PubMed Health⌕ Search

Biomedical subjects

E G Tuddenham

Publications and source records attributed to E G Tuddenham.

At least 19 recordsLinked to original sources
PubMed1999

Inhibition of tissue factor-dependent and -independent coagulation by cell surface expression of novel anticoagulant fusion proteins.

BACKGROUND: Thrombotic vascular occlusion occurs in disorders of diverse etiology, including atherosclerosis, vasculitis, and disseminated intravascular coagulation. The same process results in hyperacute rejection of renal allografts transplanted into sensitized patients and remains a major problem in experimental xenotransplantation. METHODS: We have previously described the design and expression of several genetic constructs encoding novel fusion proteins with anticoagulant properties. They are based on two naturally occurring soluble anticoagulant proteins, human tissue factor pathway inhibitor (hTFPI) and the leech protein hirudin, which act early and late in the clotting cascade, respectively. We report the expression of human hTFPI-CD4 on the surface of immortalized porcine endothelial cells (IPEC), and show that it functions across the species divide as evidenced by the binding of membrane-expressed porcine tissue factor (pTF)-human factor VIIa complexes. RESULTS: Using a human plasma recalcification clotting assay, we distinguished between pTF-dependent and pTF-independent fibrin generation, and we have demonstrated that expression of hTFPI-CD4 on IPEC effectively prevented pTF-dependent clotting. Moreover, we show that when hTFPI-CD4 was co-expressed with the hirudin construct, the procoagulant properties of in vitro cultured, activated IPEC were almost completely abolished. CONCLUSIONS: These results suggest that these novel anticoagulant molecules may prove useful therapeutic agents for gene therapy or for transgenic expression in animals whose organs may be used for cliniCal xenotransplantation.

Animals↗
PubMed1998

Expression of hirudin fusion proteins in mammalian cells: a strategy for prevention of intravascular thrombosis.

BACKGROUND: Intravascular thrombosis occurs in disorders of diverse pathogeneses, including allograft and xenograft rejection. In this in vitro study, we describe an approach for tethering the specific thrombin inhibitor hirudin to plasma membranes as part of a genetic strategy for regulating intravascular coagulation. METHODS AND RESULTS: An HLA class I leader sequence was fused with hirudin linked to domains 3 and 4 of human CD4 and intracytoplasmic sequence from either CD4 or human P-selectin. The constructs were transfected into mouse fibroblasts, Chinese hamster ovary (CHO)-K1 cells, immortalized porcine endothelial cells (IPECs), and a pituitary secretory cell line (D16/16). Thrombin binding to the hirudin fusion proteins expressed on fibroblasts and CHO-K1 cells could be blocked by an anti-hirudin monoclonal antibody and by pretreatment of thrombin with either the synthetic tripeptide thrombin inhibitor PPACK or native hirudin. Hirudin expression significantly modified the procoagulant phenotype of IPECs in human plasma, leading to prolongation of clotting times. Hirudin-CD4-P-selectin fusion proteins accumulated in adrenocorticotropic hormone-containing granules in D16/16 cells, with no cell surface expression except on activation with phorbol ester, when hirudin relocated to the outer membrane. CONCLUSIONS: Hirudin fusion proteins were expressed on mammalian cells, where they reduced local thrombin levels and inhibited fibrin formation. Regulated expression was achieved on activated cells by use of the cytoplasmic sequence from P-selectin. In vivo, these fusion proteins may prove useful transgenic or gene therapy agents for preventing intravascular thrombosis.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Enhanced expression of urokinase receptor induced through the tissue factor-factor VIIa pathway in human pancreatic cancer.

Overexpression of tissue factor (TF) is characteristically observed in advanced pancreatic cancer and has been associated with invasion and metastasis. Functional responses of TF activation are here investigated using as a model system the human pancreatic cancer cell lines SW979 (which overexpresses TF) and MIAPaCa2 (which does not express detectable levels). After stimulation of these cell lines with factor VIIa (FVIIa), the only known TF ligand, expression of urokinase receptor (uPAR) gene was up-regulated in SW979 cells in a dose-dependent manner but not in MIAPaCa2 cells. Interestingly, urokinase (uPA) and its specific inhibitor PAI-1 were not up-regulated. Exposure to functionally inactivated FVIIa did not show any effect on uPAR expression on SW979 cells despite binding to TF with higher efficiency. The neutralizing anti-TF antibody 5G9 blocked the FVIIa-induced up-regulation of uPAR completely, whereas hirudin failed to block this up-regulation. Treatment of SW979 cells with Factor Xa did not up-regulate the expression of uPAR gene, whereas treatment with FVII induced the same level of enhanced uPAR gene expression as that with FVIIa. In the matrigel invasion assay, enhanced invasion of SW979 cell line induced by FVIIa was completely inhibited by anti-TF antibody and alpha2-antiplasmin. Moreover, the endogenous levels of uPAR gene expression were significantly correlated with the level of TF gene expression in 19 human cancer cell lines (P < 0.05). These data suggest that up-regulation of uPAR expression by tumor cells leading to tumor invasion is induced through the TF-FVIIa pathway rather than TF-initiated thrombin generation. This is the first report that TF may be one of the key receptors that can up-regulate expression of the plasminogen activator receptor in human cancer cells to enhance tumor invasion and metastasis.

Breast Neoplasms↗
PubMed1998

Exclusion of the first EGF domain of factor VII by a splice site mutation causes lethal factor VII deficiency.

We have studied a family with homozygous lethal, blood coagulation factor VII (FVII) deficiency. To identify the mutation responsible for the deficiency, exons 2 to 8 and the intron-exon junctions of their FVII genes were amplified from peripheral white blood cell DNA by polymerase chain reaction and screened by single-strand conformational polymorphism analysis. The fragment showing aberrant mobility was cloned and sequenced. We detected a single point mutation, a homozygous G to A substitution at nucleotide position 6070, in the invariant GT dinucleotide at the 5' splice site of intron 4. Homozygosity was confirmed by loss of a site for the restriction endonuclease Mlu I. Analysis of the splicing pattern of ectopic transcripts in lymphocytes in the parents revealed that this mutation is associated with skipping of exon 4, which produces an mRNA encoding FVII with an in-frame deletion of the first epidermal growth factor-like domain (EGF 1). Transient transfection of COS-7 cells with an expression vector containing the triangle upEGF 1 FVII cDNA shows that this mutant protein is not expressed. The identification of the molecular basis of the FVII deficiency in this family allowed mutation-specific prenatal diagnosis to be performed in a subsequent pregnancy. In this family complete FVII deficiency is associated with a severe bleeding diathesis but no developmental abnormalities, lending weight to the hypothesis that fetal FVII is not required for the putative angiogenic functions of tissue factor in humans.

Animals↗
PubMed1998

Coagulation factor VII Gln100 --> Arg. Amino acid substitution at the epidermal growth factor 2-protease domain interface results in severely reduced tissue factor binding and procoagulant function.

We have used recombinant mammalian expression and purification of the factor VII (FVII) variant Gln100 --> Arg (Q100RFVII) to study FVII deficiency in subjects with this mutation. Q100RFVII was secreted poorly in comparison with wild-type FVII (WTFVII) in a stable mammalian expression system, and purified variant protein was found to have undetectable clotting activity. Following activation by immobilized factor Xa, Q100RFVIIa had amidolytic activity similar to WTFVIIa in the absence of soluble tissue factor (sTF); however, unlike WTFVIIa no typical increase in activity was seen after addition of sTF. In a factor X activation assay using relipidated transmembrane truncated tissue factor (residues 1-243), Q100RFVIIa showed less than 5% of the ability of WTFVIIa to activate factor X. We performed direct binding analysis of WT and Q100RFVII/FVIIa to immobilized sTF using surface plasmon resonance, and severely reduced binding of both non-activated and activated Q100RFVII to sTF was seen, indicating a pronounced defect in tissue factor (TF) interaction with this variant. In the sTF-FVIIa crystal structure the candidate residue Gln100 is not in contact with TF but is at the epidermal growth factor 2-protease domain interface. We suggest that the mutation results in a global fold change severely reducing tissue factor interaction; mutation of FVII residues not directly involved in the interaction with TF may still result in variant FVII unable to take part in the initiation of coagulation.

Amino Acid Substitution↗
PubMed1998

The factor VIII Structure and Mutation Resource Site: HAMSTeRS version 4.

Since 1996 the HAMSTeRS (Haemophilia A Mutation, Search, Test and Resource Site) WWW site has provided an online resource for access to data on the molecular pathology of haemophilia A, replacing previous text editions of the Haemophilia A Database published in Nucleic Acids Research . This report describes the continued development of the site (version 4), and in particular the expansion of factor VIII (FVIII) structure-related features. Access to the mutation database itself, both for searching the listings and for submission of new mutations, is via custom-designed forms: more powerful Boolean searches of the point mutations in the database are also available. During 1997 a total of 22 novel missense mutations were reported, increasing the total number of unique variants now described to 252 (238 in exonic sequences and 14 at intronic splice junctions). Currently, a total of 586 individual reports with associated phenotypic data are available for searching by any category including phenotype. The FVIII structure section now includes a download of a FVIII A domain homology model in Protein Data Bank format and a multiple alignment of the FVIII amino-acid sequencies from four species (human, murine, porcine and canine) in addition to the virtual reality simulations, secondary structural data and FVIII animation already available. Finally, to aid navigation across this site, a clickable roadmap of the main features provides easy access to the page desired. Our intention is that continued development and updating of the site shall provide workers in the fields of molecular and structural biology with a one-stop resource site to facilitate FVIII research and education. The HAMSTeRS URL is http://europium.mrc.rpms.ac.uk

Amino Acid Sequence↗
PubMed1998

Bleeding symptoms in 27 Iranian patients with the combined deficiency of factor V and factor VIII.

Inherited deficiency of factors V and VIII is the most frequent combined coagulation defect. The cases reported so fair are mostly single cases or small series from different centres, making it difficult to evaluate the overall pattern of clinical manifestations of the combined defect. We examined at a single institution 27 Iranian patients. Mucocutaneous and post-surgical bleeding were the most frequent clinical manifestations. The presence of two defects did not make the severity of bleeding greater than that expected in patients with single coagulation defects of similar degrees.

Adolescent↗
PubMed1998

The genetic basis of inhibitor development in haemophilia A.

Inhibitor development is now the main complication of replacement therapy in haemophilia A. Given that most severely affected patients make no detectable factor VIII, it is perhaps surprising that only approximately 30% actually mount an immune response to factor VIII as a foreign antigen. Those that do mostly have major factor VIII gene lesions. The association of HLA genotype with inhibitors in patients with identical mutations is weak. Environmental factors may be more important than genetic in antibody response to factor VIII.

Antibodies↗
PubMed1998

Coronary thrombosis and the platelet glycoprotein IIIA gene PLA2 polymorphism.

Myocardial infarction results from a platelet-rich occlusive coronary thrombus. Platelet membrane glycoprotein IIb/IIIa plays an important role in platelet adhesion and aggregation. Two polymorphisms of the gene encoding the IIIa subunit. PLA1 and PLA2, have been identified. We investigated the frequency of these polymorphisms in 114 consecutive patients with a history of angina-like chest pain admitted for coronary arteriography. Forty-three of these patients had previously suffered a myocardial infarction. The PLA2 polymorphism was found in 21% of the patients with previous myocardial infarction and in 27% of the patients with angina-like chest pain but no previous myocardial infarction (p = 0.634). There was also no significant association with the extent of coronary disease. There is no evidence, therefore, from this study of an association between the PLA polymorphisms and the occurrence of myocardial infarction.

Adult↗
PubMed1998

Analysis of the F8 gene in individuals with high plasma factor VIII: C levels and associated venous thrombosis.

High FVIII:C levels have previously been shown to be an independent risk factor for thrombosis with 4.8 times higher potential risk of thrombosis in individuals with FVIII:C levels greater than 1.5 u/ml. Recently, we found that raised FVIII:C levels are largely attributable to elevated FVIII:Ag levels. The determinants of FVIII:Ag levels are unclear and might be partly genetic. The promoter of the F8 gene has recently been characterised we therefore investigated the promoter and the 3' terminus of the F8 gene for possible polymorphisms associated with raised FVIII:Ag levels in 62 selected individuals with a thrombotic tendency. We confirm previous reports that raised FVIII:C levels are largely attributable to an elevation in FVIII:Ag and this is also associated with elevation of vWF; non-O blood group: relatively short APTT and relatively low APC ratio. We screened 1140 bp of the proximal promoter including the protein binding sites identified by DNase I footprint analysis by SSCP, however no polymorphisms were identified. Direct DNA sequence analysis of the region -542 to +165 failed to identify any sequence polymorphisms. The recently described polymorphism in the polyadenylation cleavage site in the prothrombin gene associated with increased prothrombin activity prompted us to screen the region surrounding the 3' terminus of the F8 gene for polymorphisms but we found none.

Base Sequence↗
PubMed1997

A molecular model for the triplicated A domains of human factor VIII based on the crystal structure of human ceruloplasmin.

The hemophilia A mutation database lists more than 160 missense mutations: each represents a molecular defect in the FVIII molecule, resulting in the X-linked bleeding disorder hemophilia A with a clinical presentation varying from mild to severe. Without a three-dimensional FVIII structure it is in most cases impossible to explain biological dysfunction in terms of the underlying molecular pathology. However, recently the crystal structure of the homologous human plasma copper-binding protein ceruloplasmin (hCp) has been solved, and the A domains of FVIII share approximately 34% sequence identity with hCp. This advance has enabled the building of a molecular model of the A domains of FVIII based on the sequence identity between the two proteins. The model allows exploration of predictions regarding the general features of the FVIII molecule, such as the binding-sites for factor IXa and activated protein C; it has also allowed the mapping of more than 30 selected mutations with known phenotype from the database, and the prediction of hypothetical links to dysfunction in all but a few cases. A computer-generated molecular model such as that reported here cannot substitute for a crystal structure. However, until such a structure for FVIII becomes available, the model represents a significant advance in modeling FVIII; it should prove a useful tool for exploiting the increasing amount of information in the hemophilia A mutation database, and for selecting appropriate targets for investigation of the structure-function relationships via mutagenesis and expression in vitro.

Amino Acid Sequence↗
PubMed1997

The Factor VIII Mutation Database on the World Wide Web: the haemophilia A mutation, search, test and resource site. HAMSTeRS update (version 3.0).

The HAMSTeRS WWW site was set up in 1996 in order to facilitate easy access to, and aid understanding of, the causes of haemophilia A at the molecular level; previously, the first and second text editions of the database have been published in Nucleic Acids Research. This report describes the facilities originally available at the site and the recent additions which we have made to increase its usefulness to clinicians, the molecular genetics community and structural biologists interested in factor VIII. The database (version 3.0) has been completely updated with easy submission of point mutations, deletions and insertions via e-mail of custom-designed forms. The searching of point mutations in the database has been made simpler and more robust, with a concomitantly expanded real-time bioinformatic analysis of the database. A methods section devoted to mutation detection has been added, highlighting issues such as choice of technique and PCR primer sequences. Finally, a FVIII structure section gives access to 3D VRML (Virtual Reality Modelling Language) files for any user-definable residue in a FVIII A domain homology model based on the crystal structure of human caeruloplasmin, together with secondary structural data and a sound+video animation of the model. It is intended that the general availability of this model will assist both in interpretation of causative mutations and selection of candidate residues forin vitromutagenesis. The HAMSTeRS URL is http://europium.mrc.rpms.ac.uk.

Computer Communication Networks↗
PubMed1997

Factor VIII gene mutations found by a comparative study of SSCP, DGGE and CMC and their analysis on a molecular model of factor VIII protein.

Screening of the factor VIII (FVIII) gene which spans 186 kb and codes for 26 exons, was originally hampered by its size but is now feasible because rapid DNA scanning methodologies have been developed. The present study for the first time directly compares the three most widely applied screening methods, denaturing gradient gel electrophoresis (DGGE), single-stranded conformational polymorphism (SSCP) and chemical mismatch cleavage (CMC) for their sensitivity of mutation detection in a selected group of ten haemophilia A patients. Nine of these patients are known to be cross-reacting material positive and eight exhibited a mild to moderate phenotype. Of the ten patients screened, we identified mutations in nine by all three screening methods. Of the mutations characterised, two are previously unpublished. T to C (S373P) and G to A (D525N). In one mildly affected haemophiliac, we identified a second T to C sequence change in the 5' untranslated region at -601 bp, probably having no effect on FVIII gene expression. Modelling studies were performed on those mutations lying within the A domains of FVIII (D525N, R527W, I566T) to study the possible effect of these mutations on structure and/or function. When the three methods are performing optimally and have been standardised, our experience is that CMC and DGGE are equally efficient at sequence variation detection while SSCP is slightly less sensitive.

Computer Simulation↗
PubMed1997

Factor VIII gene analysis in Japanese CRM-positive and CRM-reduced haemophilia A patients by single-strand conformation polymorphism.

Haemophilia A is the most common X-linked blood coagulation disorder; it is caused by deficiency of factor VIII activity (FVIII:C). Half of the affected patients do not have detectable levels of FVIII protein in their plasma, whereas about 5% have normal levels of the FVIII antigen (FVIII:Ag) (> 50 u/dl), and are called cross-reacting material (CRM) positive (CRM+ or A+). About 45% of patients have reduced levels of the FVIII:Ag (1-50 u/dl), classified as CRM reduced (CRM[R] or A[R]). We screened the FVIII gene of 13 Japanese patients (five CRM+ and eight CRM[R]) by single-strand conformation polymorphism, and identified 11 different mutations in 13 patients by analysing all 26 exons (Trp255Cys, Tyr473Cys, Gly479Arg, Arg531His, Thr667Arg, Arg1689Cys, Arg1941Gln, Arg2150His, Arg2159Cys, Thr2245Ala and Gly2285Val). Seven mutations were identified in the A domains (four in the A2 domain). All the mutations are point mutations resulting in missense codons. Four mutations (Trp255Cys, Thr667Arg, Thr2245Ala and Gly2285Val) have not been described previously.

Enzyme-Linked Immunosorbent Assay↗
PubMed1997

The locus for combined factor V-factor VIII deficiency (F5F8D) maps to 18q21, between D18S849 and D18S1103.

Combined factor V-factor VIII deficiency (F5F8D) is a rare, autosomal recessive coagulation disorder in which the levels of both coagulation factor V and coagulation factor VIII are diminished. In order to map and subsequently clone the gene responsible for this phenotype, DNAs from 19 families (16 from Iran, 2 from Pakistan, and 1 from Algeria) with a total of 32 affected individuals were collected for a genomewide linkage search using genotypes of highly informative DNA polymorphisms. All pedigrees except two contained at least one consanguineous marriage. A maximum LOD score (Zmax) of 14.82 for theta = .02 was generated with marker D18S1129 in 18q21; LOD scores > 9 were obtained for several other markers-D18S849, D18S1103, D18S64, and D18S862. Multipoint analysis resulted in Zmax = 18.91 for the interval between D18S1129 and D18S64. Informative recombinants placed the locus for F5F8D between D18S849 and D18S1103, in an interval of approximately 1 cM. These results are similar to the recently reported linkage of this disease to chromosome 18q in Jewish families (Nichols et al. 1997) and provide evidence that the same gene is responsible for all F5F8D among human populations. The difference in clinical severity of the phenotype in unrelated families, as well as the failure to detect a specific haplotype of DNA polymorphisms in the consanguineous Iranian families, suggests the existence of different molecular defects in the F5F8D gene. There exists an apparently gap-free contig with CEPH YACs linking the two markers on either side of the critical region. Positional cloning efforts are now in progress to clone the F5F8D gene.

Chromosome Mapping↗
PubMed1997

HLA genotype of patients with severe haemophilia A due to intron 22 inversion with and without inhibitors of factor VIII.

Molecular genetic studies have shown that development of antibodies to factor VIII (inhibitors) occurs most frequently in patients with severe haemophilia due to major gene lesions including inversions, stop codons and large deletions. Previous studies of HLA type were performed on inhibitor and non-inhibitor patients with diverse uncharacterized mutations which may have confounded detection of significant associations. We therefore selected a group of patients with a single mutation type, the prevalent intron 22 inversion, with or without inhibitors, to determine HLA genotype. Seventy-one such patients, 42 without and 29 with inhibitors (13 high, 9 low and 7 transient responders) were genotyped for MHC Class I HLA-A, -B, -C and Class II HLA-DQA, -DQB and -DRB loci. No strong correlation of any HLA-allele to inhibitor or non-inhibitor status was found. However, alleles of the haplotype HLA-A3, HLA-B7, HLA-C7, HLA-DQA0102, HLA-DQB0602, HLA-DR15 occurred more often in inhibitor patients. Since the alleles of this extended haplotype are common in the North European population only a very strong association would achieve statistical significance. Further studies of groups of patients similar to those studied here will be needed to confirm or exclude this association.

Alleles↗