Innovative spiral electrode configuration for enhancement of electrocoagulation-flotation.

The performance of electrocoagulation-flotation (ECF) process can profoundly be affected by the reactor design and electrode configuration. These may, in turn, influence the removal efficiency, flow hydrodynamic, floc formation, and flotation/settling characteristics. The present work aimed at developing a new spiral electrode configuration to enhance the ECF process. To do so, the impacts of parameters such as energy consumption, removal efficiency of the contaminants from industrial wastewater with a composition of turbidity, emulsified oil, and heavy metals (Si, Zn, Pb, Ni, Cu, Cr, and Cd), as well as stirring speed and foaming have been investigated. Comparison was also made between the experimental results of the new electrode configuration with the conventional rectangular cell with plate electrode configuration with the same volume and electrode surface area. The findings revealed that energy consumption of the spiral electrode configuration within the operating times of 10, 20, 30, 32, 48, and 70 min, was approximately 20% lower compared to that of the conventional ECF. Moreover, the maximum and minimum removal efficiency of 97% and 60% were obtained for turbidity and TOC for the stirring speed of 500 rpm and Reynolds number of 10,035, respectively. Finally, the formed gas bubbles tilted toward the center due to the enhanced flow hydrodynamic which resulted in substantial reduction of foam formation.

Identification of a modified coagulation factor X with enhanced activation properties as potential hemostatic agent.

Hemophilia A and hemophilia B are X-linked inherited bleeding disorders caused by a deficiency of coagulation factor VIII and IX, respectively. Standard of care is prophylactic factor replacement therapy; however, the development of neutralizing antibodies against these factors represents serious complications underlining the need for alternative treatment approaches. Human coagulation factor X has a central role within the blood coagulation system making it an attractive target for the development of alternative treatment strategies for patients with hemophilia. This study focuses on a modified variant of the human coagulation factor X with enhanced hemostatic bypass activity due to insertion of a factor IX derived activation sequence. This molecule design leads to the direct activation of the modified factor X protein by factor XIa allowing it to bypass the need for coagulation factor VIIIa/factor IXa. The modified variant was able to correct in-vitro activated partial prothrombin time of human and murine factor VIII/factor IX deficient plasma. Furthermore, reduced blood loss in factor VIII knock-out mice was observed after intravenous application of the modified factor X variant. In conclusion, these data suggest that the factor X variant described here could potentially serve as a bypassing agent independent of the inhibitor status of hemophilia patients. However, more research is needed to further investigate the potential of this molecule.

FXIIa inhibitor rHA-Infestin-4: Safe thromboprotection in experimental venous, arterial and foreign surface-induced thrombosis.

Haemostasis including blood coagulation is initiated upon vessel wall injury and indispensable to limit excessive blood loss. However, unregulated pathological coagulation may lead to vessel occlusion, causing thrombotic disorders, most notably myocardial infarction and stroke. Furthermore, blood exposure to foreign surfaces activates the intrinsic pathway of coagulation. Hence, various clinical scenarios, such as extracorporeal membrane oxygenation, require robust anticoagulation consequently leading to an increased bleeding risk. This study aimed to further assess the antithrombotic efficacy of the activated factor XII (FXIIa) inhibitor, rHA-Infestin-4, in several thrombosis models. In mice, rHA-Infestin-4 decreased occlusion rates in the mechanically-induced arterial (Folt's) and the FeCl3 -induced venous thrombosis model. rHA-Infestin-4 also protected from FeCl3 -induced arterial thrombosis and from stasis-prompted venous thrombosis in rabbits. Furthermore, rHA-Infestin-4 prevented occlusion in the arterio-venous shunt model in mice and rabbits where thrombosis was induced via a foreign surface. In contrast to heparin, the haemostatic capacity in rabbits was unaffected by rHA-Infestin-4. Using rodent and non-rodent species, our data demonstrate that the FXIIa inhibitor rHA-Infestin-4 decreased arterial, venous and foreign surface-induced thrombosis without affecting physiological haemostasis. Hence, we provide further evidence that targeting FXIIa represents a potent yet safe antithrombotic treatment approach, especially in foreign surface-triggered thrombosis.

The G534E polymorphism of the gene encoding the factor VII-activating protease is associated with cardiovascular risk due to increased neointima formation.

The G534E polymorphism (Marburg I [MI]) of factor VII-activating protease (FSAP) is associated with carotid stenosis and cardiovascular disease. We have previously demonstrated that FSAP is present in atherosclerotic plaques and it is a potent inhibitor of vascular smooth muscle proliferation and migration in vitro. The effect of wild-type (WT)- and MI-FSAP on neointima formation in the mouse femoral artery after wire-induced injury was investigated. Local application of WT-FSAP led to a 70% reduction in the neointima formation, and this effect was dependent on the protease activity of FSAP. MI-FSAP did not inhibit neointima formation in vivo. This is due to a reduced proteolytic activity of MI-FSAP, compared to WT-FSAP, toward platelet-derived growth factor BB, a key mediator of neointima development. The inability of MI-FSAP to inhibit vascular smooth muscle accumulation explains the observed linkage between the MI-polymorphism and increased cardiovascular risk. Hence, FSAP has a protective function in the vasculature, and analysis of MI polymorphism is likely to be clinically relevant in restenosis.

Increased potency of recombinant VWF D'D3 albumin fusion proteins engineered for enhanced affinity for coagulation factor VIII.

BACKGROUND: We have recently reported on a recombinant von Willebrand factor (VWF) D'D3 albumin fusion protein (rD'D3-FP) developed to extend the half-life of coagulation factor VIII (FVIII) for the treatment of hemophilia A. Based on predictive modelling presented in this study, we hypothesized that modifying rD'D3-FP to improve FVIII interaction would reduce exchange with endogenous VWF and provide additional FVIII half-life benefit. OBJECTIVES: The aim of this study was to identify novel rD'D3-FP variants with enhanced therapeutic efficacy in extending FVIII half-life. METHODS: Through both directed mutagenesis and random mutagenesis using a novel mammalian display platform, we identified novel rD'D3-FP variants with increased affinity for FVIII (rVIII-SingleChain) under both neutral and acidic conditions and assessed their ability to extend FVIII half-life in vitro and in vivo. RESULTS: In rat preclinical studies, rD'D3-FP variants with increased affinity for FVIII displayed enhanced potency, with reduced dose levels required to achieve equivalent rVIII-SingleChain half-life extension. In cell-based imaging studies in vitro, we also demonstrated reduced dissociation of rVIII-SingleChain from the rD'D3-FP variants within acidic endosomes and more efficient co-recycling of the rD'D3-FP/rVIII-SingleChain complex via the FcRn recycling system. CONCLUSIONS: In summary, at potential clinical doses, the rD'D3-FP variants provide marked benefits with respect to dose levels and half-life extension of co-administered FVIII, supporting their development for use in the treatment of hemophilia A.

FVIII half-life extension by coadministration of a D'D3 albumin fusion protein in mice, rabbits, rats, and monkeys.

A novel mechanism for extending the circulatory half-life of coagulation factor VIII (FVIII) has been established and evaluated preclinically. The FVIII binding domain of von Willebrand factor (D'D3) fused to human albumin (rD'D3-FP) dose dependently improved pharmacokinetics parameters of coadministered FVIII in all animal species tested, from mouse to cynomolgus monkey, after IV injection. At higher doses, the half-life of recombinant FVIII (rVIII-SingleChain) was calculated to be increased 2.6-fold to fivefold compared with rVIII-SingleChain administered alone in rats, rabbits, and cynomolgus monkeys, and it was increased 3.1-fold to 9.1-fold in mice. Sustained pharmacodynamics effects were observed (ie, activated partial thromboplastin time and thrombin generation measured ex vivo). No increased risk of thrombosis was observed with coadministration of rVIII-SingleChain and rD'D3-FP compared with rVIII-SingleChain alone. At concentrations beyond the anticipated therapeutic range, rD'D3-FP reduced the hemostatic efficacy of coadministered rVIII-SingleChain. This finding might be due to scavenging of activated FVIII by the excessive amount of rD'D3-FP which, in turn, might result in a reduced probability of the formation of the tenase complex. This observation underlines the importance of a fine-tuned balance between FVIII and its binding partner, von Willebrand factor, for hemostasis in general.

Genetic fusion to albumin improves the pharmacokinetic properties of factor IX.

Haemophilia B is a X-chromosome linked disease characterised by a deficiency of functionally active coagulation Factor IX (FIX). Patients with severe haemophilia B at risk of recurrent bleeding are treated approximately twice a week in a prophylactic setting by application of FIX concentrates. To increase convenience and compliance of the therapy it is desirable to reduce the dosing frequency by improving the pharmacokinetic properties of FIX. Here a concept of rFIX (recombinant factor IX) albumin fusion proteins (rIX-FPs) with cleavable linker peptides derived from the FIX activation sequence is presented. Constructs of the genetic fusion of FIX to albumin via cleavable linkers were expressed in mammalian cells and characterised after purification. In vitro activation studies with FXIa demonstrated that cleavage of the linker and the activation peptide proceeded comparably well. In a clotting assay the rIX-FPs with cleavable linker showed a 10- to 30-fold increase in the molar specific clotting activity compared to fusion proteins with non-cleavable linkers. Furthermore, in-vivo recovery, terminal half-life and the AUC of rIX-FPs in rats and rabbits as determined by FIX antigen measurements were significantly increased compared to rFIX (BeneFIX). In FIX deficient (FIX(-/-)) mice the in-vivo recovery and the AUC were also significantly increased. The efficacy in reducing bleeding time was shown in FIX(-/-) mice by a tail tip bleeding model. The results suggest that rIX-FPs with a cleavable linker between FIX and albumin are a promising concept that may support the use of the albumin fusion technology to extend the half-life of FIX.

Prolonged in-vivo half-life of factor VIIa by fusion to albumin.

For the treatment of haemophilia patients with inhibitors, recombinant factor VIIa (rFVIIa) is available as a therapeutic option to control bleeding episodes with a good balance of safety and efficacy. However, the short in-vivo half-life of approximately 2.5 hours makes multiple injections necessary, which is inconvenient for both physicians and patients. Here we describe the generation of a recombinant FVIIa molecule with an extended half-life based on genetic fusion to human albumin. The recombinant FVII albumin fusion protein (rVII-FP) was expressed in mammalian cells and upon activation displayed a FVII activity close to that of wild type FVIIa. Pharmacokinetic studies in rats demonstrated that the half-life of the activated recombinant FVII albumin fusion protein (rVIIa-FP) was extended six- to seven-fold compared with wild type rFVIIa. The in-vitro and in-vivo efficacy was evaluated and was found to be comparable to a commercially available rFVIIa (NovoSeven((R))). The results of this study demonstrate that it is feasible to develop a half-life extended FVIIa molecule with haemostatic properties very similar to the wild-type factor.

The Coagulation Factor XIIa Inhibitor rHA-Infestin-4 Improves Outcome after Cerebral Ischemia/Reperfusion Injury in Rats.

BACKGROUND AND PURPOSE: Ischemic stroke provokes severe brain damage and remains a predominant disease in industrialized countries. The coagulation factor XII (FXII)-driven contact activation system plays a central, but not yet fully defined pathogenic role in stroke development. Here, we investigated the efficacy of the FXIIa inhibitor rHA-Infestin-4 in a rat model of ischemic stroke using both a prophylactic and a therapeutic approach. METHODS: For prophylactic treatment, animals were treated intravenously with 100 mg/kg rHA-Infestin-4 or an equal volume of saline 15 min prior to transient middle cerebral artery occlusion (tMCAO) of 90 min. For therapeutic treatment, 100 mg/kg rHA-Infestin-4, or an equal volume of saline, was administered directly after the start of reperfusion. At 24 h after tMCAO, rats were tested for neurological deficits and blood was drawn for coagulation assays. Finally, brains were removed and analyzed for infarct area and edema formation. RESULTS: Within prophylactic rHA-Infestin-4 treatment, infarct areas and brain edema formation were reduced accompanied by better neurological scores and survival compared to controls. Following therapeutic treatment, neurological outcome and survival were still improved although overall effects were less pronounced compared to prophylaxis. CONCLUSIONS: With regard to the central role of the FXII-driven contact activation system in ischemic stroke, inhibition of FXIIa may represent a new and promising treatment approach to prevent cerebral ischemia/reperfusion injury.

Marburg I polymorphism of factor VII--activating protease: a prominent risk predictor of carotid stenosis.

BACKGROUND: Atherothrombosis is a main pathomechanism in the evolution of vessel stenosis and is counteracted by endogenous fibrinolysis. Recently, the plasmatic serine protease "factor seven-activating protease" (FSAP) was recognized as a potent activator of prourokinase in vitro. The Marburg I polymorphism of FSAP impairs this potential and may thus facilitate arterial thrombosis. METHODS AND RESULTS: This analysis of the Bruneck Study involved 810 men and women aged 40 to 79 years. The ultrasound-based atherosclerosis progression model (5-year follow-up) permits differentiation between early atherogenesis and the advanced stenotic stages of carotid artery disease. The FSAP Marburg I polymorphism was found in 37 subjects (carriage rate 4.4%). Individuals with this genetic variant showed a prominently reduced in vitro capacity to activate prourokinase. No relation was found to exist between the Marburg I polymorphism and early atherogenesis. In contrast, it emerged as a strong and independent risk predictor of incident/progressive carotid stenosis (multivariate odds ratio [95%CI], 6.6 [1.6 to 27.7]). This finding equally applied to subjects with and without co-segregation of the Marburg II polymorphism. The risk profile of advanced atherogenesis further includes cigarette smoking, high lipoprotein(a), the factor V Leiden mutation, low antithrombin III, high fibrinogen, and diabetes. CONCLUSIONS: In concert with other genetic and acquired conditions known to interfere with coagulation or fibrinolysis, the Marburg I polymorphism of FSAP, which attenuates its capacity to activate prourokinase, is a significant risk predictor for the evolution and progression of carotid stenosis.

Non-clinical pharmacokinetics and pharmacodynamics of rVIII-SingleChain, a novel recombinant single-chain factor VIII.

INTRODUCTION: rVIII-SingleChain (CSL627), a novel recombinant coagulation factor VIII (FVIII), is under investigation in a phase I/III clinical programme (AFFINITY) for the treatment of haemophilia A. Non-clinical studies were conducted to investigate the pharmacokinetic/pharmacodynamic profile of rVIII-SingleChain in comparison with full-length recombinant FVIII. MATERIALS AND METHODS: Binding affinity of rVIII-SingleChain for von Willebrand factor was investigated by surface plasmon resonance analysis. The pharmacokinetic profile of rVIII-SingleChain was compared with a marketed full-length recombinant FVIII concentrate (Advate(®)) in haemophilia A mice, von Willebrand factor knock-out mice, Crl:CD (SD) rats, rabbits and cynomolgus monkeys. Systemic FVIII activity or antigen levels were recorded. Procoagulant activity was measured in an FeCl3-induced arterial occlusion model and by recording thrombin generation activity (ex vivo) after administration of 200-250 IU/kg rVIII-SingleChain or full-length FVIII to haemophilia A mice. RESULTS: rVIII-SingleChain displayed a high affinity for von Willebrand factor (KD=44 pM vs. 139 pM for full-length recombinant FVIII). In all animal species tested, rVIII-SingleChain had more favourable pharmacokinetic properties than full-length recombinant FVIII: clearance was decreased and area under the curve and terminal half-life were enhanced vs. full-length recombinant FVIII, while in vivo recovery and volume of distribution were equivalent. rVIII-SingleChain showed a prolonged thrombin generation potential and prolonged procoagulant activity vs. full-length recombinant FVIII in an FeCl3-induced arterial occlusion model. CONCLUSIONS: rVIII-SingleChain had a higher affinity for von Willebrand factor than full-length recombinant FVIII and displayed favourable pharmacokinetic/pharmacodynamic properties in non-clinical models.

Preclinical efficacy and safety of rVIII-SingleChain (CSL627), a novel recombinant single-chain factor VIII.

INTRODUCTION: The preclinical efficacy and safety of rVIII-SingleChain (CSL627), a novel recombinant single-chain factor VIII, was assessed in a series of animal studies. MATERIALS AND METHODS: In the tail-clip bleeding model, hemophilia A mice were injected with escalating doses (1-150 IU/kg) of rVIII-SingleChain, B-domain deleted (BDD) rFVIII (ReFacto AF(®)), or full-length rFVIII products (Advate(®), Helixate(®)). Total blood loss and the percentage of animals in which hemostasis occurred were assessed in this observer-blinded, randomized study. In a second non-randomized study in hemophilia A mice, thromboelastographic analysis, thrombin generation, and activated partial thromboplastin time assays were performed. General safety and toxicity were assessed in three animal species, including determination of the prothrombotic potential of rVIII-SingleChain in a rabbit venous thrombosis model. RESULTS: Under acute bleeding conditions, the effect of rVIII-SingleChain on total blood loss and hemostasis was indistinguishable from BDD and full-length rFVIII. rVIII-SingleChain and full-length rFVIII (both 20 IU/kg) corrected thromboelastographic parameters, activated partial thromboplastin time, and thrombin generation to a similar degree in hemophilia A mice. In a thrombosis model, the effect of rVIII-SingleChain on thrombus incidence was non-significant and comparable to BDD rFVIII at doses up to 500 IU/kg. Treatment with rVIII-SingleChain did not cause anaphylactic reaction or local intolerance in safety and toxicity studies, and demonstrated an excellent overall safety profile. CONCLUSIONS: rVIII-SingleChain showed convincing hemostatic efficacy and excellent tolerability in animal studies, warranting continued investigation in human Phase I/III trials (AFFINITY).

Structure-function analysis of factor VII activating protease (FSAP): sequence determinants for heparin binding and cellular functions.

Factor VII activating protease (FSAP) is associated with cardiovascular diseases and liver fibrosis. To understand the regulation of its proteolytic activity we have characterized recombinant FSAP-mutants over-expressed in HEK-293 cells. The secreted FSAP-protein concentration correlated inversely with the enzymatic activity of the FSAP-mutants. Over-expression of enzymatically active FSAP decreased cell viability, whereas inactive variants were expressed and secreted in adequate amounts. The naturally occurring G534E-variant exhibited reduced proteolytic activity. The DeltaEGF-3 mutant showed diminished binding to and activation by heparin. Hence, regulation of FSAP activity is dependent on its EGF-3 domain and over-expression of active variants induces cell death.

Technical considerations for the performance of Nucleic acid Amplification Technology (NAT). The NAT Task Force Group.

The complexity of Nucleic acid Amplification Technology (NAT(1)), comprising sample preparation, amplification and detection methods, requires specific design considerations for both the laboratory and the procedures utilized in such testing. The purpose of this paper is to establish technical considerations for the performance of NAT. These include the collection, handling and assay of specimens and the design of laboratories to routinely and reliably detect low levels of nucleic acid sequences. The sensitivity of NAT due to the exponential amplification of nucleic acids makes contamination a major concern from specimen collection to sample detection. Therefore, laboratories need to be designed to prevent and control contamination through adequate equipment and appropriate workflow. These technical considerations should provide a basis for establishing a robust and reproducible NAT system.