Knockdown of coagulation factor VII has potential anticoagulant and anti-inflammatory effects in a mouse model of group A streptococcal sepsis.

Infections originating from subcutaneous tissues are among the most common invasive infections caused by group A streptococcus (GAS) and associated with systemic coagulation activation. The role of intrinsic coagulation factors on GAS virulence has recently been determined, but the role of the extrinsic coagulation factor VII is unknown. Using a mouse model, in which GAS-sepsis emerges from a subcutaneous infection, we show that FVII is a negative acute phase protein. F7 knockdown using antisense oligonucleotides resulted in an attenuated systemic coagulation activation and inflammatory response in septic animals. The findings indicate FVII's ability to modify the host response.

Keel venom: Rhabdophis subminiatus (Red-Necked Keelback) venom pathophysiologically affects diverse blood clotting pathways.

Venoms are evolutionary novelties that have real-world implications due to their impact upon human health. However, relative to the abundant studies of elapid and viperid snake venoms, fewer investigations have been undertaken on those of rear-fanged snakes as they are more problematic for obtaining venom. While most rear-fanged venomous snakes are not considered to be of great medical importance, several species are capable of producing fatalities. Most notable among these are snakes from the genus Rhabdophis, the Asian "keelback" snakes. Prior work have described potent procoagulant toxicity suggesting Factor X and prothrombin activation, but did not investigate the ability to activate other clotting factors. Here we show that in addition to activating both Factor X and prothrombin (with prothrombin twice that of FX), the venom of Rhabdophis subminiatus is able to more potently activate Factor VII (ten times that of prothrombin), while also activating FXII and FIX equipotently to prothrombin, and with FXI also activated but at a much lower level. The ability to activate FVII represents a third convergent evolution of this trait. The Australian elapid clade of [Oxyuranus (taipans) + Pseudonaja (brown snakes)] was the first identified to have evolved this trait. and only recently was it shown to be independently present in another lineage (the Central American viperid species Porthidium volcanicum). In addition, the abilities to activate FXI and FXII are also convergent between R. subminiatus and P. volcanicum, but with R. subminiatus being much more potent. By testing across amphibian, avian, and mammalian plasmas we demonstrate that the venom is potently procoagulant across diverse plasma types. However, consistent with dietary preference, R. subminiatus venom was most potent upon amphibian plasma. While a Rhabdophis antivenom is produced in Japan to treat R. tigrinus envenomings, it is scarce even within Japan and is not exported. As this genus is very wide-ranging in Asia, alternate treatment options are in need of development. Hence we tested the ability of candidate, broad-spectrum enzyme inhibitors to neutralize R. subminiatus venom: marimastat was more effective than prinomastat but both marimastat and prinomastat were significantly more effective than DMPS (2,3-Dimercapto-1-propanesulfonic acid). The findings of this study shed light on the evolution of these fascinating rear-fanged snakes as well as explored their systemic effects upon blood coagulation and point to potential treatment options for the rare, but potentially lethal encounters.

Coagulation factors VII, IX and X are effective antibacterial proteins against drug-resistant Gram-negative bacteria.

Infections caused by drug-resistant "superbugs" pose an urgent public health threat due to the lack of effective drugs; however, certain mammalian proteins with intrinsic antibacterial activity might be underappreciated. Here, we reveal an antibacterial property against Gram-negative bacteria for factors VII, IX and X, three proteins with well-established roles in initiation of the coagulation cascade. These factors exert antibacterial function via their light chains (LCs). Unlike many antibacterial agents that target cell metabolism or the cytoplasmic membrane, the LCs act by hydrolyzing the major components of bacterial outer membrane, lipopolysaccharides, which are crucial for the survival of Gram-negative bacteria. The LC of factor VII exhibits in vitro efficacy towards all Gram-negative bacteria tested, including extensively drug-resistant (XDR) pathogens, at nanomolar concentrations. It is also highly effective in combating XDR Pseudomonas aeruginosa and Acinetobacter baumannii infections in vivo. Through decoding a unique mechanism whereby factors VII, IX and X behave as antimicrobial proteins, this study advances our understanding of the coagulation system in host defense, and suggests that these factors may participate in the pathogenesis of coagulation disorder-related diseases such as sepsis via their dual functions in blood coagulation and resistance to infection. Furthermore, this study may offer new strategies for combating Gram-negative "superbugs".

Site-Specific 64Cu Labeling of the Serine Protease, Active Site Inhibited Factor Seven Azide (FVIIai-N3), Using Copper Free Click Chemistry.

A method for site-specific radiolabeling of the serine protease active site inhibited factor seven (FVIIai) with 64Cu has been applied using a biorthogonal click reaction. FVIIai binds to tissue factor (TF), a trans-membrane protein involved in hemostasis, angiogenesis, proliferation, cell migration, and survival of cancer cells. First a single azide moiety was introduced in the active site of this 50 kDa protease. Then a NOTA moiety was introduced via a strain promoted azide-alkyne reaction and the corresponding conjugate was labeled with 64Cu. Binding to TF and the stability was evaluated in vitro. TF targeting capability of the radiolabeled conjugate was tested in vivo by positron emission tomography (PET) imaging in pancreatic human xenograft cancer mouse models with various TF expressions. The conjugate showed good stability (>91% at 16 h), an immunoreactivity of 93.5%, and a mean tumor uptake of 2.1 ± 0.2%ID/g at 15 h post injection. In conclusion, FVIIai was radiolabeled with 64Cu in single well-defined position of the protein. This method can be utilized to prepare conjugates from serine proteases with the label at a specific position.

Differing mechanisms of thrombin generation in live haematological and solid cancer cells determined by calibrated automated thrombography.

: Calibrated automated thrombography (CAT) is emerging as a reliable tool for real-time estimation of thrombin generation potential. There is a clinical need for knowledge about the pathways underlying the thrombotic phenotype of different malignancies. Cells from solid (e.g. pancreatic cancer; n = 7) and malignant haematological cell lines (e.g. multiple myeloma; n = 5) were evaluated for thrombin generation, using CAT, with the addition of control plasma (NormTrol; Helena Biosciences, Gateshead, UK)) or plasma deficient in coagulation factors VII and XII. In addition, tissue factor (TF) cell surface expression was determined by flow cytometry. In platelet-free plasma, thrombin generation in all cancer cell lines was cell concentration dependent, with the pancreatic cancer line CFPAC-1 producing the highest thrombin of 220 nmol/l at 5 × 10-cells/ml concentration. Lag times and times to peak reflected most significant differences out of all thrombin generation parameters measured and were inversely correlated with cell surface TF surface expression. Solid tumour cell lines had higher thrombin peaks, faster lag times, and a thrombin generation profile of overall greater magnitude than haematological cell lines. In the absence of factor VII in platelet-free plasma, thrombin generation in solid pancreatic cancer cell lines was significantly reduced unlike in haematological cell lines. However, in the absence of factor XII, thrombin generation was reduced more in haematological cells but had little or no effect on solid cell lines. The CAT assay identified characteristic differences in thrombin generation kinetics between solid tumour and haematological cancer cell lines, of which lag time and time to peak correlated with TF cell surface expression.

Development of lipid nanoparticle formulations of siRNA for hepatocyte gene silencing following subcutaneous administration.

Recently developed lipid nanoparticle (LNP) formulations of siRNA have proven to be effective agents for hepatocyte gene silencing following intravenous administration with at least three LNP-siRNA formulations in clinical trials. The aim of this work was to develop LNP-siRNA systems for hepatocyte gene silencing that can be administered subcutaneously (s.c.). Three parameters were investigated, namely LNP size, residence time of the polyethylene glycol (PEG)-lipid coating and the influence of hepatocyte-specific targeting ligands. LNP sizes were varied over the range of 30 to 115 nm in diameter and PEG-lipid that dissociates rapidly (PEG-DMG) and slowly (PEG-DSG) were employed. In mice, results show that large (~80 nm) LNP exhibited limited accumulation in the liver and poor Factor VII (FVII) gene silencing at 1mg siRNA/kg body weight. Conversely, small (~30 nm) LNP systems showed maximal liver accumulation yet still had minimal activity. Interestingly, intermediate size (~45 nm) LNP containing PEG-DSG exhibited nearly equivalent liver accumulation as the smaller systems following s.c. administration but reduced FVII levels by 80% at 1mg siRNA/kg body weight. Smaller systems (~35 nm diameter) containing either PEG-DMG or PEG-DSG were less active; however addition of 0.5 mol.% of a GalNAc-PEG lipid to these smaller systems improved activity to levels similar to that observed for the ~45 nm diameter systems. In summary, this work shows that appropriately designed LNP-siRNA systems can result in effective hepatocyte gene silencing following s.c administration.

Prothrombin complex concentrates as reversal agents for new oral anticoagulants: lessons from preclinical studies with Beriplex.

Although new oral anticoagulants (NOACs) represent an advance in anticoagulant therapy over vitamin K antagonists (VKAs), they nevertheless have a low, but significant risk for bleeding complications. Reversal agents for VKAs, such as prothrombin complex concentrates (PCCs), are currently being evaluated in preclinical studies for NOAC reversal. This article reviews the preclinical data for the most extensively studied PCC for NOAC reversal, Beriplex, a 4-factor PCC. The results from the Beriplex studies are also compared with those obtained with other reversal agents, including different nonactivated PCCs, activated PCCs, and recombinant activated factor VII.

Pharmacodynamics of recombinant activated factor VII and plasma-derived factor VII in a cohort of severe FVII deficient patients.

Recombinant activated factor VII (rFVIIa) and plasma-derived factor VII (pdFVII) are used to prevent bleedings in severe FVII deficient patients, despite their short half-lifes. It is suggested that FVII levels of 15-20 IU/dL are sufficient to maintain hemostasis. We analyzed the pharmacodynamic effects of FVII substitution therapy in the Nijmegen Hemostasis Assay (NHA) that simultaneously measures thrombin and plasmin generation. Ten severe FVII deficient patients were treated with 20 µg/kg rFVIIa or 25 IU/kg pdFVII in a cross-over design. Thrombin generation lag-time (TG-LT) was identified as an effect-response parameter. Pharmacodynamic analysis using a maximum effect model showed 50% reduction of the TG-LT effect at ~2 IU/dL FVII activity for both rFVIIa and pdFVII. The FVII activity to obtain TG-LT comparable to the upper limit of normal range in healthy controls (4 min) was given by the effective concentration (ECnormal), showing sufficient hemostasis at 3-4 IU/dL FVII activity. No association was seen between FVII activity and other thrombin or plasmin generation parameters as measured by NHA. In conclusion, 3-4 IU/dL FVII activity seems sufficient to maintain hemostasis in patients with severe FVII deficiency during prophylaxis. These data may suggest a potential value for measurement of TG-LT in the monitoring of FVII(a) therapy.

Reversal of dabigatran anticoagulation by prothrombin complex concentrate (Beriplex P/N) in a rabbit model.

BACKGROUND: One limitation of the direct thrombin inhibitor dabigatran is the lack of specific antidotes that allow acute bleeding events to be managed or urgent interventional procedures performed. Prothrombin complex concentrates (PCCs) have served as a standard treatment for the reversal of coumarin anticoagulation. OBJECTIVES: This study was designed to determine in an animal model whether a PCC (Beriplex P/N) can effectively reverse the effects of dabigatran. An additional objective was to evaluate markers of dabigatran-associated bleeding diathesis. METHODS: Anesthetized rabbits were treated with 0.4 mg kg(-1) dabigatran followed by PCC doses of 20, 35 or 50 IU kg(-1) or placebo. After a standardized kidney incision, volume of blood loss and time to hemostasis were determined. RESULTS: From an initial mean of 29 mL, blood loss progressively declined by 5.44 mL with a 95% confidence interval (CI) of 2.21-8.67 mL per 10 IU kg(-1) increment in PCC dose (P = 0.002). At a PCC dose of 50 IU kg(-1) blood loss was fully normalized. Increasing PCC doses shortened the median time to hemostasis from 20.0 to 5.7 min (P < 0.001). The rate of hemostasis was nearly trebled with each 10 IU kg(-1) increment in PCC dose (rate ratio, 2.89; CI, 1.64-5.09). CONCLUSIONS: In this animal study, PCC showed potential as an agent for reversing the effects of dabigatran. Further investigation is warranted.

Hemostatic properties of the FVIIa analog NN1731.

Recombinant activated coagulation factor VII (rFVIIa) has proven to be a useful prohemostatic agent in patients with hemophilia and antibody inhibitors. It has also been used off-label in other settings. A major mechanism of its hemostatic efficacy is its ability to activate factor X on the surface of activated platelets in a tissue factor (TF)-independent manner. Novel analogs of FVIIa have been designed to have greater platelet-surface activity in the hope that these will be more active and reliable hemostatic agents. The analog NN1731 (vatreptacog alfa) was designed to have greater activity in the absence of TF, with the same substrate specificity as FVIIa. Surprisingly, it also binds to a greater number of sites on activated platelets by an, as yet, unknown mechanism. This molecule exhibits greater potency than FVIIa in biochemical assays, in vitro and animal models, and in early phase clinical trials.

Recombinant activated coagulation factor VII and prothrombin complex concentrates are equally effective in reducing hematoma volume in experimental warfarin-associated intracerebral hemorrhage.

BACKGROUND AND PURPOSE: Based on an experimental model of warfarin-associated intracerebral hemorrhage, we investigated whether the rapid reversal of anticoagulation using prothrombin complex concentrates (PCC) or recombinant activated coagulation factor VII (rFVIIa) reduces hematoma volume. METHODS: Mice were orally pretreated with warfarin (2 mg/kg). Intracerebral hemorrhage was induced by collagenase injection into the right striatum. Forty-five minutes later, PCC (100 IE/kg), rFVIIa (1 mg/kg), or an equal volume of saline was administered intravenously. Hematoma volume after 24 hours was quantified using a photometric hemoglobin assay. RESULTS: International normalized ratio was 4.3±0.4 in saline-treated mice, 0.9±0.1 in rFVIIa mice, and 1.4±0.2 in PCC mice. Intracerebral hemorrhage volume was 29.0±19.7 µL in the saline group (n=7), 8.6±4.3 µL in the rFVIIa group (n=6), and 6.1±1.8 µL in the PCC group (n=7; analysis of variance between-group differences P=0.004; post hoc rFVIIa versus saline P=0.021; PCC versus saline P=0.007). No significant difference was found between PCC- and rFVIIa-treated animals. CONCLUSIONS: Our results suggest that PCC and rFVIIa are equally effective in restoring coagulation and preventing excessive hematoma growth in acute warfarin-associated intracerebral hemorrhage.

Recombinant factor VIIa analog NN1731 (V158D/E296V/M298Q-FVIIa) enhances fibrin formation, structure and stability in lipidated hemophilic plasma.

INTRODUCTION: The bypassing agent recombinant factor VIIa (rFVIIa) is efficacious in treating bleeding in hemophilia patients with inhibitors. Efforts have focused on the rational engineering of rFVIIa variants with increased hemostatic potential. One rFVIIa analog (V158D/E296V/M298Q-FVIIa, NN1731) improves thrombin generation and clotting in purified systems, whole blood from hemophilic patients and factor VIII-deficient mice. METHODS: We used calibrated automated thrombography and plasma clotting assays to compare effects of bypassing agents (rFVIIa, NN1731) on hemophilic clot formation, structure, and ability to resist fibrinolysis. RESULTS: Both rFVIIa and NN1731 shortened the clotting onset and increased the maximum rate of fibrin formation and fibrin network density in hemophilic plasma clots. In the presence of tissue plasminogen activator, both rFVIIa and NN1731 shortened the time to peak turbidity (TTPeak(tPA)) and increased the area under the clot formation curve (AUC(tPA)). Phospholipids increased both rFVIIa and NN1731 activity in a lipid concentration-dependent manner. Estimated geometric mean concentrations of rFVIIa and NN1731 producing similar onset, rate, TTPeak(tPA), and AUC(tPA) as seen with 100% factors VIII and IX were: 24.5, 74.3, 29.7, and 37.1 nM rFVIIa, and 8.6, 31.2, 9.0, and 11.3 nM NN1731, respectively. In each case, the NN1731 concentration was significantly lower than rFVIIa. CONCLUSIONS: These findings suggest that like rFVIIa, NN1731 improves the formation, structure, and stability of hemophilic clots. Higher lipid concentrations may facilitate assessment of both rFVIIa and NN1731 activity. NN1731 appears likely to support rapid clot formation in tissues with high endogenous fibrinolytic activity.

Clearance of rFVIIa and NN1731 after intravenous administration to Beagle dogs.

AIM: NN1731 is a recombinant activated factor VII (rFVIIa) analogue with enhanced activity. The objective of the present study was to evaluate the clearance mechanisms of rFVIIa and NN1731 after intravenous administration to Beagle dogs. METHODS: The study was performed in Beagle dogs administered with a single dose of 5.4 nmol/kg rFVIIa or NN1731 intravenously. Plasma samples collected up to 12-h post-administration were analysed using three different assays to determine FVIIa clot activity (FVIIa:C), total FVIIa antigen, and levels of FVIIa-antithrombin (AT) complexes. Pharmacokinetic parameters were determined by use of standard non-compartmental and non-linear mixed effects methods. RESULTS: For both compounds, complex formation with AT accounted for the observed difference between the activity and the antigen curves and constituted 60-70% of the total clearance. The clearance of rFVIIa and NN1731 was estimated to be 73 and 214 mL/h/kg, respectively, accordingly, AT complex formation occurred around three times faster for NN1731. The difference in activity observed in the initial phase, resulting in distribution half-lives of 0.71 and 0.22 h for rFVIIa and NN1731, was mainly caused by the 3-fold difference in clearance. The terminal half-life of rFVIIa and NN1731 was estimated to be 2.1 and 2.5 h, respectively. The non-compartmental analysis resulted in almost identical parameters. CONCLUSION: The present study demonstrates that the difference between the activity and the antigen profiles of rFVIIa and NN1731 in Beagle dogs is the result of complex formation with AT which constitutes a major pathway for the clearance of rFVIIa activity.

FVII dependent coagulation activation in citrated plasma by polymer hydrogels.

Polymer hydrogels containing positively charged functional groups were used to investigate the critical material and biological components of FVII activation and subsequent fibrin formation in citrated plasma. A FVIIa ELISA confirmed the ability of the polymer to induce FVII activation and provided insight into the material parameters which were influential in this activation. Experiments utilizing coagulation factor depleted and inhibited plasmas indicated that FVII, FX, FII, and FI are all vital to the process outlining the general mechanism of fibrin formation from the onset of FVII activation. Dynamic mechanical analysis and swelling experiments were used to establish a critical correlation between polymer microstructure and FVII activation.

Recombinant activated factor VII: mechanisms of action and current indications.

Recombinant activated factor VII (rFVIIa) was initially developed to overcome the limitations of existing treatments for patients with congenital hemophilia and inhibitors. Clinical success in this arena led to experimental use in other coagulopathies characterized by impaired thrombin generation, and approval for use in acquired hemophilia, factor VII deficiency, and Glanzmann's thrombasthenia soon followed. Extensive research and growing experience in the clinical setting has increased our knowledge of rFVIIa and improved our understanding of how its mechanism of action leads to effective bleeding control. This article reviews current understanding of the mechanisms of action of rFVIIa before providing a brief overview of clinical experience to date in its licensed indications. The agent's safety profile is also examined, along with recent advances in rFVIIa dosing and storage that may help to improve both clinical outcomes and patient quality of life.

Factor VIIa analog has marked effects on platelet function and clot kinetics in blood from patients with hemophilia A.

To evaluate the hemostatic effects of NN1731 and rFVIIa, an ex-vivo study in hemophilia patients used the Hemodyne Hemostasis Analysis System (HAS) to measure platelet contractile force (PCF), clot elastic modulus (CEM), and force onset time (FOT), and the Haemoscope Thrombelastograph (TEG) to measure reaction time (R), kinetics time (K), and maximum amplitude (MA). Blood samples from 10 healthy volunteers and 10 Factor VIII-deficient patients of varying severity (mild, moderate, severe), were spiked with rFVIIa and NN1731 (both 0.64 and 1.28 microg/ml, respectively) and analyzed to characterize platelet function and clot kinetics. There was wide variability in the rFVIIa response. NN1731 had greater and more consistent effects on PCF, CEM, FOT, R, and K relative to rFVIIa, in all hemophilia groups. The lowest NN1731 concentration (0.64 microg/ml) shortened R and FOT, and increased CEM and PCF more than rFVIIa 1.28 microg/ml. NN1731 normalized clotting parameters equivalent to values obtained in healthy volunteers. FOT and R were highly correlated (r = 0.96). No correlation was observed between CEM and MA. NN1731 produced less variable, more pronounced and predictable ex-vivo hemostatic effects on PCF, CEM, FOT, R and K than rFVIIa in all hemophilia groups. HAS and TEG assays provided similar estimates of FOT and R, however CEM appeared to be more sensitive than MA to changes in clot firmness.

Thrombin generation and platelet activation induced by rFVIIa (NovoSeven) and NN1731 in a reconstituted cell-based model mimicking haemophilia conditions.

Replacement therapy with factor VIII (FVIII) and factor IX (FIX) is routinely used in haemophilia patients with haemophilia A and B, respectively, while recombinant activated FVII (rFVIIa) has proven to induce haemostasis in haemophilia patients with inhibitors. To evaluate the effect of therapeutic intervention in patients with residual factor activities, the effects of increasing concentrations of rFVIIa or NN1731 on thrombin generation and platelet activation were measured in a cell-based model system mimicking severe, moderate and mild haemophilia A or B. Purified monocytes stimulated to express tissue factor and non-activated platelets from peripheral blood of healthy donors were incubated with a mixture of purified human coagulation factors in the absence or presence of increasing concentrations of FVIII or FIX. Sub-samples were analysed for thrombin activity and platelet activation measured as exposure of P-selectin by flow cytometry. Dose-dependent increases in thrombin generation and platelet activation were observed following increasing concentrations of rFVIIa or NN1731 in both haemophilia A- and B-like conditions. At 25 nm rFVIIa, which nears the peak levels in patient plasma after 90 microg kg(-1) intravenous dosing, the effects on maximum thrombin generation rate (maxTG) at 1-10% FVIII were comparable to those at 100% and 200% FVIII in the absence of rFVIIa. Normalization of maxTG required 500 nm rFVIIa and 25 nm NN1731 or 25-100 nm rFVIIa and 5 nm NN1731 in severe or moderate/mild haemophilia A and haemophilia B, respectively. This suggests that NN1731 holds its promise as a future bypassing agent for haemophilia patients with and without inhibitors.