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Biomedical subjects

I R Peake

Publications and source records attributed to I R Peake.

At least 91 records · Page 5Linked to original sources
PubMed1983

The interaction between factor VIII clotting antigen (VIIICAg) and phospholipid.

The interaction between factor VIII clotting antigen (VIIICAg) and phospholipid (PL) was studied using a two-site solid phase immunoradiometric assay (IRMA) for VIII CAg. Incubation (2 h, 37 degrees C) of normal plasma, cryoprecipitate or factor VIII concentrate with Diagen PL (0.5 mg/unit VIIICAg) resulted in 80-90% loss of IRMA-measurable VIIICAg. No loss of factor VIII related antigen (VIIIRAg) or factor VIII clotting activity (VIIIC) was seen. Treatment of factor VIII concentrate with purified PLs showed greatest VIIICAg reduction with phosphatidylserine, less with phosphatidylethanolamine, and very little with phosphatidylcholine. The action of phospholipase-C (PL-C) on VIIICAg-PL complexes was investigated, with enzyme activity being monitored by thin-layer chromatography. Treatment of normal plasma, cryoprecipitate or factor VIII concentrate with PL-C (5 u/unit VIIICAg) resulted in 25%, 25% and 30% increases in VIIICAg. No increase in VIIIC or VIIIRAg was seen. Preincubation of factor VIII concentrate with PL, followed by PL-C incubation, resulted in 70-80% recovery of measurable VIIICAg. Incubation of 'activated' prothrombin complex with PL-C increased VIIICAg by 44% (Autoplex) and 80% (FEIBA), indicating VIIICAg-PL complexes are present. Incubation of factor VIII concentrate with fresh platelet lysate led to a reduction in VIIICAg (100 u/dl to 21 u/dl). In fresh platelet lysate alone low VIIICAg levels were detectable (0.71 X 10(-3) u/10(9) plt). After PL-C incubation VIIICAg levels increased to 9.8 X 10(-3) u/10(9) plt) (14-fold increase). Thus VIIICAg in platelets may be hidden by VIIICAg-PL complexes.

Antigens↗
PubMed1983

Studies on the stability of factor VIII coagulant antigen (VIII:CAg) in the presence of VIII:C antibodies.

The stability of factor VIII coagulant antigens (VIII:CAg) at 56 degrees C was investigated using an immunoradiometric assay for VIII:CAg. In normal or CRM+ haemophilic plasmas VIII:CAg was rapidly inactivated at 56 degrees C. VIII:CAg in spontaneous VIII:C inhibitor plasmas and in post-treatment samples from haemophiliacs with VIII:C inhibitor was resistant to inactivation at 56 degrees C, indicating the presence of heat stable VIII:CAg-anti VIII:CAg complexes. In vitro VIII:CAg-anti VIII:CAg complexes were formed by incubation of diluted VIII:C antibodies and normal plasma and the stability of these complexes at 56 degrees C was studied. Haemophilic VIII:CAg antibodies formed heat stable immune complexes over a narrow range of inhibitor dilutions whilst some spontaneous VIII:CAg antibodies formed these stable complexes over a much wider range of dilutions emphasizing the difference in the properties of these antibodies.

Antibodies↗
PubMed1983

An electroblotting technique for the detection of factor VIII/von Willebrand factor multimers in plasma.

A new, relatively simple technique has been developed in order to study the multimers of factor VIII/von Willebrand factor (VIII/vWF). It involves electrophoresis on SDS agarose gels and electrophoretic transfer (electroblotting) of the separated protein bands onto nitrocellulose membranes, to which they are non-covalently bound. VIII/vWF multimers are then detected by 125I-labelled antibodies to VIII/vWF, and autoradiography. Optimum electrophoretic transfer occurred at 0.5 A, for 18 h, on 0.8% agarose gels, thus enabling detection of the multimeric profile of VIII/vWF in 5 microliters of normal plasma. The multimeric profile for haemophilia A patients was identical to that for normal plasma. In plasma from patients with von Willebrand's disease (vWd), various patterns were seen, with a preponderance of smaller multimers in type II (atypical) vWd, similar to those seen in cryosupernatant. Heterogeneity within a particular type of vWd was also evident. Investigation of commercial factor VIII concentrates showed the presence of 'doublets' of VIII/vWF. Unlike other reported techniques, the rapid transfer and fixing of the protein bands to the nitrocellulose, minimizes loss of resolution, and handling of the paper is easier. It is possible to cut a sample electrophoresis strip into several areas, for incubation with different antibodies. Preliminary experiments also suggest that double antibody techniques are possible, or even removal of a first radiolabelled antibody by low pH, and then incubation of the separated proteins with a second, unrelated antibody.

Autoradiography↗
PubMed1981

Factor VIII clotting antigen (VIIICAg) in haemophilia measured by two immunoradiometric assays (IRMA) using different antibodies, and the measurement of inhibitors to procoagulant factor VIII (VIIIC) by IRMA.

Factor VIII clotting antigen (VIIICAg) was measured by immunoradiometric assay (IRMA) using two different antibodies. Both antibodies arose in polytransfused severe haemophiliacs and had similar titres against VIIIC. In 12 normal plasmas there was no significant difference in VIIICAg values obtained (VIIICAg (AbI) = VIIICAg (AbII)). In the majority of 15 severe haemophiliacs tested VIIICAg was undetectable by both antibodies. In 28 mild to moderate haemophiliacs VIIICAg (AbII) was significantly greater than VIIICAg (AbI) (P less than 0.01) suggesting different antigenic determinants. The difference, however, was small and does not affect diagnosis of haemophilia. A modified IRMA has been used to measure anti VIIIC inhibitors by competition of the inhibitor with 125I labelled VIIICAg antibodies for common antigenic determinants. Using an inhibitor of 225 Bethesda units as a standard, results by IRMA of inhibitors in severe haemophiliacs have been similar to those obtained by clotting assay, but with a sensitivity of 0.01 u/ml suggesting the possible use in the detection of weak inhibitors.

Antibodies↗
PubMed1981

Carrier detection in haemophilia a by immunological measurement of factor VIII related antigen (VIIIRAg) and factor VIII clotting antigen (VIIICAg).

23 obligate carriers of mild and severe haemophilia A and 26 normal females were bled on three occasions, and their plasmas assayed for procoagulant factor VIII (VIIIC), factor VIII related antigen (VIIIRAg) and factor VIII clotting antigen (VIIICAg). A comparison of the ratios VIIIC/VIIIRAg and VIIICAg/VIIIRAg indicated that, although the two ratios gave the same proportional misclassification of carriers as normals (four of 23), the latter ratio showed greater discriminatory power when an unequal variances predictive method was used to calculate likelihood ratios (for carrier status). This greater power was shown to be due to a greater reproducibility between visits for the VIIICAg/VIIIRAg ratio. Discrimination was considerably better when the median of the three median values for each variable was analysed, compared to the median value obtained at the first visit. There was also no statistical difference between VIIICAg/VIIIRAg (or VIIIC/VIIIRAg) ratios obtained from carriers of severe compared to mild haemophilia.

Antigens↗
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↗
PubMed1980

Dual diagnosis of prenatal haemophilia A by measurement of fetal factor VIIIC and VIIIC antigen (VIIICAg).

Procoagulant factor VIII (VIIIC) and procoagulant factor VIII antigen (VIIICAg) were measured in 20 samples of pure fetal blood obtained by fetoscopy at mid-term from non-haemophilic fetuses. VIIIC levels ranged from 25 to 89 U/dl (mean 43.5) and VIIICAg from 11 to 43 U/dl (mean 22.3). Samples from 39 consecutive fetuses at risk of haemophilia A gave results indicating haemophilia in 11, and these pregnancies were terminated at the parents' request. Of the remaining 28, 18 have resulted in normal births, 8 still await delivery, and 2 pregnancies were terminated for non-haematological reasons. The results confirm that pure fetal blood can be aspirated fetoscopically for plasma assays of both VIIIC and VIIICAg. Dual measurement enhances the reliability of predicting or excluding haemophilia A, and makes prenatal diagnosis a dependable option for known or potential carriers seeking this information.

Antigens↗
PubMed1980

Factor VII and fibrinolytic response to deamino-8-D-argenine vasopressin in normal subjects and dissociate response in some patients with haemophilia and von Willebrand's disease.

Deamino-8-D-argenine vasopressin (DDAVP) was given by intravenous infusion to normal subjects, haemophiliacs and patients with von Willebrand's disease (vWd) and the factor VIII and plasminogen activator response was studied. In normal subjects and most patients with mild haemophilia and mild (intermediate) von Willebrand's disease there was an increase in plasminogen activator and all factor VIII related activities. In patients with mild vWd the prolonged bleeding time was shortened by DDAVP despite only a modest rise in factor VIII related Ristocetin cofactor activity (VIIIR:RiCoF). Sub-groups of patients have been characterized in whom atypical responses was observed. In two brothers with clinically severe haemophilia, but with 5--6 u/dl procoagulant factor VIII (VIIIC), there was an increase in VIIIC but no rise of the corresponding antigen, suggesting increased release of an antigenically abnormal poorly functioning molecule. A patient with intermediate vWd was studied in whom neither DDAVP, adrenaline infusion, nor venous occlusion resulted in an increase in either plasminogen activator or factor VIII related antigen (VIIRAg), although there was a significant increase in VIIIC. In a further patient with severe vWd, DDAVP failed to elicit any plasminogen activator or VIII response. The results obtained from these two patients suggested that in some individuals the presumed endothelial cell abnormality in vWd may be more extensive than a defect in VIIIRAg synthesis. Sub-groups of patients have been identified for whom treatment with factor VIII concentrates would be more appropriate than DDAVP prior to minor surgery.

Adolescent↗
PubMed1980

Characterization of procoagulant activity produced by cultures of human monocytes and lymphocytes separated in colloidal silica-polvinylpyrrolidone gradients.

Human peripheral blood leucocytes were examined for their ability to synthesize procoagulant activity in tissue culture. A method was developed utilizing silica coated with polyvinylpyrrolidone (Percoll) to separate monocytes from lymphocytes prior to culture. Coagulation activity was demonstrated in all mononuclear cell cultures after 24 hours incubation. This activity was inhibited by phospholipase C which suggested that tissue thromboplastin was the major source of activity. Specific immunoradiometric assays failed to demonstrate synthesis of factor VIII related antigen (FVIIIRAG) or factor VIII coagulant antigen. The results indicate that under conditions of this study factor VIII was not synthesized in mononuclear cell cultures.

Cell Separation↗
PubMed1979

An immunoradiometric assay for procoagulant factor VIII antigen: results in haemophilia, von Willebrand's disease and fetal plasma and serum.

An immunoradiometric assay (IRMA) has been developed based on the inhibitor which arose in a polytransfused severe haemophiliac. The two-site IRMA measures antigens closely associated with the procoagulant parts of the factor VIII complex, which are termed FVIIC antigens or FVIIICAG. FVIIICAG was present in normal plasma and also, at a slightly lower concentration, in normal serum. In 37 patients with haemophilia A, 36 had FVIIICAG levels of less than 10% of the normal plasma pool. In patients with von Willebrand's disease the levels of FVIIIC and FVIIICAG were in good agreement, both before and after treatment with cryoprecipitate or DDAVP. FVIIICAG was relatively stable in plasma at 37 degrees C and could also be detected in cord and fetal serum. The assay is of potential value for detecting reduced levels of factor VIII, for carrier detection and for the prenatal diagnosis of haemophilia.

Antigens↗
PubMed1979

Synthesis of procoagulant factor VIII, factor VIII related antigen and other coagulation factors by the isolated perfused rat liver.

The synthesis of factor VIII and other coagulation factors has been studied using an isolated, perfused rat liver. Synthetic function of the liver was validated by adding [35S]L-methionine to the perfusion medium and performing two-dimensional crossed immunoelectrophoresis and autoradiography on samples obtained during perfusion. Progressive incorporation of radioactivity into plasma proteins was demonstrated. This was inhibited by cycloheximide. Coagulation factor assays demonstrated synthesis of factors II, IX and X and of factor V and procoagulant factor VIII (VIIIC). Synthesis of factor VIII related antigen (VIIIRAg), measured in an immunoradiometric assay, was not significantly demonstrated. Addition of warfarin to the perfusion medium inhibited the synthesis of factors II, IX and X but not of factors V and VIII. Cycloheximide completely inhibited synthesis of all coagulation factors but actinomycin acted only after a latent period. Reticuloendothelial cell blockade was attempted by adding ethionine to the perfusion medium or by administration of Indian ink to the donor animals prior to removal of the livers. In these instances synthesis of factor V and factor VIIIC was inhibited but not that of factors II, IX and X. The results confirmed the functional capacity of the isolated liver for synthesizing proteins and the vitamin K dependent coagulation factors, and suggested similar kinetic features for the synthesis of factors V and VIIIC. Failure to detect significant VIIIRAg synthesis in these experiments is consistent with the hypothesis that this protein is released by vascular endothelial cells throughout the body and is activated or joined to VIIIC or stimulates its production in the liver.

Animals↗
PubMed1978

Immunoradiometric assay of procoagulant factor-VIII antigen in plasma and serum and its reduction in haemophilia. Preliminary studies on adult and fetal blood.

An immunoradiometric assay (I.R.M.A.) for antihaemophilic factor (factor VIII) has been developed which is based on a human inhibitor to procoagulant factor VIII (FVIIIC) which arose in a polytransfused haemophiliac. The assay detected and measured the antigen related to FVIIIC (FVIIICAg) in normal plasma and serum but failed to detect any antigen in samples from five patients with severe haemophilia. In a sixth patient with severe haemophilia a non-parallel dose-response curve was obtained. FVIIICAg was also present in plasma and serum from six samples of cord blood and in two samples of fetal serum obtained after termination of pregnancy. The assay may, therefore, be used to measure FVIIICAg in small serum samples and provides a potential practical method for prenatal diagnosis of haemophilia.

Adult↗