Quantification of plasma-derived blood coagulation factor VIII by real-time biosensor measurements

Plasma-derived blood coagulation factor VIII was analyzed in real time using biosensor technology. Monoclonal antibodies directed against the heavy and against the light chain of factor VIII were immobilized on different carboxymethyl dextran surfaces. Different factor VIII concentrations were injected over the antibody surfaces in parallel and response levels were determined from the dissociation phase at a fixed time after sample injection. Serial dilutions of plasma-derived factor VIII with known concentrations determined by a commercial FVIIIC:Ag ELISA were used as standards. A quantification limit of 0.9 I.U./ml with antibody 530p and 1.5 I.U./ml with antibody 531p was calculated. Intra-assay precision expressed as percent coefficient of variation was below 10% for concentrations above 0.6 I.U./ml. Inter-assay precision for antibody 530p was below 20% for concentrations higher than 0.6 I.U./ml. For 531p, inter-assay precision was below 10% for concentrations higher than 2 I.U./ml. A sensor chip lifetime in respect to regeneration of at least 100 cycles for both antibodies was found. The small sample requirement of 35 μl allows fast analysis of different FVIII products and the use of two monoclonal antibodies directed against two different FVIII domains provides additional information about the integrity of the FVIII molecule.     

Structure determination of the major asparagine-linked sugar chain of human factor VIII--von Willebrand factor

N-glycosidically-linked glycans released by hydrazinolysis of human factor VIII/von Willebrand factor (FVIII/vWf) were separated by high-voltage electrophoresis. Five fractions were obtained, one of them representing 60% of the total amount of the N-glycosidically-linked glycans of FVIII/vWf. On the basis of the carbohydrate composition, methylation analysis and 500 MHz 1H-NMR spectroscopy, we describe the primary structure of this major glycan which is of the monosialylated and monofucosylated biantennary N-acetyllactosaminic type.     

凝血因子IX基因剔除小鼠的培育历史与建系方法

目的　培育凝血因子IX基因剔除小鼠近交系。方法　采用全同胞兄妹对交配 ,每代选用第一胎小鼠留种 ,记录繁殖性能 ;第 8代开始筛选纯合子。结果　凝血因子IX基因剔除小鼠已近交培育到第 12代 ,交配年龄 70 - 90d ,平均每窝产仔数 5只以上 ,离乳数 3只以上。结论　纯合子FIX剔除基因小鼠在子代鼠中能稳定遗传     

A novel core fractionation process of human plasma by expanded bed adsorption chromatography

Current plasma fractionation technology combines ethanol precipitation with packed bed chromatography. We have developed a novel core fractionation process comprising five expanded bed adsorption (EBA) chromatographic steps on high-density modified agarose/tungsten carbide beads. Plasma was first chromatographed on two diethyl amino-ethyl (DEAE)-tungsten carbide agarose adsorbents (respective mean particle diameters of d_v(0.5) = 190 and 37 μm) to isolate at 50 to 80% recovery a fraction containing 4 to 7 IU/ml factor II (FII), factor IX (FIX), and factor X (FX) (specific activity >1 IU/mg) and another enriched in FVIII and von Willebrand factor (vWF) (∼1 IU/ml and 0.6 IU/mg, respectively). The flow-through was adsorbed on 4% agarose-10% tungsten carbide beads coupled with an acidic mixed-mode ligand to isolate an 80% pure immunoglobulin G (IgG) at a 93% step recovery. A highly purified α1-antitrypsin was isolated at 95% step recovery by adsorbing the flow-through on 4% epoxy-crosslinked agarose-10% tungsten carbide adsorbent material coupled with a cationic ligand. Isolation of 98% pure albumin was achieved at a 99% step recovery by pH 4.5 adsorption of the flow-through on 6% agarose-10% tungsten carbide beads coupled with an acidic mixed-mode ligand. EBA may represent a feasible alternative core plasma fractionation tool.     

Isolation of the factor VIII–von Willebrand factor complex directly from plasma by gel filtration

A high capacity gel filtration system was developed with the purpose of isolating factor VIII (FVIII) and von Willebrand factor (vWF) directly from plasma in significantly higher yields than obtained by cryoprecipitation, the technique most commonly used to recover FVIII–vWF from human plasma. After laboratory-scale gel filtration of plasma, a FVIII-containing fraction was collected containing about 90% of FVIII in the applied plasma and with almost tenfold higher purity than that obtained by cryoprecipitation. The gel filtration step has been scaled up for use as the initial step in the manufacturing process for a FVIII preparation (Nordiate).     

The treatment of hemophilia A: from protein replacement to AAV-mediated gene therapy

Factor VIII (FVIII) is an essential component in blood coagulation, a deficiency of which causes the serious bleeding disorder hemophilia A. Recently, with the development of purification level and recombinant techniques, protein replacement treatment to hemophiliacs is relatively safe and can prolong their life expectancy. However, because of the possibility of unknown contaminants in plasma-derived FVIII and recombinant FVIII, and high cost for hemophiliacs to use these products, gene therapy for hemophilia A is an attractive alternative to protein replacement therapy. Thus far, the adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. It is likely that hemophilia will be the first genetic disease to be cured by somatic cell gene therapy.     

Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos

Transgenic rabbits provide a useful biological model for the study of the regulation of mammalian genes. However, transgene integration efficiency has generally been low. Here we present a first attempt to increase the integration rate of exogenous DNA into the rabbit genome, using a double pronuclei microinjection method. Pronuclear stage rabbit embryos were recovered from superovulated NZW females, 19–20 h after hCG injection. About 5 μg/mL of exogenous DNA solution was microinjected either into one pronucleus (single microinjection, SM) or into both pronuclei (double microinjected, DM). The transgene consisted of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb cDNA of the human clotting factor VIII (hFVIII), and 4.6 kb that of 3′ flanking sequences of the mWAP gene. The in vitro survival of DM embryos to the blastocyst stage was lower than that of SM embryos (68 vs. 89%). Similar results were obtained using EGFP as a control gene construct. However, there was no difference in the percentage of embryos that developed into live offspring using DM (25%) vs. SM (26%). The integration frequency of mWAP-hFVIII into the genome of transgenic rabbits was 3.3% (1/30) upon SM and 8.1% (4/49) at DM (p < 0.05). All founders transmitted the transgene to their offspring in a Mendelian fashion. The SM founder female secreted 87.4 μg/mL rhFVIII in milk, with an activity of 0.594 lU/mL. The DM founder female produced 118 μg/mL rhFVIII, with activity values of 18 lU/mL. This is the first report of transgenic rabbit production using a double microinjection technique. Our preliminary results suggest that this method can increase the efficiency of production of transgenic rabbit founders, giving a higher integration rate than single microinjection.     

Virus removal capacity at varying ionic strength during nanofiltration of AlphaNine® SD

Nanofiltration is incorporated into the manufacturing processes of many protein biopharmaceuticals to enhance safety by providing the capacity to retain pathogens while allowing protein drugs to pass through the filter. Retention is mainly a function of size; however, the shape of the pathogen may also influence retention. The ability of the Viresolve^® Pro nanofilter to remove different sized viruses during the manufacture of a Coagulation Factor IX (Alphanine^® SD) was studied at varying ionic strength, a process condition with the potential to affect virus shape and, hence, virus retention. Eight viruses were tested in a scale-down of the nanofiltration process. Five of the viruses (EMCV, Reo, BVDV, HIV, PRV) were nanofiltered at normal sodium processing conditions and three (PPV, HAV and WNV) were nanofiltered at higher and lower sodium. Representative Reduction Factors for all viruses were ≥4.50 logs and removal was consistent over a wide range of ionic strength.     

erythro-β-hydroxyaspartic acid in bovine factor IX and factor X

To localize β-hydroxyaspartic acid in factor IX and factor X the two proteins were cleaved with cyanogen bromide and trypsin, respectively. Peptides containing β-hydroxyaspartic acid were isolated and subjected to Edman degradation. The phenylthiohydantoin derivative of β-hydroxyaspartic acid was identified by HPLC in position 3 in the factor IX fragment and in position 1 in the factor X fragment. This corresponds to position 64 in factor IX and position 63 in the light chain of factor X. The assignments were confirmed by subtractive Edman degradation and with the dansyl method.     

Purification, identification, and characterization of elastase on erythrocyte membrane as factor IX-activating enzyme

In our previous papers, we reported that factor IX (F-IX), when activated by erythrocyte membranes, causes coagulation. We report on purification, identification, and characterization of F-IX-activating enzyme extracted from human erythrocyte membranes. The enzyme whose amino acid sequence is almost in accord with neutrophil elastase was found in normal erythrocyte membrane. The molecular mass was slightly smaller than that of neutrophil elastase. The content of the enzyme in erythrocyte membranes was estimated to be 3.0-3.7 ng per 10(6)erythrocytes. The F-IX sites cleaved by the enzyme were slightly different from those by the ordinary coagulation reaction. The ability of F-IX cleaved by the enzyme to cause coagulation was estimated to be approximately 1/10 as high as that of the F-IX cleaved by activated F-XI. These findings provide evidence that F-IX is activated by erythrocyte membrane, which may serve as a triggering mechanism for blood coagulation.     

Purification of porcine plasma factor VIII using chromatographic methods

Factor VIII was purified from porcine plasma using adsorption on aluminium hydroxide with CM-cellulose as a filtration aid, cold ethanol precipitation, and two anion-exchange (Q-Sepharose fast flow) chromatographies. The final product was purified 264-fold and had a specific activity of 10 U mg^–1. The method is suitable to produce purified porcine FVIII by an easy process where all steps can be scaled up. The final product is free of von Willebrand factor that is responsible for the main side effects in patients. Finally, this method can be used to obtain purified porcine plasma FVIII for use in haemophilic patients with inhibitors.     

Encapsulation of the peptide Ac-Glu-Thr-Lys-Thr-Tyr-Phe-Trp-Lys-NH2 into polyvinyl alcohol biodegradable formulations—Effect of calcium alginate

It has been recently reported that the peptide Ac-Glu-Thr-Lys-Thr-Tyr-Phe-Trp-Lys-NH₂, analogue of the Glu1811-Lys1818 region of A3 light chain of blood coagulation factor VIII, presents in vitro significant anticoagulant activity. The encapsulation of this peptide into different polyvinyl alcohol formulations is examined here. The formulations were prepared using polyvinyl alcohol cross-linked with either boric acid or glutaraldehyde, giving a series of twelve different hydrogels. In case of PVA-boric acid method, a small percentage of sodium alginate was used in order to avoid bead's agglomeration. In that case, the most efficient encapsulation of the octapeptide (74%) was achieved with 0.2% (w/w) sodium alginate. It was also observed that the increase in sodium alginate percentage leads to beads with increased peptide release time, ranging from 60 to 90 min at 0.02% and 1% (w/w) sodium alginate respectively. The water holding of the PVA gels was estimated to be 27% regardless of the cross-linking reagent used, while it was increased with increasing sodium alginate concentration and reached about 60% for 1% sodium alginate. The longer octapeptide release, at 120 min, was observed with PVA-glutaraldehyde hydrogel, with encapsulation efficiency comparable to those obtained with boric acid, indicating that this hydrogel may be further used in drug delivery systems.     

Light Chain of Factor VIII Is Sufficient for Accelerating Cleavage of von Willebrand Factor by ADAMTS13 Metalloprotease

We previously demonstrated that coagulation factor VIII (FVIII) accelerates proteolytic cleavage of von Willebrand factor (VWF) by A disintegrin and metalloprotease with thrombospondin type 1 repeats (ADAMTS13) under fluid shear stress. In this study, the structural elements of FVIII required for the rate-enhancing effect and the biological relevance of this cofactor activity are determined using a murine model. An isolated light chain of human FVIII (hFVIII-LC) increases proteolytic cleavage of VWF by ADAMTS13 under shear in a concentration-dependent manner. The maximal rate-enhancing effect of hFVIII-LC is ∼8-fold, which is comparable with human full-length FVIII and B-domain deleted FVIII (hFVIII-BDD). The heavy chain (hFVIII-HC) and the light chain lacking the acidic (a3) region (hFVIII-LCΔa3) have no effect in accelerating VWF proteolysis by ADAMTS13 under the same conditions. Although recombinant hFVIII-HC and hFVIII-LCΔa3 do not detectably bind immobilized VWF, recombinant hFVIII-LC binds VWF with high affinity (K_D, ∼15 nm). Moreover, ultra-large VWF multimers accumulate in the plasma of fVIII^−/− mice after hydrodynamic challenge but not in those reconstituted with either hFVIII-BDD or hFVIII-LC. These results suggest that the light chain of FVIII, which is not biologically active for clot formation, is sufficient for accelerating proteolytic cleavage of VWF by ADAMTS13 under fluid shear stress and (patho) physiological conditions. Our findings provide novel insight into the molecular mechanism of how FVIII regulates VWF homeostasis.     

Purification of horse immunoglobulin isotypes based on differential elution properties of isotypes from protein A and protein G columns

Elution properties of horse immunoglobulin isotypes from protein A and protein G columns were examined. IgGa and IgGb isotypes were bound to protein A and protein G columns and were eluted by adjusting the pH of the elution buffer from 8.0 to 2.0. IgGc bound to protein G column but not to protein A column while IgG(T) bound to both columns. IgM and IgA apparently appeared not to bind to either column. New methods for purification of serum isotypes were developed using protein A and protein G columns as well as formerly established methods. Using these methods, it was possible to obtain purified isotypes for establishment of immunological assays for practical clinical use.     

Quantifying vitamin K-dependent holoprotein compaction caused by differential γ-carboxylation using high-pressure size exclusion chromatography

This study uses high-pressure size exclusion chromatography (HPSEC) to quantify divalent metal ion (X²⁺)-induced compaction found in vitamin K-dependent (VKD) proteins. Multiple X²⁺ binding sites formed by the presence of up to 12 γ-carboxyglutamic acid (Gla) residues are present in plasma-derived FIX (pd-FIX) and recombinant FIX (r-FIX). Analytical ultracentrifugation (AUC) was used to calibrate the Stokes radius (R) measured by HPSEC. A compaction of pd-FIX caused by the filling of Ca²⁺ and Mg²⁺ binding sites resulted in a 5 to 6% decrease in radius of hydration as observed by HPSEC. The filling of Ca²⁺ sites resulted in greater compaction than for Mg²⁺ alone where this effect was additive or greater when both ions were present at physiological levels. Less X²⁺-induced compaction was observed in r-FIX with lower Gla content populations, which enabled the separation of biologically active r-FIX species from inactive ones by HPSEC. HPSEC was sensitive to R changes of approximately 0.01 nm that enabled the detection of FIX compaction that was likely cooperative in nature between lower avidity X²⁺ sites of the Gla domain and higher avidity X²⁺ sites of the epidermal growth factor 1 (EGF1)-like domain.     

Cofactor Activity in Factor VIIIa of the Blood Clotting Pathway Is Stabilized by an Interdomain Bond between His281 and Ser524 Formed in Factor VIII

The factor VIII (FVIII) crystal structure suggests a possible bonding interaction of His²⁸¹ (A1 domain) with Ser⁵²⁴ (A2 domain), although the resolution of the structure (∼4 Å) does not firmly establish this bonding. To establish that side chains of these residues participate in an interdomain bond, we prepared and examined the functional properties of a residue swap variant (H281S/S524H) where His²⁸¹ and Ser⁵²⁴ residues were exchanged with one another and a disulfide-bridged variant (H281C/S524C) where the two residues were replaced with Cys. The latter variant showed efficient disulfide bonding of the A1 and A2 domains. The swap variant showed WT-like FVIII and FVIIIa stability, which were markedly reduced for H281A and S524A variants in an earlier study. The disulfide-bridged variant showed ∼20% increased FVIII stability, and FVIIIa did not decay during the time course measured. This variant also yielded 35% increased thrombin peak values compared with WT in a plasma-based thrombin generation assay. Binding analyses of H281S-A1/A3C1C2 dimer with S524H-A2 subunit yielded a near WT-like affinity value, whereas combining the variant dimer or A2 subunit with the WT complement yielded ∼5- and ∼10-fold reductions, respectively, in affinity. Other functional properties including thrombin generation potential, FIXa binding affinity, K_m for FX of FXase complexes, thrombin activation efficiency, and down-regulation by activated protein C showed similar results for the two variants compared with WT FVIII. These results indicate that the side chains of His²⁸¹ and Ser⁵²⁴ are in close proximity and contribute to a bonding interaction in FVIII that is retained in FVIIIa.