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C V Prowse

Publications and source records attributed to C V Prowse.

At least 55 records · Page 3Linked to original sources
PubMed1987

Evaluation of monoclonal antibodies to vWf antigen for use in autoradiographing vWf multimer analysis.

Nine monoclonal antibodies (MAb, coded ESvWF 1-5, 7-10) to human von Willebrand's factor (vWf) have been studied for their labelling characteristics with 125I and their ability to demonstrate vWf multimers by autoradiography after discontinuous SDS electrophoresis on agarose and agarose/acrylamide gels in plasma from normal and von Willebrand's disease (vWD) patients. Of ESvWF 1, 2, 4, 7, 8 and 9, ESvWF 2 gave autoradiographs most similar to those obtained with a polyclonal antibody. The others gave much fainter staining of all bands suggesting that the epitope detected by ESvWF 2 is either more common or, less easily altered by SDS than the epitopes identified by the other MAb's. ESvWF 2 gave a different staining pattern with a IIC vWD patient than that obtained using a polyclonal antibody. The most striking result, however, was shown by ESvWF 3, 5 and 10 which failed to stain the lower band of the lower molecular weight multimer. This suggests that the lower triplet band, unlike the central and upper bands of the triplet, does not contain the epitope identified by these three MAb's. With these reagents it has been possible to show, for the first time, that epitope differences do exist between the constituent bands of the vWf triplet.

Antibodies, Monoclonal↗
PubMed1987

Immunoaffinity purification of factor VIII complex.

Murine monoclonal antibodies to human von Willebrand factor (vWf) were immobilised on Sephacryl S-1000. Various solutes were screened for their ability to elute 125I-vWf from the immobilised antibodies. The most effective solutions were then tested to determine which allowed retention of factor VIII procoagulant activity (VIII:C) and activity of vWf measured by platelet aggregation in the presence of ristocetin (Ristocetin cofactor activity R. cof.). Finally, F VIII complex was purified from both plasma and cryoprecipitate by immunoaffinity chromatography under the selected conditions. The product had a specific activity of 45 units of VIII:C per mg of protein and 60 units of R. cof. per mg representing a 4000-fold purification from plasma. The fibrinogen and fibronectin content were each less than 4% of the total protein with vWf accounting for 60% of the total protein in the final product. Multimer analysis of the product showed a similar pattern to normal plasma and contamination by murine monoclonal antibody was less than 300 ng per mg of protein. A novel product is thus obtained containing both clinically relevant VIII:C and R. cof. in a single vial whilst using only one specific monoclonal antibody.

Antibodies, Monoclonal↗
PubMed1987

Studies on the effect of heat treatment on the thrombogenicity of factor IX concentrates in dogs.

This study examines the effects of heat treatment for 72 h at 80 degrees C on the potential thrombogenicity of lyophilized human coagulation factor IX concentrates. Since heating generated minor amounts of thrombin, concentrate was prepared with antithrombin III addition prior to heat treatment. Changes in coagulation parameters were followed prior to and after infusion of 100 iu/kg of heated and unheated concentrates to dogs. All batches produced a transient fall in platelet count during infusion and a delayed rise in plasma fibrinopeptide A, accompanied by a minor prolongation of the activated partial thromboplastin time. Such changes were less marked for heated batches. Control infusion of a 'failed' factor IX concentrate showed an additional fall in fibrinogen, rise in fibrin degradation products and a more rapid rise in fibrinopeptide A, while thrombin infusion caused an even more dramatic intravascular coagulation. These studies indicated no increase in the potential thrombogenicity of freeze dried factor IX concentrates as a result of heat treatment.

Albumins↗
PubMed1987

Assessment of a new centrifugal plasmapheresis machine: the SPC-600.

An approach to the full assessment of new plasmapheresis machines is outlined. This involves testing for changes in donated blood, donors and blood components using fresh donor blood and during full procedures in an animal model prior to performing full procedures in human volunteers. Using this approach, a new centrifugal plasmapheresis machine, the SPC-600, was assessed. No adverse effects associated with the use of the machine were detected in the initial studies. When used for plasmapheresis of volunteers, 500 ml of plasma was obtained in 43 +/- 4 min without adverse effects on donors. The plasma product contained normal levels of plasma proteins, including factor VIII, and 20 +/- 6 X 10(9)/1 platelets. There was no evidence of activation of complement or coagulation systems, and the factor VIII yield in cryoprecipitate prepared from single donations of plasma was equivalent to that observed for standard whole blood donations.

Animals↗
PubMed1986

Absorption of heparin, LMW heparin and SP54 after subcutaneous injection, assessed by competitive binding assay.

Unfractionated heparin, pentosan polysulphate (SP54) and the low molecular weight heparins CY216 and CY222 were injected subcutaneously at a minimum of weekly intervals into 5 healthy volunteers. The dose was 75 mg in all cases. Concentrations of administered glycosaminoglycan in serial plasma samples and voidings of urine were measured using a competitive binding assay, and biological activity was assessed in plasma using APTT and anti-Xa clotting assays. There was wide individual variation in the absorption of unfractionated heparin as indicated both by the maximal plasma concentrations reached 2-3 h after injection and by the area under the concentration vs. time curve. The efficiency of absorption increased and the individual variation decreased with decreasing molecular weight of the administered glycosaminoglycan. Urinary excretion correlated with plasma concentration, and recovery in the urine also increased with decreasing molecular weight. Similar patterns of uptake and clearance were indicated by the APTT and competitive binding assays, but anti-Xa clotting activity could be detected in the plasma after clearance of the administered glycosaminoglycan.

Absorption↗
PubMed1986

Reductive amination for solid-phase coupling of protein. A practical alternative to cyanogen bromide.

For coupling proteins to Sephacryl gels periodate oxidation of these gels was investigated as an alternative method to cyanogen bromide activation. Optimum conditions were studied with respect to periodate concentration, time of oxidation, pH and type of coupling buffer, concentration of protein, temperature and time of protein uptake, and protein leakage after coupling. The effects of sodium cyanoborohydride and ascorbic acid as reducing agents, and of manganese ions as a potential catalyst were investigated. Using the derived optimum conditions, stable solid-phased antibodies were produced in high yield and used to adsorb factor VIII from plasma. These gels were stable for many weeks, as was the intermediate oxidised gel. Reductive amination for coupling proteins to oxidised Sephacryl gels results in increased binding and lower leakage than is obtained with cyanogen bromide activated agarose.

Acrylic Resins↗
PubMed1986

Immunopurification of human coagulation factor IX using monoclonal antibodies.

This study examines the suitability of four recently characterised monoclonal antibodies (MAbs) for the immunoaffinity purification of human coagulation factor IX (FIX) from plasma concentrates. Initial studies using 125I-FIX indicated that appreciable amounts of bound FIX could be eluted from immobilised MAbs with 0.2 M glycine, 50% ethanediol pH 10 (buffer N). Further studies with FIX concentrates showed that buffer N eluted FIX without compromising the activity of the zymogen. Although FIX was eluted from all four MAbs with this buffer, the best yield (82%) was obtained with MAb ESN-3. ESN-3 bound 40 to 60 iu FIX per mg MAb when immobilised on Sepharose 4B. After washing, column elution with buffer N yielded FIX at 100-200 iu/mg. The purity of the product was confirmed by sodium-dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting. The product contained no detectable mouse IgG (less than 3%) and less than 1% FII, X, or protein C.

Antibodies, Monoclonal↗
PubMed1986

Monoclonal antibodies and the haemostasis laboratory: current position.

In recent years research laboratories have produced a wide variety of monoclonal antibodies to haemostatic proteins. While these have proven very useful in fundamental research they have not, as yet, been widely used as routine reagents. This article reviews the range of monoclonal antibodies currently available and discusses their potential applications in routine laboratory practice.

Antibodies, Monoclonal↗
PubMed1986

The relationship of soluble immune complexes, insulin antibodies and insulin-anti-insulin complexes to platelet and coagulation factors in type 1 diabetic patients with and without proliferative retinopathy.

The possible correlation between soluble immune factors and platelet and coagulation factors has been evaluated in Type 1 diabetic patients with and without proliferative retinopathy, and in non-diabetic controls. Soluble immune complexes, platelet factor IV (PF4), beta-thromboglobulin, fibrinogen, factor VIII related antigen and anti-thrombin III were significantly increased in Type 1 diabetic patients with retinopathy as compared to non-diabetic controls. Fibrinogen and anti-thrombin III were also higher in those patients with retinopathy compared to those without retinopathy. A significant correlation was found between positive values of soluble immune complexes and increased levels of PF4 and beta-thromboglobulin in diabetic patients with retinopathy. The presence of soluble immune complexes and insulin-anti-insulin complexes was associated with a significantly greater number of elevated haemostatic factors in retinopathic patients. Our findings suggest that the interaction of platelets and soluble immune complexes or insulin-anti-insulin complexes may be pathologically relevant to the development of diabetic retinopathy.

Adult↗
PubMed1985

The measurement of heparin and other therapeutic sulphated polysaccharides in plasma, serum and urine.

The competitive binding assay described will specifically and accurately measure concentrations of administered heparin in biological fluids with a sensitivity of 60 ng ml-1. Neither endogenous glycosaminoglycans, nor plasma proteins such as ATIII and PF4 interfere in the assay. Semi-synthetic highly sulphated heparinoids and LMW heparin can also be measured. Using this assay heparin clearance followed simple first-order kinetics over the dose range 100-5,000 units, but the half-life was strongly dose-dependent. There was good correlation with heparin activity measurements by APTT and anti-Xa clotting assays. Plasma concentrations were measurable for at least 5 h following subcutaneous injection of 10,000 units of heparin. Excretion in the urine could be followed after all but the lowest intravenous dose. This assay, used in conjunction with measurements of heparin anticoagulant activity, will be valuable in the elucidation of mechanisms of action of heparin and the heparinoids, and in the assessment and management of problems related to heparin therapy.

Antithrombin III↗
PubMed1985

Enhancement of factor VIII-von Willebrand factor ristocetin cofactor activity by monoclonal antibodies.

Five monoclonal antibodies to human von Willebrand factor were selected for characterization from 18 produced in murine hybridomas. All showed a high and specific affinity for human von Willebrand factor (vWf) but exhibited little if any cross-reaction with sera from other species. The antibodies defined four epitopes on vWf, none of which were involved in platelet binding. Binding of two distinct antibodies at one of these epitopes was associated with enhancement of the rate of vWf-dependent platelet agglutination in the presence of ristocetin. This effect was more noticeable when cryosupernatant plasma was used in place of normal plasma as the source of vWf, and was not explicable simply in terms of antibody-induced cross-linking of vWf.

Animals↗
PubMed1985

Metabolism of sodium pentosan polysulphate in man measured by a new competitive binding assay for sulphated polysaccharides--comparison with effects upon anticoagulant activity, lipolysis and platelet alpha-granule proteins.

Three human volunteers were injected with a range of doses of pentosan polysulphate, SP54, i.v. or s.c. A competitive binding assay (CBA) for sulphated polysaccharides was used to detect circulating SP54 after doses as low as 1 mg i.v. and a linear relationship was observed between the peak plasma concentration of SP54 measured by CBA and the administered dose. A comparison was made between the clearance of SP54 measured by CBA and its anticoagulant and lipolytic activities. SP54 was detectable by CBA after doses which caused no alteration in activated partial thromboplastin time (APTT) or anti-factor Xa activity but after which a small increase of lipase activity was measurable. After SP54 at 10 mg i.v. or 100 mg s.c. anti-factor Xa activity was 4-6 times greater than would be expected from the in vitro activity of the concentrations of SP54 measured by CBA. Like heparin and other heparin analogues, SP54 caused an increase in plasma concentrations of platelet factor 4 (PF4) without a concomitant rise in beta-thromboglobulin (beta-TG). It is concluded that the newly developed CBA will provide a more sensitive means than conventional bioassays for the determination of plasma concentrations of SP54.

Adult↗
PubMed1985

Macroaggregate formation in optimal additive red cells.

The formation of macroaggregates weighing up to 9 g was observed in optimal additive red cells. Such aggregates, with a mean wet weight of 3 g, formed progressively during storage and were present in up to 85% of units. They were composed of leucocyte and platelet debris, together with some fibrin. Macroaggregates formation was halved by less stringent centrifugation during preparation and was reduced by use of an optimal additive system in which the additive solution contained citrate. Extra mixing during transfer of the additive solution only delayed aggregate formation. Partial leucocyte depletion or addition of 200,000 KIU of the enzyme inhibitor aprotinin did not prevent macroaggregate formation.

Blood Preservation↗
PubMed1984

Monoclonal antibodies directed against human alpha-thrombin and the thrombin-antithrombin III complex.

Human alpha-thrombin was poorly immunogenic in Balb/c mice. Nevertheless, following fusion of spleen cells from a responding mouse with NS-1 cells, 8 mouse monoclonal antibodies against alpha-thrombin were isolated, and 6 were characterised. Five of these were isotype IgG2a, and one was IgG1. One, EST 1, bound thrombin only minimally, and was directed against a neoantigen on the thrombin-ATIII (T-AT) complex. This antibody also recognised a site on prothrombin, though with much lower affinity. Its binding was markedly temperature-dependent, indicating a requirement for molecular mobility. A second antibody, EST 4, would not bind the T-AT complex. It inhibited both the clotting and amidase activities of thrombin, and modification of the active site histidine, but not the active site serine, reduced the affinity constant of binding to EST 4. This antibody appears to be directed against an epitope in the vicinity of the enzyme active site. The epitopes for EST 1 and EST 4 were both remote from those of the other monoclonal antibodies, EST 2, 6, 7 and 8. These four competed with each other for binding to thrombin, and all inhibited clotting but not amidase activity. Thrombin binding was not affected by modification of the active site, though formation of the T-AT complex reduced the affinity of binding to EST 6 and EST 8. These monoclonals recognise epitopes in the region of the fibrinogen binding site.

Animals↗