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T W Barrowcliffe

Publications and source records attributed to T W Barrowcliffe.

At least 127 records · Page 7Linked to original sources
PubMed1983

Standardization of Factor VIII-IV. Establishment of the 3rd International Standard for Factor VIII: C concentrate.

An international collaborative study was carried out to establish a replacement for the current (2nd) international standard for Factor VIII:C, concentrate. Twenty-six laboratories took part, of which 17 performed one-stage assays, three performed two-stage assays and six used both methods. The proposed new standard, an intermediate purity concentrate, was assayed against the current standard, against a high-purity concentrate and against an International Reference Plasma, coded 80/511, previously calibrated against fresh normal plasma. Assays of the proposed new standard against the current standard gave a mean potency of 3.89 iu/ampoule, with good agreement between laboratories and between one-stage and two-stage assays. There was also no difference between assay methods in the comparison of high-purity and intermediate purity concentrates. In the comparison of the proposed standard with the plasma reference preparation, the overall mean potency was 4.03 iu/ampoule, but there were substantial differences between laboratories, and the two-stage method gave significantly higher results than the one stage method. Of the technical variables in the one-stage method, only the activation time with one reagent appeared to have any influence on the results of this comparison of concentrate against plasma. Accelerated degradation studies showed that the proposed standard is very stable. With the agreement of the participants, the material, in ampoules coded 80/556, has been established by the World Health Organization as the 3rd International Standard for Factor VIII:C, Concentrate, with an assigned potency of 3.9 iu/ampoule.

Antigens↗
PubMed1983

Studies of the heterogeneity of antithrombin III concentrates.

The biological activities of antithrombin III (At III) concentrates, prepared by several manufacturers for clinical use, have been compared by three assay methods, and their heparin-binding properties studied by crossed immunoelectrophoresis and heparin affinity chromatography. Concentrates from two of the four manufacturers showed discrepancies between assay methods, with concentrations by heparin co-factor assays significantly lower than those by immunological and progressive antithrombin methods. Heterogeneity was also found by heparin binding studies, with about half the total At III antigen in these concentrates being unable to bind to heparin. These results confirm previous findings of heterogeneity in At III concentrates and show that some concentrates contain substantial amounts of altered At III molecules in which the heparin-binding site has been denatured but the thrombin-neutralizing site left largely intact.

Antigens↗
PubMed1983

Binding to phospholipid protects factor VIII from inactivation by human antibodies.

The addition of purified factor IXa and phospholipid to factor VIII concentrate protected the VIII:C from inactivation by human antibodies. This protective effect was shown to be due largely to the phospholipid. Addition of phospholipid alone gave substantial protection against even high-titer antibodies, as shown by measurements of thrombin generation and VIII:C assays. Increasing concentrations of phospholipid led to significant reductions in the amount of VIII C:Ag detected by an IRMA method, up to 70% of the original VIII C:Ag being "lost" at the highest concentration of phospholipid. These results indicate that phospholipid binding plays an important part in the procoagulant activity of factor VIII and that human antibodies to VIII:C are directed largely at the phospholipid binding site. The addition of phospholipid to factor VIII concentrates could have important clinical applications in the treatment of hemophiliacs with antibodies to factor VIII.

Antibodies↗
PubMed1982

Studies of anti-Xa activity in human plasma. II: The role of lipoproteins.

The major plasma inhibitor of factor Xa is thought to be anti-thrombin III (At III). However, adsorption of plasma by aluminium hydroxide (A1(OH)3) increases its rate of neutralisation 7-8 fold, and this 'fast-acting' anti-Xa activity has been shown to be independent of At III. Gel filtration of plasma indicated that the anti-Xa activity after A1(OH)3 adsorption was located largely in the high molecular weight (greater than 200,000) fractions, which contain most of the plasma lipoproteins. Purified lipoproteins of very low-density (VLDL), low-density (LDL) and high density (HDL) were prepared by ultracentrifugation and their anti-Xa activities measured before and after adsorption by A1(OH)3. Both LDL and HDL had significant anti-Xa activities by clotting and amidolytic assays. A1(OH)3 adsorption of LDL and HDL gave a marked increase in anti-Xa clotting activity and a decrease in amidolytic activity. Incubation of the adsorbed lipoproteins with phospholipase enzymes destroyed the anti-Xa activity, and prior incubation of Factor Xa with Ca++ and phospholipid protected it against inactivation, indicating that the anti-Xa activity of the adsorbed lipoproteins is mediated via binding of Xa to phospholipid in the lipoproteins. These results indicate that lipoproteins, especially LDL and HDL, are responsible for the increased anti-Xa activity of plasma after A1(OH)3 adsorption. These lipoproteins appear to contain high affinity phospholipid binding sites for Xa which are revealed by A1(OH)3 adsorption.

Adsorption↗
PubMed1982

Effects of heparin oligosaccharides with high affinity for antithrombin III in experimental venous thrombosis.

The in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 microgram/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays. It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

Animals↗
PubMed1982

A comparison of pentosan polysulphate (SP54) and heparin. I: Mechanism of action on blood coagulation.

The effects of SP54 on inhibition of thrombin, factor Xa and factor IXa, in the presence and absence of antithrombin III (At III), have been examined and compared to those of heparin. SP54 potentiated inhibition of thrombin and Xa by purified At III, but crossed immunoelectrophoresis data indicated that these effects were mediated by binding to the enzyme, rather than to At III. Relatively high concentrations of SP54 were required for inhibition of thrombin and Xa in plasma, but at concentrations less than 2 micrograms/ml there was a marked suppression of the intrinsic activation of factor X. This effect was shown to be independent of At III, and to be due largely to inhibition of factor IXa. Prothrombin activation by factor Xa and phospholipid was also suppressed by SP54 in the absence of At III, and its effect on the APTT was also shown to be independent of At III. It is concluded that a relatively low concentrations the anticoagulant actions of SP54 are mainly due to these At III-independent pathways.

Animals↗
PubMed1982

A comparison of pentosan polysulphate and heparin. II: Effects of subcutaneous injection.

A comparison has been made between the effects of pentosan polysulphate (SP54) and mucosal heparin following subcutaneous injection in man. Unlike heparin, pentosan polysulphate has relatively little effect in vivo as measured by anti-factor Xa clotting assay and none by an anti-Xa amidolytic assay (S-2222). However, pentosan polysulphate is at least as potent as heparin on a weight basis in producing activation of lipoprotein lipase, shortening of the euglobulin clot lysis time and impairing the generation of factor Xa. Our data indicate that pentosan polysulphate has more marked effects in vivo than in vitro, that the action of the drug on clotting is mediated mainly via an At III-independent pathway, and that its effects are not confined to the coagulation system.

Blood Coagulation↗
PubMed1981

Studies in man and experimental animals of a low molecular weight heparin fraction.

In vitro and in vivo studies were carried out on a commercially prepared low molecular weight heparin fraction. By APTT assay the fraction had a specific activity of half that of unfractionated mucosal heparin, yet retained full potency by anti-Xa assay (both clotting and chromogenic substrate). When administered intravenously to human volunteers, the anti-Xa/APTT ratio remained the same as it was in vitro. However, after subcutaneous injection, the ratio increased and anti-Xa activity could not be fully neutralized ex vivo by PF4. The fraction was as effective as unfractionated heparin in preventing experimental serum-induced thrombosis, suggesting that a heparin fraction with high specific activity by anti-Factor Xa assay compared to APTT activity may be an effective drug for the prophylaxis of venous thrombosis.

Adult↗
PubMed1981

Factor VIII-related activities in therapeutic concentrates.

Recent studies have suggested that factor VIII exists in plasma as a complex containing two distinct antigens (VIII R:Ag and VIII C:Ag) and two different biological activities (VIII:C and VIII R:RCo). Concentrations of these four entities have been measured in all the different types of therapeutic material used for treatment of hemophilia and von Willebrand's disease. In all materials, the ratio of VIII R:Ag to VIII:C was greater than 1: however, there were significant differences between concentrates, and the lowest ratio was found in cryoprecipitate. In VIII R:Ag assays, the freeze-dried concentrates gave nonparallel dose-response curves compared with plasma, indicating alterations in molecular form during purification. Values for VIII C:Ag were much lower than the corresponding VIII R:Ag values, and ratios of VIII C:Ag to VIII:C were close to 1 in many of the concentrates, suggesting that the low yields of VIII:C in some production processes could result from losses of VIII:C molecules rather than from denaturation. Measurement of VIII R:RCo by a platelet counting method gave lower values in most concentrates than by an aggregometry technique. It is proposed that the platelet counting method may reflect closely the biological activity of the concentrates in treatment of von Willebrand's disease.

Antigens↗
PubMed1981

Studies of phospholipid reagents used in coagulation I: some general properties and their sensitivity to factor VIII.

Some general properties of phospholipid reagents have been studied, particularly in relation to their use in detection and measurement of Factor VIII deficiency. There was wide variation among reagents in their total phospholipid concentration, and in the degree of contamination with tissue factor and oxidation products. Although clotting times of normal and haemophilic plasma were affected by change of phospholipid concentration, presence of oxidation products, and order of addition of reagents, these factors did not appear to influence the efficiency of detection of the Factor VIII defect. The method of activation had a definite influence on sensitivity to Factor VIII deficiency, both the type of activator and the time of activation being important. In Factor VIII one-stage assays, use of different phospholipids with the same substrate plasma gave significant differences in precision, but the estimates of potency of a Factor VIII concentrate against a plasma standard were not significantly different.

Blood Coagulation↗
PubMed1981

Studies of phospholipid reagents used in coagulation II: factors influencing their sensitivity to heparin.

This study demonstrates the many technical variables which exist in the use of APTT reagents to detect and measure heparin. With the same phospholipid reagent different activation regimes produced significant differences in heparin sensitivity. The concentration of phospholipid was also an important influence; decreasing concentration led to increasing sensitivity, and the highest sensitivity was given by omitting the phospholipid altogether. Other factors which influenced the sensitivity to heparin were buffering of the reagents and the presence of oxidation products in the phospholipid. The results emphasize the need for standardisation of at least some of these variables if the APTT is to be used to determine the therapeutic range of heparin.

Animals↗
PubMed1980

Studies of heparin binding to antithrombin III by crossed immunoelectrophoresis.

The technique of crossed immunoelectrophoresis has been used to study the binding to purified antithrombin III (At III) of heparin and other mucopolysaccharides. The technique was unable to detect differences among samples of whole heparin from various manufacturers, but proved useful in studying the binding of heparin fractions; at the same molarities, low and high molecular weight heparin fractions displayed equal binding ability to At III. A semi-synthetic heparin analogue showed no evidence of binding to At III, but a sample of heparan sulphate did interact with At III at a concentration 3 times that of heparin. Samples of purified At III from four different manufacturers all displayed heterogeneity with respect to heparin binding. A proportion of the total At III did not bind to heparin and, in one sample, this non-binding material constituted about 40% of the total. An antiserum made against purifed At III contained antibodies with different cross-reactivities against heparin bound and non-heparin bound At III.

Animals↗