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Biomedical subjects

T W Barrowcliffe

Publications and source records attributed to T W Barrowcliffe.

At least 19 recordsLinked to original sources
PubMed1992

Standardisation of factor VIII--V. Calibration of the 2nd International Standard for Factor VIII and von Willebrand factor activities in plasma.

The proposed 2nd International Standard for Factor VIII and von Willebrand Factor activities in plasma, NIBSC code 87/718, was assayed against the 1st IRP, 80/511, and against fresh normal plasma, in 21 laboratories. There were no significant differences between the various assay methods for factor VIII antigen, von Willebrand factor antigen, and von Willebrand factor ristocetin co-factor activity. For factor VIII clotting activity there was a significant difference between the results of one-stage and two-stage assays. Plasma 87/718 has now been established by the WHO Expert Committee on Biological Standardisation as the 2nd IS for factor VIII and vWF in plasma with the following potencies: VIII:C 0.60 IU/ampoule; VIII:Ag 0.91 IU/ampoule; vWF:Ag 0.91 IU/ampoule; vWF/RCo 0.84 IU/ampoule.

Blood Chemical Analysis↗
PubMed1992

Interaction of factor VIII with phospholipids: role of composition and negative charge.

Radiolabelled human anti-FVIII:C antibody was affinity-purified according to its ability to bind to factor VIII-phospholipid (FVIII-PL) complexes, yielding a fraction directed against the phospholipid binding-site (PL-site antibody). This antibody was used as a specific probe for FVIII binding to PL vesicles containing a variety of natural and synthetic PLs. Of purified PLs tested for FVIII binding, phosphatidyl serine (PS) and phosphatidic acid (PA) were highly active, phosphatidyl inositol (PI) much less so, and both phosphatidyl ethanolamine (PE) and phosphatidyl choline (PC) inactive: the apparent dissociation constant (Kd app) for FVIII binding to PS:PC vesicles showed a strong dependence on PS content. Free-flow electrophoresis of vesicles confirmed FVIII binding to PS:PC required both net negative charge and specific head-group: neither PS vesicles given a positive charge with stearylamine nor PC vesicles made negative with dicetyl phosphate bound FVIII. It is concluded that the negative charge required for FVIII binding must be presented on the phospholipid surface in the correct orientation: phosphatidyl serine supplies this charge in coagulant-active PL preparations.

Amines↗
PubMed1992

Inhibition of thrombin generation by heparin and low molecular weight (LMW) heparins in the absence and presence of platelet factor 4 (PF4).

The ability of several low molecular weight (LMW) heparins and unfractionated heparin (UFH) to inhibit thrombin generation, and their anti-Xa and anti-IIa activities, were measured in the absence and presence of platelet factor 4 (PF4). The LMW heparins studied were 2-5 times less potent, on a weight basis, than UFH as inhibitors of thrombin generation in platelet-poor plasma; the inhibition of thrombin generation by LMW heparins correlated better with their anti-IIa activity (r = 0.98) than with their anti-Xa activity (r = 0.69). At low concentrations of PF4, the activity of LMW heparins in the thrombin generation test was neutralized less than that of UFH, but at higher PF4 concentrations all their activities could be neutralized except in anti-Xa assays. These observations support the hypothesis that anti-IIa activity is important for inhibition of thrombin generation by LMW heparins in vitro. However, when all the anti-IIa activity of LMW heparins was neutralized by PF4, considerable inhibitory activity remained in thrombin generation and anti-Xa assays, indicating that a portion of the anti-Xa activity of LMW heparins also contributes towards inhibition of thrombin generation.

Factor Xa Inhibitors↗
PubMed1991

Anti-Xa clotting activities in different hepatic-triglyceride lipase preparations from post-heparin plasma.

Two different preparations of hepatic triglyceride lipase (HTGL) with comparable lipolytic activities, purified from post-heparin human plasma, were assessed for their anti-Xa activities by two clotting and one chromogenic method. Preparation 1, prepared by heparin affinity followed by ion exchange chromatography, did not contain antithrombin III and exhibited no anti-Xa activity in any of the assay systems. Preparation 2, prepared by two consecutive heparin affinity chromatography steps, was active in all three assay systems, and was shown to contain antithrombin III (AT III). Addition of purified AT III to preparation 1 did not result in the anti-factor Xa activity of preparation 2, and monoclonal antibodies to AT III did not antagonize the activity of preparation 2. These results show that the anti-Xa activity of some HTGL preparations is neither due to the lipase itself nor to the content of AT III, but suggest, that it could be due to contamination with another protein, which binds to heparin sepharose columns but is removed during ion exchange chromatography. Most likely the effect is due to the extrinsic pathway inhibitor (EPI), also called lipoprotein-associated coagulation inhibitor (LACI), which has recently been shown to be released by heparin.

Antibodies↗
PubMed1991

Experimental studies on a recombinant hirudin, CGP 39393.

The in vitro anticoagulant activities of recombinant desulphatohirudin (r-hirudin) were studied in the activated partial thromboplastin time (APTT) and the thrombin generation test systems. In the APTT, at concentrations below 5 micrograms/ml, r-hirudin showed a dose-response curve. At concentrations above 5 micrograms/ml, the plasma became unclottable, but in the thrombin generation test, at least 10 micrograms/ml of r-hirudin was required for full inhibition of thrombin generation. The antithrombotic effect was assessed using a rabbit venous stasis model; 150 micrograms/kg r-hirudin completely prevented thrombus formation at 10 and 20 min stasis. At this full antithrombotic dose, the mean bleeding time ratio measured in a rabbit ear template model, was not prolonged over control values. At higher doses, the bleeding time ratios were higher than those observed for the same dosage of heparin. These data indicate that while r-hirudin is an effective antithrombotic agent, antithrombotic doses have to be carefully titrated to avoid excessive bleeding.

Animals↗
PubMed1991

Proteolysis of factor VIII heavy chain polypeptides in plasma and concentrates.

Factor VIII heavy chain (FVIII HC) polypeptides have been studied in both normal plasma and FVIII concentrates on exposure to three coagulation proteases. FVIII samples were incubated with labelled affinity-purified anti-FVIII Fab' fragments, immunocomplexes formed were visualized by autoradiography after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and apparent relative molecular masses (Mr) of each band assigned. FVIII HC polypeptides were detected in all types of samples, including plasma, without further purification. Normal plasma contained a range of polypeptides with the largest dominant band at a net apparent Mr of 250-300 kD, and the smallest at 80-90 kD: the bands visualized correspond to the 90-210 kD HC species seen on conventional analysis of purified FVIII. No bands were produced from samples of haemophilic plasma. Treatment of plasma or FVIII concentrate with low concentrations (1 IU/ml) of thrombin removed the 250-300 kD and other intermediate bands, intensified then removed the 80-90 kD polypeptide and produced a band at 40-50 kD. Thrombin-associated rise and fall in FVIII clotting activity by one-stage assay correlated with intensity of the 80-90 kD polypeptide. A polypeptide of Mr 40-50 kD was also produced after incubation with activated factor X: activated factor VII plus thromboplastin had no effect on HC structure. FVIII polypeptides were visualized in prothrombin complex concentrates, with a more degraded profile seen in a deliberately 'activated' product.

Biological Products↗
PubMed1991

Variable inactivation of human factor VIII from different sources by human factor VIII inhibitors.

The source of human factor VIIII (FVIII) had a marked effect on the inhibitory activity of a panel of eight human FVIII inhibitors. Use of conventional FVIII concentrates gave lower inhibitor titres whereas a monoclonal antibody purified FVIII concentrate gave titres similar to or greater than those with plasma. Addition of phospholipid (PL) protected highly purified FVIII against inhibition. The content of PL-bound FVIII in concentrates may account for the observed differences.

Animals↗
PubMed1990

Factor VIII heavy chain polypeptides in plasma and concentrates.

Factor VIII polypeptides in plasma and FVIII concentrates have been analysed by an electrophoretic technique based on that of Weinstein et al (1981). Samples were complexed with radiolabelled anti-FVIII Fab', and the immunocomplexes visualized by SDS-polyacrylamide electrophoresis. The technique visualized FVIII heavy chain polypeptides in all types of samples, including plasma, without further purification. Fresh or frozen normal plasma (collected into protease inhibitors) contained a range of polypeptides with the largest dominant band at an apparent Mr of 250-300 kDa, and the smallest at 80-90 kDa: no bands were produced from samples of severe haemophilic plasma. Cryoprecipitate had a similar polypeptide distribution to normal plasma, but intermediate purity FVIII concentrates showed more degraded patterns which varied between products: the 250-300 kDa bands were reduced or absent, the 80-90 kDa bands were more pronounced than in plasma, and in one product a polypeptide was seen at approximately 40-50 kDa. In some products heat treatment for viral inactivation increased the proportion of smaller FVIII polypeptides. Highly-purified FVIII concentrate derived from plasma was also degraded relative to plasma FVIII, and two products obtained by recombinant DNA technology both showed degraded, though slightly different, profiles. The native structure of FVIII in fresh plasma appears heterogeneous with a predominance of higher Mr forms: these are degraded to a greater or lesser extent during concentrate production, dependent on the manufacturing processes used.

Blood Preservation↗
PubMed1990

High molecular weight aggregate content of heated and unheated factor VIII products determined by fast-protein liquid chromatography.

The molecular-weight distribution of proteins in factor VIII concentrates has been analysed by fast-protein liquid chromatography. The proportion of high-molecular-weight (HMW) aggregates in one product increased on freeze-drying and heating, with fibrinogen and fibronectin being the main protein components of the HMW peak. In all other concentrates, the HMW peak was less than or equal to 5% of the total protein content and there were no differences in HMW content according to purity or method of viral inactivation.

Chromatography, Liquid↗
PubMed1990

Monoclonal antibodies against antithrombin III and their application in an immunoradiometric assay.

Monoclonal antibodies (MAbs) to antithrombin III (AT III) have been produced and characterized, and a two-site immunoradiometric assay (IRMA) developed, using one of the MAbs as a 'catcher' antibody and a radiolabelled affinity-purified rabbit antibody for detection. The IRMA could be performed in one day and the optimum concentration range of AT III was 1-100 ng/ml. Assays on AT III concentrates by the IRMA method showed a good correlation with AT III antigen values measured by the immunoelectrophoresis method (Laurell), the IRMA results averaging 94.3% of the Laurell values. Two of the MAbs (11 and 16) inhibited heparin cofactor but not AT III progressive activity, and the binding of AT III to MAb 11, used for the IRMA, was blocked by heparin. These results indicate that MAb 11 is directed at or near to the heparin binding site, and could therefore be useful in the study of structural aspects of this site in normal and genetically abnormal AT III.

Animals↗
PubMed1989

The effect of calcium chloride on anti-Xa activity of heparin and its molecular weight fractions.

The anti-Xa activities of unfractionated heparin (UFH) and nine low molecular weight heparins (LMWH) have been measured in the presence and absence of 3 mM CaCl2, using bovine Factor Xa, purified human ATIII and an amidolytic assay. The addition of CaCl2 increased the activity of UFH by 93%, but the effect on LMWH was less, ranging from -20% to +55%. Studies of gel filtration fractions of UFH showed marked Mr dependence of the CaCl2 effect in the range 4,000-12,000. The differences among the various LMW heparins with respect to the effect of CaCl2 were closely correlated with the amount of polysaccharide above an Mr of 6,500. Kinetic studies confirmed the potentiation of the activity of UFH with bovine Xa and showed an even more marked effect using human Xa.

Antithrombin III↗
PubMed1989

Procoagulant activity of T lymphoblastoid cells in extrinsic and intrinsic coagulation systems.

We have measured the procoagulant activity (PCA) of four T lymphoblastoid cell lines (Jurkat, CEM, HSB-2 and Molt 4) as well as normal peripheral blood T lymphocytes, before and after stimulation with phytohaemagglutinin (PHA), using clotting and amidolytic methods. Of the four cell lines only one, Jurkat, gave enhanced PCA after stimulation with PHA. This activity was shown to be tissue factor-like by its dependence on factor VII in plasma and in an amidolytic assay with purified factors VII and X. Jurkat was also the only one of the four cell lines to secrete interleukin-2. All four cell lines promoted the generation of large amounts of thrombin in platelet-free plasma in glass tubes. This activity was dependent on the presence of plasma factor VIII, and was probably due to phospholipids in the cell membranes. Normal T lymphocytes gave intrinsic PCA in the thrombin generation test which was only 15% of that of the lymphoma cells. These results show that some T lymphocytes can develop PCA in both intrinsic and extrinsic systems and this should be taken into account in studies of the PCA of mixed leukocyte populations.

Blood Coagulation↗
PubMed1989

The relative antithrombotic effectiveness of heparin, a low molecular weight heparin, and a pentasaccharide fragment in an animal model.

The antithrombotic efficacy of unfractionated heparin (UFH), a low molecular weight heparin (LMWH) and a synthetic pentasaccharide (PENTA) has been compared in an animal model for stasis thrombosis. We have also compared the relative ability of these three agents to impair thrombin generation in vitro and in vivo, and measured their effects on anti-Xa activity and thrombin clotting times. UFH, LMWH and PENTA all had the capacity to impair thrombogenesis, although there were marked differences in their relative effectiveness. Reduction of thrombin generation to 20% of control values was closely correlated with the prevention of thrombosis after 20 minutes' stasis, but this was only achieved with UFH. The same dry weight dose of LMWH or PENTA reduced thrombin generation to about half control values, and neither significantly impaired thrombus formation after 20 minutes' stasis. Impaired thrombin generation correlated better than anti-Xa activity with prevention of stasis thrombosis. In this model, UFH was clearly superior to LMWH and PENTA as an antithrombotic agent.

Animals↗
PubMed1989

The anticoagulant activity of heparin: measurement and relationship to chemical structure.

For many years the anticoagulant activity of heparin has been estimated by coagulation assays, in which the prolongation of clotting times by heparin is measured under various conditions. More recently, assays have been developed which measure the inhibitory action of heparin on isolated coagulation enzymes, notably Factor Xa and thrombin, using specific amidolytic peptide substrates. The anticoagulant activity of heparin arises primarily from its ability to bind to antithrombin III (AT III), altering the conformation and enhancing the activity of this major protease inhibitor. Passage of heparin through an immobilised AT III column yields two fractions: a high affinity fraction with 300-350 iu mg-1 anticoagulant activity, comprising one-third of the total, and a low affinity fraction with an activity of less than 10 iu mg-1, comprising the remaining two-thirds. Studies in several laboratories have demonstrated that a specific pentasaccharide sequence is required for AT III binding. The authors have shown that the presence or absence of this sequence can be detected by high-field proton NMR, thus providing a semi-quantitative method for a functionally important group. A second major influence on anticoagulant activity is molecular weight distribution. Studies in the authors' laboratory on a series of fractions of 5000-35,000 showed that whereas anticoagulant activity in APTT clotting assays decreased with decreasing molecular weight (Mr), activity in anti-Xa assays was maintained or increased in the low Mr fractions. However, in vivo studies showed that high affinity fragments with anti-Xa activity only were poor antithrombotic agents. It appears that the presence of the AT III binding site alone is not sufficient for full antithrombotic activity; an extra length of polysaccharide chain of at least 15 residues is required. Molecular weight distribution is readily assessed by HPLC, although the lack of suitable reference materials hampers assignment of absolute molecular weights. Important determinants of anticoagulant activity can now be assessed by physicochemical techniques but, at present, these techniques are not precise enough to replace anticoagulant assays as predictors of in vivo behaviour.

Heparin↗
PubMed1989

Inhibition of interleukin-2 secretion by factor VIII concentrates: a possible cause of immunosuppression in haemophiliacs.

The inhibitory effect of factor VIII concentrate products on IL-2 secretion by human T-cells was investigated. The six products used widely in the U.K. showed very different activities varying from almost total inhibition to no significant effect. There appeared to be no obvious relationship between inhibitory activity and protein composition but factor VIII itself was not responsible for the effect as affinity purified products were entirely non-inhibitory. The two wet-heated products were most inhibitory whereas dry-heated products were less inhibitory or non-inhibitory. However, a wet-heated version of a non-inhibitory dry-heated product was also non-inhibitory, suggesting that the composition of the concentrate rather than anti-viral treatment is important for immunosuppressive activity. A product treated by the solvent/detergent procedure showed considerable inhibitory activity. Immunoglobulin and albumin products did not inhibit IL-2 secretion to any significant extent, but factor IX concentrates were inhibitory. We suggest that inhibition of IL-2 secretion by factor VIII concentrates may be related to the immunosuppression observed in haemophiliacs treated with high dose factor VIII products and that our results should be considered by clinicians and manufacturers of factor VIII products.

Antibodies, Monoclonal↗