIIa heavy chain structure from free FVIIa and sTF/FVIIa complex MD simulations shows that similarly to human the activation loops 1-3, TF-binding helix, 94-shunt, and Ca2+-binding region of lamprey FVIIa are all significantly less dynamic and structurally stabilized by sTF-binding (Fig. S7).

Fig. 6. Conformational flexibility of FVIIa during MD simulation.

Fig. 6

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The average root mean square deviation (RMSD) (A) and root mean square fluctuation (RMSF) (B) of residue Cα atom distances over the MD trajectory structures of free FVIIa (black) or sTF-bound FVIIa (red) are plotted versus residue number, for human and lamprey.

Conservation of allosteric protein network pathways in lamprey sTf/FVIIa

The dynamic cross-correlation matrix (DCCM) displays calculated correlations in the movements of residue Cα pairs across MD trajectories. Comparison of residue-residue Cα DCCMs for free verses sTF-bound FVIIa shows that for both human and lamprey, the presence of sTF dramatically increases both positive and negative correlated motions of FVIIa heavy chain residue pairs (Fig. 7). Both human and lamprey sTF-bound FVIIa heavy chain show dramatic increases in positive correlations within the SP domain and between SP and EGF2 domains, along with corresponding regions of anti-correlated motions. Moreover, near identity is observed in the residue pairs and sub-regions within human and lamprey FVIIa heavy chains that show significant TF-induced changes in dynamic correlated and anti-correlated residue motions.

Fig. 7. Conservation in TF-induced changes in the residue-residue covariance matrix for FVIIa serine protease during MD simulation.

Fig. 7

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Maps of the dynamic cross-correlation matrices (DCCM) show the extent of correlation for all residue Cα pairs of human (A) free FVIIa and (B) sTF-bound FVIIa, and lamprey (C) free FVIIa and (D) sTf-bound FVIIa. FVIIa residues along the axes of each DCCM map are represented by FVIIa domain schematics. The DCCM diagonal represents the correlations between covalently bonded residues, while off-diagonal regions provide information of correlations across non-covalently bonded residues. Motion occurring along the same direction is represented by positive correlation (blue, +1.0), whereas anti-correlated motion is represented by negative correlation (red, -1.0).

Correlated-residue clustering and dynamic network analysis have elucidated pathways of protein allostery [48–52]. We applied such analysis [53] to free FVIIa and sTF-bound FVIIa MD trajectories to investigate pathways of allostery from the TF-binding region to FVIIa active site (Fig.8). Dynamic correlated motions between residue centers of mass pairs, shown as nodes, and the strength of the measured correlation interdependences, shown as connecting edges, identify allosteric communication pathways in the protein network. The results demonstrate how conserved the long-range allosteric signal propagation is from the TF contact point through the SP domain to the catalytic residues in human and lamprey (Fig. 8 and Fig. S8). Five most-optimal paths (Table S8) overlap onto two nearly identical pathways of signal propagation from TF-binding helix residue (source), human Met306 or lamprey Asp351, to active site (sink) Asp/His ([Fig. 8, A and C] Pathway I) or Ser ([Fig. 8, B and D] Pathway II) residues. Our optimal allosteric pathway result for Hu-sTF/FVIIa is very similar to that previously published using different simulation methods and analysis algorithm [12, 54]. As for human, the shortest source-to-sink paths in lamprey free FVIIa are considerably longer than for sTf/FVIIa demonstrating significantly weaker correlated motions in the absence of Tf. Together, our findings reveal molecular mechanisms of allosteric activation shared by human and extant vertebrate lamprey Tf/FVIIa sequences and conserved since the Gla domain-linked FVIIa trypsin-like serine protease emerged functionally in Tf-mediated vertebrate blood coagulation.

Fig. 8. Conserved pathway propagation of long-range allosteric signal from the TF interaction point to the FVIIa active site.

Fig. 8

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Visualization of the five most-optimal pathways of long-range allosteric signal propagation within human (A,B) and lamprey (C,D) TF-bound FVIIa heavy chain using the dynamic network analysis tool WISP [53] in VMD [68] to determine the dynamic interdependence between residues in FVIIa across superposed MD simulation structure trajectories. Correlated motion among residue pairs, represented as nodes (spheres) positioned at the residue center of mass, and the interdependence in correlated motion among nodes, represented as connecting edges (spline lines) with associated numeric values reflecting the strength (thickness) of each edge. Pathway propagation from M306 in human and D351 in lamprey, to active site Asp/His residues [Pathway I] or to active site residue Ser [Pathway II], follows nearly identical pathways in human and lamprey TF-bound FVIIa heavy chain and along pathways of exclusively conserved residues. Pathway I: TF-contact point → Cys → Phe → Met → Thr → Asp/His; and Pathway II: TF-contact point → Cys → Ala → Tyr → Val → Ser. Pathway correlation strength is represented in color, from red (strongest) to yellow, and line thickness.

DISCUSSION

Lampreys are jawless fish whose ancestors diverged from the vertebrate lineage more than 500 mya [55]. Previous studies in lamprey indicate the clotting system is a simpler version of the mammalian system [1, 19, 20, 22, 23]. Our functional and molecular data support that the extrinsic pathway is intact in lamprey. We identified a single f7 cDNA, consistent with the identification of a f7 in Lethenteron japonicum lamprey [23] that shares 98.8% sequence identity in the SP domain with Pm-FVII. As additional reconstituted partial FVII sequences have been reported from P. marinus draft-genome bioinformatic searches [22], paralogs of Pm-FVII cannot be ruled out. Without complete sequence, further understanding if duplicate gene products function redundantly in clotting or have novel function is lacking. However, we find these SP domain sequences have higher similarities to vertebrate FXs, and moreover, in alignments of candidate lamprey FVII and functionally validated zebrafish FVII [40] sequences, the Pm-FVII SP domain showed the greatest sequence identity to zebrafish (50.9%). Together, these data and our lamprey tissue expression data, support reporting the identification of complete tf and f7 cDNAs from lamprey Petromyzon marinus and studies presented herein.

In human FVIIa, Met306 plays a pivotal role in TF interaction [10] and allosteric enhancement of FVIIa [42, 44]. Met306Asp substitution, which does not affect binding affinity for FVIIa, abrogates TF’s allosteric effects on FVIIa [43, 54, 56] by disrupting hydrogen bonding networked communication between the TF-binding interface and FVIIa active site [56] and/or changing Cys340-Cys368 disulfide structure [16]. The corresponding critical allosteric communication residue in Pm-Tf/FVIIa, Asp351, plays a similar pivotal role in protein dynamic networks to the FVIIa active site (Fig. 8 and Fig. S8) with a surrounding species-specific context that facilitates significant stabilization and reduced dynamic flexibility of FVIIa, just as in human. Likely, substitution in lamprey that would disrupt the Asp351 and helix 352-357 hydrogen bonding network or impart structural effects on Cys381-Cys409 at would have a similar detrimental effect as human Met306Asp substitution. Intriguingly, PT clotting data shows inhibited cross-species TF/FVIIa activation. Examinations of the Hu- and Pm-sTF/FVIIa interface (Fig. 4A–D, Table S5) show interacting residues are similar in some cases, yet very different in others, and predict significant unfavorable cross-species interactions or sidechain clash (for example Pm-Tf Trp43 and Hu-FVII Phe278 clash). Unfavorable contacts and repulsive electrostatics account for significant cross-species specificities observed in vertebrate TF/FVIIa, fish to mammals (reviewed, [57]). Together with relative binding energy determinations, such data suggests vertebrate TF and FVIIa have co-evolved to preserve interaction.

Full-length TF increases the enzymatic efficiency of FVIIa in FX activation by more than ~106-fold (summarized, [58]), while the extracellular domain of TF (sTF) shows only a fraction of full-length TF activity [59]. While complete understanding remains elusive, a few studies have proposed additional mechanisms by which TF affects FVIIa efficiency. Association of TF with FVIIa was shown to significantly reorient the active-site groove relative to the membrane surface [60, 61]. Simulation studies characterizing TF/FVIIa interactions with modeled membranes suggest TF transmembrane and linker region interactions with the lipid bilayer optimize active site height and orientation above membranes [62]. Additionally, several studies demonstrated contributions of TF residues to a FX exosite, directly interacting with FX [63–66]. Therefore, in addition to the TF-induced allosteric effects on FVIIa in complex formation, the role of TF in membrane binding, in active site orientation above the membrane, and FX exosite binding are thought to contribute to the 106-fold enhancement of FVIIa activity.

Our analyses of correlated residue motions in TF-bound FVIIa and protein dynamic networks of sTF/FVIIa elucidate how remarkably similar is the long-range allosteric signal propagation from the TF contact point through the SP domain to active site residues in human and lamprey complexes. Moreover, despite low identity between interacting TF and FVIIa sub-domains, residues involved in long-range allosteric signal propagation are highly conserved. The strikingly conservation of FVIIa side-chains constituting optimal pathways of allosteric signaling spans mammals to lamprey. Together, these findings suggest that the critical evolutionary precedent for extrinsic system TF/FVIIa-initiated blood coagulation from a common vertebrate ancestor includes conservation of allosteric TF-mediated affects on FVIIa.

Supplementary Material

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ESSENTIALS.

  • Tissue factor (TF) enhances factor VIIa (FVIIa) activity through structural and dynamic changes.

  • We analyzed conservation of TF-activated FVIIa allosteric networks in extant vertebrate lamprey.

  • Lamprey Tf/FVIIa molecular dynamics show conserved Tf-induced structural/dynamic FVIIa changes.

  • Lamprey Tf activation of FVIIa allosteric networks follows molecular pathways similar to human.

Acknowledgments

This research was supported by National Heart, Lung and Blood Institute of the National Institutes of Health grants HL086766 (M.A. Grant) and HL119322 (W.C. Aird), and Visiting Scientist Awards from Mount Dessert Island Biological Laboratory (M.A. Grant and W.C. Aird).

Footnotes

Addendum

D.L. Beeler, M.A. Grant and W.C. Aird carried out experiments. M.A. Grant conceived and carried out the computational biology, molecular dynamics simulations, and protein dynamic network analyses. W.C. Aird and M.A. Grant analyzed and discussed the data and wrote the manuscript. All authors critically reviewed and revised the manuscript.

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.

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