Mutations of TFPI-binding exosites on factor VII cause bleeding phenotypes in factor VII deficiency.

Tissue factor (TF) pathway inhibitor (TFPI) is a Kunitz-type anticoagulation protein that inhibits activated factor VII (FVIIa)/TF complex. Incidentally, many different F7 gene variants, including TFPI-binding exosite mutations, have been reported in patients with congenital FVII deficiency and clinical bleeding variabilities. Here, TFPI-binding exosites (R147 and K192) on FVII zymogen were selectively disrupted to understand their roles in the pathogenesis of bleeding phenotypes. Expression of recombinant FVII variants (R147A, K192A, and R147A/K192A) demonstrated markedly reduced secretion of FVII owing to intracellular retention in the endoplasmic reticulum, as demonstrated by upregulation of the unfolded protein response genes in all FVII variants. FVII variants showed a similar FVII activation pattern and FVIIa amidolytic activity than FVII wild-type (WT). In contrast to FVII activation, R147A and K192A showed a 90% reduction in FX activation relative to WT, whereas the R147A/K192A variant demonstrated a 99% decrease in FX activation. The clotting time was markedly prolonged with R147A and K192A than WT, and no FVII coagulant activity was detected in R147A/K192A. In addition, the thrombin generation assay revealed a significant prolongation of lag time in all FVII variants. Our study explains how mutations of TFPI-binding exosites of FVII can lead to bleeding phenotypes in individuals carrying these aberrancies.

Activation of ß1 integrins and caveolin-1 by TF/FVIIa promotes IGF-1R signaling and cell survival.

The tissue factor/coagulation factor VIIa (TF/FVIIa) complex induces transactivation of the IGF-1 receptor (IGF-1R) in a number of different cell types. The mechanism is largely unknown. The transactivation leads to protection from apoptosis and nuclear translocation of the IGF-1R. The aim of this study was to clarify the signaling pathway between TF and IGF-1R after FVIIa treatment with PC3 and DU145 prostate or MDA-MB-231 breast cancer cells as model systems. Protein interactions, levels, and phosphorylations were assessed by proximity ligation assay or flow cytometry in intact cells and by western blot on cell lysates. The transactivation of the IGF-1R was found dependent on TF/FVIIa-induced activation of ß1-integrins. A series of experiments led to the conclusion that the caveolae protein caveolin-1 prevented IGF-1R activation in resting cells via its scaffolding domain. TF/FVIIa/ß1-integrins terminated this inhibition by activation of Src family kinases and subsequent phosphorylation of caveolin-1 on tyrosine 14. This phosphorylation was not seen after treatment with PAR1 or PAR2 agonists. Consequently, the protective effect of FVIIa against apoptosis induced by the death receptor agonist TRAIL and the de novo synthesis of cyclin D1 induced by nuclear IGF-1R accumulation were both significantly reduced by down-regulation of ß1-integrins or overexpression of the caveolin-1 scaffolding domain. In conclusion, we present a plausible mechanism for the interplay between TF and IGF-1R involving FVIIa, ß1-integrins, Src family proteins, and caveolin-1. Our results increase the knowledge of diseases associated with TF and IGF-1R overexpression in general but specifically of TF-mediated signaling with focus on cell survival.

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.

Mitigation of T-cell dependent immunogenicity by reengineering factor VIIa analogue.

Vatreptacog alfa (VA), a recombinant activated human factor VII (rFVIIa) variant with 3 amino acid substitutions, was developed to provide increased procoagulant activity in hemophilia patients with inhibitors to factor VIII or factor IX. In phase 3 clinical trials, changes introduced during the bioengineering of VA resulted in the development of undesired anti-drug antibodies in some patients, leading to the termination of a potentially promising therapeutic protein product. Here, we use preclinical biomarkers associated with clinical immunogenicity to validate our deimmunization strategy applied to this bioengineered rFVIIa analog. The reengineered rFVIIa analog variants retained increased intrinsic thrombin generation activity but did not elicit T-cell responses in peripheral blood mononuclear cells isolated from 50 HLA typed subjects representing the human population. Our algorithm, rational immunogenicity determination, offers a broadly applicable deimmunizing strategy for bioengineered proteins.

The In Vitro Effects of Pentamidine Isethionate on Coagulation and Fibrinolysis.

Pentamidine is bis-oxybenzamidine-based antiprotozoal drug. The parenteral use of pentamidine appears to affect the processes of blood coagulation and/or fibrinolysis resulting in rare but potentially life-threatening blood clot formation. Pentamidine was also found to cause disseminated intravascular coagulation syndrome. To investigate the potential underlying molecular mechanism(s) of pentamidine's effects on coagulation and fibrinolysis, we studied its effects on clotting times in normal and deficient human plasmas. Using normal plasma, pentamidine isethionate doubled the activated partial thromboplastin time at 27.5 µM, doubled the prothrombin time at 45.7 µM, and weakly doubled the thrombin time at 158.17 µM. Using plasmas deficient of factors VIIa, IXa, XIa, or XIIa, the concentrations to double the activated partial thromboplastin time were similar to that obtained using normal plasma. Pentamidine also inhibited plasmin-mediated clot lysis with half-maximal inhibitory concentration (IC50) value of ~3.6 µM. Chromogenic substrate hydrolysis assays indicated that pentamidine inhibits factor Xa and plasmin with IC50 values of 10.4 µM and 8.4 µM, respectively. Interestingly, it did not significantly inhibit thrombin, factor XIa, factor XIIIa, neutrophil elastase, or chymotrypsin at the highest concentrations tested. Michaelis-Menten kinetics and molecular modeling studies revealed that pentamidine inhibits factor Xa and plasmin in a competitive fashion. Overall, this study provides quantitative mechanistic insights into the in vitro effects of pentamidine isethionate on coagulation and fibrinolysis via the disruption of the proteolytic activity of factor Xa and plasmin.

Gene-based FVIIa prophylaxis modulates the spontaneous bleeding phenotype of hemophilia A rats.

A sizable proportion of hemophilia inhibitor patients fails immune tolerance induction and requires bypass agents for long-term bleed management. Recombinant human-activated coagulation Factor VII (rhFVIIa) is an on-demand bypass hemostatic agent for bleeds in hemophilia inhibitor patients. Prophylactic use of rhFVIIa may enable sustained hemostatic management of inhibitor patients, but the critical relationship of rhFVIIa circulating levels and clinical outcome in that setting remains unclear. To address this in vivo, we used the rat hemophilia A (HA) model that exhibits spontaneous bleeds and allows longitudinal studies with sufficient statistical power. We simulated activated Factor VII (FVIIa) prophylaxis by adeno-associated virus (AAV) gene transfer of a rat FVIIa transgene. Compared with naive HA animals, rat FVIIa continuous expression affected the overall observed bleeds, which were resolved with on-demand administration of recombinant rat FVIIa. Specifically, although 91% of naive animals exhibited bleeds, this was reduced to 83% and 33% in animals expressing less than 708 ng/mL (<14 nM) and at least 708 ng/mL (≥14 nM) rat FVIIa, respectively. No bleeds occurred in animals expressing higher than 1250 ng/mL (>25 nM). Rat FVIIa expression of at least 708 ng/mL was also sufficient to normalize the blood loss after a tail vein injury. Continuous, AAV-mediated rat FVIIa transgene expression had no apparent adverse effects in the hemostatic system of HA rats. This work establishes for the first time a dose dependency and threshold of circulating FVIIa antigen levels for reduction or complete elimination of bleeds in a setting of FVIIa-based HA prophylaxis.

Human platelets express endothelial protein C receptor, which can be utilized to enhance localization of factor VIIa activity.

Essentials Factor VIIa binds activated platelets to promote hemostasis in hemophilia patients with inhibitors. The interactions and sites responsible for platelet-FVIIa binding are not fully understood. Endothelial cell protein C receptor (EPCR) is expressed on activated human platelets. EPCR binding enhances the efficacy of a FVIIa variant and could impact design of new therapeutics. SUMMARY: Background High-dose factor VIIa (FVIIa) is routinely used as an effective bypassing agent to treat hemophilia patients with inhibitory antibodies that compromise factor replacement. However, the mechanism by which FVIIa binds activated platelets to promote hemostasis is not fully understood. FVIIa-DVQ is an analog of FVIIa with enhanced tissue factor (TF)-independent activity and hemostatic efficacy relative to FVIIa. Our previous studies have shown that FVIIa-DVQ exhibits greater platelet binding, thereby suggesting that features in addition to lipid composition contribute to platelet-FVIIa interactions. Objectives Endothelial cell protein C receptor (EPCR) also functions as a receptor for FVIIa on endothelial cells. We therefore hypothesized that an interaction with EPCR might play a role in platelet-FVIIa binding. Methods/results In the present study, we used flow cytometric analyses to show that platelet binding of both FVIIa and FVIIa-DVQ is partially inhibited in the presence of excess protein C or an anti-EPCR antibody. This decreased binding results in a corresponding decrease in the activity of both molecules in FXa and thrombin generation assays. Enhanced binding to EPCR was sufficient to account for the increased platelet binding of FVIIa-DVQ compared with wild-type FVIIa. As EPCR protein expression has not previously been shown in platelets, we confirmed the presence of EPCR in platelets using immunofluorescence, flow cytometry, immunoprecipitation, and mass spectrometry. Conclusions This work represents the first demonstration that human platelets express EPCR and suggests that modulation of EPCR binding could be utilized to enhance the hemostatic efficacy of rationally designed FVIIa analogs.

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.

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.

Associations of activated coagulation factor VII and factor VIIa-antithrombin levels with genome-wide polymorphisms and cardiovascular disease risk.

ESSENTIALS: Essentials A fraction of coagulation factor VII circulates in blood as an activated protease (FVIIa). We evaluated FVIIa and FVIIa-antithrombin (FVIIa-AT) levels in the Cardiovascular Health Study. Polymorphisms in the F7 and PROCR loci were associated with FVIIa and FVIIa-AT levels. FVIIa may be an ischemic stroke risk factor in older adults and FVIIa-AT may assess mortality risk. SUMMARY: Background A fraction of coagulation factor (F) VII circulates as an active protease (FVIIa). FVIIa also circulates as an inactivated complex with antithrombin (FVIIa-AT). Objective Evaluate associations of FVIIa and FVIIa-AT with genome-wide single nucleotide polymorphisms (SNPs) and incident coronary heart disease, ischemic stroke and mortality. Patients/Methods We measured FVIIa and FVIIa-AT in 3486 Cardiovascular Health Study (CHS) participants. We performed a genome-wide association scan for FVIIa and FVIIa-AT in European-Americans (n = 2410) and examined associations of FVII phenotypes with incident cardiovascular disease. Results In European-Americans, the most significant SNP for FVIIa and FVIIa-AT was rs1755685 in the F7 promoter region on chromosome 13 (FVIIa, ß = -25.9 mU mL-1 per minor allele; FVIIa-AT, ß = -26.6 pm per minor allele). Phenotypes were also associated with rs867186 located in PROCR on chromosome 20 (FVIIa, ß = 7.8 mU mL-1 per minor allele; FVIIa-AT, ß = 9.9 per minor allele). Adjusted for risk factors, a one standard deviation higher FVIIa was associated with increased risk of ischemic stroke (hazard ratio [HR], 1.12; 95% confidence interval [CI], 1.01, 1.23). Higher FVIIa-AT was associated with mortality from all causes (HR, 1.08; 95% CI, 1.03, 1.12). Among European-American CHS participants the rs1755685 minor allele was associated with lower ischemic stroke (HR, 0.69; 95% CI, 0.54, 0.88), but this association was not replicated in a larger multi-cohort analysis. Conclusions The results support the importance of the F7 and PROCR loci in variation in circulating FVIIa and FVIIa-AT. The findings suggest FVIIa is a risk factor for ischemic stroke in older adults, whereas higher FVIIa-AT may reflect mortality risk.

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.

Biochemical characterization of LR769, a new recombinant factor VIIa bypassing agent produced in the milk of transgenic rabbits.

BACKGROUND: The bypassing agent factor VII (FVIIa) is a first-line therapy for the treatment of acute bleeding episodes in patients with haemophilia and high-titre inhibitors. FVIIa is a highly post-translationally modified protein that requires eukaryotic expression systems to produce a fully active molecule. A recombinant FVIIa was produced in the milk of transgenic rabbits to increase expression and provide an efficient, safe and affordable product after purification to homogeneity (LR769). AIM: To present the biochemical and functional in vitro characteristics of LR769. RESULTS: Mass spectrometric analyses of the intact protein and of heavy and light chains revealed a fully activated, mature and properly post-translationally modified protein notably regarding N/O-glycosylations and γ-carboxylation. Primary structure analysis, performed by peptide mapping, confirmed 100% of the sequence and the low level or absence of product-derived impurities such as oxidized, deamidated and glycated forms. Low levels of aggregates and fragments were observed by different chromatographic methods. Higher order structure investigated by circular dichroism showed appropriate secondary/tertiary structures and conformational change in the presence of Ca2+ ions. Finally, activated partial thromboplastin time and thrombin generation assays showed the ability of LR769 to decrease coagulation time and to generate thrombin in haemophiliac-A-plasmas, even in the presence of inhibitors. CONCLUSION: The innovative expression system used to produce LR769 yields a new safe and effective rhFVIIa for the treatment of haemophilia A or B patients with inhibitors.

Gene Delivery of Activated Factor VII Using Alternative Adeno-Associated Virus Serotype Improves Hemostasis in Hemophiliac Mice with FVIII Inhibitors and Adeno-Associated Virus Neutralizing Antibodies.

While therapeutic expression of coagulation factors from adeno-associated virus (AAV) vectors has been successfully achieved in patients with hemophilia, neutralizing antibodies to the vector and inhibitory antibodies to the transgene severely limit efficacy. Indeed, approximately 40% of mice transduced with human factor VIII using the AAV8 serotype developed inhibitory antibodies to factor VIII (FVIII inhibitor), as well as extremely high titers (≥1:500) of neutralizing antibodies to AAV8. To correct hemophilia in these mice, AAV9, a serotype with low in vitro cross-reactivity (≤1:5) to anti-AAV8, was used to deliver mouse-activated factor VII (mFVIIa). It was found that within 6 weeks of systemic administration of 2 × 1013 particles/kg of AAV9/mFVIIa, hemophiliac mice with FVIII inhibitors and neutralizing antibodies (NAb) to AAV8 achieved hemostasis comparable to that in wild-type mice, as measured by rotational thromboelastometry. A level of 737 ng/mL mFVIIa was achieved after AAV9/mFVIIa adminstration compared to around 150 ng/mL without vector treatment, and concomitantly prothrombin time was shortened. Tissues collected after intra-articular hemorrhage from FVIII-deficient mice and mice with FVIII inhibitors were scored 4.7 and 5.5, respectively, on a scale of 0-10, indicating significant pathological damage. However, transduction with AAV9/mFVIIa decreased pathology scores to 3.6 and eliminated hemosiderin iron deposition in the synovium in most mice. Collectively, these results suggest that application of alternative serotypes of AAV vector to deliver bypassing reagents has the potential to correct hemophilia and prevent hemoarthrosis, even in the presence of FVIII inhibitor and neutralizing antibodies to AAV.

Engineering Styrene Monooxygenase for Biocatalysis: Reductase-Epoxidase Fusion Proteins.

The enantioselective epoxidation of styrene and related compounds by two-component styrene monooxygenases (SMOs) has targeted these enzymes for development as biocatalysts. In the present work, we prepare genetically engineered fusion proteins that join the C-terminus of the epoxidase (StyA) to the N-terminus of the reductase (StyB) through a linker peptide and demonstrate their utility as biocatalysts in the synthesis of Tyrain purple and other indigoid dyes. A single-vector expression system offers a simplified platform for transformation and expansion of the catalytic function of styrene monooxygenases, and the resulting fusion proteins are self-regulated and couple efficiently NADH oxidation to styrene epoxidation. We find that the reductase domain proceeds through a sequential ternary-complex mechanism at low FAD concentration and a double-displacement mechanism at higher concentrations of FAD. Single-turnover studies indicate an observed rate constant for FAD-to-FAD hydride transfer of ~8 s-1. This step is rate limiting in the styrene epoxidation reaction and helps to ensure that flavin reduction and styrene epoxidation reactions proceed without wasteful side reactions. Comparison of the reductase activity of the fusion proteins with the naturally occurring reductase, SMOB, and N-terminally histidine-tagged reductase, NSMOB, suggests that the observed changes in catalytic mechanism are due in part to an increase in flavin-binding affinity associated with the N-terminal extension of the reductase.

An activated factor VII variant with enhanced tissue factor-independent activity speeds wound healing in a mouse hemophilia B model.

UNLABELLED: Essentials Disorders of hemostasis can lead to delayed and defective wound healing. In hemophilia B (HB) mice, 7 days of Factor (F)IX or VIIa are needed to normalize wound healing. One dose of a highly active FVIIa variant (DVQ) restored normal wound closure time in HB mice. Coagulation factors with enhanced activity may acquire biological effects not due to hemostasis. SUMMARY: Introduction We have previously reported that hemophilia B (HB) mice have delayed healing of cutaneous wounds and alterations in wound histology. Administration of a single dose of either factor IX or recombinant activated FVII (rFVIIa) (NovoSeven) prior to wounding did not improve wound closure time or histology. The FVIIa analog DVQ (V158D, E296V and M298Q mutations) was designed to have higher tissue factor-independent activity than rVIIa. We hypothesized that a single dose of DVQ would be more effective in restoring wound healing in HB mice. Methods Cutaneous punch wounds were made on the backs of HB and wild-type mice, and the time to wound closure was monitored. HB mice were treated with a dose of rFVIIa (10 mg kg(-1) ) or DVQ (1 mg kg(-1) ) that corrected the tail bleeding time. Skin samples were taken at various time points after wounding, fixed, and stained, and the histology was examined. Results As previously reported, wound closure times in HB mice given one dose of rFVIIa were not improved over those in untreated HB mice. Surprisingly, healing times in HB mice treated with an equally hemostatic dose of DVQ were normalized to that in wild-type mice. However, DVQ did not correct all histologic abnormalities in HB mice. Conclusions As the doses of DVQ and rFVIIa were chosen to support comparable levels of hemostasis, our data suggest that the improved healing seen with DVQ is not solely attributable to its hemostatic activity. It is possible that the improved wound healing arises through the effect of DVQ on cell signaling mechanisms.

Tissue factor-dependent pathway in severe preeclampsia revisited: a Brazilian cohort study.

Previously we investigated the tissue factor (TF)-dependent coagulation pathway and key haemostatic cofactors in white women with preeclampsia (P-EC) and suggested that plasma factor VII (FVII) levels can differentiate women with P-EC from healthy nonpregnant women or normal pregnant women, at the same trimester, with high sensitivity, specificity, positive and negative predictive values. Here we re-examine the TF-dependent pathway in a large cohort of Brazilian women. A total of 240 women were studied. These included healthy nonpregnant women (n = 79), normotensive pregnant women (n = 80) and women with severe P-EC (n = 81). Commercially available enzyme-linked immunosorbent assays were used to measure plasma FVII, activated factor VII (FVIIa), TF and tissue factor pathway inhibitor (TFPI). All study participants were matched for age. Pregnant women (with/without P-EC) were matched for gestational age and parity. Plasma levels of FVII, FVIIa and TFPI were significantly increased in women with severe P-EC compared with healthy nonpregnant women (P < 0.01) or normotensive pregnant women (P < 0.01). FVIIa was also higher in normotensive pregnant women compared with nonpregnant women (P < 0.01). However, no such significant trends were observed for plasma TF levels (P = 0.074). In conclusion, circulating FVII, FVIIa and TFPI were significantly elevated in women with severe P-EC in the absence of comparable changes in plasma TF levels. The present work is in agreement with our previous report on FVII levels in white women with P-EC. Thus, this lends further support to the notion that plasma FVII levels are potentially valuable diagnostic marker for P-EC, irrespective of ethnicity.

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.