SEARCH · PubMed Health

Results for “FACTOR VIII”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 451 records · Page 25Linked to original sources

PubMed2006

Disulfide bond-stabilized factor VIII has prolonged factor VIIIa activity and improved potency in whole blood clotting assays.

BACKGROUND: Genetically engineered disulfide bonds in B-domain-deleted factor (F) VIII variants (C662-C1828 FVIII and C664-C1826 FVIII) improve FVIIIa stability by blocking A2 domain dissociation because the new disulfide covalently links the A2 and A3 domains in FVIIIa. AIM: The aim of this study was to assess the hypothesis that these variants have physiologically relevant properties because of prolonged thrombin generation and improved clot formation in whole blood. METHODS: Clot-formation properties in whole blood were measured in thromboelastogram assays. The thrombin generation capabilities of the wild-type (WT) FVIII and FVIII variants were determined, and half-lives of FVIIIa variants were determined in fresh whole blood serum. RESULTS: Thromboelastogram assays were performed with fresh, severe hemophilia whole blood reconstituted with variant and WT FVIII. The two disulfide bond-stabilized FVIII variants and WT FVIII had comparable clotting times at all studied concentrations. However, when compared with WT FVIII at low concentrations, the two FVIII variants required only 10% as much FVIII to achieve comparable clot-formation rates, clot-formation times and clot firmness values. The differences between WT and FVIII variants were quite pronounced at low FVIII concentrations. Measurement of the endogenous thrombin potential in FVIII-deficient plasma supplemented with these FVIII variants confirmed that the disulfide bond-stabilized variants supported high levels of thrombin generation at lower concentrations than did WT FVIII. During the course of clot generation in whole blood, the disulfide bond-stabilized FVIIIa variants had approximately 5-fold increased half-lives relative to WT FVIIIa. CONCLUSION: C662-C1828 FVIII and C664-C1826 FVIII have physiologically relevant superior clot-forming properties in a whole blood environment, most likely due to the increased half-life of these FVIIIa variants in whole blood.

Blood Coagulation↗

PubMed1997

Dissimilar interaction of factor VIII with endothelial cells and lipid vesicles during factor X activation.

A localized and regulated cascade of proteolytic events is a prerequisite for normal haemostasis. The activation of factor X by activated factor IX (factor IXa) in the presence of activated factor VIII (factor VIIIa) is essential for the formation of a fibrin clot at sites of vascular injury. We observed sustained activation of factor X on the surface of vascular endothelial cells, whereas, in agreement with others, on synthetic negatively charged phospholipid vesicles and activated blood platelets factor X activation is transient and starts to decline a few minutes after the onset of the reaction. We examined the mechanism responsible for these differences in factor X activation. Procoagulant membrane and solution were analysed separately for the occurrence of factor VIII and its activation fragments. On negatively charged phospholipid vesicles, on dissociation of factor VIIIa, the 67 kDa light-chain fragment remains associated with the lipid membrane. As a result, factor VIII-binding sites remain occupied, and dampening of factor X activation occurs. In contrast, on monolayers of endothelial cells, no residual factor VIIIa fragments associated with the cell membrane were observed. During endothelial-cell-mediated activation of factor X, accumulation of factor VIIIa fragments was observed in the solution phase only. This finding suggests that, on endothelial cells, factor VIII-binding sites remain accessible for further factor VIII binding, guaranteeing sustained activation of factor X. These data demonstrate that the nature of the procoagulant membrane contributes to the regulation of the cofactor activity of factor VIII and thereby affects the progress of factor X activation.

Binding Sites↗

PubMed1993

The behaviour of different factor VIII concentrates in a chromogenic factor X-activating system.

A chromogenic factor Xa generation method has been developed for comparing the co-factor activity of factor VIII concentrates at physiological factor VIII concentrations (1 iu/ml). In the presence of thrombin all concentrates gave similar rapid rates of factor Xa generation, but in the absence of thrombin there were major differences between the rates of Xa generation between different products. High purity products, particularly those prepared by monoclonal antibody purification from plasma and recombinant sources, gave more rapid Xa generation than most intermediate-purity products. There was a very strong correlation between the rate of Xa generation and the difference in factor VIII potency by one-stage and two-stage assays. These results suggest the possible presence of small amounts of activated factor VIII in some concentrates, but differences in von Willebrand factor content could also contribute towards the different rates of factor Xa generation observed.

Blood Chemical Analysis↗

PubMed2005

Intracellular interaction of von Willebrand factor and factor VIII depends on cellular context: lessons from platelet-expressed factor VIII.

We have previously reported that ectopically expressed factor VIII (FVIII) is stored within platelets and is released upon platelet activation. Studies by others in various cell lines have suggested that having von Willebrand factor (VWF) coexpression is necessary for FVIII granular storage and for its secretion. We tested the importance of VWF coexpression for ectopic storage of FVIII in platelets and for its bioavailability. Transgenic mice expressing platelet-specific FVIII were crossed onto a VWF-/- background. Antigenic levels of platelet FVIII in these mice were nearly unchanged whether VWF was present or not. Whole-blood clotting times and FeCl3 carotid artery injury correction demonstrated that platelet FVIII demonstrably improved the bleeding diathesis in FVIIInull mice independent of the platelets' VWF status. Immunogold electron microscopy demonstrated that platelet FVIII is stored in platelet alpha-granules independent of the presence of VWF. It appears that FVIII's interaction with VWF and its intracellular transportation, storage, and secretion differ greatly depending on the cell type. The molecular basis for these differences now needs to be elucidated.

Animals↗

PubMed1986

Disappearance of factor VIII:C antibodies in patients with haemophilia A upon frequent administration of factor VIII in intermediate or low dose.

In 18 haemophilia A patients with antibodies against factor VIII:C (F VIII:c) the effect of regular treatment with factor VIII (F VIII) in intermediate or low dose was studied. All patients with previous maximal F VIII:c antibody levels between 5 and 60 Bethesda Units per millilitre (BU/ml) showed a decrease of antibody level and normal F VIII recovery within 1-2 months. From nine patients with previous maximal antibody levels above 60 BU/ml four showed a decrease of antibody level within 2-26 months. In four young patients F VIII prophylactic therapy was started or continued as soon as there was evidence of F VIII:c antibody activity. In three of these patients F VIII recovery normalized within a few months.

Adolescent↗

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↗

PubMed2002

The canine factor VIII 3'-untranslated region and a concatemeric hepatocyte nuclear factor 1 regulatory element enhance factor VIII transgene expression in vivo.

If gene therapy is to be an effective treatment modality for hemophilia A, therapeutic levels and tissue-restricted expression of factor VIII (FVIII) must be achieved through optimization of transgene expression. To this end, we incorporated three types of sequence elements into a canine B domain-deleted FVIII transgene cassette and individually evaluated their effect on FVIII transgene expression. Functional FVIII activity was initially assessed in vitro and hydrodynamic injection of the different transgene constructs into mice was subsequently used as a model to compare in vivo expression of the various modified transgenes. Our results demonstrate that in vitro transgene expression is, in these studies, not a good predictor of in vivo transgene performance. In vivo analysis of a hybrid tissue-restricted promoter element, consisting of a concatemer of five hepatocyte nuclear factor 1 (HNF-1) consensus-binding motifs juxtaposed to the human FVIII proximal promoter, indicates that it is as efficient at mediating expression of the FVIII protein as the cytomegalovirus promoter. Addition of the full-length canine FVIII 3'-UTR also enhances transgene expression of FVIII in vivo. Sequence analysis of the canine FVIII 3'-UTR and human FVIII 3'-UTR indicates that the former lacks instability sequences and may therefore be more effective in stabilizing FVIII mRNA. Subsequent inclusion of FVIII introns 16 and 17 into the natural locations of the transgene disrupted mRNA processing and abolished expression of the FVIII protein. Introduction of intron 17 proximal to the FVIII cDNA did not enhance in vivo expression of canine FVIII from the transgene.

3' Untranslated Regions↗

PubMed1988

The natural history of factor VIII:C inhibitors in patients with hemophilia A: a national cooperative study. II. Observations on the initial development of factor VIII:C inhibitors.

During a 4-year multicenter cooperative study of acquired factor VIII inhibitors in persons with hemophilia A, new inhibitors were detected in 31 of 1,306 patients who entered the study without an inhibitor or the history of an inhibitor. The incidence of new inhibitors was eight per 1,000 patient-years of observation. The factor VIII:C level before inhibitor development was less than or equal to 0.03 U/mL in 29 individuals and 0.06 U/mL and 0.07 U/mL in the remaining two. Factor VIII:Ag levels were measured in 27 individuals and were less than 0.03 U/mL in 23 and 0.05 to 0.11 U/mL in the remaining four. Maximum inhibitor levels ranged from 1.0 to 9,044 Bethesda U/mL. In seven patients under the age of 20, relatively weak inhibitors (none higher than 4.3 Bethesda U/mL) were detected on only a single occasion despite continued factor VIII challenge. In the other 24 patients with inhibitors detected on multiple occasions, 50% had appeared by age 20 and 71% by age 30. Seventeen of the 31 inhibitors, including 12 of 15 with maximum values greater than 10 Bethesda U/mL, developed within 75 exposure days to factor VIII.

Age Factors↗

PubMed1992

Epitope mapping of human factor VIII inhibitor antibodies by site-directed mutagenesis of a factor VIII polypeptide.

Previous epitope mapping studies of human factor VIII (FVIII) inhibitor antibodies with heavy chain specificity localized epitopes to the amino-terminal half of the FVIII A2 domain. In this report we have used unidirectional deletion analysis and site-directed mutagenesis to identify a minimum length polypeptide and amino acid residues that contribute to the FVIII conformation recognized by these antibodies. Bacterial expression plasmids were exploited to demonstrate that a FVIII polypeptide of approximately 150 residues is required to generate a common heavy chain epitope(s). Another series of plasmids were constructed that synthesize: a FVIII polypeptide containing an internal deletion; four polypeptides with single residue substitutions; two polypeptides with triple residue changes; and a quadruple amino acid replacement within one polypeptide. The relative reactivities of the wild-type and mutant FVIII polypeptides were tested by immunoblotting, inhibitor neutralization assays and ELISA with a variety of human FVIII inhibitor auto- and alloantibodies. These techniques illustrate that the internal deletion mutant and one of the relatively conservative amino acid substitution triple mutants, mutant 389, resulted in significantly decreased immunoreactivity. The data identify FVIII Glu389,390,391 as three critical components of an epitope for human FVIII inhibitor antibodies and identify a major inhibitory epitope involved in the immune response to FVIII.

Amino Acid Sequence↗

PubMed1987

Inhibitor to factor VIII in a non-haemophilic patient: evaluation of the response to DDAVP and the in vitro kinetics of factor VIII. A case report.

We report a case of inhibitor to factor VIII in a non-haemophilic patient. Immunosuppressive therapy with azathioprine was started, but without any advantage. Evaluation of the kinetics of exogenous factor VIII in vitro showed a rapid but incomplete neutralization of factor VIII. Following s.c. 1-deamino-8-D-arginine vasopressin (DDAVP) administration, a large and prolonged increase in factor VIII:C and von Willebrand factor antigen occurred together with complete inhibitor saturation. Therefore DDAVP may represent an important tool in the management of the bleeding episodes in these patients and evaluation of its suitability in the management of these patients should be carried out.

Blood Coagulation Disorders↗

PubMed1993

A factor VIII neutralizing monoclonal antibody and a human inhibitor alloantibody recognizing epitopes in the C2 domain inhibit factor VIII binding to von Willebrand factor and to phosphatidylserine.

A neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 micrograms/ml for IgG and 0.2 micrograms/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 micrograms/ml by IgG and 6.3 micrograms/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 micrograms/ml.

Antibodies, Monoclonal↗

PubMed2001

Structural and functional characteristics of the B-domain-deleted recombinant factor VIII protein, r-VIII SQ.

Recombinant factor VIII SQ (r-VIII SQ), ReFacto, is a recombinant factor VIII product similar to the smallest active factor VIII protein found in plasma-derived factor VIII (p-VIII) concentrates. The protein comprises two polypeptide chains of 80 and 90 kDa and lacks the major part of the heavily glycosylated B-domain i.e. amino acids Gln744 to Ser1637. r-VIII SQ retains six potential glycosylation sites for N-linked oligosaccharides at asparagine residues 41, 239, 582, 1685, 1810 and 2118. We describe a thorough comparison of the characteristics of r-VIII SQ with those of p-VIII. The primary and secondary structures of r-VIII SQ were in good agreement with that of B-domain-deleted p-VIII (p-VIII-LMW) as shown by SDS-PAGE, Western blotting with antifactor VIII antibodies, tryptic mapping, amino acid sequence analysis and circular dichroism spectroscopy. A few divergences also existed. Thus r-VIII SQ was shown to contain a small amount of the single chain primary translation product of 170 kDa and also the product specific sequence of 14 amino acids, the SQ-link, in the C-terminal end of the 90 kDa chain. It was shown that r-VIII SQ had a high specific activity of about 14,000 IU VIII:C/mg as determined by use of a chromogenic substrate assay. The r-VIII SQ protein was comparable to p-VIII forms with a retained B-domain, in terms of potency measured by a chromogenic substrate or a two-stage clotting assay, in interactions with thrombin, and with activated protein C (APC) in combination with Protein S. The ability of r-VIII SQ to participate as a cofactor in factor Xa generation in a mixture of factors IXa and X, phospholipid and calcium was in conformity with that of p-VIII. Furthermore r-VIII SQ had a good binding capacity for phospholipid vesicles and von Willebrand factor (vWF) as shown in gel filtration studies. The same kinetics in binding to von Willebrand factor was found for r-VIII SQ and p-VIII as determined by real-time biospecific interaction analysis (BIA) with use of the BIAcore instrument. The apparent association rate constant was 4 x 10(6) M(-1)s(-1). Two dissociation rate constants were found, 1 X 10(-2)s(-1) and 4 x 10(-4)s(-1). The results extend the present knowledge that the factor VIII B-domain is dispensable for the factor VIII cofactor function in hemostasis.

Binding Sites↗

PubMed1996

Induction of human factor VIII inhibitors in rats by immunization with human recombinant factor VIII: a small animal model for humans with high responder inhibitor phenotype.

Hemophilia A is a clotting disorder that is due to reduced or absent coagulation factor VIII (FVIII) activity. In approximately 25% of people with severe hemophilia A, standard treatment with intravenous plasma-derived or recombinant FVIII (rFVIII) induces anti-FVIII antibodies that inhibit FVIII activity (inhibitors). We describe the development of a rat model to study the formation of inhibitors. Immunization of rats with human rFVIII in adjuvant induced an anti-human rFVIII antibody response characteristic of an anti-FVIII inhibitor response in hemophilia A patients. The rats exhibited a rapid, polyclonal secondary antibody response to human rFVIII. These antibodies were reactive against epitopes located in the heavy and light chains. All the rFVIII-immunized rats developed antibodies against the FVIII C2 domain, a region of major reactivity in hemophilia A patients with inhibitors. Furthermore, competition ELISAs demonstrated that rat and human anti-FVIII antibodies recognized identical or overlapping epitopes of the FVIII molecule. The rat anti-FVIII antibodies also functioned as human FVIII inhibitors with titers ranging from 120 to 2048 Bethesda Units (B.U.). We propose that this rat model may be useful to investigate immune responses to FVIII and may lead to better therapies for FVIII inhibitors.

Animals↗

PubMed1985

Sequential administration of human and porcine factor VIII for surgical treatment of a parotid tumour in a patient with a factor VIII inhibitor.

The sequential use of human and porcine factor VIII for the treatment of a patient with a moderately high titre (38 Bethesda units) factor VIII inhibitor enabled us to provide adequate haemostatic cover for 17 days. In this period a pleomorphic adenoma of the parotid gland was removed. Bleeding did not occur and wound healing was uneventful.

Adenoma, Pleomorphic↗

PubMed1981