Purification and Autoactivation Method for Recombinant Coagulation Factor VII.

Recombinant coagulation factor VII is a very important and complex protein employed for treatment of hemophiliac patients (hemophilia A/B) who develop inhibitors antibodies to conventional treatments (FVIII and FIX). The rFVII is a glycosylated molecule and circulates in plasma as zymogen of 50 kDa. When activated the molecule is cleaved to 20-30 kDa and has a half-life of about 3 h, needing to be processed fast and efficiently until freeze-drying. Here, we describe a very simple and fast purification sequence for rFVII using affinity FVII Select resin and a dialysis system that can be easily scaled up.

Production of coagulation factor VII in human cell lines Sk-Hep-1 and HKB-11.

Recombinant factor VII (rFVII) is the main therapeutic choice for hemophilia patients who have developed inhibitory antibodies against conventional treatments (FVIII and FIX). Because of the post-translational modifications, rFVII needs to be produced in mammalian cell lines. In this study, for the first time, we have shown efficient rFVII production in HepG2, Sk-Hep-1, and HKB-11 cell lines. Experiments in static conditions for a period of 96 h showed that HepG2-FVII produced the highest amounts of rhFVII, with an average of 1843 ng/mL. Sk-hep-1-FVII cells reached a maximum protein production of 1432 ng/mL and HKB-11-FVII cells reached 1468 ng/mL. Sk-Hep-1-rFVII and HKB-11-rFVII were selected for the first step of scale-up. Over 10 days of spinner flask culture, HKB-11 and SK-Hep-1 cells showed a cumulative production of rFVII of 152 µg and 202.6 µg in 50 mL, respectively. Thus, these human cell lines can be used for an efficient production of recombinant FVII. With more investment in basic research, human cell lines can be optimized for the commercial production of different bio therapeutic proteins.

Expression and purification of recombinant human coagulation factor VII fused to a histidine tag using Gateway technology.

BACKGROUND: Factor VII (FVII) is a plasma glycoprotein that participates in the coagulation process leading to the generation of fibrin. The aim of this study was to construct, express and purify recombinant FVII fused to a polyhistidine (his) tag using Gateway technology. METHODS: To construct the entry clone, blunt-end FVII cDNA and subsequent polymerase chain reaction (PCR) product isolated from a HepG2 cell line was TOPO-cloned into a pENTR TOPO vector. To construct the expression clone, a LR recombination reaction was carried out between the entry clone and destination vector, pDEST26. Chinese hamster ovary (CHO) cells were transfected with 1 microg of DNA of PDEST26-FVII using the FuGENE HD transfection reagent. Two cell lines that permanently expressed recombinant FVII were established. The expression of recombinant FVII was confirmed by reverse transcriptase PCR and enzyme-linked immunosorbent assay. Culture medium containing his-tagged FVII was added to the nickel-nitrilotriacetic acid resin column and bound protein was eluted. The purified protein was detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The biological activity of the recombinant FVII was determined by a prothrombin time assay using FVII-depleted plasma. RESULTS: The results showed that human recombinant FVII was successfully cloned and the accuracy of the nucleotide sequence of the gene and its frame in the vector were confirmed by DNA sequencing. Stable clones transfected with the construct expressed FVII mRNA and related protein but no expression was detected in the CHO cells containing an empty vector. A protein of about 52 KDa was detected in SDS-PAGE and was further confirmed by western blot analysis. A three-fold decrease in clotting time was observed using this recombinant FVII. CONCLUSION: As far as we are aware, this is the first report of expression of recombinant FVII fused with a his-tag through Gateway technology. The next steps, including large scale expression, purification, activation and stabilisation, are underway.

Establishment of the human coagulation factor VII concentrate European Pharmacopoeia biological reference preparation batch 1.

For the potency assay of human coagulation factor VII concentrate preparations according to the European Pharmacopoeia (Ph. Eur.) a reference preparation calibrated in International Units (IU) is needed. Currently, the 1st International Standard (97/592, potency: 6.3 IU/ampoule) but no Ph. Eur. reference preparation is available. A collaborative study was run to calibrate a candidate Ph. Eur. Biological Reference Preparation (BRP) for human coagulation factor VII concentrate against the 1st International Standard; the BRP is intended to be used as working standard. A candidate BRP batch 1 was produced from a plasma-derived human factor VII concentrate preparation available on the European market. It fulfilled the requirements of a BRP with regard to precision and homogeneity of fill, residual water content and stability. In addition, the content of activated factor VII was low. Sixteen laboratories from 9 countries participated in the collaborative study. The potency of the candidate BRP was determined using the participants' chromogenic assay based on the Ph. Eur. and their in-house clotting assay, if available. The statistical model used for analysis of the results from most laboratories was the maximum likelihood of the parallel line model following a logarithmic transformation of the responses. In the chromogenic assay, a potency estimate of 8.2 IU/vial (+/-3.7%) was obtained for the candidate BRP. Results from the clotting assay were lower and less homogenous (6.7 IU/vial+/-11.6%). The results from the collaborative study showed that the candidate BRP is suitable as a reference standard for the chromogenic assay according to the Ph. Eur. It was adopted by the Ph. Eur. Commission in March 2006 as official Ph. Eur. BRP for this purpose.

Characterization of mild coagulation factor VII deficiency: activity and clearance of the Arg315Trp and Arg315Lys variants in the Cys310-Cys329 loop (c170s).

BACKGROUND AND OBJECTIVES: Arginine 315 in factor VII (FVII) belongs to a solvent-exposed loop involved in direct interaction with the co-factor (tissue factor, TF), in transmission of TF-induced effects and potentially in FVIIa inactivation. Natural FVII variants at position 315 provide peculiar models for structure-function studies. DESIGN AND METHODS: We characterized a mild coagulation FVII deficiency associated with reduced FVII activity (26%) and antigen (67%). Mutations were searched by FVII gene sequencing. FVII variants were created by mutagenesis of FVII cDNA and characterized through expression in HEK293 cells followed by functional studies. FVII antigen in media was estimated by immunoassay while FVII activity was assessed by prothrombin-time based and FXa generation assays. FVII variants were injected into mice to investigate their recovery and half-life. One-way ANOVA was used to test statistical significance. RESULTS: The patient was double heterozygous for a novel R315W mutation and for the R304Q substitution (FVII Padua) previously demonstrated to impair TF binding. The recombinant 315W-FVII was normally expressed in medium but showed a markedly reduced coagulant function (52%) and activity towards factor X (FX) in plasma (34%). Moreover, the 315W-FVII showed significantly decreased recovery of the protein (20%) and a slightly shorter half-life (8.6 min) as compared to wt-FVII (50% and 10.7 min). We also studied the conservative R315K change that was responsible for low recovery (20%) and a decreased half-life (7 min) of a FVII variant with virtually normal FVII antigen and activity levels. INTERPRETATION AND CONCLUSIONS: These findings suggest a dual role of R315 for FVII function and clearance, and indicate that substitutions at this position have appreciable effects on human FVII biology, compatible with residual FVII function and thus with mild FVII deficiency.

Similar molecular interactions of factor VII and factor VIIa with the tissue factor region that allosterically regulates enzyme activity.

Tissue factor (TF) binds the zymogen (VII) and activated (VIIa) forms of coagulation factor VII with high affinity. The structure determined for the sTF-VIIa complex [Banner, D. W., et al. (1996) Nature 380, 41-46] shows that all four domains of VIIa (Gla, EGF-1, EGF-2, and protease) are in contact with TF. Although a structure is not available for the TF-VII complex, the structure determined for free VII [Eigenbrot, C., et al. (2001) Structure 9, 675-682] suggests a significant conformational change for the zymogen to enzyme transition. In particular, the region of the protease domain that must contact TF has a conformation that is altered from that of VIIa, suggesting that the VII protease domain interacts with TF in a manner different from that of VIIa. To test this hypothesis, a panel of 12 single-site sTF mutants, having substitutions of residues observed to contact the proteolytic domain of VIIa, have been evaluated for binding to both zymogen VII and VIIa. Affinities were determined by surface plasmon resonance measurements using a noninterfering anti-TF monoclonal antibody to capture TF on the sensor chip surface. Dissociation constants (K(D)) measured for binding to wild-type sTF are 7.5 +/- 2.4 nM for VII and 5.1 +/- 2.3 nM for VIIa. All of the sTF mutants except S39A and E95A exhibited a significant decrease (>2-fold) in affinity for VIIa. The changes in affinity measured for VII or VIIa binding with substitution in sTF were comparable in magnitude. We conclude that the proteolytic domain of both VII and VIIa interacts with this region of sTF in a nearly identical fashion. Therefore, zymogen VII can readily adopt a VIIa-like conformation required for binding to TF.

Cloning and expression of rat coagulation factor VII.

Smaller and widely available animals such as rats are commonly used to evaluate antithrombotic drug candidates in vivo. However, the isolation and purification of FVII from rats and other species is very challenging because they are present in extremely low levels in plasma (approximately 10 nM). Furthermore, purification of FVII from other coagulation factors present in the plasma such as prothrombin, factor IX and factor X can often be very challenging and labor-intensive. To facilitate studies on the role of the extrinsic pathway of coagulation in rats, a full-length cDNA-encoding rat factor VII was isolated using polymerase-mediated DNA amplification using a rat liver cDNA library. The cDNA codes for a 41-residue signal/propeptide region, followed by a 405-residue mature protein consisting of the light chain with gamma-carboxy glutamic acid (gla) including epidermal growth factor domains (EGF) and the heavy chain with the serine protease catalytic domain. Rat factor VII cDNA was transfected into human embryonic kidney 293 cells and several cell lines that constitutively express rat factor VII were established. The media from the stable lines expressing recombinant rat FVII were rapidly screened for functional activity and were found to normalize clotting time of FVII-depleted human plasma. The supernatants were also functionally active in the presence of tissue factor in chromogenic assays by measuring FVIIa activation using a tripeptide chromogenic substrate and in a two-stage, coupled assay measuring the generation of FXa. Recombinant rat FVII may be an important new tool in the development of novel antithrombotic drugs.

Expression and purification of recombinant rabbit factor VII.

To facilitate studies of the in vivo role of the extrinsic pathway of coagulation in experimental hemostasis and thrombosis, a full-length cDNA-encoding rabbit factor VII was isolated using polymerase chain reaction-mediated DNA amplification from plaque-purified lambda gt11 phage. Repeated DNA sequencing of both full-length rabbit factor VII cDNA and shorter cDNA fragments verified four changes in the previously reported amino acid sequence of mature rabbit factor VII, now predicted to be 405 amino acids in length. Rabbit factor VII cDNA was transfected into human embryonic kidney 293 cells and a cell line that permanently expressed rabbit recombinant factor VII was established. Rabbit recombinant factor VII was purified from tissue culture media using a combination of barium citrate precipitation, DEAE-sepharose FF chromatography, benzamidine agarose, and affinity chromatography using a sheep antirabbit factor VII polyclonal antibody. The purity and authenticity of rabbit recombinant factor VII was confirmed by polyacrylamide gel electrophoresis and Western blot analysis. Homogeneous rabbit recombinant factor VII was fully active biologically as determined by prothrombin time assay in factor FVII-depleted plasmas, of both human and rabbit origin, using either human or rabbit thromboplastin. Rabbit recombinant factor VII should prove useful for future in vivo investigations of experimental coagulopathies.

Tissue factor regulates plasminogen binding and activation.

Tissue factor (TF) has been implicated in several important biologic processes, including fibrin formation, atherogenesis, angiogenesis, and tumor cell migration. In that plasminogen activators have been implicated in the same processes, the potential for interactions between TF and the plasminogen activator system was examined. Plasminogen was found to bind directly to the extracellular domain of TF apoprotein (amino acids 1-219) as determined by optical biosensor interaction analysis. A fragment of plasminogen containing kringles 1 through 3 also bound to TF apoprotein, whereas isolated kringle 4 and miniplasminogen did not. Expression of TF on the surface of a stably transfected Chinese hamster ovary (CHO) cell line stimulated plasminogen binding to the cells by 70% more than to control cells. Plasminogen bound to a site on the TF apoprotein that appears to be distinct from the binding site for factors VII and VIIa as judged by a combination of biosensor and cell assays. TF enhanced two-chain urokinase (tcuPA) activation of Glu-plasminogen, but not of miniplasminogen, in a dose-dependent, saturable manner (half maximal stimulation at 59 pmol/L). TF apoprotein induced an effect similar to that of relipidated TF, but a relatively higher concentration of the apoprotein was required (half maximal stimulation at 3.8 nmol/L). The stimulatory effect of TF on plasminogen activation was confirmed when plasmin formation was examined directly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In accord with this, TF inhibited fibrinolysis by approximately 74% at a concentration of 14 nmol/L and almost totally inhibited the binding of equimolar concentrations of plasminogen to human umbilical vein endothelial cells and human trophoblasts. Further, CHO cells expressing TF inhibited uPA-mediated fibrinolysis relative to a wild-type control. TF apoprotein and plasminogen were found to colocalize in atherosclerotic plaque. These data suggest that plasminogen localization and activation may be modulated at extravascular sites through a high-affinity interaction between kringles 1 through 3 of plasminogen and the extracellular domain of TF.

Defective vaccinia virus as a biologically safe tool for the overproduction of recombinant human secretory proteins.

We have described recently the construction of a defective vaccinia virus (VV) lacking the essential D4R open reading frame and have shown furthermore the selection of a complementing cell line providing the essential D4R gene product. The D4R gene belongs to the group of early transcribed vaccinia genes preventing a virus defective in D4R from entering into the intermediate and late phase of replication under noncomplementing conditions. Here we show that this property, which is unique among the group of so called nonreplicating poxviruses, is helpful for the production of (secretable) recombinant human proteins. Recombinant VV based on a D4R-defective parental strain expressing cDNAs coding for the human blood coagulation factors VII and XI produced significantly more recombinant protein than the corresponding recombinants based on wild-type VV. Moreover, the complementing cell line RK-D4R-44.20 was a more effective production cell system for both vD4 and wild-type VV recombinants compared to wild-type RK-13 cells. Surprisingly, recombinant human factor VII was more efficiently produced with the defective vaccinia recombinant even under noncomplementing conditions, suggesting that persistence of the early phase of vaccinia replication in combination with a delayed host shutoff is advantageous for the overproduction of certain recombinant proteins using the VV expression system.

Review of conformation-specific affinity purification methods for plasma vitamin K-dependent proteins.

There are seven known vitamin K-dependent proteins in blood. These proteins require calcium ion for expressing their full biological activities. Calcium ion also induces conformational changes in this class of proteins. Taking advantage of the ligand induced conformational changes, a number of unique approaches of affinity chromatography have been developed. These methodologies have been very useful tools for both the purification and for understanding the structure-function relationships of this class of proteins. One method is the use of metal ion dependent immunoaffinity chromatography. The antigen can be dissociated from the antibodies with either the removal or addition of calcium ion under physiological conditions. The other method is pseudoaffinity chromatography. This method uses conventional ion-exchange or hydrophobic resin and manipulates the mobilities of the proteins on these resins by the presence or absence of calcium ions. Researchers working with other calcium binding proteins or other proteins that are known to undergo ligand induced conformational changes may benefit from the experience of these unique conformation-specific affinity chromatography approaches.

No detectable alterations in immunogenicity following terminal severe dry-heat treatment of high-purity factor VIII (Liberate) and factor IX (HP9) concentrates.

We used monoclonal antibody ELISAs, antigen molecular size distribution, competition ELISA and neonatal mouse immune tolerance methods to detect potential neoantigen formation and increased immunogenicity following severe dry-heat treatment of high-purity factor VIII (Liberate) and factor IX concentrates. To provide positive controls, concentrates were heated in solution (70 degrees C for 2 h) to produce denaturation on purpose. The competition ELISA applied to factor IX proved particularly useful for quantifying differences between the positive control and the dry-heated/unheated concentrates. None of the test systems employed by us indicated any detectable neoantigen formation or any alteration in immunogenicity following terminal severe dry-heat treatment of the high-purity concentrates, and this finding is supported by clinical experience so far.

Activated factor X and thrombin formation triggered by tissue factor on endothelial cell matrix in a flow model: effect of the tissue factor pathway inhibitor.

The procoagulant subcellular matrix of stimulated endothelial cells that contains tissue factor (TF) was used to investigate the mechanism by which TF pathway inhibitor (TFPI) inhibits thrombin formation initiated by TF/factor VIIa (FVIIa) under flow conditions. Purified coagulation factors VII, X, and V and prothrombin were perfused at a wall shear rate of 100 s-1 through a flow chamber containing a coverslip covered with matrix of cultured human umbilical vein endothelial cells. This resulted in a TF- and FVII-dependent FXa and thrombin generation as measured in the effluent at the outlet of the system. Inhibition of this TF/FVIIa-triggered thrombin formation by TFPI purified from plasma was dependent on the amount of TF present on the endothelial cell matrix. The rate of prothrombinase assembly and steady-state levels of thrombin formation were decreased by TFPI. Because persistent albeit decreased steady-state levels of thrombin formation occurred in the presence of TFPI, we conclude that plasma-TFPI does not inhibit FXa present in the prothrombinase complex. The addition of FIX and FVIII to perfusates containing FVII and FX increased the FXa generation on endothelial matrices, and counteracted the inhibition of thrombin formation on endothelial cell matrices by TFPI. Our data provide further evidence for the hypothesis that the rapid inactivation of TF/FVIIa by TFPI in combination with the absence of either FVIII or FIX causes the bleeding tendency of patients with hemophilia A or B.

Expression and localization of tissue factor-based procoagulant activity (PCA) in pigeon monocyte-derived macrophages.

Monocyte-derived macrophages, focal to initiation and progression of atherosclerosis, have been implicated in thrombotic complication of this disease. In the present study we demonstrated tissue factor based procoagulant activity in cultured macrophages from the White Carneau pigeon following endotoxin (1-2 micrograms/ml) stimulation. This macrophage procoagulant activity paralleled activity obtained with pigeon brain homogenate. We used Enzyme-Linked Coagulation Assay (ELCA), an ultrasensitive microtiter plate assay, to measure procoagulant activity in these cells. Through the use of clotting factors purified from pigeon plasma, procoagulant activity could be detected with as few as 1-3 cells. Tissue factor antigen, detected through the use of immunogold labelling in conjunction with a polyclonal antibody which was highly specific to human tissue factor, was distributed uniformly over the plasma membrane of the endotoxin-stimulated cells. These studies suggest that this procoagulant activity might play an important role in the pathobiology of atherosclerosis in White Carneau pigeons by initiating fibrin polymerization and thus leading to thrombotic complications of the disease.

Specific molecular interaction sites on factor VII involved in factor X activation.

Factor VII, a serine-protease zymogen, and tissue factor, the cellular receptor/coenzyme, are the protein components of the macromolecular complex which initiates the extrinsic pathway of the coagulation cascade. Previous studies were directed to the identification of functional sites on factor VII which mediate factor X activation, employing a series of potentially inhibitory synthetic peptides representing the primary structure of factor VII and antibodies to selected peptides. The involvement of at least four high-affinity interactive regions [factor VII (44-50), (196-229), (285-305) and (376-396) peptides] on the surface of factor VII was clearly demonstrated. The minimal sequences for the expression of inhibitory activity of these four molecular recognition domains on factor VII were identified using short and overlapping peptides. The short factor VII-(206-218)-peptide (most inhibitory peptide in the sequence 196-229 on factor VII) inhibited the binding of factor VII to the tissue-factor-expressing J82 cell line. Furthermore, radiolabeled [Tyr201] factor VII-(199-221)-peptide, with a tyrosine substituted for the normal tryptophan residue, was specifically bound to J82 cells, and also the binding of the radiolabeled peptide to this cell line was specifically inhibited by a monoclonal antibody to tissue factor, confirming that the interaction site for tissue factor on factor VII is present within the peptide sequence 196-229. Kinetic analyses suggested that the regions represented by factor VII-(285-305)- and factor VII-(376-396)-peptides are involved in factor X recognition and the chemical cross-linking of the radiolabeled peptides resulted in specific binding to factor X, confirming that these two regions on factor VII represent the substrate-recognition site. Furthermore, these radiolabeled peptides specifically interact with the heavy chain of factor X, suggesting that the complementary binding region for the substrate-recognition site on factor VII are present on the heavy chain of factor X.

A new method for factor VII deficient substrate preparation and coagulation studies in malaria.

A simplified technique using DEAE-cellulose chromatography for the preparation of factor VII deficient substrate was developed in order to reduce the high cost of individual factor VII assay in the routine coagulation laboratory. The substrate prepared from cryo-removed human and bovine plasma had a high correlation (r = 0.9929) with two of the most popular imported commercial substrates available (DADE, Ortho). When compared several other imported commercial substrates of equal quality, the prepared substrate was 3,000 to 6,000 times cheaper. Using the prepared factor VII deficient substrate along with other commercial substrates available, two hundred and fifty patients with malaria (fifty cases of P. vivax and two hundred cases of P. falciparum) were studied for coagulation and fibrinolysis abnormalities. Only P. falciparum infections showed prolonged PT and aPTT which correlated with the degree of parasitemia (r = 0.0972). Factors V, VII, and IX were the most sensitive parameters in the expression of coagulation defects and most coagulation abnormalities were due to liver involvement. Plasmin activity was normal in P. vivax patients but it was significantly increased in P. falciparum patients with > 5% parasitemia. Only two of the complicated cases of P. falciparum patients showed the evidence of DIC.