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Biomedical subjects

T W Barrowcliffe

Publications and source records attributed to T W Barrowcliffe.

At least 55 records · Page 3Linked to original sources
PubMed1995

Antithrombins Southport (Leu 99 to Val) and Vienna (Gln 118 to Pro): two novel antithrombin variants with abnormal heparin binding.

We report the characterization of three variant antithrombins with reduced heparin binding as the primary abnormality. Two of these variants, antithrombin Southport (Leu 99 to Val, 2759 C to G) and antithrombin Vienna (Gln 118 to Pro, 5349 A to C) were novel, whereas the third, Pro 41 to Leu, has been previously described as antithrombin Basel. All three variants exhibited reduced binding for heparin on crossed immunoelectrophoresis and in a quantitative monoclonal antibody-based assay. The mutations were characterized by direct sequence analysis of enzymatically amplified genomic DNA and all affected individuals were heterozygous for the mutations. These three mutations do not occur at the sites of the basic amino acids directly involved in heparin binding nor do they result in a change in charge of the affected residue. It seems probable that they reduce heparin affinity either by perturbing the initial contact site involved in the heparin-binding domain (Arg 47, Arg 129 and possibly Arg 24), or by preventing the subsequent heparin-induced conformational change.

Adult↗
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↗
PubMed1995

Factor VIII concentrates and the immune system--laboratory investigations.

Evidence suggests that haemophiliacs treated with factor VIII concentrates show abnormalities in immune functions. The basis of this is not clear, but some factor VIII concentrates down-regulate Fc receptors on monocytes which may explain the impaired function of these cells. Some concentrates inhibit lymphocyte proliferation and interleukin-2 secretion by human T-cell lines and peripheral blood lymphocytes. They can also inhibit activity of other cytokines such as interleukin-4 and interleukin-5 and secretion of cytokines such as interleukin-1 and granulocyte macrophage colony stimulating factor. These effects are product-related and vary from total inhibition to virtually no detectable inhibition. Of particular significance is that the degree of inhibition is not related to the purity or gross protein composition of the products. The inhibitory activity is not due to factor VIII itself as antibody affinity purified factor VIII products are entirely non-inhibitory. The main inhibitory protein components appear to be of approximately 200 kDa and 60 kDa (by gel filtration). Recent evidence suggests that transforming growth factor-beta (TGF-beta), derived from the plasma used for fractionation, is a major contaminant of 'inhibitory' concentrates and is responsible for the effects, observed in vitro, of concentrates on cytokine secretion or activity. The levels of TGF-beta varied between products and correlated with inhibition of interleukin-2 secretion from stimulated T-cells. The presence of TGF-beta in concentrates may therefore explain the immunosuppression observed in recipients of these products. Correlation of the inhibitory effects with clinically important consequences such as increased susceptibility to infections or decreased CD4 counts also remains to be established.

Factor VIII↗
PubMed1995

A collaborative study of proposed European Pharmacopoeia reference preparations of low molecular mass heparin.

A European collaborative study, in which 16 laboratories participated, was carried out to assess the performance of the European Pharmacopoeia (EP) monograph methods for anticoagulant activities (anti-Xa and anti-IIa assays) of low molecular mass (LMM) heparin and to assess the suitability of six candidate materials as the EP working standard for LMM heparin. There was good interlaboratory agreement for both types of assays as indicated by most gcy's being less than 10%, indicating acceptable performance of the EP assay methods. All the candidate preparations gave dose-response curves parallel to the 1st International Standard for Low Molecular Weight heparin and to each other. All preparations, possibly with the exception of E and F, gave similar performance as measured by interlaboratory agreement and would be suitable as working standards. Based on these data, preparations A, B, C and D have been established by the EP as official EP Biological Reference Preparations and they will be issued as successive batches.

Anticoagulants↗
PubMed1994

Role of N- and C-terminal amino acids in antithrombin binding to pentasaccharide.

We have used a monoclonal antibody-based binding procedure to determine the dissociation constants of the interactions between the essential antithrombin-binding pentasaccharide and a series of 13 distinct N- and C-terminal antithrombin substitution mutation variants with defective binding interaction with heparin. The reduction in binding affinity of the pentasaccharide with the N-terminal variants (with substitution mutations Pro-41-->Leu, Arg-47-->Cys and His, Leu-99-->Val and Phe, Gln-118-->Pro, Arg-129-->Gln) compared to normal antithrombin, Kd 200 nM, ranged from 15-984-fold and was generally less than 150-fold. Reduced binding affinity is assumed to arise mostly by perturbation, direct or indirect, of the initial contact of pentasaccharide with basic residues of antithrombin. Surprisingly, the binding interaction of the pentasaccharide with the C-terminal variants (with substitution mutations in or near strand 1C/4B, Phe-402-->Leu, Cys, and Ser, Ala-404-->Thr, Pro-407-->Thr, Pro-429-->Leu) was more uniformly and yet substantially (135-482-fold) decreased, despite the spatial separation between the site of mutation and the proposed primary contact site of the pentasaccharide. These results demonstrate that strand 1C/4B region integrity is required for optimum interaction with the pentasaccharide, suggesting its involvement in transmission of the induced conformation change required for high affinity binding.

Amino Acids↗
PubMed1993

Evaluation of factor VIII deficient plasmas.

Immunodepleted plasmas from Organon Teknika, Dade, Stago, Diagen and the Scottish National Blood Transfusion Service (SNBTS), and haemophilic plasma from Immuno were compared by several laboratories with haemophilic plasma from local donors as substrates in one-stage factor VIII assays. Five clinical plasma samples and four concentrates were assayed against British Standard plasma and International Standard concentrate. Potencies of all plasma samples were not significantly different from those with local haemophilic plasma for Immuno, Organon Teknika, Stago and SNBTS substrates. Dade differed from haemophilic on one sample and Diagen on three. Buffer blank times and slopes of standard lines were similar with all substrates. A positive drift between the beginning and end of the assay was found with the Immuno substrate and a negative drift with the Organon Teknika substrate. In the concentrate study results for all substrate plasmas were not significantly different from haemophilic on the intermediate purity and conventional high purity products. On the monoclonal and recombinant products, there was a tendency for the immunodepleted substrates to give lower potencies than the haemophilic, and significant differences were found with Dade and Stago on the monoclonal concentrate, and with Dade, Stago and Diagen on the recombinant concentrate. Overall, this study indicates that most commercially available substrate plasmas are suitable as replacements for locally collected haemophilic plasma in one-stage assays of clinical samples, and of intermediate purity and conventional high purity concentrates. For assays of very high purity concentrates (monoclonal and recombinant), haemophilic plasma is preferable as some immunodepleted plasmas give low results.

Hemophilia A↗
PubMed1993

Heparin binding affinity of normal and genetically modified antithrombin III measured using a monoclonal antibody to the heparin binding site of antithrombin III.

The inhibitory activity of the plasma serine proteinase inhibitor antithrombin III (AT III) is enhanced about 1000-fold upon binding to heparin. We have determined the dissociation constants, Kd, of 48.8 nM for the heparin-AT III interaction, of 175 nM for the specific pentasaccharide-AT III interaction, and of 13 microM for the low-affinity heparin-AT III interaction, using a binding assay based on a monoclonal antibody (MAb) that recognizes an epitope at or close to the heparin binding site of AT III. The heparin binding affinities and proportions of normal and variant AT III in plasma from patients with mutations of AT III have been quantitated for the first time using the binding assay. Substitution mutations in three regions of AT III have been investigated: (i) mutations in the reactive site loop affecting Ala382, Arg393, and Ser394 have no discernible effect on heparin binding; (ii) mutations in the previously identified N-terminal heparin binding region, affecting Arg47, Leu99, and Arg129, produce variant AT III molecules with heparin affinities reduced 11-924-fold, the largest reduction being observed for the substitution mutation Arg47-Cys in Padua 2, which has an affinity of 65.6 microM; (iii) mutations in the hydrophobic regions around strand 1C of the C terminus, affecting Phe402, Ala404, Asn405, Pro407, and Pro429, have pleiotropic effects that include the production of reduced amounts of low-affinity AT III with dissociation constants from 6 to 43 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
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↗
PubMed1993

Antithrombotic activity and its relationship to in vitro anticoagulant effects.

Antithrombotic activities of low molecular weight heparins (LMWHs) in a venous stasis thrombosis model, when compared to unfractionated heparin (UFH), correlated better with anti-IIa activities and inhibition of thrombin generation than with anti-Xa activities. The relative antithrombotic potencies of LMWHs were closer to inhibitory activity in thrombin generation with platelet-poor plasma than platelet-rich plasma. Comparing activities in intrinsic and extrinsic systems, higher concentrations of heparins were required in vitro to inhibit intrinsic thrombin generation than extrinsic thrombin production; however, higher doses were required to prevent in vivo thrombotic events initiated via the extrinsic pathway.

Animals↗
PubMed1993

Viral inactivation vs biological activity.

The application of viral inactivation techniques to blood products, while preserving their biological functions, presents considerable problems, especially for coagulation factor concentrates, which are mostly impure preparations containing large quantities of plasma proteins and lipids. The three main methods which have been used are dry heat, solvent/detergent treatment and pasteurisation. Many factor VIII concentrates which have been produced using these methods display evidence of FVIII activation, with higher one-stage than two-stage potencies, more rapid FXa generation, and increased lower mol wt. polypeptides. Immunogenicity of FVIII is a particular concern, and recently a switch from dry heat to pasteurisation with one product was associated with an increased incidence of antibodies to FVIII in haemophilic recipients. Viral inactivation processes could also induce changes in non-FVIII components and these may be partly responsible for the immunosuppressive actions of some concentrates, observed both in vitro and in vivo. For factor IX concentrates thrombogenicity is a major potential hazard, and studies in the UK showed that the introduction of a viral inactivation step for one product increased its thrombotic potential, which had to be countered by addition of coagulation inhibitors. Coagulation inhibitor concentrates such as antithrombin III can be inactivated by pasteurisation, but this produces some heat denatured material which may be undesirable for the recipient.

Biological Products↗