Factor VIIa-AT Complex Is an Independent Prognostic Marker of Disseminated Intravascular Coagulation.

OBJECTIVE: The factor VIIa-Antithrombin (VIIa-AT) complex is a relatively new biomarker associated with the activation of the extrinsic coagulation pathway. Since disseminated intravascular coagulation (DIC) is primarily driven by issue factor (TF)-induced extrinsic coagulation activation, the plasma level of factor VIIa-AT, via its role as an activation marker of the extrinsic pathway, could be a potential marker for DIC. The clinical significance of extrinsic coagulation markers, including factor VIIa-AT, in DIC was investigated. METHODS: The extrinsic coagulation markers, including factor VIIa-AT, TF, factor VII, and antithrombin (AT), were measured in 148 patients clinically suspicious for DIC. Multiple linear regression and Cox proportional-hazard analysis were conducted to evaluate both contributing factors and the prognostic power of the markers. RESULTS: The factor VIIa-AT complex, factor VII, and AT levels were significantly lower in the overt-DIC group and gradually decreased according to the severity of DIC based on the DIC scores. On the contrary, TF was significantly higher in the overt-DIC group. The factor VII level was revealed as a significant independent contributor to the factor VIIa-AT level. Upon multivariable Cox proportional-hazard analysis, the factor VIIa-AT complex showed the highest hazard ratio (3.41; 95% confidence interval 1.11-10.44). CONCLUSION: The factor VIIa-AT complex reflects the severity of DIC and is an independent prognostic factor of DIC. Our findings hint at the potential of the factor VIIa-AT complex to be used as a complementary marker to well-established biomarkers such as AT.

Human acute Chagas disease: changes in factor VII, activated protein C and hepatic enzymes from patients of oral outbreaks in Pará State (Brazilian Amazon).

Oral transmission of Chagas disease has been increasing in Latin American countries. The present study aimed to investigate changes in hepatic function, coagulation factor levels and parasite load in human acute Chagas disease (ACD) secondary to oral Trypanosoma cruzi transmission. Clinical and epidemiological findings of 102 infected individuals attended in the State of Pará from October 2013 to February 2016 were included. The most common symptoms were fever (98%), asthenia (83.3%), face and limb edema (80.4%), headache (74.5%) and myalgia (72.5%). The hepatic enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of 30 ACD patients were higher compared with controls, and this increase was independent of the treatment with benznidazole. Moreover, ACD individuals had higher plasma levels of activated protein C and lower levels of factor VII of the coagulation cascade. Patients with the highest parasite load had also the most increased transaminase levels. Also, ALT and AST were associated moderately (r = 0.429) and strongly (r = 0.595) with parasite load respectively. In conclusion, the present study raises the possibility that a disturbance in coagulation and hepatic function may be linked to human ACD.

Human acute Chagas disease: changes in factor VII, activated protein C and hepatic enzymes from patients of oral outbreaks in Pará State (Brazilian Amazon)

Oral transmission of Chagas disease has been increasing in Latin American countries. The present study aimed to investigate changes in hepatic function, coagulation factor levels and parasite load in human acute Chagas disease (ACD) secondary to oral Trypanosoma cruzi transmission. Clinical and epidemiological findings of 102 infected individuals attended in the State of Pará from October 2013 to February 2016 were included. The most common symptoms were fever (98%), asthenia (83.3%), face and limb edema (80.4%), headache (74.5%) and myalgia (72.5%). The hepatic enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of 30 ACD patients were higher compared with controls, and this increase was independent of the treatment with benznidazole. Moreover, ACD individuals had higher plasma levels of activated protein C and lower levels of factor VII of the coagulation cascade. Patients with the highest parasite load had also the most increased transaminase levels. Also, ALT and AST were associated moderately (r = 0.429) and strongly (r = 0.595) with parasite load respectively. In conclusion, the present study raises the possibility that a disturbance in coagulation and hepatic function may be linked to human ACD.

Tissue factor mediates microvesicles shedding from MDA-MB-231 breast cancer cells.

Extracellular vesicles, such as microvesicles (MVs), were identified as important players in tumor progression and acquisition of an aggressive phenotype. Tissue factor (TF) is a transmembrane protein that initiates the blood coagulation cascade. In tumor cells, TF has been associated with aggressiveness and cancer progression. Previous studies demonstrate that TF is incorporated into MVs secreted by tumor cells; however, it is unknown whether TF is actively involved in the release of MVs. Here, we investigated the influence of TF expression on the release of MVs. TF silencing was achieved through CRISPR/Cas9 approaches in the human breast cancer cell line, MDA-MB-231. TF knockout in MDA-MB-231 cells efficiently reduced TF-dependent signaling and procoagulant activity. Remarkably, silencing of TF caused a significant decrease in the number of MVs released by MDA-MB-231 cells. We also observed an increase in actin-positive membrane projections in TF knockout cells and a reduction in RhoA expression when compared to TF-expressing cells. Treatment of MDA-MB-231 cells with the RhoA-ROCK signaling pathway inhibitor, fasudil, significantly reduced the release of MVs. Taken together, our results suggest a novel and relevant role for TF in tumor biology by playing an active role in the MVs secretion.

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.

Non-neoplastic Portal Vein Thrombosis in HCV Cirrhosis Patients: Is it an Immuno-Inflammatory Disorder?

BACKGROUND AND AIMS: Portal vein thrombosis (PVT) is a critical complication in cirrhotic patients. We explored the role of the activated factor VII-antithrombin (FVIIa-AT) complex and enhanced monocytic tissue factor (TF) expression in the development and prediction of non-neoplastic PVT in cirrhotic patients. MATERIAL AND METHODS: A total of 30 HCV-cirrhosis patients were included in our study. They were compared to 35 cirrhotic patients without PVT, 15 non-cirrhotic patients with PVT, and 15 healthy controls. The plasma level of the FVIIa-AT complexes was analyzed by ELISA. MIF CD142, CD86, and HLA-DR on monocytes (CD14) were determined by flow cytometry. RESULTS: Compared with cirrhotic patients without PVT, cirrhotic patients with PVT had comparable plasma values of FVIIa, AT, and the FVIIa-AT complex. However, they had significantly lower values compared to non-cirrhotic patients with PVT and healthy controls. Cirrhotic patients with PVT had increased monocytic TF expression (MIF CD142) compared to non-PVT cirrhotic patients and healthy controls [86.5 (93.5) vs. 18 (32.0) and 11.0 (6.0), respectively; p < 0.001 for each]. However, cirrhosis PVT could not be distinguished from non-cirrhosis PVT. The area under the ROC curve of MIF CD142 was 0.759 (0.641- 0.876; p = 0.000) at an optimal cut-off value of 45, which yielded a sensitivity of 60% and a specificity of 77.1%, as well as a PPV and NPV of 69.2% for each. CONCLUSION: Enhanced expression of monocytic TF may have a role in the development and prediction of non-neoplastic PVT in HCV-cirrhosis patients. Large multicenter studies are necessary to validate our results.

Low plasma FVII:C and activated FVII as predictive markers for overt disseminated intravascular coagulation.

In sepsis, binding of factor VII (FVII:C) and activated factor VII (FVIIa) with tissue factor is the key step of coagulation resulting in disseminated intravascular coagulation (DIC). We conducted a prospective cohort study among 47 septic patients, aged 8 months to 18.8 years. They were initially divided into three groups of no DIC (n=27), non-overt DIC (n=14) and overt DIC (n=6). Blood samples were collected at 0, 24 and 48 hours (h) after the onset of sepsis. At the onset of sepsis, FVII:C tended to be lower in the non-overt DIC [median 57 % (interquartile range [IQR] 41-80)] and overt DIC groups [33 % (23-52)] than that in the no DIC group [65 % (44-87)]. Whereas FVIIa tended to be lower in the overt DIC group [1.29 % (0.50-4.19)] than those in the non-overt DIC [3.01 % (1.01-5.24)] and no DIC groups [2.49 % (1.14-3.13)]. At 24 h, FVII:C was significantly lower in the non-overt DIC [57 % (41-101)] and overt DIC groups [31 % (28-49)] than that in the no DIC group [83 % (70-102)]. While FVIIa was significantly lower in the overt DIC group [2.15 % (0.86-3.96)] than that in the no DIC group [3.83 % (2.90-5.46)]. Using FVII:C <65 % or FVIIa <3 % at 24 h among patients without hepatic dysfunction to determine overt DIC at 24 h, the sensitivity was 83.9 % and 77.4 %, respectively, and the specificity was both 83.3 %. Patients with low FVII:C and low FVIIa at 24 h after the onset of sepsis had a 20.8-fold (95 % confidence interval [CI], 2.0-213.0, p=0.010) and 14.4-fold (95 %CI, 1.5-142.4, p=0.023) chance of overt DIC.

The various assays for measuring activity states of factor VIIa in plasma and therapeutic products: Diagnostic value and analytical usefulness in various pathophysiological states.

The key coagulation factor FVII, and its activated form FVIIa, present a major interest for their role at the initiation phase of blood coagulation, and because they can activate all blood coagulation cascade, through the extrinsic, but also the intrinsic pathway. Blood activation initiated through FVII is first presented, as it is understood nowadays. Measurement of FVII and FVIIa were of main interest for epidemiological studies, but FVIIa contribution to assay results was only deduced. The introduction of specific FVIIa assays, functional or immunoassays, allowed measuring directly FVIIa without any interference of non-activated FVII, or other coagulation factors or their activated forms. The various methods available, and their characteristics are presented, with a special focus on two assays developed by our group for FVIIa (a clotting one and a chromogenic one). The FVIIa clotting assay shows evident superiority for measuring its activity in plasma, in pathophysiological conditions. The normal range is <2.5ng/ml, which represents less than 0.5% of the FVII protein. FVIIa is elevated in some pathological states. The chromogenic assay is of interest for assigning the potency of FVIIa concentrates, as it has a higher dynamic range. Both assays are fully automatable on laboratory instruments, and standardized in a satisfactory manner thanks to the use of the FVIIa concentrate WHO International Standard (NIBSC). The various applications and usefulness of FVIIa laboratory assays are discussed, for the measurement of therapeutic products, or for following recoveries in treated patients, including hemophiliacs with inhibitors, patients with severe bleeding risk (liver diseases, surgery, trauma, …), and lastly for measurement of its activity in therapeutic products.

Factor VIIa-antithrombin complex: a possible new biomarker for activated coagulation.

The activation of the extrinsic coagulation pathway occurs after endothelial injury when the tissue factor (TF), a transmembrane protein located outside the vasculature, binds factor VII (FVII) or activated FVII (FVIIa). Once formed, the TF-VIIa complex activates both factor IX and X and initiates the coagulation process. The TF-VIIa complex is inhibited by both TF pathway inhibitor (TFPI) and antithrombin (AT). The interaction between TF-VIIa and AT induces FVIIa-AT complex formation, which is released into the plasma. Because AT reacts with FVIIa only when it is bound to TF, the circulating levels of FVIIa-AT reflect the degree of exposure of TF to blood. Preliminary clinical studies have shown higher plasma levels of FVIIa-AT complex both in patients with a prior arterial or venous thrombotic event. Increased plasma levels of FVIIa-AT have also been reported in a number of other prothrombotic conditions - antiphospholipid antibodies, solid and hematological malignancies, pre-eclampsia (PE), obesity and cardiac surgery. However, most of the studies published so far are retrospective and with a limited sample size. Larger prospective clinical studies are needed to confirm these findings and to assess the prognostic role of this possible new biomarker for activated coagulation.

Inactivated antithrombins as fondaparinux antidotes: a promising alternative to haemostatic agents as assessed in vitro in a thrombin-generation assay.

In the absence of specific antidote to fondaparinux, two modified forms of antithrombin (AT), one recombinant inactive (ri-AT) and the other chemically inactivated (chi-AT), were designed to antagonise AT-mediated anticoagulants, e. g. heparins or fondaparinux. These inactive ATs were previously proven to effectively neutralise anticoagulant activity associated with heparin derivatives in vitro and in vivo, as assessed by direct measurement of anti-FXa activity. This study was undertaken to evaluate in vitro the effectivity of inactive ATs to reverse anticoagulation by heparin derivatives and to compare them with non-specific fondaparinux reversal agents, like recombinant-activated factor VII (rFVIIa) or activated prothrombin-complex concentrate (aPCC), in a thrombin-generation assay (TGA). Addition of fondaparinux (3 µg/ml) to normal plasma inhibited thrombin generation by prolonging lag time (LT) as much as 244 % and lowering endogenous thrombin potential (ETP) to 17 % of their control (normal plasma) values. Fondaparinux-anticoagulant activity was reversed by ri-AT and chi-AT, as reflected by the corrections of LT up to 117 % and 114 % of its control value, and ETP recovery to 78 % and 63 %, respectively. Unlike ri-AT that had no effect on thrombin generation in normal plasma, chi-AT retained anticoagulant activity that minimises its reversal capacity. However, both ATs were more effective than rFVIIa or aPCC at neutralising fondaparinux and, unlike non-specific antidotes, inactive ATs specifically reversed AT-mediated anticoagulant activities, as suggested by their absence of procoagulant activity in anticoagulant-free plasma.

Elaborating European Pharmacopoeia monographs for biotherapeutic proteins using substances from a single source.

For more than twenty years, the European Pharmacopoeia (Ph. Eur.) monographs for biotherapeutic proteins have been elaborated using the multisource approach (Procedure 1), which has led to robust quality standards for many of the first-generation biotherapeutics. In 2008, the Ph. Eur. opened up the way towards an alternative mechanism for the elaboration of monographs (Procedure 4-BIO pilot phase), which is applied to substances still under patent protection, based on a close collaboration with the Innovator company, to ensure a harmonised global standard and strengthen the quality of the upcoming products. This article describes the lessons learned during the P4-BIO pilot phase and addresses the current thinking on monograph elaboration in the field of biotherapeutics. Case studies are described to illustrate the standardisation challenges associated with the complexity of biotherapeutics and of analytical procedures, as well as the approaches that help ensure expectations are met when setting monograph specifications and allow for compatibility with the development of biosimilars. Emphasis is put on monograph flexibility, notably by including tests that measure process-dependent microheterogeneity (e.g. glycosylation) in the Production section of the monograph. The European Pharmacopoeia successfully concluded the pilot phase of the P4-BIO during its 156th session on 22-23 November 2016.

Activation of the Blood Coagulation System in Patients with Chronic Spontaneous Urticaria.

BACKGROUND: Ninty-five percent of chronic spontaneous urticaria (CSU) patients presented with signs of thrombin generation, and autologous plasma skin tests score positive. The aim of this study was to assess the initiators of blood coagulation that lead to thrombin generation and fibrinolysis in CSU patients. METHODS: The plasma level of activated factor VII, activator factor XII, fragment F1+2, and D-dimer were measured and analyzed in 103 patients with CSU and 76 control subjects. RESULTS: Mean D-dimer plasma levels were higher in patients than controls (0.41 ± 0.44 µg/mL vs. 0.21 ± 0.26 µg/ mL; p < 0.001). Mean F1+2 plasma levels were higher in patients than controls (11.17 ± 17.65 nM vs. 5.97 ± 9.42 nM; p = 0.048). Mean FVIIa plasma levels were higher in patients than controls (4.09 ± 4.22 ng/mL vs. 2.97 ± 1.59 ng/mL; p = 0.031). However, no significant difference was found on FXIIa plasma levels. On the other hand, all the coagulation factors (D-dimer, FVIIa, and F1+2) were significantly correlated with disease severity. CONCLUSIONS: The extrinsic pathway of the clotting cascade is activated in CSU and is correlated with the disease severity. The involvement of the coagulation pathway in CSU opens new perspectives for a better understanding of the pathogenesis and treatment of the disease.

Decreased plasma levels of activated factor VII in patients with deep vein thrombosis.

BACKGROUND: The initiating trigger in the development of deep vein thrombosis (DVT) remains unidentified. It has been suggested that tissue factor (TF)-bearing microparticles play a key role, which indicates a role for the TF pathway in the initiation of DVT. OBJECTIVE: To assess the role of the TF pathway in the initiation of venous thrombosis, we measured plasma levels of factor VII and VIIa in patients with acute DVT and in controls. METHODS: We included 148 patients diagnosed with acute DVT and 179 controls in this study. Antigen levels of FVII and FVIIa were measured by using assays recently developed in our laboratory. RESULTS: Median FVII levels in patients were 109.8% (interquartile range [IQR] 86.0-153.2) compared with 102.2% (IQR 76.1-141.7) in controls. Individuals with FVII levels in the upper quartile had a 1.6-fold increased risk for the presence of a DVT (odds ratio 1.6, 95% confidence interval 0.8-3.1). Median FVIIa levels in patients were 50.2 ng mL(-1) (IQR 25.2-86.1) compared with 96.6 ng mL(-1) (69.9-168.9) in controls. Individuals with FVIIa levels in the lowest quartile had a > 5-fold increased risk for the presence of a DVT (odds ratio 5.5, 95% confidence interval 2.8-10.6). Both risks did not change substantially after adjustment for potential confounders. CONCLUSION: Decreased plasma levels of FVIIa in patients with deep vein thrombosis may indicate ongoing consumption of FVIIa and suggest a contributory role for TF in venous thrombus formation.

Visualizing cancer and response to therapy in vivo using Cy5.5-labeled factor VIIa and anti-tissue factor antibody.

We have developed a specific technique for imaging cancer in vivo using Cy5.5-labeled factor VIIa (fVIIa), clotting-deficient FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-tissue factor (TF) antibody. FVIIa is the natural ligand for TF. We took advantage of the fact that vascular endothelial cells (VECs) in cancer, but not normal tissue, aberrantly express TF due to its induction by vascular endothelial growth factor (VEGF). Under physiological conditions, TF is expressed by stromal cells and outer blood vessel layers (smooth muscle and adventitia), but not by VECs. We hypothesized that labeled fVIIa or anti-TF antibodies could be used to image the tumor vasculature in vivo. To test this, Cy5.5-labeled fVIIa, FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-TF antibody were developed and administered to athymic nude mice carrying xenografts including glioma U87EGFRviii, pancreatic cancer ASPC-1 and Mia PaCa-2, and squamous cell carcinoma KB-V1. Cy5.5 labeled with these targeting proteins specifically localized to the tumor xenografts for at least 14 days but unconjugated Cy5.5 did not localize to any xenografts or organs. This method of imaging TF in the tumor VECs may be useful in detecting primary tumors and metastases as well as monitoring in vivo therapeutic responses.

Comparative Biochemical and Functional Studies on a Branded Human Recombinant Factor VIIa and a Biosimilar Equivalent Product.

Recombinant factor VIIa (rFVIIa; NovoSeven, Novo Nordisk, Copenhagen, Denmark) is used to control bleeding in patients with hemophilia. A generic version of FVIIa was developed by AryoGen (Tehran, Iran). This study compared the composition and functional activities of AryoSeven and NovoSeven. Each product was compared at equigravimetric (1 mg/mL) stock solution for protein content. The proteomic profile was obtained using surface-enhanced laser desorption ionization mass spectrometry. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was carried out to determine the protein profile and Western blotting was performed using a polyclonal rabbit antihuman FVIIa antibody. The FVIIa-related antigen was also measured using a commercially available enzyme-linked immunosorbent assay method. Functional assay included the prothrombin time correction in FVII-deficient plasma. The protein content was comparable in 2 products and the mass spectra analysis showed a single peak at 50 kDa in all products. The SDS-PAGE and immunoblotting studies were comparable. Both products exhibited similar coagulant properties in different assays.

Pharmacodynamics and pharmacokinetics during the transition from warfarin to rivaroxaban: a randomized study in healthy subjects.

AIMS: This study investigated relevant pharmacodynamic and pharmacokinetic parameters during the transition from warfarin to rivaroxaban in healthy male subjects. METHODS: Ninety-six healthy men were randomized into the following three groups: warfarin [international normalized ratio (INR) 2.0-3.0] transitioned to rivaroxaban 20 mg once daily (od; group A); warfarin (INR 2.0-3.0) followed by placebo od (group B); and rivaroxaban alone 20 mg od (group C) for 4 days. Anti-factor Xa activity, inhibition of factor Xa activity, prothrombin time (PT), activated partial thromboplastin time, HepTest, prothrombinase-induced clotting time, factor VIIa activity, factor IIa activity, endogenous thrombin potential and pharmacokinetics were measured. RESULTS: An additive effect was observed on the PT and PT/INR during the initial transition period. The mean maximal prolongation of PT was 4.39-fold [coefficient of variation (CV) 18.03%; range 3.39-6.50] of the baseline value in group A, compared with 1.88-fold (CV 10.35%; range 1.53-2.21) in group B and 1.57-fold (CV 9.98%; range 1.37-2.09) in group C. Rivaroxaban had minimal influence on the PT/INR at trough levels. Inhibition of factor Xa activity, activated partial thromboplastin time and endogenous thrombin potential were also enhanced, but to a lesser extent. In contrast, the effects of rivaroxaban on anti-factor Xa activity, HepTest and prothrombinase-induced clotting time were not affected by pretreatment with warfarin. CONCLUSIONS: Changes in pharmacodynamics during the transition from warfarin to rivaroxaban vary depending on the test used. A supra-additive effect on PT/INR is expected during the initial period of transition, but pretreatment with warfarin does not influence the effect of rivaroxaban on anti-factor Xa activity.

N-/O-glycosylation analysis of human FVIIa produced in the milk of transgenic rabbits.

Human coagulation factor VIIa is a glycoprotein that promotes haemostasis through activation of the coagulation cascade extrinsic pathway. Most haemophilia A/B patients with inhibitors are treated by injection of plasma-derived or recombinant FVIIa. The use of recombinant products raises questions about the ability of the host cell to produce efficiently post-translationally modified proteins. Glycosylation is especially critical considering that it can modulate protein safety and efficacy. The present paper reports the N-/O-glycosylation pattern of a new recombinant human factor VIIa expressed in the mammary glands of transgenic rabbits. Glycosylation was investigated by chromatography and advanced mass spectrometry techniques for glycan identification and quantitation. Mass spectrometry (MS)/MS analyses were performed to confirm the glycan structures as well as the position and branching of specific monosaccharides or substituents. The two N-glycosylation sites were found to be fully occupied mostly by mono- and bi-sialylated biantennary complex-type structures, the major form being A(2)G(2)S(1). Some oligomannose/hybrid structures were retrieved in lower abundance, the major ones being GlcNAcα1,O-phosphorylated at the C6-position of a Man residue (Man-6-(GlcNAcα1,O-)phosphate motif) as commonly observed on lysosomal proteins. No immunogenic glycotopes such as Galili (Galα1,3Gal) and HD antigens (N-glycolylneuraminic acid (NeuGc)) were detected. Concerning O-glycosylation, the product exhibited O-fucose and O-glucose-(xylose)(0, 1, 2) motifs as expected. The N-glycosylation consistency was also investigated by varying production parameters such as the period of lactation, the number of consecutive lactations and rabbit generations. Results show that the transgenesis technology is suitable for the long-term production of rhFVIIa with a reproducible glycosylation pattern.

Association between increased FVIIa-antithrombin complex/FVIIa ratio and pre-eclampsia.

OBJECTIVE: Activated factor VII-antithrombin (FVIIa-AT) complexes can be used to reflect the degree of intravascular exposure of tissue factor (TF). The aim of the present case-control study was to evaluate FVIIa-AT plasma levels during normal pregnancy and in pre-eclampsia (PE). METHODS: One hundred and five pregnant women were enrolled and namely n = 30 in the first (T1), n = 30 in the second (T2), n = 30 in the third (T3) trimester of pregnancy and n = 15 with PE. FVIIa-AT complexes were determined using a specific ELISA (Diagnostica Stago, Asnieres, France). RESULTS: FVIIa-AT complexes were significantly higher in pregnant (119 ± 24 pM) than in healthy (102 ± 12 pM, p = 0.001) women. No difference in FVIIa-AT levels between T3 women and with PE was observed. Interestingly, women with PE had significantly higher FVIIa-AT/FVIIa ratio than women during T3 (2.01 ± 0.44 versus 1.50 ± 0.29, p = 0.001). CONCLUSION: FVIIa-AT complexes plasma levels differed significantly between normal pregnancy and non-pregnant women. Moreover, FVIIa-AT/FVIIa ratio was higher in patients with PE than in normal pregnant women.

Factor VIIa-antithrombin complexes in patients with non-neoplastic portal vein thrombosis with and without cirrhosis.

INTRODUCTION: Portal vein thrombosis (PVT) is caused by local and systemic prothrombotic risk factors. In this case-control study, we evaluated the use of the Factor VIIa-antithrombin complex (FVIIa-AT) complex assay as a hypercoagulability marker in patients with PVT. METHODS: Two different groups of cases were considered: (i) n = 12 noncirrhotic PVT patients, (ii) n = 33 cirrhotic patients with PVT. Controls were sex and age-matched healthy volunteers and cirrhotic subjects without PVT, respectively. RESULTS: Levels of the FVIIa-AT complex were significantly higher in noncirrhotic PVT subjects (132 ± 32 pM) than in healthy volunteers (108 ± 18 pM, P = 0.04). No significant difference in FVIIa-AT complexes was seen between cirrhotic patients with (64 ± 20 pM) or without (61 ± 24 pM) PVT. A linear correlation was seen between FVIIa-AT and FVIIa in noncirrhotic PVT subjects. In cirrhotic patients, FVIIa-AT complexes depended on both FVIIa and AT. CONCLUSION: These results confirm the utility of the FVIIa-AT assay in identifying the hypercoagulable state of noncirrhotic patients because of a previous thrombotic event.