A thrombophilic allele of clotting Factor VII/VIIa promoting recurrent pulmonary emboli, clinical details, and a structural model of the altered protein: a case report.

BACKGROUND: The clotting or hemostasis system is a meticulously regulated set of enzymatic reactions that occur in the blood and culminate in formation of a fibrin clot. The precisely calibrated signaling system that prevents or initiates clotting originates with the activated Factor Seven (FVIIa) complexed with tissue factor (TF) formed in the endothelium. Here we describe a rare inherited mutation in the FVII gene which is associated with pathological clotting. CASE PRESENTATION: The 52-year-old patient, with European, Cherokee and African American origins, FS was identified as having low FVII (10%) prior to elective surgery for an umbilical hernia. He was given low doses of NovoSeven (therapeutic Factor VIIa) and had no unusual bleeding or clotting during the surgery. In fact, during his entire clinical course he had no unprovoked bleeding. Bleeding instances occurred with hemostatic stresses such as gastritis, kidney calculus, orthopedic surgery, or tooth extraction, and these were handled without factor replacement. On the other hand, FS sustained two unprovoked and life-threatening instances of pulmonary emboli, although he was not treated with NovoSeven at any time close to the events. Since 2020, he has been placed on a DOAC (Direct Oral Anticoagulant, producing Factor Xa inhibition) and has sustained no further clots. POSSIBLE MECHANISM OF (UNAUTHORIZED) FVII ACTIVATION: FS has a congenitally mutated FVII/FVIIa gene, which carries a R315W missense mutation in one allele and a mutated start codon (ATG to ACG) in the other allele, thus rendering the patient effectively homozygous for the missense FVII. Structure based comparisons with known crystal structures of TF-VIIa indicate that the patient's missense mutation is predicted to induce a conformational shift of the C170's loop due to crowding of the bulky tryptophan to a distorted "out" position (Fig. 1). This mobile loop likely forms new interactions with activation loop 3, stabilizing a more active conformation of the FVII and FVIIa protein. The mutant form of FVIIa may be better able to interact with TF, displaying a modified serine protease active site with enhanced activity for downstream substrates such as Factor X. CONCLUSIONS: Factor VII can be considered the gatekeeper of the coagulation system. Here we describe an inherited mutation in which the gatekeeper function is altered. Instead of the expected bleeding manifestations resulting from a clotting factor deficiency, the patient FS suffered clotting episodes. The efficacy of the DOAC in treating and preventing clots in this unusual situation is due to its target site of inhibition (anti-Xa), which lies downstream of the site of action of FVIIa/TF.

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.

BACKGROUND: Tissue factor (TF), a transmembrane glycoprotein, plays a profound role in the formation of the tissue factor-factor VIIa (TF-FVIIa) complex that initiates factor Xa (FXa) generation followed by thrombin activation and clot formation. Previous reports suggest that TF-FVIIa coagulant activity at the cell surface may be affected by various processes, including changes in cholesterol content and posttranslational modifications of TF. Numerous studies were conducted but yielded inconclusive results about the effect of cholesterol on TF expression. OBJECTIVE: The present study aimed to understand how cholesterol affects structural modulations on the tissue factor-factor VIIa-factor Xa ternary complex (TF-FVIIa-FXa). Additionally, we aimed to illustrate the effect of palmitoylation on the Cys245 residue of TF and understand its structural implications on the TF-FVIIa-FXa. METHODS: We set up the following 4 systems in different lipid environments: TF-FVIIa-FXa in POPC:POPS (CS), TF-FVIIa-FXa in POPC:POPS:CHOL (CSL), Palmitoylated TF-FVIIa-FXa in POPC:POPS:CHOL (CSLP), and Palmitoylated TF-FVIIa-FXa in POPC:CHOL (CLP), respectively, and subjected them to molecular dynamics simulation. RESULTS: Hydrogen-bond and contact probability analysis were performed between various important domains of TF-FVIIa-FXa and notable novel interactions: Asn93FVIIa:L-Lys48TF, Arg178FVIIa:H-Asp95FXa:B, Lys20FVIIa:H-Glu193FXa:A, Arg178FVIIa:H-Asp97FXa:B, and Arg153FVIIa:H-Gln135FXa:B have been reported. The protein stability study implies that the CS and CLP systems are thermodynamically less stable than CSL and CSLP systems. CONCLUSION: Analysis of molecular dynamic simulation data suggests that the presence of cholesterol and palmitoylation may contribute to structural rigidity, stability, and compactness of key domains of TF-FVIIa-FXa by augmenting protein-protein and protein-lipid interactions.

Extracellular vesicles from amniotic fluid, milk, saliva, and urine expose complexes of tissue factor and activated factor VII.

BACKGROUND: Tissue factor (TF) is expressed in the adventitia of the vessel wall and on extracellular vesicles (EVs) in body fluids. TF and activated coagulation factor (F) VII(a) together form the so-called extrinsic tenase complex, which initiates coagulation. AIM: We investigated whether EVs in amniotic fluid, milk, saliva, and urine expose functional extrinsic tenase complexes that can trigger coagulation. METHODS: Milk, saliva, and urine were collected from healthy breastfeeding women (n = 6), and amniotic fluid was collected from healthy women undergoing routine amniocentesis (n = 7). EVs were isolated from body fluids by size exclusion chromatography (SEC) and clotting experiments were performed in the presence and absence of antibodies against TF and FVIIa in normal plasma and in FVII-deficient plasma. The ability of body fluids to generate FXa also was determined. RESULTS: Amniotic fluid, milk, saliva, and urine triggered clotting of normal plasma and of FVII-deficient plasma, which was almost completely inhibited by an anti-FVII antibody and to a lesser extent by an anti-TF antibody. Fractionation of body fluids by SEC showed that only the fractions containing EVs triggered clotting in normal plasma and FVII-deficient plasma and generated FXa, which again was almost completely inhibited by an anti-FVII antibody and partially by an anti-TF antibody. CONCLUSION: Here we show that EVs from amniotic fluid, milk, saliva, and urine expose complexes of TF and FVIIa (i.e., extrinsic tenase complexes) that directly activate FX. Based on our present findings we propose that these EVs from normal body fluids provide hemostatic protection.

Structural model of tissue factor (TF) and TF-factor VIIa complex in a lipid membrane: A combined experimental and computational study.

Tissue factor (TF) is a membrane protein involved in blood coagulation. TF initiates a cascade of proteolytic reactions, ultimately leading to the formation of a blood clot. The first reaction consists of the binding of the coagulation factor VII and its conversion to the activated form, FVIIa. Here, we combined experimental, i.e. quartz crystal microbalance with dissipation monitoring and neutron reflectometry, and computational, i.e. molecular dynamics (MD) simulation, methods to derive a complete structural model of TF and TF/FVIIa complex in a lipid bilayer. This model shows that the TF transmembrane domain (TMD), and the flexible linker connecting the TMD to the extracellular domain (ECD), define the location of the ECD on the membrane surface. The average orientation of the ECD relative to the bilayer surface is slightly tilted towards the lipid headgroups, a conformation that we suggest is promoted by phosphatidylserine lipids, and favours the binding of FVIIa. On the other hand, the formation of the TF/FVIIa complex induces minor changes in the TF structure, and reduces the conformational freedom of both TF and FVIIA. Altogether we describe the protein-protein and protein-lipid interactions favouring blood coagulation, but also instrumental to the development of new drugs.

Structure of human factor VIIa-soluble tissue factor with calcium, magnesium and rubidium.

Coagulation factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains and a protease domain. FVIIa binds three Mg2+ ions and four Ca2+ ions in the GLA domain, one Ca2+ ion in the EGF1 domain and one Ca2+ ion in the protease domain. Further, FVIIa contains an Na+ site in the protease domain. Since Na+ and water share the same number of electrons, Na+ sites in proteins are difficult to distinguish from waters in X-ray structures. Here, to verify the Na+ site in FVIIa, the structure of the FVIIa-soluble tissue factor (TF) complex was solved at 1.8 Šresolution containing Mg2+, Ca2+ and Rb+ ions. In this structure, Rb+ replaced two Ca2+ sites in the GLA domain and occupied three non-metal sites in the protease domain. However, Rb+ was not detected at the expected Na+ site. In kinetic experiments, Na+ increased the amidolytic activity of FVIIa towards the synthetic substrate S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) by ∼20-fold; however, in the presence of Ca2+, Na+ had a negligible effect. Ca2+ increased the hydrolytic activity of FVIIa towards S-2288 by ∼60-fold in the absence of Na+ and by ∼82-fold in the presence of Na+. In molecular-dynamics simulations, Na+ stabilized the two Na+-binding loops (the 184-loop and 220-loop) and the TF-binding region spanning residues 163-180. Ca2+ stabilized the Ca2+-binding loop (the 70-loop) and Na+-binding loops but not the TF-binding region. Na+ and Ca2+ together stabilized both the Na+-binding and Ca2+-binding loops and the TF-binding region. Previously, Rb+ has been used to define the Na+ site in thrombin; however, it was unsuccessful in detecting the Na+ site in FVIIa. A conceivable explanation for this observation is provided.

Conformational Plasticity-Rigidity Axis of the Coagulation Factor VII Zymogen Elucidated by Atomistic Simulations of the N-Terminally Truncated Factor VIIa Protease Domain.

The vast majority of coagulation factor VII (FVII), a trypsin-like protease, circulates as the inactive zymogen. Activated FVII (FVIIa) is formed upon proteolytic activation of FVII, where it remains in a zymogen-like state and it is fully activated only when bound to tissue factor (TF). The catalytic domains of trypsin-like proteases adopt strikingly similar structures in their fully active forms. However, the dynamics and structures of the available corresponding zymogens reveal remarkable conformational plasticity of the protease domain prior to activation in many cases. Exactly how ligands and cofactors modulate the conformational dynamics and function of these proteases is not entirely understood. Here, we employ atomistic simulations of FVIIa (and variants hereof, including a TF-independent variant and N-terminally truncated variants) to provide fundamental insights with atomistic resolution into the plasticity-rigidity interplay of the protease domain conformations that appears to govern the functional response to proteolytic and allosteric activation. We argue that these findings are relevant to the FVII zymogen, whose structure has remained elusive despite substantial efforts. Our results shed light on the nature of FVII and demonstrate how conformational dynamics has played a crucial role in the evolutionary adaptation of regulatory mechanisms that were not present in the ancestral trypsin. Exploiting this knowledge could lead to engineering of protease variants for use as next-generation hemostatic therapeutics.

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

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

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.

Apolipoprotein C-III Strongly Correlates with Activated Factor VII-Anti-Thrombin Complex: An Additional Link between Plasma Lipids and Coagulation.

Activated factor VII-anti-thrombin (FVIIa-AT) complex is a potential biomarker of pro-thrombotic diathesis reflecting FVIIa-tissue factor (TF) interaction and has been associated with mortality in patients with coronary artery disease (CAD). Previous data indicated plasma lipids as predictors of FVIIa-AT variability, and plasma lipoproteins as potential stimulators of the coagulation cascade. Our aim was to evaluate the relationships between FVIIa-AT plasma concentration and a broad apolipoprotein profile (including ApoA-I, ApoB, ApoC-III and ApoE). Within the framework of the observational Verona Heart Study, we selected 666 subjects (131 CAD-free and 535 CAD, 75.4% males, mean age: 61.1 ± 10.9 years) not taking anticoagulant drugs and for whom plasma samples were available for both FVIIa-AT assay and a complete lipid profile. Plasma concentration of FVIIa-AT levels significantly and directly correlated with total and high-density lipoprotein cholesterol, triglycerides, ApoA-I, ApoC-III and ApoE levels. ApoC-III showed the strongest correlation (R = 0.235, p = 7.7 × 10-10), confirmed in all the sub-group analyses (males/females and CAD/CAD-free). Only ApoC-III remained associated with FVIIa-AT plasma concentration, even after adjustment for sex, age, CAD diagnosis, body mass index, renal function, smoking status, lipid-lowering therapies and FVIIa levels. The APOC3 gene locus-tagging polymorphism rs964184, previously linked with cardiovascular risk and plasma lipids by genome-wide association studies, was associated with both ApoC-III and FVIIa-AT plasma concentration. Our results indicate a strong association between ApoC-III and FVIIa-AT levels, thereby suggesting that an increased ApoC-III concentration may identify subjects with a pro-thrombotic diathesis characterized by an enhanced TF-FVIIa interaction and activity.

Contribution of allosteric disulfide in the structural regulation of membrane-bound tissue factor-factor VIIa binary complex.

Two distinct populations, active and cryptic forms of tissue factor (TF), reside on the cell surface. Apart from phospholipid contribution, various models have been introduced to explain decryption/encryption of TF. The proposed model, the switching of Cys186-Cys209 bond of TF, has become the matter of controversy. However, it is well accepted that this disulfide has an immense influence upon ligand factor VIIa (FVIIa) for its binding. However, molecular level understanding for this remains unveiled due to lack of detailed structural information. In this regard, we have performed the molecular dynamic study of membrane-bound TF/TF-FVIIa in both the forms (±Cys186-Cys209 allosteric disulfide bond), individually. Dynamic study depicts that disulfide bond provides structural rigidity of TF in both free and ligand-bound forms. This disulfide bond also governs the conformation of FVIIa structure as well as the binding affinity of FVIIa toward TF. Significant differences in lipid-protein interaction profiles of both the forms of TF in the complex were observed. Two forms of TF, oxidized and reduced, have different structural conformation and behave differentially toward its ligand FVIIa. This disulfide bond not only alters the conformation of GLA domain of FVIIa in the vicinity but allosterically regulates the conformation of the distantly located FVIIa protease domain. We suggest that the redox status of the disulfide bond also governs the lipid-mediated interactions with both TF and FVIIa. Communicated by Ramaswamy H. Sarma.

In vitro characterization of MOD-5014, a novel long-acting carboxy-terminal peptide (CTP)-modified activated FVII.

INTRODUCTION: Recombinant FVIIa (rFVIIa) is an effective treatment for haemophilia through frequent administration. However, the short half-life of rFVIIa decreases its prophylactic ability to reduce bleeding. Carboxy-terminal peptide (CTP)-modified FVIIa (MOD-5014) is a long-acting rFVIIa developed for on-demand treatment of haemophilia using either an intravenous or subcutaneous injection with the aim of less frequent administrations, as well as for prophylactic use. AIM: The comprehensive evaluation of the activity MOD-5014 vs commercially available rhFVIIa, as well as their interaction with cofactors and inhibitors. METHODS: The in vitro characterization included clotting activity, affinity by surface plasmon resonance, cleavage of synthetic substrates, thrombin generation (TG) and rotation thromboelastometry. RESULTS: Reduced specific activity was obtained for MOD-5014 compared to rhFVIIa, while both compounds demonstrated comparable affinity to tissue factor (TF). MOD-5014 showed reduced TG when spiked at a similar concentration as rhFVIIa, suggesting that an increased concentration might be needed in a clinical setting to provide initial haemostatic effect. MOD-5014 demonstrated a slightly lower affinity for binding to activated platelets and slightly lower proteolytic activity on the platelet surface, possibly as the fusion of CTP has the potential to sterically hinder binding to both the platelet membrane and to protein substrates. Both compounds showed a similar dose-dependent stimulatory effect on clot formation, and both showed a similar deactivation pattern following incubation with TF pathway inhibitor (TFPI), antithrombin and heparin. CONCLUSION: The comparable in vitro activity of MOD-5014 and rhFVIIa paves the way for in vivo pharmacology evaluations of MOD-5014 in preparation for clinical studies.

Factor Xa and VIIa inhibition by tissue factor pathway inhibitor is prevented by a monoclonal antibody to its Kunitz-1 domain.

Essentials Activated FVII (FVIIa) and FX (FXa) are inhibited by tissue factor pathway inhibitor (TFPI). A monoclonal antibody, mAb2F22, was raised against the N-terminal fragment of TFPI (1-79). mAb2F22 bound exclusively to the K1 domain of TFPI (KD ∼1 nm) and not to the K2 domain. mAb2F22 interfered with inhibition of both FVIIa and FXa activities and restored clot formation. SUMMARY: Background Initiation of coagulation is induced by binding of activated factor VII (FVIIa) to tissue factor (TF) and activation of factor X (FX) in a process regulated by tissue factor pathway inhibitor (TFPI). TFPI contains three Kunitz-type protease inhibitor domains (K1-K3), of which K1 and K2 block the active sites of FVIIa and FXa, respectively. Objective To produce a monoclonal antibody (mAb) directed towards K1, to characterize the binding epitope, and to study its effect on TFPI inhibition. Methods A monoclonal antibody, mAb2F22, was raised against the N-terminal TFPI(1-79) fragment. Binding data were obtained by surface plasmon resonance analysis. The Fab-fragment of mAb2F22, Fab2F22, was expressed and the structure of its complex with TFPI(1-79) determined by X-ray crystallography. Effects of mAb2F22 on TFPI inhibition were measured in buffer- and plasma-based systems. Results mAb2F22 bound exclusively to K1 of TFPI (KD ~1 nm) and not to K2. The crystal structure of Fab2F22/TFPI (1-79) mapped an epitope on K1 including seven residues upstream of the domain. TFPI inhibition of TF/FVIIa amidolytic activity was neutralized by mAb2F22, although the binding epitope on K1 did not include the P1 residue. Binding of mAb2F22 to K1 blocked TFPI inhibition of the FXa amidolytic activity and normalized hemostasis in hemophilia human A-like plasma and whole blood. Conclusion mAb2F22 blocked TFPI inhibition of both FVIIa and FXa activities and mapped a FXa exosite for binding to K1. It reversed TFPI feedback inhibition of TF/FVIIa-induced coagulation and restored clot formation in FVIII-neutralized human plasma and blood.

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

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. 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 humans and extant vertebrate jawless fish (lampreys), where blood coagulation emerged more than 500 million years ago, is unknown and of considerable interest. Objective To model the 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.

Suppressive Role of Tissue Factor Pathway Inhibitor-α in Platelet-Dependent Fibrin Formation under Flow Is Restricted to Low Procoagulant Strength.

Tissue factor pathway inhibitor-alpha (TFPI-α) is a Kunitz-type serine protease inhibitor, which suppresses coagulation by inhibiting the tissue factor (TF)/factor VIIa complex as well as factor Xa. In static plasma-phospholipid systems, TFPI-α thus suppresses both factor Xa and thrombin generation. In this article, we used a microfluidics approach to investigate how TFPI-α regulates fibrin clot formation in platelet thrombi at low wall shear rate. We therefore hypothesized that the anticoagulant effect of TFPI-α in plasma is a function of the local procoagulant strength-defined as the magnitude of thrombin generation under flow, due to local activities of TF/factor VIIa and factor Xa. To test this hypothesis, we modulated local coagulation by microspot coating of flow channels with 0 to 100 pM TF/collagen, or by using blood from patients with haemophilia A or B. For blood or plasma from healthy subjects, blocking of TFPI-α enhanced fibrin formation, extending from a platelet thrombus, under flow only at <2 pM coated TF. This enhancement was paralleled by an increased thrombin generation. For mouse plasma, genetic deficiency in TFPI enhanced fibrin formation under flow also at 0 pM TF microspots. On the other hand, using blood from haemophilia A or B patients, TFPI-α antagonism markedly enhanced fibrin formation at microspots with up to 100 pM coated TF. We conclude that, under flow, TFPI-α is capable to antagonize fibrin formation in a manner dependent on and restricted by local TF/factor VIIa and factor Xa activities.

Passivating Injured Endothelium with Kinexins in Thrombolytic Therapy.

Without conjunctive administration of an anticoagulant, endothelial injury-induced thrombosis is resistant to thrombolysis and prone to re-thrombosis. We hypothesized that co-delivery of recombinant tissue plasminogen activator (rtPA) with annexin V-containing anticoagulants that specifically target the injured endothelium may passivate the thrombogenic elements of the vascular injury site and enhance rtPA-induced thrombolysis. In this study, the effects of conjunctive administration of Kinexins (Kunitz inhibitor-annexin V fusion proteins) with rtPA on thrombolysis were determined in vitro and in vivo. Thromboelastometry showed that both TAP-A (tick anticoagulant peptide-annexin V fusion protein; an inhibitor of factor Xa [FXa] and prothrombinase) and A-6L15 (annexin V-6L15 fusion protein; an inhibitor of tissue factor/FVIIa) exerted concentration-dependent (10-100 nM) effects on clot formation, with TAP-A being several folds more potent than A-6L15 in whole blood. Combination of TAP-A or A-6L15 with rtPA (1 µg/mL) led to decrease in lysis index, suggesting conjunctive enhancement of thrombolysis by combined use of rtPA with TAP-A or A-6L15. In a rat cremaster muscle preparation subjected to photochemical injury, conjunctive administration of rtPA and TAP-A significantly restored tissue perfusion to 56%, which is approximately two fold of that by rtPA or TAP-A alone. Near-infrared fluorescence images demonstrated local retention of a fluorescent A-6L15-S288 at the injury site, suggesting a targeting effect of the fusion protein. Pharmacokinetic analysis showed that 123I-labelled TAP-A and A-6L15 had initial distribution half-lives (T1/2α) of approximately 6 minutes and elimination half-lives (T1/2ß) of approximately 2.3 hours. In conclusion, Kinexins were potentially useful adjunctive agents with rtPA thrombolytic therapy especially for thrombosis induced by endothelial injury.

Structural modulation of factor VIIa by full-length tissue factor (TF1-263): implication of novel interactions between EGF2 domain and TF.

Tissue factor (TF)-mediated factor VII (FVII) activation and a subsequent proteolytic TF-FVIIa binary complex formation is the key step initiating the coagulation cascade, with implications in various homeostatic and pathologic scenarios. TF binding allosterically modifies zymogen-like free FVIIa to its highly catalytically active form. As a result of unresolved crystal structure of the full-length TF1-263-FVIIa binary complex and free FVIIa, allosteric alterations in FVIIa following its binding to full-length TF and the consequences of these on function are not entirely clear. The present study aims to map and identify structural alterations in FVIIa and TF resulting from full-length TF binding to FVIIa and the key events responsible for enhanced FVIIa activity in coagulation. We constructed the full-length TF1-263-FVIIa membrane bound complex using computational modeling and subjected it to molecular dynamics (MD) simulations. MD simulations showed that TF alters the structure of each domain of FVIIa and these combined alterations contribute to enhanced TF-FVIIa activity. Detailed, domain-wise investigation revealed several new non-covalent interactions between TF and FVIIa that were not found in the truncated soluble TF-FVIIa crystal structure. The structural modulation of each FVIIa domain imparted by TF indicated that both inter and intra-domain communication is crucial for allosteric modulation of FVIIa. Our results suggest that these newly formed interactions can provide additional stability to the protease domain and regulate its activity profile by governing catalytic triad (CT) orientation and localization. The unexplored newly formed interactions between EGF2 and TF provides a possible explanation for TF-induced allosteric activation of FVIIa.

Identification of the integrin-binding site on coagulation factor VIIa required for proangiogenic PAR2 signaling.

The tissue factor (TF) pathway serves both hemostasis and cell signaling, but how cells control these divergent functions of TF remains incompletely understood. TF is the receptor and scaffold of coagulation proteases cleaving protease-activated receptor 2 (PAR2) that plays pivotal roles in angiogenesis and tumor development. Here we demonstrate that coagulation factor VIIa (FVIIa) elicits TF cytoplasmic domain-dependent proangiogenic cell signaling independent of the alternative PAR2 activator matriptase. We identify a Lys-Gly-Glu (KGE) integrin-binding motif in the FVIIa protease domain that is required for association of the TF-FVIIa complex with the active conformer of integrin ß1. A point mutation in this motif markedly reduces TF-FVIIa association with integrins, attenuates integrin translocation into early endosomes, and reduces delayed mitogen-activated protein kinase phosphorylation required for the induction of proangiogenic cytokines. Pharmacologic or genetic blockade of the small GTPase ADP-ribosylation factor 6 (arf6) that regulates integrin trafficking increases availability of TF-FVIIa with procoagulant activity on the cell surface, while inhibiting TF-FVIIa signaling that leads to proangiogenic cytokine expression and tumor cell migration. These experiments delineate the structural basis for the crosstalk of the TF-FVIIa complex with integrin trafficking and suggest a crucial role for endosomal PAR2 signaling in pathways of tissue repair and tumor biology.

Engineering of a membrane-triggered activity switch in coagulation factor VIIa.

Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa.

Multiplexed silicon photonic sensor arrays enable facile characterization of coagulation protein binding to nanodiscs with variable lipid content.

Interactions of soluble proteins with the cell membrane are critical within the blood coagulation cascade. Of particular interest are the interactions of γ-carboxyglutamic acid-rich domain-containing clotting proteins with lipids. Variability among conventional analytical methods presents challenges for comparing clotting protein-lipid interactions. Most previous studies have investigated only a single clotting protein and lipid composition and have yielded widely different binding constants. Herein, we demonstrate that a combination of lipid bilayer nanodiscs and a multiplexed silicon photonic analysis technology enables high-throughput probing of many protein-lipid interactions among blood-clotting proteins. This approach allowed direct comparison of the binding constants of prothrombin, factor X, activated factor VII, and activated protein C to seven different binary lipid compositions. In a single experiment, the binding constants of one protein interacting with all lipid compositions were simultaneously determined. A simple surface regeneration then facilitated similar binding measurements for three other coagulation proteins. As expected, our results indicated that all proteins exhibit tighter binding (lower Kd ) as the proportion of anionic lipid increases. Interestingly, at high proportions of phosphatidylserine, the Kd values of all four proteins began to converge. We also found that although koff values for all four proteins followed trends similar to those observed for the Kd values, the variation among the proteins was much lower, indicating that much of the variation came from the kinetic binding (kon) of the proteins. These findings indicate that the combination of silicon photonic microring resonator arrays and nanodiscs enables rapid interrogation of biomolecular binding interactions at model cell membrane interfaces.

Molecular determinants involved in differential behaviour between soluble tissue factor and full-length tissue factor towards factor VIIa.

During blood-coagulation, the transmembrane protein tissue factor (TF) binds to its ligand, factor (F)VII, activating and allosterically modifying it to form a mature active binary complex (TF-FVIIa). Although the extracellular domain of TF (sTF) can bind to FVII, it fails to activate it. Binding of TF with FVIIa only partially enhances FVIIa proteolytic activity. Our previous kinetic study revealed that sTF has a lower binding capacity with FVIIa compared to membrane bound full-length (fl)TF. The reason behind this incapability of FVII activation and reduced catalytic activity remains unexplored due to the lack of an flTF crystal structure. Here we employed a comparative dynamic study between sTF-FVIIa in solution and flTF-FVIIa in a membrane system to give probable explanations for the differential behaviour of these complexes. Based on potential of mean force and interaction energy calculations, the binding affinities between sTF and FVIIa are weaker than those of the flTF-FVIIa complex. We further observed domain-wise less stability, reduced height, and thus less inter and intra-domain interaction between the sTF and FVIIa complexes. We detected higher fluctuation among the inter-atomic distances of the catalytic triad (CT) residues in sTF-FVIIa over the flTF-FVIIa complex. The flTF-FVIIa complex forms two major interactions between EGF2 and TF. We showed the enhanced activity of the flTF-FVIIa complex over the sTF-FVIIa complex, which is guided by mainly two interactions between EGF2 and TF. Due to the lack of these interactions, sTF-FVIIa somehow forms a less stable binary complex and could not react upon binding its substrates (FIX, FX). Our study, for the first time, provides a possible explanation of the distinct behaviour of the two forms of TF (sTF and flTF) towards its only ligand FVII/FVIIa.