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Biomedical subjects

A R Hubbard

Publications and source records attributed to A R Hubbard.

At least 37 records · Page 2Linked to original sources
PubMed1989

The effect of phospholipase C on plasma factor VII.

The proposed link between a circulating factor VII-phospholipid complex and the risk of cardiovascular disease (CVD) has stimulated us to investigate the effect of phospholipase C (PLC) on the factor VII (FVII) activity in plasma from healthy individuals. PLC caused a rapid fall in FVII activity which was larger with heparinized than with citrated plasma. EDTA inhibited the PLC effect so emphasizing the involvement of divalent cations. PLC dependent loss of FVII activity varied widely between individuals, showed a highly significant correlation with plasma triglyceride concentrations, and was always greater in post-prandial compared to fasting plasma samples. Experiments using pure recombinant FVIIa and plasma depleted of FVII by adsorption indicated that loss of FVII activity only occurred in the simultaneous presence of absorbed plasma, FVIIa and PLC. Preincubation of PLC with adsorbed plasma before adding FVIIa did not lead to loss of FVII activity. It appears that PLC may act on lipoproteins already bound to FVII, in order to inhibit FVII activity. Other results indicated that competition between different plasma components (lipoproteins) in binding to FVII may govern the extent of the PLC dependent reduction in FVII activity.

Citrates↗
PubMed1988

Standardization of protein C in plasma: establishment of an international standard.

An international collaborative study, involving 18 laboratories, was carried out to establish an international standard for protein C in plasma. The proposed standard, which consisted of a freeze-dried ampouled plasma preparation coded 86/622, was assayed against fresh normal plasma and the participants' local standards. Protein C activity assays were placed in four groups, depending on the method of activation and detection of protein C. The combined potencies (units per ampoule) for the proposed international standard were: thrombin activation/clotting assays, 0.86; thrombin activation/chromogenic assays, 0.81; snake venom activation/clotting assays, 0.81 and snake venom activation/chromogenic assays, 0.82. Measurement of protein C antigen gave potency estimates of 0.81 and 0.82 unites per ampoule for the Laurell electroimmunoassay and ELISA techniques, respectively. The good agreement in potency estimates between the different methods indicates that the overall combined figure (226 assays) for the international standard of 0.82 international units per ampoule should serve for all methods. Accelerated degradation studies have indicated that the standard should be suitably stable when stored at -20 degrees C. The freeze-dried plasma 86/622 has been established by the WHO Expert Committee on Biological Standardization as the 1st International Standard for Protein C in Plasma; with an assigned unitage of 0.82 international units per ampoule.

Blood Coagulation Tests↗
PubMed1987

Inhibition of the tissue factor-factor VII complex: involvement of factor Xa and lipoproteins.

Inhibition of the procoagulant activity of a tissue factor-Factor VII (TF-FVII) complex by Al(OH)3-adsorbed plasma (AP) was found to require the presence of Factor Xa (FXa). Inhibitory activity seems to be generated through the interaction of FXa with a component in AP rather than with the TF-FVII complex. Quantitation of inhibitor activity was carried out using an amidolytic assay for TF-FVII activity. Incubation of AP with various antisera demonstrated that the inhibition was mainly associated with the presence of apolipoprotein B (apo B) rather than alpha 2-macroglobulin or antithrombin III. Purified lipoprotein-rich fractions prepared from AP, using density gradient ultracentrifugation, all contained some inhibitory activity. Incubation with anti-apo B greatly reduced the inhibitor in the very low density lipoprotein (VLDL)- and low density lipoprotein (LDL)-rich fractions but had essentially no effect on inhibition by the high density lipoprotein (HDL) fraction, which was rich in apo A. The inhibitory activity of AP was 60% that of normal plasma and this correlated well with the relative apo A and apo B concentrations. It is proposed that inhibition requires the interaction of FXa with plasma lipoproteins or associated components and that the product of this interaction is then able to bind to and inhibit the TF-FVII complex.

Adsorption↗
PubMed1986

Anticoagulant activities of pentosan polysulphate (Hémoclar) due to release of hepatic triglyceride lipase (HTGL).

Subcutaneous injections of 50 mg pentosan polysulphate (Hémoclar) were given to normal volunteers and the effects on anti-Factor Xa activity, thrombin generation and lipase release measured. Concentrations of pentosan polysulphate were measured by a competitive binding assay and the mean peak level found to be 1.6 micrograms/ml. Anti-Xa clotting activity rose to 0.034 iu/ml and thrombin generation induced by lipid peroxides was inhibited by approximately 50%. Neither of these effects could be accounted for by the direct action of pentosan polysulphate at the concentrations measured. Pentosan polysulphate was very effective in releasing lipase, approximately 70-80% of the total enzyme activity being due to hepatic triglyceride lipase (HTGL). In vitro addition of purified HTGL to plasma markedly enhanced anti-Xa clotting activity, and caused a 70% inhibition of lipid peroxide induced thrombin generation. Anti-Xa activity of post-injection plasma was increased rather than neutralised by addition of polybrene, and this effect could be mimicked by addition of polybrene to plasma containing pentosan polysulphate and purified HTGL. It is concluded that, when given in low doses subcutaneously, pentosan polysulphate acts as an indirect anticoagulant, its major effects being due to release of HTGL.

Adult↗
PubMed1986

Inhibition of tissue thromboplastin-mediated blood coagulation.

Factors affecting the inhibition of tissue thromboplastin (TP)-mediated blood coagulation have been investigated. Human brain thromboplastin progressively loses procoagulant activity when incubated in the presence of defibrinated plasma and CaCl2. Inhibition is maximal at a CaCl2 concentration of 1.5 mM during incubation and involves the calcium dependent binding of a plasma component(s) to the TP-FVII complex, preventing the activation of FX. Chelation of calcium ions using EDTA releases active TP and FVII from the inhibited complex. No inhibition occurs during incubation of TP with Al (OH)3 adsorbed plasma and calcium ions unless a Factor VII concentrate (or purified FVII and FX) is also present. Incubation of TP with antithrombin III-deficient plasma and calcium ions also leads to inhibition. Moreover, purified AT III cannot substitute for adsorbed plasma in producing TP inhibition. The data are consistent with the presence in plasma of a potent AT III independent inhibitor of TP-mediated blood coagulation.

Blood Coagulation↗
PubMed1985

Standardization of low molecular weight heparins: a collaborative study.

A collaborative study was carried out, in which eight laboratories each assayed eight low molecular weight (LMW) heparins against the International Standard (IS) for heparin. APTT assays and three types of anti-Xa method were used. The results of this study showed that: LMW heparins cannot be validly assayed against the IS by APTT or anti-Xa methods. Potencies of LMW heparins vs. the IS differed considerably between the four types of assay method used and also between different laboratories using the same type of method. Adoption of a single LMW heparin standard would improve validity, improve inter-laboratory variation, and largely abolish the differences between the three types of anti-Xa method. However, since calibration of a LMW heparin standard against the IS would give potencies that differ widely by the different assay methods, a single assay method such as the anti-Xa amidolytic, plasma, would need to be chosen for this calibration.

Antithrombins↗
PubMed1985

Neutralisation of heparan sulphate and low molecular weight heparin by protamine.

The neutralisation by protamine sulphate (PS) of heparan sulphate (HS), a low molecular weight heparin (LMWH), and a reference preparation of unfractionated heparin (UH), was studied by activated partial thromboplastin time (APTT) and anti-Xa clotting assays. UH was most easily neutralised in the APTT assay by PS (on a weight for weight basis), followed by LMWH and HS. The neutralisation of APTT activity by PS closely followed the loss of activity in the anti-Xa clotting assay, when plasma was used as the source of At III. When the anti-Xa clotting assay was carried out using purified At III in place of plasma, HS and LMWH were neutralised by much lower amounts of PS and resembled UH neutralisation more closely. Resistance of HS anti-Xa activity to PS neutralisation decreased with increasing plasma dilution. The presence of bovine albumin with purified At III concentrate increased the resistance of HS to PS neutralisation. It is concluded that PS binding to UH, HS and LMWH is probably related more to their degree of sulphation than molecular weight and that non-specific interactions between PS and plasma proteins inhibit the binding of PS to HS and LMWH.

Factor X↗
PubMed1984

Anticoagulant properties in vitro of heparan sulphates.

The anticoagulant properties in vitro of eight heparan sulphate preparations were studied using clotting (APTT, anti-Xa) and amidolytic (anti-Xa, anti-thrombin) assays. Activities ranged from very low levels (less than 5 iu/mg) up to values similar to those of heparin. Activities measured by APTT assay showed the best correlation with the sulphate to carboxylate ratio of the heparan sulphates. Highest activities were obtained in the anti-Xa clotting assay, these being approximately two-fold greater than activities in the anti-Xa amidolytic assay. Five of the heparan sulphate preparations were readily neutralised by protamine sulphate, whereas the three heparans with the lowest sulphate to carboxylate ratio were much more resistant to neutralisation. After fractionating each heparan sulphate into At III-binding and non-binding material, it was found that the anti-coagulant properties were associated only with the former. It is concluded that these properties are dependent on the activation of At III.

Animals↗
PubMed1980

Organic-acid transport in resealed haemoglobin-containing human erythrocyte 'ghosts'.

The transport of organic acids across the membrane of resealed haemoglobin-containing erythrocyte 'ghosts' prepared by a dialysis technique has been studied. The present work forms part of studies directed towards the use of erythrocyte cellular carriers in enzyme-replacement therapy of inherited metabolic diseases. Oxalic acid, glycollic acid and glyoxylic acid were taken as representative of aliphatic acids of low molecular mass and benzoic and cinnamic acids as representative of unsubstituted aromatic acids. These selected acids are important in the diseases with which the present work is concerned. Comparison of influx and efflux transport characteristics showed that erythrocyte 'ghosts' retain transport properties closely similar to those of normal erythrocytes. Rapid transport was observed with all organic acids studied and there was a linear relationship between initial amount of influx and external concentration of aliphatic acid. Saturation of the transport system was not observed up to 1 mM external concentration, and the presence of plasma in the external medium had no effect on transport characteristics. Transport in intact erythrocytes and prepared erythrocyte 'ghosts' from patients with hyperoxaluria was also studied.

Biological Transport↗
PubMed1980

In vivo life span of resealed rabbit erythrocyte 'ghosts'.

Resealed erythrocyte 'ghosts' have been proposed as biodegradable in vivo carriers for exogenous enzymes in the therapy of inherited metabolic diseases. Extended animal studies are essential prior to clinical application. The survival of rabbit erythrocyte 'ghosts' has been studied to find a suitable animal model. In vitro experiments have shown that incorporation of macromolecules into rabbit erythrocyte 'ghosts' is much lower than into human erythrocyte 'ghosts'. This could be related to the continuous cell destruction during 'ghost' preparation procedures. Rapid destruction of 'ghosts' in the circulation was obtained in all experiments particularly in the first minutes and hours. Cells surviving this initial period showed a longer life span and kinetics of further cell destruction followed a logarithmic function similar to that of normal erythrocytes. The experimental use of rabbits appears limited to studies where rapid uptake of the enzyme is required and other animal models must be studied when prolonged survival of the erythrocyte 'ghosts' is required, e.g., as the site of substrate degradation in the circulation.

Animals↗
PubMed1979

In vitro studies on resealed erythrocyte ghosts as protein carriers.

Resealed erythrocyte ghosts have been proposed as in vivo carriers for enzyme replacement therapy of inherited metabolic diseases. In comparative studies of methods for reversible hypotonic haemolysis of erythrocytes, a five-fold increased entrapment of human serum albumin was obtained by use of a dialysis procedure instead of direct dilution. The percentage incorporation of protein was also affected by varying mixing procedure, haematocrit, lysis, and resealing times but not by varying buffer composition or added protein concentrations over a wide range. Higher protein entrapment was observed with time-expired blood compared to fresh blood and this may be ascribed to increased osmotic fragility of the erythrocyte membrane in stored cells. Haemolysed and resealed ghosts prepared by any method used were smaller than normal erythrocytes as measured with a Coulter Counter and with a fluorescence-activated cell sorter, and protein entrapment reduced the ghost size further.

Blood Preservation↗