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PubMed2006

In vitro differentiation of mouse ES cells into hepatocytes with coagulation factors VIII and IX expression profiles.

Coagulation factors II, V, VII, VIII, IX and X are produced by hepatocytes. So factors VIII and IX deficiencies, which result in hemophilia A and B, have the potential to respond to cellular replacement therapy. Embryonic stem (ES) cells provide a unique source for therapeutic applications. Here, E14 mouse ES cells have been induced into hepatocytes in vitro. Morphology revealed that ES-derived hepatic-like cells were round or polyhedral shaped with distinct boundary of individual cells, and some arranged in trabeculae. These cells expressed endodermal- or liver-specific mRNA--transthyretin (TTR), alpha1-anti-trypsin (AAT), alpha-fetoprotein (AFP), albumin (ALB), glucose-6-phoshpatase (G6P) and tyrosine aminotransferase (TAT). Approximately (85.1 +/- 0.5)% of the ES-derived cells was stained positive green with ICG uptake. These cells were also stained magenta as a result of PAS reaction. In this paper, expression of coagulation factors VIII and IX mRNA in the ES-derived cells is documented. Therefore, ES cells might be developed as substitute donor cells for the therapy of coagulation factor deficiencies.

Animals↗
PubMed2005

Low-density lipoprotein receptor-related protein polymorphisms in patients with elevated factor VIII coagulant activity and venous thrombosis.

Elevated factor VIII coagulant activity (FVIII:C) levels (>150 IU/dl) represent a prevalent independent risk factor for venous thromboembolism (VTE). Low-density lipoprotein receptor-related protein (LRP) is involved in factor VIII clearance in vivo, and elevated FVIII:C was a feature of the LRP knockout mouse model. Three coding polymorphisms of LRP1 (exon 3, C766T; exon 14, A217V; and exon 39, D2080N), together with an insertion/deletion polymorphism within the first intron of lipoprotein receptor-associated protein (LRPAP1), have been identified. In addition, LRP1 2080D was recently reported to be associated with increased plasma FVIII:C levels in normal individuals. In this study, we investigated the role of these four polymorphisms in patients with objectively confirmed VTE and elevated FVIII:C levels. In our control group, genotype distributions were consistent with previous reports. Neither the allele frequencies nor genotype distributions at LRP1 A217V, LRP1 D2080N and LRPAP1 intron 1 were significantly different between the elevated FVIII:C and control groups. In contrast to previous reports, we found no effect of LRP1 D2080N genotype on plasma FVIII:C levels in normal individuals. More importantly, prevalence of the LRP1 2080D allele was not increased in the group of patients with high FVIII:C and VTE. We conclude that LRP1 and LRPAP1 polymorphisms are not responsible for high FVIII:C levels in patients with VTE.

Adult↗
PubMed1982

Immunologic studies of factor VIII coagulant activity (VIII:C). 2. Factor VIII in selected vertebrates.

Cross reactive antigens to factor VIII have been measured in a range of vertebrate phyla. VIII coagulant antigen (VIII:CAg) measured using a human antibody was in general, much lower than reported values of VIII coagulant activity (VIII:C) except in simians and the guinea pig. The dose response curve for the assay was parallel in all cases. Factor VIII-related antigen (VIIIR:Ag) measured using rabbit antibody to human VIIIR:Ag was low or absent in most species except simians and the dose response curve was non-parallel to the human standard except with goat plasma. Avians lacked cross-reactive material.

Animals↗
PubMed1986

DDAVP-induced increases in coagulation factor VIII and von Willebrand factor in the plasma of conscious dogs.

Infusion of the vasopressin analogue DDAVP into five normal dogs at doses of 0.1-2.0 micrograms DDAVP per kg body weight induced dose-dependent increases in the plasma content of coagulation factor VIII and von Willebrand factor. Plasma concentrations of von Willebrand factor (determined antigenically as factor VIII-related antigen and functionally as coagglutinin cofactor activity) and coagulation factor VIII were measured immediately before and at 10, 30, and 120 min after 10-min intravenous infusions of DDAVP. The greatest increases in coagulation factor VIII were produced with the 2.0 micrograms/kg dose. Ten minutes after infusion the mean increase in coagulation factor VIII was 32 units/dl (concentrations of all indices were reported relative to concentrations in a standard canine plasma pool, arbitrarily assigned a concentration of 100 units/dl) and this increase did not change significantly throughout the duration of the experiment. At 10 min post-infusion, the mean factor VIII-related antigen concentration increased 81 units/dl (dose = 2.0 micrograms/kg) and did not change significantly for the duration of the experiment. The maximum mean increase in coagglutinin cofactor activity, 141 units/dl, occurred 10 min after infusion (dose = 1.0 microgram/kg). Coagglutinin cofactor activity decreased significantly from peak activity by 120 min post-infusion.

Animals↗
PubMedDate not supplied

B-Domain deleted recombinant coagulation factor VIII modified with monomethoxy polyethylene glycol.

Recombinant coagulation factor VIII (r-VIII SQ) was chemically modified with monomethoxy poly(ethylene glycol) (mPEG). Three mPEG derivatives were used for coupling to the r-VIII SQ lysines, a mixed anhydride of monomethoxy poly(ethylene glycol) succinic acid (mPEG-SAH), monomethoxy poly(ethylene glycol) succinimidyl succinate (mPEG-SS), and monomethoxy poly(ethylene glycol) tresylate (mPEG-TRES). A consequence of the modification with all derivatives was a substantial reduction in coagulant activity, even at very low degrees of modification. A method was developed with the purpose of avoiding conjugation at certain important biological sites on the factor VIII and thereby producing conjugates with better retained activity. This was achieved by immobilizing the protein onto a solid matrix during the modification reaction. Characterization of conjugates by SDS-PAGE, western blots, interaction with von Willebrand factor (vWf), and thrombin activation/inactivation analyses was undertaken. The SDS-PAGE and western blots revealed coupling heterogeneity regarding degree of modification. The amount of factor VIII able to bind to vWf decreased with the conjugation. Thrombin activated the modified factor VIII to essentially the same extent as the reference preparation of r-VIII SQ. Inactivation of the modified factor VIII was, however, slower than inactivation of the unmodified protein. Finally, an in vitro study was performed to evaluate the influence of the mPEG modification on the protein stability in extract of porcine tissue. Despite that conjugates with low degrees of modification were included in the study, the coagulant activity was preserved to a significantly higher extent in all incubation mixtures containing conjugates compared to that with unmodified protein.

Animals↗
PubMed1989

Inhibition of human coagulation factor VIII by monoclonal antibodies. Mapping of functional epitopes with the use of recombinant factor VIII fragments.

The epitopes of four monoclonal antibodies against coagulation Factor VIII were mapped with the use of recombinant DNA techniques. Full-length Factor VIII cDNA and parts thereof were inserted into the vector pSP64, permitting transcription in vitro with the use of a promoter specific for SP6 RNA polymerase. Factor VIII DNA inserts were truncated from their 3'-ends by selective restriction-enzyme digestion and used as templates for 'run-off' mRNA synthesis. Translation in vitro with rabbit reticulocyte lysate provided defined radiolabelled Factor VIII fragments for immunoprecipitation studies. Two antibodies are shown to be directed against epitopes on the 90 kDa chain of Factor VIII, between residues 712 and 741. The 80 kDa chain appeared to contain the epitopes of the other two antibodies, within the sequences 1649-1778 and 1779-1840 respectively. The effect of antibody binding to these sequences was evaluated at two distinct levels within the coagulation cascade. Both Factor VIII procoagulant activity and Factor VIII cofactor function in Factor Xa generation were neutralized upon binding to the region 1779-1840. The antibodies recognizing the region 713-740 or 1649-1778, though interfering with Factor VIII procoagulant activity, did not inhibit in Factor Xa generation. These findings demonstrate that antibodies that virtually inhibit Factor VIII in coagulation in vitro are not necessarily directed against epitopes involved in Factor VIII cofactor function. Inhibition of procoagulant activity rather than of cofactor function itself may be explained by interference in proteolytic activation of Factor VIII. This hypothesis is in agreement with the localization of the epitopes in the proximity of thrombin-cleavage or Factor Xa-cleavage sites.

Antibodies, Monoclonal↗
PubMed1997

Biosynthesis, assembly and secretion of coagulation factor VIII.

Factor VIII is a large complex glycoprotein that is deficient in hemophilia A. It has a domain organization consisting of A1-A2-B-A3-C1-C2 where the B domain is a heavily glycosylated region that is dispensable for procoagulant activity. Factor VIII expression is 10-to 20-fold lower than the homologous coagulation factor V. Factor VIII expression is limited due to a low level of steady-state messenger RNA in the cytoplasm and inefficient transport of the primary translation product from the endoplasmic reticulum to the Golgi apparatus. Within the secretory pathway, factor VIII is processed to a heterodimer of the heavy chain (domains A1-A2-B) in a metal ion association with the light chain (domains A3-C1-C2). Upon secretion from the cell, von Willebrand factor binds the light chain of factor VIII and stabilizes the factor, preventing degradation. Protein folding within the mammalian secretory pathway is facilitated by molecular chaperones. Within the endoplasmic reticulum, factor VIII exhibits stable interaction with protein chaperones identified as the immunoglobulin-binding protein (BiP), calnexin and calreticulin. BiP is a peptide-dependent ATPase that interacts with exposed hydrophobic surfaces on unfolded proteins or unassembled protein subunits. A potential BiP binding site within factor VIII has been identified. Mutation of a single amino acid residue in the potential BiP binding site increased the secretion efficiency of factor VIII by threefold. Interestingly, the proposed BiP binding site is adjacent to a type-1 copper binding site within the A1 domain that is required for interaction between the factor VIII A1 domain and the A3 domain. We propose that Cu(I) binds the type-1 copper ion-binding site in the A1 domain and provides the essential requirement for a stable interaction between the heavy and light chains. Calnexin and calreticulin are transmembrane and lumenal proteins, respectively, localized to the endoplasmic reticulum, which associate transiently with many soluble and membrane glycoproteins during folding and subunit assembly. The calnexin and calreticulin interaction with factor VIII occurs primarily through amino-terminal linked oligosaccharides within the heavily glycosylated factor VIII B domain and this interaction appears to be required for factor VIII secretion. The findings suggest that factor VIII cycles through interactions with BiP, calnexin and calreticulin. Although the interaction with BiP does not appear to be required for factor VIII secretion, data suggest that the calnexin and/or calreticulin interaction is required for secretion. The observations suggest a unique requirement for carbohydrate processing and calnexin/calreticulin interaction that may limit the productive secretion of factor VIII and have implications for approaches towards somatic cell gene therapy for hemophilia A.

Animals↗
PubMed2003

Coagulation factor VIII: structure and stability.

Factor VIII (FVIII), a coagulation factor in the blood, is one of the most complex proteins known today. To facilitate the rapid development of a more convenient and safer FVIII product and to improve the quality of life for hemophilia patients, this short article reviews the recent investigations on the structure, activity, and more importantly, stability of FVIII.

Animals↗
PubMed2005

The B domain of coagulation factor VIII interacts with the asialoglycoprotein receptor.

BACKGROUND: Coagulation factor VIII (FVIII) is a heavily glycosylated heterodimeric plasma protein that consists of a heavy (domains A1-A2-B) and light chain (domains A3-C1-C2). It has been well established that the clearance of FVIII from the circulation involves mechanisms that are sensitive to the low-density lipoprotein receptor (LDLR) family antagonist receptor-associated protein (RAP), including LDLR-related protein. Because FVIII clearance in the presence of a bolus injection of RAP still occurs fairly efficient, also RAP-independent mechanisms are likely to be involved. OBJECTIVES: In the present study, we investigated the interaction of FVIII with the endocytic lectin asialoglycoprotein receptor (ASGPR) and the physiological relevance thereof. METHODS AND RESULTS: Surface plasmon resonance studies demonstrated that FVIII dose-dependently bound to ASGPR with high affinity (Kd approximately 2 nM). FVIII subunits were different in that only the heavy chain displayed high-affinity binding to ASGPR. Studies employing a FVIII variant that lacks the B domain revealed that FVIII-ASGPR complex assembly is driven by structure elements within the B domain of the heavy chain. The FVIII heavy chain-ASGPR interaction required calcium ions and was inhibited by soluble D-galactose. Furthermore, deglycosylation of the FVIII heavy chain by endoglycosidase F completely abrogated the interaction with ASGPR. In clearance experiments in mice, the FVIII mean residence time was prolonged by the ASGPR-antagonist asialo-orosomucoid (ASOR). CONCLUSIONS: We conclude that asparagine-linked oligosaccharide structures of the FVIII B domain recognize the carbohydrate recognition domains of ASGPR and that an ASOR-sensitive mechanism, most likely ASGPR, contributes to the catabolism of coagulation FVIII in vivo.

Animals↗
PubMed1993

Biological risk factors for restenosis after percutaneous transluminal coronary angioplasty.

In an attempt to discern biological (such as thrombotic or fibrinolytic) risk factors in patients developing restenosis after percutaneous transluminal coronary angioplasty, the following factors were measured prior to angiography in a population of 23 patients (20 men, 3 women, mean age 57 +/- 5 yr) treated by a successful angioplasty (gain > 20% and residual stenosis < 50%) for stable angina pectoris and who had a routine angiographic restudy. The following factors were thus assessed: lipid factors: cholesterol, triglycerides, high density lipoprotein cholesterol, low density lipoprotein cholesterol, apolipoprotein AI, apolipoprotein B; coagulation factors: fibrinogen, antithrombin III, fibrinopeptide A, factor VIII coagulant, factor VIII antigen, protein C; factors of physiological fibrinolysis: plasminogen, alpha 2-antiplasmin, tissue plasminogen activator and euglobulin clot lysis time before and after venous occlusion, plasminogen activator inhibitor before venous occlusion; and factors of platelet release: beta-thromboglobulin, platelet factor 4. Also studied were clinical characteristics: age, gender, diabetes, hypertension, smoking habits, previous myocardial infarction; angiographic data: global extent of coronary artery disease, location of the stenosis in a bend or branch point, complexity of the lesion, initial and residual stenosis and treatment during follow-up. The coronary angiograms were analyzed by a computer-assisted method with automatic edge detection. On angiographic criteria, 6 patients (restenosis group) were judged to have developed a restenosis (30% decrease in diameter and/or return to a 50% stenosis). The other 17 patients (those without restenosis) were considered to have a persistent success. Apart from age (group without restenosis: 55 +/- 6; restenosis group 61 +/- 5, p < 0.04), there were no differences in clinical, angiographic or treatment variables. There were no differences in lipid factors, but significant differences were observed in hemostatic variables: fibrinogen (without restenosis: 3.18 +/- 0.83; restenosis: 3.83 +/- 0.51 milligrams, p = 0.05), tissue plasminogen activator before venous occlusion (without restenosis: 10.9 +/- 26.8; restenosis: 232.5 +/- 371.2 IU, p < 0.04), euglobulin clot lysis time after venous occlusion (without restenosis: 176.5 +/- 100.5; restenosis: 78.6 +/- 40.2 min, p < 0.05) and for marker of the platelet release: platelet factor 4 (without restenosis: 10.8 +/- 7.9; restenosis: 20.5 +/- 7.5 ng/l, p < 0.04). These findings indicate that patients developing restenosis after coronary angioplasty tend to have an imbalance in the prothrombotic-antithrombotic equilibrium prior to the procedure.

Aged↗
PubMed2003

Characterization of human hybridoma clones isolated from hemophilia patients with specificity for different domains of coagulating factor VIII.

Hemophilia A patients treated with human coagulating factor VIII (FVIII) may develop inhibitory antibodies (inhibitors). Characterization of the inhibitors at the clonal level may help exploring new therapeutic strategies. We have generated lymphoblastoid cell lines (LCLs) producing anti-FVIII antibodies from peripheral blood lymphocytes of hemophilia A patients with high inhibitor titers. We fused the anti-FVIII-positive LCLs with a heteromyeloma, to produce FVIII specific hybridomas. We determined the specificity, isotype, idiotypic and immunoglobulin (Ig) variable region heavy (VH) chain gene family profiles of the secreted antibodies (Ab) by ELISA, immunoblotting and RT-PCR. We established eight hybridomas which produced high titers of anti-FVIII Ab. All hybridomas secreted IgM Ab, associated with either kappa(5/8) or lambda(3/8) light chain. Analysis of the expressed VH genes by RT-PCR revealed that the hybridomas utilized only the VH1 (63%) or the VH3 (37%) gene families. Among the cross-reactive idiotypes (CRIs) we tested, only the VH1 and VK3b-associated CRIs were expressed by 3 hybridomas. Immunoblotting of thrombin-digested FVIII demonstrated distinct patterns of reactivity of the monoclonal Ab (MAb) secreted by the hybridomas, which recognized either the A2 domain of the Fvm heavy chain, or the light chain, or both. Our findings suggest that: a) the isotype of the anti-FVIII Ab secreted by LCLs and hybridoma clones (IgM) differs from that of anti-FVIII Ab in vivo, which are predominantly IgG4: this suggests a negative selection of the isotype-switched FVIII-specific B-cells in the periphery of these patients; b) the anti-FVIII Ab have a biased representation of the VH1 gene family, and c) somatic mutations in the VH genes coding for FVIII specificity occur in the anti-FVIII Ab response, as evidenced by lack of expression of the VH-associated CRI.

Autoantibodies↗
PubMed2000

Change of coagulation factor VIII and antithrombin III activity in bank-stored blood.

Coagulation factor VIII and antithrombin III activity were detected in 15 health donors. It was found that antithrombin III activity decreased obviously 12 h after blood drawing. It lost 56% of the activity at the 3rd day, and 70% of the activity at the 7th day. FVIII:c showed no obvious change after 24 h, until the 3rd day. It lost 40%-60% of the activity after 36 h and was reduced to the 30% of the original activity at the 5th day. Our results suggested that at the 3rd day coagulation factor VIII of bank-stored blood can be used to replenish antithrombin III, while bank-stored blood in one day can be used to replenish F VIII.

Adolescent↗
PubMed1978

Factor VIII coagulant activity in an African population in relation to a recognized standard.

The normal range of factor VIII coagulant activity (derived from log potency ratio) in some sections of the Nigerian population has been established at 0.65--5.55 iu/ml with a geometric mean of 1.90 iu/ml. This was determined against an acceptable standard (MRC Human 68/413 with activity o.66 iu/ml). The distribution of the potency ratio was log normal. The level was not affected by age or an abnormal haemoglobin (Hb A + S or A + C). The mean activity in females was significantly higher than the mean value in male subjects. With the use of a stable standard, our results show that the conclusions of some previous studies in respect of some of the parameters such as population distribution but which did not use a recognized standard, were valid. Within the age limits of our subjects, age did not affect the population level of factor VIII coagulant activity.

Adolescent↗
PubMed2003

LMAN1 is a molecular chaperone for the secretion of coagulation factor VIII.

Combined deficiency of both coagulation factors (F)V and VIII is a rare autosomal recessive bleeding disorder caused by null expression of LMAN1 (previously termed ERGIC-53) in a majority of affected individuals. Previously, a requirement for a functional LMAN1 cycling pathway between the ER and Golgi was demonstrated for efficient secretion of FV and FVIII (Moussalli et al. J Biol Chem 1999; 274: 32569), however, the molecular nature of the interaction between LMAN1 and its cargo was not characterized. Using coimmunoprecipitation of LMAN1 and FVIII from transfected HeLa and COS-1 cells, we demonstrate an interaction between LMAN1 and FVIII in vivo. The interaction was mediated via high mannose-containing asparagine-linked oligosaccharides that are densely situated within the B domain of FVIII, as well as protein-protein interactions. These results are interpreted based on the recent determination of the crystal structure of the carbohydrate recognition domain of LMAN1.

Factor V Deficiency↗
PubMed1987

Factor VIII coagulant moiety binds to platelets by binding to phospholipids of the platelet membrane.

Washed human platelets were incubated with commercial factor VIII concentrate, or with purified factor VIII coagulant moiety. Platelets were then washed again and lysed by sonication. VIII:Ag and vWf:Ag were measured in the platelet lysate prior to and after incubation of the lysate with phospholipase C (PL-C). Platelet bound VIII:Ag was significantly higher after incubation of washed platelets with factor VIII concentrate than after incubation with buffer. Platelet bound VIII:Ag was further increased when platelets had been incubated with concentrate in the presence of thrombin and collagen. In contrast, only a slight increase in platelet bound vWf:Ag was observed after incubation of platelets with concentrate. When washed platelets had been incubated with factor VIII coagulant moiety, also significantly more platelet bound VIII:Ag was observed than after incubation with buffer. Measurable VIII:Ag, but not vWf:Ag, increased significantly after incubation of the platelet lysate with PL-C. When intact washed platelets had been treated with PL-C prior to the incubation with concentrate, binding of VIII:Ag to platelets was nearly completely abolished. Our data suggest that the factor VIII coagulant moiety binds to phospholipids of the platelet membrane and thereby contributes to the assembly of the factor X activating complex on the platelet surface.

Blood Platelets↗
PubMed1986

Characterization of four monoclonal antibodies to factor VIII coagulant protein and their use in immunopurification of factor VIII.

Four monoclonal anti-VIII:C antibodies were obtained from the fusion of the splenocytes of one Balb/C mouse with a specific activity ranging from 2.3 to 45,000 U/mg when purified from ascitic fluid. Only one antibody was able to inhibit completely Factor VIII:C in normal plasma. The four antibodies could bind Factor VIII:CAg in plasma and commercial concentrate both in liquid and solid phase, and were suitable for immunopurification of Factor VIII:C. Three antibodies competed with polyclonal anti-VIII:CAg Fab' in a liquid phase IRMA, and all of them were able to displace their own binding to Factor VIII:CAg. Competition studies between monoclonal antibodies for the binding to Factor VIII:CAg were performed and showed the recognition of different epitopes and various functional impact. These studies indicate that at least one antibody, with the lowest anti-VIII:C titer clearly recognizes a different epitope of VIII:C than those recognized by the others. Affinity constants ranged from 10(9) to 10(10) l/mole.

Animals↗
PubMedDate not supplied

A mathematical model of coagulation factor VIII kinetics.

It appears that the binding of coagulation factor VIII to von Willebrand factor in plasma stabilizes the otherwise highly labile factor VIII. A mathematical model of factor VIII kinetics has been developed based upon this proposed effect of factor VIII binding. The model's kinetic parameter values have been estimated by fitting the model to data available in the medical literature. The model gives accurate quantitative predictions of the elevated steady-state concentrations of factor VIII in clinical conditions associated with the acute phase reaction and in pregnancy, the decreased steady-state concentrations of factor VIII in females heterozygous for hemophilia, the decreased steady-state concentrations of factor VIII in patients with type 1 (heterozygous) and type 3 (homozygous) von Willebrand disease, and the variable half-life of factor VIII in factor replacement therapy for hemophilia and von Willebrand disease.

Computer Simulation↗