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G Kemball-Cook

Publications and source records attributed to G Kemball-Cook.

At least 37 records · Page 2Linked to original sources
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

Structural determination of lipid-bound human blood coagulation factor IX.

Human coagulation factor IX (FIX) is a serine protease which binds to a negatively charged phospholipid surface in the presence of Ca ions (Ca2+). FIX two-dimensional (2-D) crystals were obtained by the lipid layer crystallisation technique under near physiological conditions. The 2-D projection map of the protein was calculated to a resolution of 3 nm using electron crystallographic analysis. The structural organisation of membrane-bound FIX is discussed and compared with the known X-ray crystallographic data.

Binding Sites↗
PubMed1998

The molecular basis for cross-reacting material-positive hemophilia A due to missense mutations within the A2-domain of factor VIII.

Factor VIII (FVIII) is the protein defective in the bleeding disorder hemophilia A. Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. To determine the mechanistic basis of the genetic defects within the A2-domain for FVIII function we constructed six mutations within the FVIII cDNA that were previously found in five CRM-positive hemophilia A patients (R527W, S558F, I566T, V634A, and V634M) and one CRM-reduced hemophilia A patient (DeltaF652/3). The specific activity for each mutant secreted into the conditioned medium from transiently transfected COS-1 cells correlated with published data for the patients plasma-derived FVIII, confirming the basis of the genetic defect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated FVIII protein radiolabeled in COS-1 cells showed that all CRM-positive mutant proteins were synthesized and secreted into the medium at rates similar to wild-type FVIII. The majority of the DeltaF652/3 mutant was defective in secretion and was degraded within the cell. All mutant FVIII proteins were susceptible to thrombin cleavage, and the A2-domain fragment from the I566T mutant had a reduced mobility because of use of an introduced potential N-linked glycosylation site that was confirmed by N-glycanase digestion. To evaluate interaction of FVIII with factor IXa, we performed an inhibition assay using a synthetic peptide corresponding to FVIII residues 558 to 565, previously shown to be a factor IXa interaction site. The concentration of peptide required for 50% inhibition of FVIII activity (IC50) was reduced for the I566T (800 mumol/L) and the S558F (960 mumol/L) mutants compared with wild-type FVIII (> 2,000 mumol/L). N-glycanase digestion increased I566T mutant FVIII activity and increased its IC50 for the peptide (1,400 mumol/L). In comparison to S558F, a more conservative mutant (S558A) had a sixfold increased specific activity that also correlated with an increased IC50 for the peptide. These results provided support that the defects in the I566T and S558F FVIII molecules are caused by steric hindrance for interaction with factor IXa.

Animals↗
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↗
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

Homology modelling of the catalytic domain of early mammalian protein C: evolution of structural features.

By means of a novel cDNA-based strategy employing the maximum parsimony principle, we have previously deduced probable amino acid sequences for the catalytic domains of the early mammalian ancestors of each of the five extant vitamin K-dependent serine proteases of coagulation, and for their common ancestor from a still earlier stage of vertebrate evolution. In the present study, we employed one of these sequences to construct a molecular model of the catalytic domain of early mammalian protein C and to explore its functional architecture. Following the domain's progression from the common ancestor of the vitamin K-dependent serine proteases toward extant human protein C, this novel application of homology modelling to a reconstructed amino acid sequence has allowed us to trace the evolution of structural features in a vital coagulation protein.

Amino Acid Sequence↗
PubMed1997

High prevalence of elevated factor VIII levels in patients referred for thrombophilia screening: role of increased synthesis and relationship to the acute phase reaction.

A recent report from the Leiden Thrombophilia Survey identified high factor VIII activity levels as an independent risk factor for venous thromboembolism in a population survey. As the study measure for factor VIII was a one-stage coagulation assay, and since markers for the acute phase reaction were not assessed, it remained uncertain whether the increase was due to a constitutional increased rate of synthesis, to circulating activated factor VIII, or to an acute phase response. We added factor VIII activity assay (FVIII:C), factor VIII antigen (FVIII:Ag), vWF antigen (vWF:Ag), ABO blood group, fibrinogen and C-reactive protein to our routine thrombophilia screen of patients referred because of unexplained thromboembolism. Elevated FVIII:C (> 1.5 iu/ml) emerged as the single commonest abnormality detected in 25.4% of a group of 260 such patients. FVIII:C and FVIII:Ag were highly correlated (p = 0.003), showing that this represented a true increase in FVIII. In 4 of 46 patients this was clearly attributable to an acute phase reaction. Eleven others showed minor elevation of ESR and one of CRP. Neither FVIII:C or FVIII:Ag showed significant correlation with fibrinogen, ESR or C-reactive protein by non parametric analysis. Although there was an excess of patients with B blood group (known to be associated with FVIII:C levels which are approximately 15% higher than those in blood group O), this could not account for the marked elevation of factor VIII observed in these patients. We conclude that factor VIII activity assay should be a routine part of thrombophilia screening. We are investigating the cause of the increased synthesis, initially by means of family studies and linkage analysis with polymorphic markers of the FVIII locus. We postulate that it may be constitutive in some cases and in others an abnormal or exaggerated response to inflammatory stimuli.

ABO Blood-Group System↗
PubMed1997

Human tissue factor pathway inhibitor fused to CD4 binds both FXa and TF/FVIIa at the cell surface.

Tissue factor pathway inhibitor (TFPI) is one of the main regulators of the tissue factor (TF) pathway of coagulation. To tether human TFPI to the cell surface, full length or truncated TFPI lacking the third Kunitz domain were fused with domains three and four and the carboxy-terminal sequence of human CD4. Constructs were transfected into a mouse fibroblast cell line and individual clones were checked for expression using monoclonal antibodies directed against the first two TFPI Kunitz domains and against CD4. Specific human FXa binding was detected by flow cytometry using an anti-FX polyclonal antibody, and inhibition of FXa proteolytic activity was verified by chromogenic substrate assay using S-2765. In addition, TFPI-CD4-expressing cells, preincubated with FXa, specifically bound human TF-FVIIa complexes as revealed with an anti-human TF polyclonal antibody. No functional difference was observed between full length or truncated TFPI-CD4. These results demonstrate that functionally intact TFPI can be tethered to the cell surface. Genetic manipulation of, for example, endothelial cells leading to the stable expression of TFPI may inhibit the development of coronary artery heart disease following cardiac allotransplantation, and may inhibit thrombosis in the context of xenotransplantation.

Animals↗
PubMed1996

The haemophilia A mutation search test and resource site, home page of the factor VIII mutation database: HAMSTeRS.

In order to facilitate easy access to and aid understanding of the causes of haemophilia A at the molecular level we have constructed HAMSTeRS, the third release of the factor VIII mutation database and the first release of this database that may be accessed and interrogated over the internet through a World Wide Web browser. The database also presents a review of the structure and function of factor VIII and the molecular genetics of haemophilia A, a real time update of the biostatistics of each parameter in the database, a molecular model of the A1, A2 and A3 domains of the factor VIII protein (based on the crystal structure of caeruloplasmin) and a bulletin board for discussion of issues in the molecular biology of factor VIII.

Databases, Factual↗
PubMed1996

In vivo studies of activated porcine factor VIII.

The haemostatic effectiveness of activated FVIII was compared to that of non-activated FVIII in a cross-over study in a canine model of haemophilia. Activation of FVIII in porcine concentrate was achieved by the addition of 3 x 10(-5) IU thrombin per ml of concentrate, which gave consistent increases in 1-stage FVIII activity of 13- to 14-fold and slow decay. The haemostatic effect was monitored by measurements of the cuticle bleeding time 10 and 45 min after infusion and there were no consistent differences between the activated and non-activated concentrates. One-stage factor VIII assays on plasmas 5 min after infusion showed identical mean values for activated and non-activated concentrates, indicating that most of the higher activity observed in vitro had disappeared rapidly from the circulation. These results suggest that controlled activation of FVIII by thrombin, which increases its activity in 1-stage assays, is unlikely to be of therapeutic benefit. For therapeutic concentrates which may contain small amounts of activated FVIII, the 1-stage assay may be an unreliable guide to their therapeutic effect.

Animals↗
PubMed1996

Pharmacokinetic in vivo comparison using 1-stage and chromogenic substrate assays with two formulations of Hemofil-M.

In a study to demonstrate the safety and pharmacokinetics (half-life and recovery) of two different method M purified AHF (Hemofil-M) concentrates processed in the USA and Spain, two different methods of factor VIII assay (one-stage clotting and chromogenic) have been compared in vivo. The study was a single centre blinded, randomised, crossover study. Twelve patients with severe haemophilia A (VIII:C < 2 u/dl) were divided into two subgroups of six. None had received factor VIII concentrate within 48 h preceding the study. Twenty-four pharmacokinetic studies were performed in the 12 patients. Each subgroup received two different lots of study material (US and Spanish) at a dose of 50 u/kg seven days apart. A second randomisation was nominal potency, high: 1000 u or mid: 500 u per vial. The potency label was a one-stage clotting assay using the mega I standard. A standard pharmacokinetic study was performed over 24 h and each blinded sample was analysed in duplicate by a one-stage clotting (aPTT) and a chromogenic (Chromogenix AB; CS) assay at the Royal Free and NIBSC. Pharmacokinetic modelling was performed. The mean label for Hemofil-M using the chromogenic substrate assay was 79% that using the one stage assay (Mega I standard). The recovery was 17-28% higher measured by chromogenic compared to the clotting assay. Since most clinicians use the clotting assay, potency labelling using the chromogenic assay, will overestimate predicted Hemofil-M recovery by as much as 25%.

Biological Assay↗
PubMed1995

Factor VIIa and the extracellular domains of human tissue factor form a compact complex: a study by X-ray and neutron solution scattering.

The four-domain structure of human factor VIIa and the two-domain structure of tissue factor form a tight complex to initiate blood coagulation. By solution scattering, the mean X-ray and neutron radii of gyration RG (which determine macro-molecular elongation) were found to be 3.25 nm, 2.13 nm and 3.14 nm (+/- 0.13 nm) for factor VIIa, the extracellular region of tissue factor and their complex in that order. The mean cross-sectional radii of gyration RXS were 1.33 nm, 0.56 nm and 1.42 nm (+/- 0.13 nm) in that order. The mean lengths were 10.3 nm, 7.7 nm and 10.2 nm in that order. The data show that, in solution, the free proteins have extended domain structures, and the complex is formed by a compact side-by-side alignment of the two proteins along their long axes. The high binding affinity of tissue factor for factor VIIa may thus be accounted for by the occurrence of many intermolecular contacts in the complex.

Animals↗
PubMed1995

Measurement of activated factor IX in factor IX concentrates: correlation with in vivo thrombogenicity.

Current in vitro tests for thrombogenicity of FIX concentrates used for prothrombin complex concentrates (PCCs), are of little value when applied to high purity FIX (HP FIXs). In the present study, we have developed a chromogenic assay for activated FIX (FIXa) and evaluated its ability to predict in vivo thrombogenic potential of HP FIXs in a modified Wessler stasis model. Among the HP FIXs, only 1 out of 7 products had no detectable FIXa; this product also showed no in vivo thrombogenicity. In the other 6 products, FIXa content ranged from 0.15-1.2 U/1000 in FIX, and all showed some evidence of in vivo thrombogenicity, with mean thrombus scores ranging from 0.25-4. There was a significant positive correlation (r = 0.55, p < 0.02) between FIXa levels and in vivo thrombogenicity of HP FIXs. NAPTT data were not significantly correlated with the in vivo results and the TFCT also showed no direct correlation with the mean thrombus score. These results indicate that HP FIXs may still carry a small residual thrombotic risk and measurement of FIXa content of these products may be a better predictor of thrombogenicity than the current in vitro tests.

Animals↗
PubMed1994

Surface plasmon resonance studies of the interaction between factor VII and tissue factor. Demonstration of defective tissue factor binding in a variant FVII molecule (FVII-R79Q).

The blood coagulation cascade is initiated when vessel injury allows factor VII (FVII) to form a complex with tissue factor (TF). Complete deficiency of FVII causes a lethal bleeding diathesis, but individuals with moderately reduced FVII levels are often asymptomatic. Some of these individuals have circulating partially functional FVII, as a result of point missense mutations in critical parts of the molecule. One such mutation has been reported at position 79 in the first epidermal growth factor-like (EGF) domain of FVII, where an arginine residue has been replaced by glutamine. There is controversy as to whether or not this mutation reduces the affinity of the FVII/TF interaction compared to wild-type FVII. To address this problem, we have expressed recombinant FVII-R79Q and subjected it to detailed biochemical analysis. One-stage FVII:C assays show the variant FVII to have reduced activity with respect to the wild type. Rates of autoactivation and activation by FXa to the two-chain molecule were identical for wild-type and variant FVII. The Vmax for FX activation was lower for the mutant as measured using an amidolytic assay for FX activity. In contrast, the Km for FX was lower for the variant than the wild-type molecule. Peptidyl substrate hydrolysis was virtually identical for both variant and normal FVIIa in the presence and absence of TF. The variant has reduced affinity for TF as measured by surface plasmon resonance. FVII-R79Q has an association rate constant (kassoc) one-fifth of that of normal FVII, but a similar kdiss, resulting in a decrease in the affinity of the enzyme for its cofactor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

High-level production of human blood coagulation factors VII and XI using a new mammalian expression vector.

Recombinant human proteins are generally recovered in low yields from mammalian tissue culture following transfection with commercially available vectors. We have constructed a novel vector containing both the neomycin-resistance-encoding gene (neo) as a dominant selectable marker, and the dihydrofolate reductase-encoding gene (DHFR) to enable amplification of transfected DNA followed by stable expression in mammalian cell lines. Levels of 5 micrograms/ml of the coagulation proteins, factor VII (FVII) and factor XI (FXI), have been achieved in serum-free media. N-terminal sequencing of the purified proteins, and of their separated chains after proteolytic activation, demonstrated correct processing of the recombinant products. In addition, the ratios of clotting activity to antigen for each are close to unity, and the recombinant and plasma-derived proteins had identical mobilities upon electrophoresis in the presence of SDS. The vector described will be of use for the synthesis of recombinant proteins, both wild-type and variants produced by site-directed mutagenesis, especially where complex post-translational modification of the protein makes it essential to use mammalian cells.

Animals↗
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↗