Nathalie Enjolras

Two novel mutations in EGF‐like domains of human factor IX dramatically impair intracellular processing and secretion

Summary.  We investigated the mechanisms responsible for severe factor IX (FIX) deficiency in two cross‐reacting material (CRM)‐negative hemophilia B patients with a mutation in the first and second epidermal growth factor (EGF) domains of FIX (C71Y and C109Y, respectively). We have determined the kinetics of mutant FIX biosynthesis and secretion in comparison with wild‐type FIX (FIXwt). In transfected cells, FIXwt was retrieved as two intracellular molecular forms, rapidly secreted into the culture medium. One appeared to be correctly N‐glycosylated, and corresponded to a form trafficking between the endoplasmic reticulum (ER) and Golgi apparatus. The other corresponded to the mature form, ready to be secreted, exhibiting correct N‐glycosylation and sialylation. In contrast, the two mutants, FIXC71Y and FIXC109Y, were not secreted from the cells and did not accumulate intracellularly. Relative to FIXwt, they were retained longer in the ER and were only N‐glycosylated. In addition, the intracellular concentration of the FIX mutants increased when ALLN, an inhibitor of cysteine proteases and of the proteasome degradation pathway, was added to the culture medium. Both the FIX mutants and FIXwt were associated in the ER with the 78‐kDa glucose‐regulated protein (GRP78/BiP) and calreticulin (CRT), though the amount of CRT associated with the two mutants was twice as strong as with FIXwt. These results strongly suggest that chaperone and lectin molecules act in concert to ensure both proper folding of FIXwt and the retention of mutant molecules.

Activated factor X cleaves factor VIII at arginine 562, limiting its cofactor efficiency

Summary.  Background: Factor VIII (FVIII) and its activated form (FVIIIa) are subject to proteolysis that dampens their cofactor function. Among the proteases that attack FVIII (activated factor X (FXa), activated protein C (APC) and plasmin), only APC cleaves within the FVIII A2 domain at R562 to fully abolish FVIII activity. Objectives: We investigated the possible involvement of the FXa cleavage at R562 within the A2 domain in the process of FVIII inactivation. Methods: An antibody (GMA012/R8B12) that recognizes the carboxy‐terminus extremity of the A2 domain (A2C) was used to evaluate FXa action. A molecule mutated at R562 was also generated to assess the functional role of this particular residue. Results and Conclusions: The appearance of the A2C domain as a function of time evidenced the identical cleavage within the A2 domain of FVIII and FVIIIa by FXa. This cleavage required phospholipids and occurred within minutes. In contrast, the isolated A2 domain was not cleaved by FXa. Von Willebrand factor and activated FIX inhibited the cleavage in a dose‐dependent manner. Mutation R562K increased both the FVIII specific activity and the generation of FXa due to an increase in FVIII catalytic efficiency. Moreover, A2C fragment could not be identified from FVIII‐R562K cleavage. In summary, this study defines a new mechanism for A2 domain‐mediated FVIII degradation by FXa and implicates the bisecting of the A2 domain at R562.

B-domain deleted factor VIII is aggregated and degraded through proteasomal and lysosomal pathways.

Factor VIII (FVIII) processing within mammalian cells is demonstrated to be much less efficient than proteins of similar size. The deletion of the B-domain from FVIII improves the level of production, due partly to the increase in mRNA synthesis. We aimed to characterise the cellular fate and the intracellular processing of the FVIII molecule devoid of B-domain. A B-domain deleted factor VIII (BDD-FVIII) possessing a furin consensus cleavage site in the connecting segment between the heavy and the light chain, was produced in CHO cell line. In such cells, FVIII was retained as two single chain products from which a majority was aggregated. The two species were located in Triton X-100 soluble (for 60-80%) and insoluble fractions (for 20-40%). The incubation of the expressing cells with tunicamycin (5 mug/ml) and the treatment of the intracellular species with a mixture of Neuraminidase and N-glycosidase-F revealed that both intracellular species were N-glycosylated. Furin over-expression neither diminished the intracellular FVIII contents nor improved its extracellular production. Intracellular FVIII was degraded through both lysosomal and proteasomal pathways as evidenced by inhibitor treatments (e.g. NH(4)Cl, leupeptin, clasto-Lactacystin beta-lactone and MG-132), pulse-chase analysis and confocal observations. This study demonstrates that a BDD-FVIII expressed in CHO cells is inefficiently processed consecutively to intracellular aggregation, proteasomal degradation, and routage to lysosomes.

Human hepatoma cell line HuH-7 is an effective cellular system to produce recombinant factor IX with improved post-translational modifications.

Recombinant factor IX (rFIX) is increasingly used to treat patients with hemophilia B. CHO (Chinese Hamster Ovary) cells are commonly used for the production of rFIX but they have a limited capacity for introducing post-translational modifications (PTM) leading to incomplete γ-carboxylation, low phosphorylation and sulfation profiles as compared with plasma-derived preparations. Imperfect PTM might have an impact on the activity of Factor IX molecule. Several studies in animal models as well as clinical trials have previously reported a lower recovery of rFIX compared to plasma-derived FIX concentrates. In the present study, we aimed to produce a rFIX having a profile of PTM similar to plasma-derived FIX, using human hepatoma cell line HuH-7. We showed that rFIX produced by HuH-7 cells followed the classical intracellular pathway before secretion. In addition, improved PTM were associated with fully active molecule compared to plasma-derived and recombinant control FIX molecules. Secreted rFIX presented as a single band at the correct molecular weight. HuH-7 cellular clones were obtained and they secreted a biologically active human FIX. FIX was then purified for a detailed evaluation of PTM. Glycosylation and sialylation profiles were similar to plasma-derived and rFIX and mass spectrometry analysis demonstrated the presence of phosphorylated and sulfated forms of rFIX. These data strongly support that HuH-7 cells may represent an effective cellular system for production of rFIX exhibiting PTM similar to plasma-derived FIX.

Biosynthesis of FVIII in megakaryocytic cells: improved production and biochemical characterization

Haemophilia A is an attractive target for gene therapy. We designed a haemophilia A gene therapy strategy involving the genetic modification of haematopoietic stem cells to achieve tissue‐specific expression of a factor VIII (FVIII) transgene in the megakaryocytic lineage. Platelets would then serve as vehicles to store the expressed FVIII and deliver the coagulation factor at the site of vascular injury. A local correction of the haemostasis defect could, therefore, be expected following platelet activation and secretion. In this study, we demonstrated that a model of haematopoietic cell lines (Dami cells) could produce a correctly processed FVIII. FVIII transgenes were placed under the control of the human platelet glycoprotein IIb (GPIIb) promoter and used for stable transfection of the Dami megakaryocytic cell line. The highest FVIII production was obtained when the FVIII transgene contained a factor IX intron 1 gene sequence inserted in the FVIII intron 1 and 13 sites. Reverse transcription polymerase chain reaction demonstrated that the splicing of these introns was complete. Recombinant FVIII (rFVIII) produced in Dami cells was a biologically active molecule (specific activity: 5664 IU/mg) that was correctly glycosylated and sulphated. This recombinant FVIII protein exhibited biochemical characteristics after deglycosylation or thrombin activation that were comparable to a commercially available B‐domainless rFVIII. These results demonstrate the advantages of a modified FVIII transgene and represent the first biochemical characterization of megakaryocyte‐produced FVIII.

Expression and characterization of a novel human recombinant factor IX molecule with enhanced in vitro and in vivo clotting activity

INTRODUCTION Hemophilia B is an inherited X-linked recessive bleeding disorder, due to a defect in human factor IX (FIX). The main treatment for hemophilia B is replacement therapy using FIX concentrates. Prophylactic treatment in severe hemophilia B is very effective but is limited by cost issues. Production of a recombinant FIX (rFIX) with enhanced clotting activity, offering the possibility of fewer infusions and fewer costs with similar efficacy, is one of the current challenges for hemophilia B treatment. The present study focused on an important amino acid sequence known to be involved in the interaction of activated FIX (FIXa) with its cofactor, activated factor VIII (FVIIIa). MATERIALS AND METHODS Using site-directed mutagenesis of glutamate E410 (c240, chymotrypsin numbering), four recombinant FIX-E410 (E410H, A, L and N) mutants were developed and produced by the human hepatoma cell line Huh-7. RESULTS The in-vitro clotting activity of mutant FIX molecules was 3 to 5-fold higher than wild-type recombinant FIX (FIX-WT). FIX-E410H compound showed the highest in-vitro procoagulant activity. Enhanced specific activity was confirmed using thrombin generation assay. FIX-E410H induced 5.2-fold higher thrombin generation than FIX-WT. In hemophilia B mice, we observed significantly higher in-vivo clotting activity and thrombin generating capacity with FIX-E410H compared to FIX-WT. We demonstrated that increased procoagulant activity of FIX-E410H was mainly explained by 2.5- fold enhanced affinity of the mutant for human FVIIIa. CONCLUSION We have engineered and characterized four improved FIX proteins with enhanced in-vitro and in-vivo activity. Future studies are required to evaluate the immunogenicity of FIX-E410.

Why patients with THBD c.1611C>A (p.Cys537X) nonsense mutation have high levels of soluble thrombomodulin?

Background Recently our group has described a new autosomal dominant bleeding disorder characterized by very high plasma levels of soluble thrombomodulin (TM). The THBD c.1611C>A (p.Cys537X) mutation in heterozygous state was found in the propositus. This mutation leads to the synthesis of a truncated TM which has lost the last three amino-acids of the transmembrane domain and the cytoplasmic tail. Objective We investigated the mechanism responsible for TM shedding in endothelial cells with THBD c.1611C>A mutation. Methods Complementary DNA of TM wild type (TM-WT) was incorporated into a pcDNA3.1 vector for transient transfection in COS-1 cells. Mutagenesis was performed to create the c.1611C<A (TM1-536) mutant and 4 other TM mutants (TM1-515, TM1-525, TM1-533 and TM1-537) with a transmembrane domain having different lengths. The effect of shear stress, metalloprotease inhibitor, certain proteases and reducing agents were tested on TM shedding. Results Western blot and immunofluorescent analysis showed that TM1-536 was produced and a certain amount of TM1-536 was anchored on the cell membrane. A significantly higher levels of soluble TM was observed in the TM1-536 cell medium in comparison with TM-WT (56.3 +/- 5.2 vs 8.8 +/- 1.6 ng/mL, respectively, p = 0.001). The shedding of TM1-536 was 75% decreased in cells cultured in the presence of a metalloprotease inhibitor. No difference was observed between TM1-536 and TM-WT shedding after cell exposure to cathepsin G, elastase, several reducing agents and high shear stress (5000 s-1). Significantly higher levels of soluble TM were observed in the cell media of TM1-533, TM1-525, TM1-515 in comparison with TM-WT (p < 0.05). Conclusion The mechanism responsible for TM shedding is complex and is not completely understood: higher sensitivity of the TM1-536 to the proteolysis by metalloproteases and a defect of synthesis due to the decreased size of the transmembrane domain might explain the high levels of soluble TM in plasma of the carriers.

In vivo efficacy of human recombinant factor IX produced by the human hepatoma cell line HuH-7.

Post‐translational modifications of the CHO‐cell‐derived‐recombinant human factor IX (FIX) currently used for the treatment of hemophilia B (HB) are different from plasma derived FIX. Our previous studies described a rFIX (HIX) having better profile of post‐translational modifications than rFIX produced by CHO cells. The aim of the study consisted to verify the improved post‐translational modifications effect of HIX on in vivo recovery.