Molecular dynamics simulations and functional characterization of the interactions of the PAR2 ectodomain with factor VIIa

Signaling of the tissue factor‐FVIIa complex regulates angiogenesis, tumor growth, and inflammation. TF‐FVIIa triggers cell signaling events by cleavage of protease activated receptor (PAR2) at the Arg36‐Ser37 scissile bond. The recognition of PAR2 by the FVIIa protease domain is poorly understood. We perform molecular modeling and dynamics simulations to derive the PAR2‐FVIIa interactions. Docking of the PAR2 Arg36‐Ser37 scissile bond to the S1 site and subsequent molecular dynamics leads to interactions of the PAR2 ectodomain with P and P′ sites of the FVIIa catalytic cleft as well as to electrostatic interactions between a stably folded region of PAR2 and a cluster of basic residues remote from the catalytic cleft of FVIIa. To address the functional significance of this interaction for PAR2 cleavage, we employed two antibodies with epitopes previously mapped to this cluster of basic residues. Although these antibodies do not block the catalytic cleft, both antibodies completely abrogated PAR2 activation by TF‐FVIIa. Our simulations indicate a conformation of the PAR2 ectodomain that limits the cleavage site to no more than 33 Å from its membrane proximal residue. Since the active site of FVIIa in the TF‐FVIIa complex is ～75 Å above the membrane, cleavage of the folded conformation of PAR2 would require tilting of the TF‐FVIIa complex toward the membrane, indicating that additional cellular factors may be required to properly align the scissile bond of PAR2 with TF‐FVIIa. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Engineering of Substrate Selectivity for Tissue Factor·Factor VIIa Complex Signaling through Protease-activated Receptor 2

The complex of factor VIIa (FVIIa) with tissue factor (TF) triggers coagulation by recognizing its macromolecular substrate factors IX (FIX) and X (FX) predominantly through extended exosite interactions. In addition, TF mediates unique cell-signaling properties in cancer, angiogenesis, and inflammation that involve proteolytic cleavage of protease-activated receptor 2 (PAR2). PAR2 is cleaved by FVIIa in the binary TF·FVIIa complex and by FXa in the ternary TF·FVIIa·FXa complex, but physiological roles of these signaling complexes are incompletely understood. In a screen of FVIIa protease domain mutants, three variants (Q40A, Q143N, and T151S) activated macromolecular coagulation substrates and supported signaling of the ternary TF·FVIIa-Xa complex normally but were severely impaired in binary TF·FVIIa·PAR2 signaling. The residues identified were located in the model-predicted S2′ pocket of FVIIa, and complementary PAR2 P2′ Leu-38 replacements demonstrated that the P2′ side chain was indeed crucial for PAR2 cleavage by TF·FVIIa. In addition, PAR2 was activated more efficiently by FVIIa T99Y, consistent with further contributions from the S2 subsite. The P2 residue preference of FVIIa and FXa predicted additional PAR2 mutants that were efficiently activated by TF·FVIIa but resistant to cleavage by the alternative PAR2 activator FXa. Thus, contrary to the paradigm of exosite-assisted cleavage of PAR1 by thrombin, the cofactor-associated protease FVIIa recognizes PAR2 predominantly by catalytic cleft interactions. Furthermore, the delineated molecular details of this substrate interaction enabled protein engineering of protease-selective PAR2 receptors that will aid further studies to dissect the roles of TF signaling complexes in vivo.     

The endothelial protein C receptor supports tissue factor ternary coagulation initiation complex signaling through protease-activated receptors

Protease-activated receptor (PAR) signaling is closely linked to the cellular activation of the pro- and anticoagulant pathways. The endothelial protein C receptor (EPCR) is crucial for signaling by activated protein C through PAR1, but EPCR may have additional roles by interacting with the 4-carboxyglutamic acid domains of procoagulant coagulation factors VII (FVII) and X (FX). Here we show that soluble EPCR regulates the interaction of FX with human or mouse tissue factor (TF)-FVIIa complexes. Mutagenesis of the FVIIa 4-carboxyglutamic acid domain and dose titrations with FX showed that EPCR interacted primarily with FX to attenuate FX activation in lipid-free assay systems. In human cell models of TF signaling, antibody inhibition of EPCR selectively blocked PAR activation by the ternary TF-FVIIa-FXa complex but not by the non-coagulant TF-FVIIa binary complex. Heterologous expression of EPCR promoted PAR1 and PAR2 cleavage by FXa in the ternary complex but did not alter PAR2 cleavage by TF-FVIIa. In murine smooth muscle cells that constitutively express EPCR and TF, thrombin and FVIIa/FX but not FVIIa alone induced PAR1-dependent signaling. Although thrombin signaling was unchanged, cells with genetically reduced levels of EPCR no longer showed a signaling response to the ternary complex. These results demonstrate that EPCR interacts with the ternary TF coagulation initiation complex to enable PAR signaling and suggest that EPCR may play a role in regulating the biology of TF-expressing extravascular and vessel wall cells that are exposed to limited concentrations of FVIIa and FX provided by ectopic synthesis or vascular leakage.     

Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity

The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215–217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215–217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity.     

Protease-activated receptor-2 regulates vascular endothelial growth factor expression in MDA-MB-231 cells via MAPK pathways

Protease-activated receptor 2 (PAR2) is a G-protein coupled receptor that is cleaved and activated by serine proteases including the coagulation protease factor VIIa (FVIIa). There is evidence that PAR2 function contributes to angiogenesis, but the mechanisms involved are poorly defined. Here we show that PAR2 activation in human breast cancer cells leads to the upregulation of vascular endothelial growth factor (VEGF). Activation of PAR2 with agonist peptide (AP), trypsin or FVIIa results in a robust increase of VEGF message and protein. Incubation of cells with PAR1-AP, PAR3-AP, PAR4-AP, or thrombin has only a modest effect on VEGF production. Cleavage blocking antibodies show that FVIIa-mediated VEGF production is PAR2 mediated. Mitogen-activated protein kinase (MAPK) pathway inhibitors U0126 and SB203580 inhibit PAR2-mediated VEGF production. Incubation of cells with PAR2-AP leads to significant extracellular regulated kinase1/2 (ERK1/2) and p38 MAPK phosphorylation and activation. Collectively, these data suggest that PAR2 signaling through MAPK pathways leads to the production of proangiogenic VEGF in breast cancer cells.     

Tissue factor mediates inflammation

The role of tissue factor (TF) in inflammation is mediated by blood coagulation. TF initiates the extrinsic blood coagulation that proceeds as an extracellular signaling cascade by a series of active serine proteases: FVIIa, FXa, and thrombin (FIIa) for fibrin clot production in the presence of phospholipids and Ca2+. TF upregulation resulting from its enhanced exposure to clotting factor FVII/FVIIa often manifests not only hypercoagulable but also inflammatory state. Coagulant mediators (FVIIa, FXa, and FIIa) are proinflammatory, which are largely transmitted by protease-activated receptors (PAR) to elicit inflammation including the expression of tissue necrosis factor, interleukins, adhesion molecules (MCP-1, ICAM-1, VCAM-1, selectins, etc.), and growth factors (VEGF, PDGF, bFGF, etc.). In addition, fibrin, and its fragments are also able to promote inflammation. In the event of TF hypercoagulability accompanied by the elevations in clotting signals including fibrin overproduction, the inflammatory consequence could be enormous. Antagonism to coagulation-dependent inflammation includes (1) TF downregulation, (2) anti-coagulation, and (3) PAR blockade. TF downregulation and anti-coagulation prevent and limit the proceeding of coagulation cascade in the generation of proinflammatory coagulant signals, while PAR antagonists block the transmission of such signals. These approaches are of significance in interrupting the coagulation-inflammation cycle in contribution to not only anti-inflammation but also anti-thrombosis for cardioprotection.     

Cortactin activation by FVIIa/tissue factor and PAR2 promotes endothelial cell migration

Cellular migration is a complex process that requires the polymerization of actin filaments to drive cellular extension. Smooth muscle and cancer cell migration has been shown to be affected by coagulation factors, notably the factor VII (FVIIa) and tissue factor (TF) complex. The present studies delineated mediators involved with the process of FVIIa/TF-induced cell migration and utilized a simple, precise, and reproducible, migration assay. Both FVIIa and protease-activated receptor-2 (PAR2)-activating peptide, SLIGRL, increased the migration rate of porcine cerebral microvascular endothelial cells (pCMVECs) overexpressing human TF. Ras homolog gene family member A (RhoA) and cortactin were upregulated during the process; expression of HIF, actin polymerization nuclear diaphanous-related formin-1 and -2 (Dia1, and Dia2) were unaffected. Gene silencing by shRNA to PAR2, RhoA, and cortactin attenuated this gene upregulation and migration induced by FVIIa/TF. Utilizing immunocellular localization, we demonstrate that during FVIIa/TF and PAR2 activation, cortactin molecules translocate from the cytoplasm to the cell periphery and assist in lamellipodia formation of pCMVECs. Overall, we demonstrate a novel regulation and role for cortactin in FVIIa/TF-mediated endothelial cell migration that occurs through a PAR2 and RhoA dependent mechanism.     

Crystal Structure of Active Site-Inhibited Human Coagulation Factor VIIa (des-Gla)

Factor VIIa (FVIIa) is a crucial haemostatic protease consisting of four distinct domains termed the Gla, epidermal growth factor-1 (EGF-1), EGF-2, and protease domains (from N- to C-terminus). The crystal structure of human FVIIa inhibited at the active site with 1, 5-dansyl-Glu-Gly-Arg-chloromethyl ketone and lacking the Gla domain has been solved to a resolution of 2.28 A. The EGF-2 and protease domains were well resolved, whereas no electron density for the EGF-1 domain was observed, suggesting a flexible arrangement or disorder within the crystal. Superposition of the protease domain of the present structure with that previously resolved in the tissue factor (TF)/FVIIai complex revealed that although overall the domain structures are similar, the EGF-2 domain is rotated by 7.5 degrees relative to the protease domain on binding TF. A single cleavage in the protease domain was found, between Arg315 and Lys316 (chymotrypsin numbering 170C-170D) in a FVII-specific insertion loop: this cleavage appeared to be essential for crystallisation. Insertion of the heavy chain N-terminal Ile153 is essentially identical in the two structures, as is the geometry of the active site residues and the inhibitor C-terminal arginine residue. Some differences are seen in the cleaved loop, but changes in TF-contact residues are generally minor. This structure supports the hypothesis that TF binding enables spatial domain arrangements in the flexible FVIIa molecule necessary for procoagulant function and furthermore that active site occupancy induces FVIIa active conformation via N-terminal insertion.     

A selective, slow binding inhibitor of factor VIIa binds to a nonstandard active site conformation and attenuates thrombus formation in vivo

The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.     

Allosteric activation of coagulation factor VIIa visualized by hydrogen exchange

Coagulation factor VIIa (FVIIa) is a serine protease that, after binding to tissue factor (TF), plays a pivotal role in the initiation of blood coagulation. We used hydrogen exchange monitored by mass spectrometry to visualize the details of FVIIa activation by comparing the exchange kinetics of distinct molecular states, namely zymogen FVII, endoproteolytically cleaved FVIIa, TF-bound zymogen FVII, TF-bound FVIIa, and FVIIa in complex with an active site inhibitor. The hydrogen exchange kinetics of zymogen FVII and FVIIa are identical indicating highly similar solution structures. However, upon tissue factor binding, FVIIa undergoes dramatic structural stabilization as indicated by decreased exchange rates localized throughout the protease domain and in distant parts of the light chain, spanning across 50A and revealing a concerted interplay between functional sites in FVIIa. The results provide novel insights into the cofactor-induced activation of this important protease and reveal the potential for allosteric regulation in the trypsin family of proteases.     

Protease-activated receptor 2-dependent phosphorylation of the tissue factor cytoplasmic domain

Tissue factor (TF) is the physiological activator of the coagulation cascade that plays pathophysiological roles in metastasis, angiogenesis, and inflammation. Downstream in coagulation, thrombin is the central protease that signals through G protein-coupled, protease-activated receptors (PARs). However, the TF-VIIa-Xa complex upstream in coagulation also activates PAR1 and 2. Here, we address the question of whether signaling of the TF initiation complex is a relevant pathway that leads to TF cytoplasmic domain phosphorylation. In heterologous expression systems and primary endothelial cells, we demonstrate that the ternary TF-VIIa-Xa complex induces TF phosphorylation specifically by activating PAR2 but not through PAR1 signaling. In addition, TF cytoplasmic domain phosphorylation is induced only by TF-dependent signaling but not by other coagulation factors in endothelial cells. Phosphorylation of the Pro-directed kinase target site Ser258 is dependent on prior phosphorylation of Ser253 by protein kinase C (PKC) alpha. TF phosphorylation is somewhat delayed and coincides with sustained PKCalpha activation downstream of PAR2 but not PAR1 signaling. Phosphatidylcholine-dependent phospholipase C is the major pathway that leads to prolonged PKCalpha recruitment downstream of PAR2. Thus, PAR2 signaling specifically phosphorylates TF in a receptor cross-talk that distinguishes upstream from downstream coagulation protease signaling.     

Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor

The protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn2+ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn2+ bind with high affinity to FVIIa outside the N-terminal gamma-carboxyglutamic acid (Gla) domain. Binding of Zn2+ to FVIIa, which was influenced by the presence of Ca2+, resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn2+ inhibition. A search for putative Zn2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn2+ binding sites in the Glu210-Glu220 Ca2+ binding loop adjacent to the so-called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn2+-mediated allosteric down-regulation of FVIIa's activity.     

Allostery in Coagulation Factor VIIa Revealed by Ensemble Refinement of Crystallographic Structures

A critical step in injury-induced initiation of blood coagulation is the formation of the complex between the trypsin-like protease coagulation factor VIIa (FVIIa) and its cofactor tissue factor (TF), which converts FVIIa from an intrinsically poor enzyme to an active protease capable of activating zymogens of downstream coagulation proteases. Unlike its constitutively active ancestor trypsin, FVIIa is allosterically activated (by TF). Here, ensemble refinement of crystallographic structures, which uses multiple copies of the entire structure as a means of representing structural flexibility, is applied to explore the impacts of inhibitor binding to trypsin and FVIIa, as well as cofactor binding to FVIIa. To assess the conformational flexibility and its role in allosteric pathways in these proteases, main-chain hydrogen bond networks are analyzed by calculating the hydrogen-bond propensity. Mapping pairwise propensity differences between relevant structures shows that binding of the inhibitor benzamidine to trypsin has a minor influence on the protease flexibility. For FVIIa, in contrast, the protease domain is “locked” into the catalytically competent trypsin-like configuration upon benzamidine binding as indicated by the stabilization of key structural features: the nonprime binding cleft and the oxyanion hole are stabilized, and the effect propagates from the active site region to the calcium-binding site and to the vicinity of the disulphide bridge connecting with the light chain. TF binding to FVIIa furthermore results in stabilization of the 170 loop, which in turn propagates an allosteric signal from the TF-binding region to the active site. Analyses of disulphide bridge energy and flexibility reflect the striking stability difference between the unregulated enzyme and the allosterically activated form after inhibitor or cofactor binding. The ensemble refinement analyses show directly, for the first time to our knowledge, whole-domain structural footprints of TF-induced allosteric networks present in x-ray crystallographic structures of FVIIa, which previously only have been hypothesized or indirectly inferred.     

Factor VIIa/tissue factor generates a form of factor V with unchanged specific activity, resistance to activation by thrombin, and increased sensitivity to activated protein C

Factor VIIa, in complex with tissue factor (TF), is the serine protease responsible for initiating the clotting cascade. This enzyme complex (TF/VIIa) has extremely restricted substrate specificity, recognizing only three previously known macromolecular substrates (serine protease zymogens, factors VII, IX, and X). In this study, we found that TF/VIIa was able to cleave multiple peptide bonds in the coagulation cofactor, factor V. SDS-PAGE analysis and sequencing indicated the factor V was cleaved at Arg679, Arg709, Arg1018, and Arg1192, resulting in a molecule with a truncated heavy chain and an extended light chain. This product (FVTF/VIIa) had essentially unchanged activity in clotting assays when compared to the starting material. TF reconstituted into phosphatidylcholine vesicles was ineffective as a cofactor for the factor VIIa cleavage of factor V. However, incorporation of phosphatidylethanolamine in the vesicles had little effect over the presence of 20% phosphatidylserine. FVTF/VIIa was as sensitive to inactivation by activated protein C (APC) as thrombin activated factor V as measured in clotting assays or by the appearance of the expected heavy chain cleavage products. The FVTF/VIIa could be further cleaved by thrombin to release the normal light chain, albeit at a significantly slower rate than native factor V, to yield a fully functional product. These studies thus reveal an additional substrate for the TF/VIIa complex. They also indicate a new potential regulatory pathway of the coagulation cascade, i.e., the production of a form of factor V that can be destroyed by APC without the requirement for full activation of the cofactor precursor.     

Manipulation of EphB2 regulatory motifs and SH2 binding sites switches MAPK signaling and biological activity

Signaling by the Eph family of receptor tyrosine kinases (RTKs) is complex, because they can interact with a variety of intracellular targets, and can potentially induce distinct responses in different cell types. In NG108 neuronal cells, activated EphB2 recruits p120RasGAP, in a fashion that is associated with down-regulation of the Ras-Erk mitogen-activated kinase (MAPK) pathway and neurite retraction. To pursue the role of the Ras-MAPK pathway in EphB2-mediated growth cone collapse, and to explore the biochemical and biological functions of Eph receptors, we sought to re-engineer the signaling properties of EphB2 by manipulating its regulatory motifs and SH2 binding sites. An EphB2 mutant that retained juxtamembrane (JM) RasGAP binding sites but incorporated a Grb2 binding motif at an alternate RasGAP binding site within the kinase domain had little effect on basal Erk MAPK activation. In contrast, elimination of all RasGAP binding sites, accompanied by the addition of a Grb2 binding site within the kinase domain, led to an increase in phospho-Erk levels in NG108 cells following ephrin-B1 stimulation. Functional assays indicated a correlation between neurite retraction and the ability of the EphB2 mutants to down-regulate Ras-Erk MAPK signaling. These data suggest that EphB2 can be designed to repress, stabilize, or activate the Ras-Erk MAPK pathway by the manipulation of RasGAP and Grb2 SH2 domain binding sites and support the notion that Erk MAPK regulation plays a significant role in axon guidance. The behavior of EphB2 variants with mutations in the JM region and kinase domains suggests an intricate pattern of regulation and target recognition by Eph receptors.     

Gene Targeting of Tissue Factor, Factor X, and Factor VII in Mice: Their Involvement in Embryonic Development

Inactivation of specific genes in mammals by gene targeting has accelerated our ability to determine gene function. Nearly all genes involved in the blood coagulation system have been knocked out in mice. Tissue factor (TF) is the main initiator of the coagulation system and functions as a cell surface receptor for coagulation factor VII (FVII). Knockout studies have shown that TF deficiency results in lethality around embryonic day (E) 8.5-10.5. The results suggest a role for TF in embryonic blood vessel development and maintenance of vascular integrity in the yolk sac. In addition, TF may be involved in the maintenance of the placental labyrinth. Factor X (FX) deficiency causes partial embryonic lethality between E11.5-12.5.FX^–/– mice that were born died from fatal neonatal bleeding. In contrast, FVII deficiency is not embryonic lethal, but FVII^–/– neonates died from hemorrhage within the first days after birth. The various lethal phenotypes of deficiencies of the different coagulation factors suggest involvement in processes beyond hemostasis. Both TF/FVIIa and FXa can trigger intracellular signaling events in certain cell types. Signaling by coagulation proteases and protease activated receptors (PARs) may have important roles in embryonic development.     

Transition state analysis of the complex between coagulation factor VIIa and tissue factor: suggesting a sequential domain-binding pathway

Injury of a blood vessel exposes membrane-bound tissue factor (TF) to blood, which allows binding of coagulation factor VIIa (FVIIa). This initiation of the coagulation cascade is dictated by a specific multi-domain interaction between FVIIa and TF. To examine the energies involved in the transition state of the FVIIa:TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with a smaller cysteine residue. Determination of Phi values in each of the positions using surface plasmon resonance measurements enabled us to characterize the transition state complex between the resulting sTF variants and FVIIa. We found that the interactions in the transition state seemed to be most pronounced between the protease domain of FVIIa and sTF while detailed specific interactions between the Gla-domain and sTF were missing. Thus, the transition state energy data indicate a sequential binding event between these two macromolecules.     

Novel interactions of large P3 moiety and small P4 moiety in the binding of the peptide mimetic factor VIIa inhibitor

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is seen as a promising target for developing new anticoagulant drugs. A novel peptide mimetic factor VIIa inhibitor, ethylsulfonamide-d-biphenylalanine-Gln-p-aminobenzamidine, shows 100-fold selectivity against thrombin in spite of its large P3 moiety, unlike previously reported FVIIa/TF selective inhibitors. X-ray crystal structure analysis reveals that the large P3 moiety, d-biphenylalanine, and the small P4 moiety, ethylsulfonamide, make novel interactions with the 170-loop and Lys192 of FVIIa/TF, respectively, accompanying ligand-induced conformational changes of the 170-loop, Gln217, and Lys192. Structural comparisons of FVIIa with thrombin and amino acid sequence comparisons among coagulation serine proteases suggest that these interactions play an important role in achieving selective inhibition for FVIIa/TF.