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E G Tuddenham

Publications and source records attributed to E G Tuddenham.

At least 91 records · Page 5Linked to original sources
PubMed1993

Crystallization and preliminary X-ray analysis of human tissue factor extracellular domain.

The extracellular domain (residues 1 to 220) of human tissue factor has been cloned and expressed in Escherichia coli and purified to isoelectric homogeneity. Single crystals suitable for X-ray analysis have been obtained by vapour diffusion. They belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with a = b = 45.2 A, c = 231.5 A, contain one molecule per asymmetric unit and diffract to 2.6 A resolution. Native and derivative data sets have been collected to 3.6 and 3.9 A, respectively.

Crystallography, X-Ray↗
PubMed1993

Structural requirements for the interaction between tissue factor and factor VII: characterization of chymotrypsin-derived tissue factor polypeptides.

Tissue Factor (TF) is the cellular receptor for coagulation Factor VII/VIIa (FVII/VIIa). TF binds to FVIIa and promotes the rapid activation of the zymogen substrates Factors IX and X (FIX and FX) to the respective serine proteinases. In order to probe structure-function relationships in TF, we have subjected the truncated membrane-bound variant, TF 1-243, to proteolytic digestion in SDS-containing gels. Three major polypeptide fragments were generated by proteolysis of TF 1-243 with chymotrypsin, producing cleavages C-terminal to residues 34, 76 and 103. All three polypeptides, TF 35-243, 77-243 and 104-243, bound biotinylated human FVII in a highly specific ligand blot assay. High-performance electrophoretic chromatography was used to isolated chymotrypsin-derived fragments of TF. These purified fragments bound FVII in ligand blots, and two of the three polypeptides exhibited much reduced, but significant, procoagulant activity in a chromogenic assay for the generation of Factor Xa in the presence of FVIIa and Ca2+. The smallest chymotrypsin-derived TF polypeptide, TF 104-243, showed reduced binding of FVII in ligand blot analyses, inhibited the activity of the full-length molecule, but had no procoagulant activity. These data suggest that a part of the binding site for FVII is contained within the TF sequence 104-243. The sequence TF 1-34 either contains a part of the FVII-binding domain or its removal leads to dysfunctional folding, disrupting binding sites elsewhere in the molecule.

Amino Acid Sequence↗
PubMed1993

Double heterozygosity for mutations in the platelet glycoprotein IX gene in three siblings with Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS) giant platelets have defective and/or deficient glycoprotein (GP) Ib/IX complexes, causing absent ristocetin-induced aggregation, defective interaction with von Willebrand factor, morphologic abnormality, and a clinical bleeding tendency. Recently several mutations have been described in the platelet GPIb alpha gene in individuals exhibiting the BSS phenotype. We have studied a family with classical BSS, and have excluded lesions at the GPIb alpha locus by restriction fragment length polymorphism linkage analysis. Analysis of the genes for two other components of the platelet GPIb:IX complex, namely GPIb beta and GPIX, showed two different missense mutations in the coding region of the GPIX gene: an A-->G transition in codon 21 results in conversion of an aspartic acid to glycine and an A-->G change in codon 45 converts an asparagine residue to serine. Three affected individuals are doubly heterozygous for these mutations, which alter conserved residues in or flanking the GPIX leucine-rich glycoprotein motif. Both mutations create new recognition sites for the enzyme Fnu 4H1; therefore, this enzyme was used to screen 60 normal subjects (120 alleles). Neither mutation was detected in any subject other than direct relatives of the affected individuals. Although low levels of GPIb were demonstrable by both flow cytometry and immunoblot analysis in an affected individual's platelets, there was no evidence of GPIX immunoreactivity. We propose that expression of abnormal GPIX prevents stable assembly of the GPIb/IX complex, causing BSS in the doubly heterozygous individuals in this family.

Amino Acid Sequence↗
PubMed1993

A G-->A substitution in an HNF I binding site in the human alpha-fetoprotein gene is associated with hereditary persistence of alpha-fetoprotein (HPAFP).

A family displaying hereditary persistence of alpha-fetoprotein (HPAFP) in adult life was detected in an antenatal screening programme for spina bifida. RFLP linkage analysis shows that the trait is linked with the albumin-AFP locus. The molecular mechanism responsible for the post-natal repression of the AFP gene is unknown. We wished to determine the molecular mechanism underlying HPAFP in this family. Sequence analysis of the 5'-flanking sequences of their gene revealed a GA substitution at position -119 associated with the trait. This substitution occurs in a potential HNF I binding site, and increases the similarity of the sequence to a consensus HNF I recognition site. In a competitive gel retardation assay the mutant sequence binds HNF I alpha more tightly than the wild type sequence. Furthermore, 5'-flanking sequences of the human AFP gene containing the G-->A substitution direct a higher level of CAT expression in transfected human hepatoma cells than the wild type sequences. We conclude that the G-->A substitution at position -119 of the AFP gene is the mutation causing HPAFP in this family. These results highlight the importance of this HNF I binding site in the developmental regulation of the AFP gene.

Adult↗
PubMed1993

Detection of missense mutations by single-strand conformational polymorphism (SSCP) analysis in five dysfunctional variants of coagulation factor VII.

Five unrelated subjects with dysfunctional coagulation factor VII (FVII) were studied in order to identify missense mutations affecting function. Exons 2 to 8 and the intron-exon junctions of their FVII genes were amplified from peripheral white blood cell DNA by PCR and screened by SSCP analysis. DNA fragments showing aberrant mobility were sequenced. The following mutations were identified: in case 1 (FVII:C < 1%, FVII:Ag 18%) a heterozygous A to G transition at nucleotide 8915 in exon 6 results in the amino acid substitution Lys-137 to Glu near the C-terminus of the FVIIa light chain; in case 2 (FVII:C 7%, FVII:Ag 47%) a heterozygous A to G transition at nucleotide 7834 in exon 5 results in the substitution of Gln-100 by Arg in the second EGF-like domain; in case 3 (FVII:C 20%, FVII:Ag 76%) a homozygous G to A transition at nucleotide position 6055 in exon 4 was detected resulting in substitution of Arg-79 by Gln in the first EGF-like domain; in case 5 (FVII:C 10%, FVII:Ag 52%) a heterozygous C to T transition at nucleotide position 6054 in exon 4 also results in the substitution of Arg79, but in this case it is replaced by Trp; case 4 (FVII:C < 1%, FVII:Ag 100%) was homozygous for a previously reported mutation (G to A) at nucleotide position 10715 in exon 8, substituting Gln for Arg at position 304 in the protease domain. Cases 1, 2 and 5 evidently have additional undetected mutations.

Amino Acid Sequence↗
PubMed1993

Symptomatic type II protein C deficiency caused by a missense mutation (Gly 381-->Ser) in the substrate-binding pocket.

A patient with recurrent deep vein thrombosis and heterozygous type II deficiency, characterized by reduced protein C activity in both amidolytic and clotting functional assays, was investigated by direct sequencing of PCR fragments derived from the coding portion of the protein C gene. AG (8856) to A transition was noted in the patient which was not present in healthy controls. This mutation is predicted to cause the substitution of Ser for Gly 381, an evolutionari'y conserved residue in the substrate binding pocket of serine-proteases (Gly 216, chymotrypsin numbering). A computer model of the structure of the serine-protease domain indicates that the properties of the altered protein C molecule can be explained on the basis of steric hindrance between the substituted serine and the substrate arginine side chains.

Adult↗
PubMed1993

A molecular model of the serine protease domain of activated protein C: application to the study of missense mutations causing protein C deficiency.

A molecular model of the serine protease domain of protein C was constructed by standard comparative methods. Individual missense mutations were inserted into the model and plausible explanations for their interference with protein C structure/function were derived through consideration of location, steric effects and protein stability. A hydrophilic cluster of many Arg and Lys residues, found adjacent to the active site cleft, is proposed to be involved in thrombomodulin and/or protein S interactions. Analysis of comparative binding studies also suggested the presence of an extended substrate binding pocket in the model.

Amino Acid Sequence↗
PubMed1993

Mutations in haemophilia A.

In the present study DNA from 281 unrelated haemophilia A patients including 15 inhibitor patients has been analysed by Southern blotting technique. Using various restriction enzymes, cloned factor VIII cDNA probes and genomic fragments we have identified 14 mutations. Six of the mutations are novel partial factor VIII gene deletions. One deletion affects exon 1, two deletions concern exon 6, another deletion, of which breakpoints are sequenced, takes part of exon 16 and two deletions affect exon 26. Besides the deletions, eight point mutations have been found at the TaqI restriction sites of exons 18, 24 and 26. Five C-->T mutations resulted in nonsense mutations, one in exon 18, one in exon 26 and three in exon 24. Two G-->A mutations caused a missense mutation in exon 24 leading to an arginine/glutamine exchange. Although two patients showed this mutation, their clinical phenotypes were different, possibly due to an additional unidentified sequence polymorphism. A G-->T mutation in exon 26 substituted the arginine with leucine. All deletions and seven of the point mutations are associated with severe disease with a detectable inhibitor in the patient with the TaqI-point mutation in exon 18. One of the G-->A mutations is associated with mild haemophilia but the patient also has developed an inhibitor. Amongst these mutations the origin of the mutation could be determined in four kindred, one of which showed maternal mosaicism.

Base Sequence↗
PubMed1992

The interaction of botrocetin with normal or variant von Willebrand factor (types IIA and IIB) and its inhibition by monoclonal antibodies that block receptor binding.

We have recently shown the existence of two distinct forms of botrocetin (one-chain and two-chain), and demonstrated that the two-chain species is approximately 30 times more active than the one-chain in promoting von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib. The N-terminal sequence of two-chain botrocetin is highly homologous to sea-urchin Echinoidin and other Ca(2+)-dependent lectins (Fujimura et al., Biochemistry 1991; 30: 1957-64). Present data indicate that purified two-chain botrocetin binds to vWF from plasmas of patients with type IIA or IIB von Willebrand disease and its interaction is indistinguishable from that with vWF from normal individuals. However, an "activated complex" formed between botrocetin and IIB vWF expresses an enhanced biological activity for binding to GP Ib whereas the complex with IIA vWF has a decreased binding activity. Among several anti-vWF monoclonal antibodies (MoAbs) which inhibit ristocetin-induced platelet aggregation and/or vWF binding to GP Ib, only two MoAbs (NMC-4 and RFF-VIII RAG:1) abolished direct binding between purified botrocetin and vWF. This suggests that they recognize an epitope(s) on the vWF molecule in close proximity to the botrocetin binding site.

Antibodies, Monoclonal↗
PubMed1992

Inactivation of factor VIII by factor IXa.

Factor VIII (FVIII) is the nonproteolytic cofactor for FIXa in the tenase complex of blood coagulation. FVIII is proteolytically activated by thrombin and FXa in vitro to form a heterotrimer with full procoagulant activity. Activated protein C inactivates thrombin-activated FVIII through cleavage adjacent to position Arg 336 in the cofactor. We have investigated the interaction of FIXa and FVIII and subjected FVIII polypeptides to N-terminal amino acid sequence analysis. Contrary to previous reports, we were unable to demonstrate the activation of FVIII by FIXa. Incubation of these two proteins at equimolar or close to equimolar concentrations resulted in the inactivation of FVIII, coincident with cleavage of the FVIII heavy chain adjacent to Arg 336 and the light chain adjacent to Arg 1719. These cleavages were detected in the presence or absence of thrombin, indicating that FIXa does not stabilize thrombin-activated FVIIIa. APC cleaved FVIII at the same position in the heavy chain, and simultaneous incubation of FVIII, APC, and FIXa did not result in stabilization of the cofactor. We conclude that FIXa does not play a role in the stabilization or activation of FVIII.

Amino Acid Sequence↗
PubMed1991

Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene.

Mutations at the factor VIII gene locus causing Haemophilia A have now been identified in many patients from many ethnic groups. Earlier studies used biased methods which detected repetitive mutations at a few CG dinucleotides. More recently rapid gene scanning methods have uncovered an extreme diversity of mutations. Over 80 different point mutations, 6 insertions, 7 small deletions, and 60 large deletions have been characterised. Repetitive mutation has been proved for at least 16 CpG sites. All nonsense mutations cause severe disease. Most missense mutations appear to cause instability of the protein, but some are associated with production of dysfunctional factor VIII molecules, thereby localising functionally critical regions of the cofactor. Variable phenotype has been observed in association with three of the latter class of genotype. This catalogue of gene lesions in Haemophilia A will be updated annually.

Base Sequence↗
PubMed1991

Haemophilia A diagnosis by analysis of a hypervariable dinucleotide repeat within the factor VIII gene.

The diagnosis of haemophilia A and the identification of carriers has greatly improved with knowledge of the structure of the gene for factor VIII. This has permitted the defect to be tracked in families by the study of restriction fragment length polymorphisms (RFLPs), irrespective of the nature of the molecular defect. However, this approach is time-consuming and the information yielded falls away as more polymorphisms are added. Within the factor VIII gene lies another source of polymorphism, a dinucleotide repeat sequence of varying length known as (CA)n. Conventional mapping localised this (CA)n repeat to intron 13. The polymerase chain reaction, used to examine (CA)n variability in genomic DNA from 25 males and 67 females, revealed eight allelic bands between n = 16 and n = 24. 91% of females were heterozygous for this repeat, and family studies showed X-linked mendelian inheritance with allelic frequencies ranging from 1% to 45%. The intron 13 (CA)n repeat is tightly linked with established RFLPs and tracks with haemophilia A in family studies. The analysis requires DNA from other family members, and relatives of sporadic cases of haemophilia A are only amenable to exclusion. Nonetheless, this intron 13 (CA)n repeat provides the most highly informative marker so far available for factor VIII gene tracking studies in haemophilia A kindreds and a result can be available within a day.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of factor VII 304-Gln: a variant molecule with reduced activity isolated from a clinically unaffected male.

Factor VII (FVII) is the plasma serine protease zymogen which, on binding to its cellular receptor tissue factor (TF), initiates blood coagulation. A 47-year-old man with no clinical bleeding tendency was found to have undetectable plasma FVII activity when tested in a one-stage assay using rabbit brain TF, but 0.3 U/mL using recombinant human TF and 1.04 U/mL FVII antigen. Variant FVII purified from his plasma showed an identical migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to wild-type zymogen. By enzyme kinetic analysis the Km of the variant using FX as a substrate was 12-fold higher than that of normal FVII. Also, the variant FVII was unable to compete with wild-type FVII for limited rabbit TF binding sites. A ligand blot procedure was used to directly demonstrate reduced binding of recombinant human TF to the variant FVII compared with normal FVII. Genetic analysis of leukocyte DNA showed a G to A mutation in the propositus' gene at codon 304 that results in the substitution of a glutamine for an arginine residue in the catalytic domain of the protease. We conclude that this region of the FVII molecule is important for its function.

Amino Acid Sequence↗
PubMed1991

Relationships of plasma viscosity, coagulation and fibrinolysis to coronary risk factors and angina.

Plasma viscosity, molecular markers of activated coagulation and fibrinolysis (fibrinopeptides A and B beta 15-42), coagulation factors (fibrinogen and factor VII) and antiplasmins were measured in 529 men aged 35-54 years and related to new angina pectoris (n = 117) and to coronary risk factors in controls without angina (n = 412). Five major risk factors (cigarette-smoking, blood pressure, cholesterol, triglyceride and body mass index) were each associated with increases in plasma viscosity, coagulation factors, and imbalance of coagulation over fibrinolysis (increased ratio of fibrinopeptide A/fibrinopeptide B beta 15-42). Increased viscosity and fibrinogen in smokers were partly reversed in ex-smokers, but the imbalance of coagulation and fibrinolysis persisted. Cholesterol and triglyceride were also associated with increased antiplasmin activity. In men with angina, only fibrinogen was elevated compared to controls. We suggest that increased plasma viscosity and an imbalance of coagulation over fibrinolysis may be mechanisms by which known risk factors promote arterial thrombosis, but are not present in stable angina.

Adult↗
PubMed1990

Characterization of a panel of monoclonal antibodies to human coagulation factor XI and detection of factor XI in Hep G2 cell conditioned medium.

We have produced a panel of ten monoclonal antibodies specific to coagulation factor XI. Western blot analysis demonstrates that 9 of these antibodies react with the heavy chain of factor XI and one with the light chain. Seven of these antibodies inhibit factor XI and factor XIa activity. We have used immobilised monoclonal antibody for the production of factor XI deficient plasma and to purify factor XI to homogeneity with high yield in a simple two-step procedure. These monoclonal antibodies were used to develop highly sensitive immunoassays capable of detecting less than 0.01 mu factor XI antigen ml-1. A strong correlation was found between antigen and activity levels in 11 patients with hereditary deficiency indicating that none was cross-reacting material positive. Cultured Hep G2 cells were found to synthesize small amounts of factor XI antigen and this could also be detected by functional assay and by western blot analysis.

Animals↗