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PubMed1991

[No HCV seroconversion in hemophilia following substitution with virus-inactivated coagulation factor VIII and IX concentrates].

Since 1986 the factor VIII and IX concentrates of the Central Laboratory, Swiss Red Cross Blood Transfusion Service have been virus inactivated with tri-(n-butyl) phosphate and Tween 80. Clinical studies had shown that both preparations were well tolerated and hemostatically effective; no HIV infection was transmitted. However, safety from transmission of non-A/non-B hepatitis could not be shown since the study included no previously untreated patients. In the meantime, a laboratory test has become available which allows retrospective testing for anti-hepatitis C antibodies in frozen sera of the study patients. 5 of the 26 patients, observed during a 2-year follow-up study, had no HCV antibodies before entering the long-term trial. During this trial, each of these 5 patients substituted an average quantity of 40,200 coagulation factor units (7500-69,000) from 45 production lots. None of these 5 patients developed anti-HCV antibodies, nor did any of them show clinical signs of infection with hepatitis. This suggests that virus inactivation using solvent/detergent treatment reduces the risk of transmission of HCV.

Blood Coagulation Disorders↗
PubMed2005

Exon skipping partially restores factor VIII coagulant activity in patients with mild hemophilia A with exon 13 duplication.

Ectopic mRNA was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) in patients with duplication of F8 gene exon 13, a mutation which has been demonstrated to be a cause of mild hemophilia A in 32% of Northern Italian subjects. Two different transcripts originate from mutated genomic DNA, due to alternative splice processes. The larger-sized transcript contains both duplicated exons 13, the smaller one contains only one exon 13. The residual FVIII:C activity which accounts for the mild hemophilia A phenotype derives from the latter transcript.

Alternative Splicing↗
PubMed1999

Synthesis of recombinant blood coagulation factor VIII (FVIII) heavy and light chains and reconstitution of active form of FVIII.

FVIII is synthesized as a single chain precursor of approximately 280 kD with the domain structure of A1-A2-B-A3-C1-C2 and it circulates as a series of metal ion-linked heterodimers that result from cleavages at B-A3 junction as well as additional cleavages within B domain. Factor VIII is converted to its active form, factor VIIIa, upon proteolytic cleavages by thrombin and is a heterotrimer composed of the A1, A2, and A3-C1-C2 subunits. A1 subunits of factor VIIIa terminates with 36 residue segment (Met337-Arg372) rich in acidic residues. This segment is removed after cleavages at Arg336 by activated protein C, which results in inactivation of the cofactor. In the present study, site-directed mutagenesis of FVIII at Arg336 to Gln336 was performed in order to produce an inactivation resistant mutant rFVIII (rFVIIIm) with an extended physiological stability. A recombinant mutant heavy chain of FVIII (rFVIII-Hm; Arg336 to Gln336) and wild-type light chain of FVIII (rFVIII-L) were expressed in Baculovirus-insect cell (Sf9) system, and a biologically active recombinant mutant FVIII (rFVIIIm) was reconstituted from rFVIII-Hm and rFVIII-L in the FVIII-depleted human plasma containing 40 mM CaCl2. The rFVIIIm exhibited cofactor activity of FVIIIa (2.85 x 10(-2) units/mg protein) that sustained the high level activity during in vitro incubation at 37 degrees C for 24 h, while the cofactor activity of normal plasma was declined steadily for the period. These results indicate that rFVIIIm (Arg336 to Gln336) expressed in Baculovirus-insect cell system is inactivation resistant in the plasma coagulation milieu and may be useful for the treatment of hemophilia A.

Animals↗
PubMed1996

Changes in factor VIII binding capacity of von Willebrand factor and factor VIII coagulant activity in two patients with type 2N von Willebrand disease after hemostatic treatment and during pregnancy.

The changes of the FVIII binding capacity of vWF after the infusion of FVIII/vWF concentrate was studied in two patients with type 2N vWD, and also during pregnancy in one of them. After infusion of FVIII or DDAVP to the patients, FVIII:C in plasma increased as expected, but it then decreased, with a markedly short half-life, of about 2 h, due to the defect in the FVIII binding capacity of vWF in plasma. However, after infusion of FVIII/vWF concentrate (40 U of FVIII:C/kg), FVIII:C increased, from 4-6 to 100-160 U/dl, more than the expected values, and decreased with the half-life expected. The FVIII binding capacity of vWF in plasma changed in parallel with the concentration of exogenous normal vWF, with a half-life of more than 24 h. During pregnancy, no increase of FVIII:C was observed, although vWF:Ag increased from 40 (before pregnancy) to 90 U/dl in plasma at 35th week of gestation. The FVIII binding capacity of vWF in plasma showed no increase during pregnancy. Accordingly, the administration of FVIII/vWF concentrate to the patient at delivery resulted in adequate hemostasis.

Deamino Arginine Vasopressin↗
PubMed2006

Detection of 95 novel mutations in coagulation factor VIII gene F8 responsible for hemophilia A: results from a single institution.

Hemophilia A (HA) is an X-linked hereditary bleeding disorder defined by a qualitative and/or quantitative factor VIII (FVIII) deficiency. The molecular diagnosis of HA is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. The putative role of the novel mutations, especially missense mutations, may be difficult to interpret as causing HA. We identified 95 novel mutations out of 180 different mutations responsible for HA in 515 patients from 406 unrelated families followed up at a single hemophilia treatment center of the Bicêtre university hospital (Assistance Publique-Hôpitaux de Paris [AP-HP], Le Kremlin-Bicêtre). These 95 novel mutations comprised 55 missense mutations, 12 nonsense mutations, 11 splice site mutations, and 17 small insertions/deletions. We therefore developed a mutation analysis based on a body of proof that combines the familial segregation of the mutation, the resulting biological and clinical HA phenotype, and the molecular consequences of the amino acid (AA) substitution. For the latter, we studied the putative biochemical modifications: its conservation status with cross-species FVIII and homologous proteins, its putative location in known FVIII functional regions, and its spatial position in the available FVIII 3D structures. The usefulness of such a strategy in interpreting the causality of novel F8 mutations is emphasized.

DNA Mutational Analysis↗
PubMed2004

Sustained transgene expression by human cord blood derived CD34+ cells transduced with simian immunodeficiency virus agmTYO1-based vectors carrying the human coagulation factor VIII gene in NOD/SCID mice.

BACKGROUND: Gene therapy is being studied as the next generation therapy for hemophilia and several clinical trials have been carried out, albeit with limited success. To explore the possibility of utilizing autologous bone marrow transplantation of genetically modified hematopoietic stem cells for hemophilia gene therapy, we investigated the efficacy of genetically engineered CD34+ cell transplantation to NOD/SCID mice for expression of human factor VIII (hFVIII). METHODS: CD34+ cells were transduced with a simian immunodeficiency virus agmTYO1 (SIV)-based lentiviral vector carrying the enhanced green fluorescent protein (eGFP) gene (SIVeGFP) or the hFVIII gene (SIVhFVIII). CD34+ cells transduced with SIV vectors were transplanted to NOD/SCID mice. Engraftment of transduced CD34+ cells and expression of transgenes were studied. RESULTS: We could efficiently transduce CD34+ cells using the SIVeGFP vector in a dose-dependent manner, reaching a maximum (99.6 +/- 0.1%) at MOI of 5 x 10(3) vector genome/cell. After transducing CD34+ cells with SIVhFVIII, hFVIII was produced (274.3 +/- 20.1 ng) from 10(6) CD34+ cells during 24 h in vitro incubation. Transplantation of SIVhFVIII-transduced CD34+ cells (5-10 x 10(5)) at a multiplicity of infection (MOI) of 50 vector genome/cell into NOD/SCID mice resulted in successful engraftment of CD34+ cells and production of hFVIII (minimum 1.2 +/- 0.9 ng/mL, maximum 3.6 +/- 0.8 ng/mL) for at least 60 days in vivo. Transcripts of the hFVIII gene and the hFVIII antigen were also detected in the murine bone marrow cells. CONCLUSIONS: Transplantation of ex vivo transduced hematopoietic stem cells by non-pathogenic SIVhFVIII without exposure of subjects to viral vectors is safe and potentially applicable for gene therapy of hemophilia A patients.

Animals↗
PubMed1991

Definition of the affinity of binding between human von Willebrand factor and coagulation factor VIII.

Factor VIII and von Willebrand factor are two plasma proteins essential for effective hemostasis. In vivo, they form a non-covalent complex whose association appears to be metal ion dependent. However, a precise definition of the nature of the molecular forces governing their association remains to be defined, as does their binding affinity. In this paper we have determined the dissociation constant and stoichiometry for Factor VIII binding to immobilized von Willebrand factor. The data demonstrate that these proteins interact saturably and with relatively high affinity. Computer assisted analyses of the Scatchard data favour a two site binding model. The higher affinity site was found to have a Kd of 62 (+/- 13) x 10(-12) M while that of the lower affinity site was 380 (+/- 92) x 10(-12) M. The density of Factor VIII binding sites (Bmax) present on von Willebrand factor was 31 (+/- 3) pM for the high affinity binding site and 46 (+/- 6) pM for the lower site, corresponding to a calculated Factor VIII: von Willebrand factor binding ratio of 1:33 and 1:23, respectively.

Binding Sites↗
PubMed1999

The gene expression of coagulation factor VIII in mammalian cell lines.

Both the full-length and B domain-deleted cDNA of factor VIII were constructed in plasmid pcDNA3, respectively, and successfully expressed in Cos-7 cells. The yield of recombinant factor VIII-deltaB (0.4 U/mL/10(6) cells/day) was approximately four times higher than that of the recombinant factor VIII. In addition, it was indicated that the gene expression of factor VIII is specific for cells from different tissues. The highest expression level was found in the hepatocellular carcinoma line SMMC-7721, followed by kidney, ovary, and lung cell lines. To compare the efficiency of gene expression of recombinant factor VIII, the factor VIII-deltaB gene was further reconstructed in different forms in the expression plasmid pCMV-dhfr for transient gene expression in Chinese hamster ovary cells. The redundant 5'- and 3'-untranslated sequences of factor VIII-deltaB were deleted. The cDNA encoding the heavy and light chains of factor VIII were constructed, respectively. Among them the high yield of the recombinant factor VIII was found in the coexpression of the heavy and light chain cDNA fragments of factor VIII. The deletion of the redundant 5'-untranslated sequence of factor VIII-deltaB was also beneficial for gene expression. As expected, the gene coexpression of factor VIII-deltaB and von Willibrand Factor cloned by the long-polymerase chain reaction method was also helpful for enhancing the expression level of recombinant factor VIII. A monoclonal antibody raised against factor VIII was prepared and used for the specific assay of recombinant factor VIII by the competitive ELISA method, the assay results were consistent with those determined by the one-stage bioassay.

Animals↗
PubMed1998

Therapy of coagulation factor VIII autoantibodies with long-term extracorporeal protein A adsorption and immunosuppression.

UNLABELLED: Acquired factor VIII (FVIII) inhibitors in non-haemophiliacs may pose serious treatment problems. MATERIALS AND METHODS: For IgG-adsorptions we utilized an automated plasma separation device and a plasma flow monitor. CASE REPORTS: A 63-year old woman showed life-threatening bleeding because of an inhibitor. After stabilization by porcine FVIII three cycles of a modified Malmoe treatment protocol were applied, followed by long-term cyclophosphamide p.o. and weekly IgG-adsorptions. Within twelve months the patient exhibited a complete remission. A 54-year old man presented a comparable history. Because of a good response after the first cycle he was subsequently switched to the long-term therapy. Up to now (> 7 months) FVIII activities and inhibitor titers remained stable (10-20% rsp. < 3 BU/ml). In both cases no FVIII substitution therapy was necessary. CONCLUSIONS: A modified Malmoe protocol combined with long-term cyclophosphamide p.o. and weekly IgG-adsorptions seems to be an efficient, safe and cost-effective regimen for non-haemophiliacs with FVIII inhibitors.

Autoantibodies↗
PubMed2002

Expression and characterization of a mutant recombinant blood coagulation factor VIII (rFVIII (m)).

In an earlier study, a site directed mutant rFVIII (rFVIII(m), Arg(336) --> Gln(336)) expressed in baculovirus-insect cell (Sf9) system was found to sustain high level activity during incubation at 37 degrees celsius for 24 h while the cofactor activity of normal plasma was declined steadily. In this study, a mutant B-domain deleted rFVIII(m), Arg(336) --> Gln(336) expressed in baculovirus-insect cell (Sf9) system was characterized for its enzymatic and chemical properties. The expressed rFVIII(m) and plasma FVIII (pFVIII) were purified by immunoaffinity column chromatography and identified by Western blot analysis. The partially purified rFVIII(m) exhibited cofactor specific activity of 2.01 x 10(3)units/mg protein. The molecular weight of rFVIII(m) ranged between 40 to 150 kDa with a major band at 150 kDa. Treatment of both rFVIII(m) and pFVIII with thrombin increased their cofactor activity in a similar pattern. Treatment of both the activated rFVIII(m) and native FVIII with APC decreased their cofactor activities, however, the former exhibited a slower decrease than the latter, although no significant difference was present. rFVIII(m) formed a complex with vWF, resulting in a stabilized form, and the lag period of thrombin-mediated activating was extended by vWF association. These results implicated that rFVIII(m) expressed in baculovirus-insect cell system had a comparable capacity as FVIII cofactor activity and might be a good candidate for the FVIII replacement therapy for hemophilia A patients.

Animals↗
PubMed1989

The interaction between human blood-coagulation factor VIII and von Willebrand factor. Characterization of a high-affinity binding site on factor VIII.

The interaction between human Factor VIII and immobilized multimeric von Willebrand Factor (vWF) was characterized. Equilibrium binding studies indicated the presence of multiple classes of Factor VIII-binding sites on vWF. The high-affinity binding (Kd = 2.1 x 10(-10) M) was restricted to only 1-2% of the vWF subunits. Competition studies with monoclonal antibodies with known epitopes demonstrated that the Factor VIII sequence Lys1673-Arg1689 is involved in the high-affinity interaction with vWF.

Amino Acid Sequence↗
PubMed2001

High concentrations of coagulation factor VIII and thrombosis: Is the factor VIII-binding domain of von Willebrand factor implicated?

The possibility that high factor VIII (FVIII) levels in thrombosis patients is principally explained by a gain of function in the FVIII-binding domain of von Willebrand factor (VWF), arising from amino acid substitution(s) or polymorphism(s), was investigated. Exons 18-24 of the VWF gene were sequenced in 13 thrombosis patients with high FVIII (> 1.50 IU/ml). No novel mutations were found. Four known polymorphisms were detected: G2615A and C2635T (Ex18), G2805A (Ex20) and G3130A (Ex22). Their frequencies showed no significant differences in a thrombosis vs. control cohort. The data suggest that amino acid substitutions/polymorphisms in the VWF-FVIII-binding domain are not the principal explanation for high FVIII in thrombosis patients.

Adolescent↗
PubMed2003

Human CD4+ T-cell epitope repertoire on the C2 domain of coagulation factor VIII.

Approximately 25% of severe hemophilia A patients develop antibodies (Ab) that neutralize the procoagulant function of factor (F)VIII (inhibitors). Autoimmune FVIII inhibitors may develop in individuals without congenital FVIII deficiency and cause acquired hemophilia. Low titers of anti-FVIII Ab may be present in hemophilia A patients without inhibitors and in healthy blood donors. FVIII-specific CD4+ T-cells drive the synthesis of anti-FVIII Ab. We examined the epitope repertoire of CD4+ T-cells from 15 healthy subjects, 10 hemophilia A patients without inhibitors, 11 hemophilia A patients with inhibitors, and six acquired hemophilia patients. Blood CD4+ T-cells were challenged in proliferation assays with a panel 16 overlapping synthetic peptides, spanning the sequence of the FVIII C2 domain. The sequence region 2291-2330 contained the most frequently and strongly recognized peptides in each of the four subject groups. Crystallographic B factor data and the location of these peptides within the three-dimensional structure of the C2 domain confirm that this region has a high degree of solvent exposure and flexibility within the peptide backbone, which are structural features typical of immunodominant universal CD4+ epitopes. Furthermore, this sequence region overlaps inhibitor-binding sites, suggesting that CD4+ T-cells recognizing peptide sequences within this region might be involved in inhibitor synthesis. The sequence regions 2191-2210 (recognized strongly by each study group except hemophilia A patients with inhibitors) and 2241-2290 (recognized primarily by acquired hemophilia patients and healthy subjects) share the same structural features, and also overlap inhibitor-binding sites. Although similar, there appear to be important differences in the CD4+ epitope repertoires of congenital and acquired hemophilia patients.

Binding Sites↗