Elizabeth Thompson

Methylenetetrahydrofolate Reductase 677 C→T Mutation and Coronary Heart Disease Risk in UK Indian Asians

Plasma homocysteine concentrations are elevated in UK Indian Asians and may contribute to twice as many coronary heart disease (CHD) deaths in this group compared with European whites. The mechanisms underlying elevated homocysteine concentrations among Indian Asians are not well understood. In this study, we have investigated the extent to which the methylenetetrahydrofolate reductase (MTHFR) 677 C →T mutation accounts for elevated plasma homocysteine and increased CHD risk in Indian Asians compared with European whites. We investigated 454 male cases (with myocardial infarction or angiographically proven CHD: 224 Indian Asians, 230 European whites) and 805 healthy male controls (381 Indian Asians, 424 European whites). Fasting homocysteine concentrations, MTHFR 677 C →T genotype, and conventional CHD risk factors were measured. The prevalence of homozygous MTHFR 677 T in Indian Asian controls was less than one third that in European white controls (3.1% versus 9.7%, P <0.001). In Indian Asians, the TT MTHFR genotype was not associated with homocysteine concentrations and was not present in any of the Asian controls with hyperhomocysteinemia (>15 &mgr;mol/L). In contrast, among European whites, the TT MTHFR genotype was strongly related to elevated plasma homocysteine concentrations and was found in 27% of the European controls with hyperhomocysteinemia. Elevated homocysteine in Indian Asian compared with European white controls was accounted for by their reduced levels of B vitamins but not by the MTHFR 677 T genotype. However, neither the TT MTHFR genotype nor B vitamin levels explained the elevated homocysteine concentrations in CHD cases compared with controls. TT MTHFR was not a risk factor for early-onset CHD in Indian Asians (odds ratio, 0.5; 95% confidence interval, 0.1 to 2.4;P =0.39), unlike in European whites (odds ratio, 2.1; 95% confidence interval, 1.1 to 4.1;P =0.02). We conclude that the MTHFR 677 T mutation does not contribute to elevated plasma homocysteine concentrations or increased CHD risk in Indian Asians compared with European whites. Our results suggest that novel genetic defects and/or environmental factors influence homocysteine metabolism in Indian Asians residing in the United Kingdom.

Antithrombin Glasgow II: alanine 382 to threonine mutation in the serpin P12 position, resulting in a substrate reaction with thrombin

A female with recurrent thrombosis was found to have a functional abnormality of antithrombin, with a ratio of functional to immunological activity in plasma of ∼ 50%. Crossed immunoelectrophoresis in the presence of heparin was normal, indicating an abnormality of the reactive site, rather than the heparin binding domain. Accordingly, the antithrombin was isolated by heparin‐Sepharose chromatography: this produced a mixture of normal and variant antithrombin, as the patient was heterozygous for the abnormality. To remove the normal component, the antithrombin was passed through a column of thrombin‐Sepharose. On sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE), prior to its application to thrombin‐Sepharose, the antithrombin migrated as a single band with identical mobility to that of normal antithrombin. After thrombin‐Sepharose, the purified variant component was proteolysed, and migrated as two components, one with a reduced and one with enhanced mobility under non‐reducing conditions. This demonstrated that the variant was unable to form stable inhibitor‐thrombin complexes and was cleaved in a substrate reaction with thrombin. One site of cleavage was unambiguously ascertained to be the Arg 393‐ Ser 394 reactive site bond, by NH2 terminal sequencing of the cleaved variant antithrombin: 10 steps beginning at the P 1’position, Ser‐Leu‐Asn‐Pro‐Asn‐Arg…., were clearly identified.

Delayed release of an abnormal fibrinopeptide A from fibrinogen Manchester: effect of the Aα 16 Arg → His substitution upon fibrin monomer polymerization and the immunological crossreactivity of the peptide

Summary. Fibrinogen Manchester is an abnormal fibrinogen with an impaired release of fibrinopeptide A (FPA) and a polymerization abnormality. In the accompanying article we have identified the amino acid substitution in fibrinogen Manchester as Aα 16 Arg → His. When fibrinogen Manchester was digested with low thrombin concentrations approximately 40–50% of the total FPA content was released at a rate similar to FPA release from normal fibrinogen. The fibrin so formed exhibited an impaired polymerization of monomers. Digestion of fibrinogen Manchester with high concentrations of thrombin for prolonged times released the remaining FPA which had an abnormal retention time when studied by high performance liquid chromatography (HPLC). This fibrinopeptide has been shown previously to contain the Aα 16 Arg → His substitution. Fibrin resulting from this exhaustive digestion had normal polymerization of monomers. The normal and substituted FPAs were isolated by HPLC and compared in a double antibody competitive‐binding assay for normal FPA. The immunological cross‐reactivity of the abnormal peptide was reduced, so that approximately 5 times more abnormal peptide was required on a molar basis to displace labelled normal FPA. Normal intact fibrinogen was 10–fold less reactive (on a half molar basis) than free normal FPA and the crossreactivity of fibrinogen Manchester was measurably less than that of normal fibrinogen. It is concluded that immunological measurement alone of FPA released from abnormal fibrinogens may not give a complete description of the kinetics of peptide release if the amino acid substitution lies within the FPA sequence. The combination of radioimmunoassay and HPLC, however, provides a powerful analytical approach that should be useful in classifying and characterizing abnormal fibrinogens.

Relationship between ex vivo anti-proteinase (factor Xa and thrombin) assays and in vivo anticoagulant effect of very low molecular weight heparin, CY222

There is uncertainty as to which activities of unfractionated heparin (UFH) and low MW heparin are responsible for their anticoagulant and antithrombotic properties. We have sought to answer this question by examining plasma samples taken during a recently conducted dose‐finding study of the low MW heparin, CY222, in haemodialysis for chronic renal failure. In this study, in vivo anticoagulant effect was assessed by measurement of plasma FPA levels. UFH was administered as a dose of 5000 iu bolus + 1500 iu/h maintenance infusion, while the effects of three doses of CY222 were studied (10000, 15000 and 20000 Institute Choay anti‐factor Xa u bolus, all with 1500 Institute Choay anti‐factor Xa u/h maintenance infusion). Anti‐factor Xa levels were determined by chromogenic substrate assay. Anti‐thrombin levels were determined by chromogenic substrate assay and by quantitation of catalysed thrombin–inhibitor complexes (using autoradiography). Analysis of the results indicate that plasma fibrinopeptide A (FPA) levels correlate with anti‐factor Xa (r= 0.45) and anti‐thrombin (substrate) (r= ‐0.63) levels of UFH, but only with the anti‐factor Xa levels (r= ‐0.41) of CY222. These results suggest that the anti‐factor Xa assay is currently the most suitable assay for monitoring low MW heparins such as CY222 in humans.

The in vitro Effect of Phytohaemagglutinin on Separated Human Bone Marrow Cells

Cells from 60 marrow aspirates were separated using columns of glass microspherules. The effect of PHA on the viability and synthesis of DNA and RNA by these cells was observed after 3 days growth. The synthesis of DNA and RNA was observed before and after culture by means of autoradiographs prepared after exposure to 3H thymidine or 3H uridine. 3H thymidine uptake was low in the initial suspensions and was not increased by the addition of PHA, 3H uridine uptake was increased in only four of 41 cultures stimulated with PHA, and there was a reduction in the number of viable cells in cultures containing PHA.

The effect of phytohaemagglutinin on human foetal cells grown in culture.

Cell suspensions of foetal thymus, liver, spleen and bone marrow have been grown in cell culture and the effect of adding PHA observed. 3H thymidine uptake by the cells after exposure for a measured time was quantitated by autoradiographic counts performed on the initial suspensions and following culture. The initial unstimulated liver and thymic cell suspensions had a high level of 3H thymidine uptake, while that of the spleen and bone marrow cells was low. After 3 days culture the unstimulated cells from the liver, thymus and bone marrow showed very little 3H thymidine uptake, while that of the spleen was slightly increased. Foetal thymic and splenic cells responded to PHA while liver and bone marrow cells did not. The findings suggest that foetal cells of similar morphology may have different origins in the reticulo‐endothelial system.

Direct analysis of plasma fibrinogen‐derived fibrinopeptides by high‐performance liquid chromatography: investigation of nine congenital fibrinogen abnormalities

Summary. A simple method has been developed for the rapid analysis of fibrinopeptides contained on fibrinogen in small anticoagulated plasma samples. Following incubation with thrombin the plasma is diluted, boiled and then studied by high performance liquid chromatography (HPLC). The three forms of FPA (AP, A, AY) and two forms of FPB (B, des Arg B) can be identified and quantified in samples of less than 200 μl. Additionally, the FPB peak height can be used to measure the plasma fibrinogen level. This method has been used to screen plasma samples with abnormal clotting times for possible congenital fibrinogen abnormalities. Results of the study of nine unrelated cases are presented. Four cases of congenital dysfibrinogenaemia were diagnosed directly from HPLC analysis alone. Fibrinogen Sheffield and Paris VI were identified as AαArgl6→His substitutions and fibrinogens London VI and Madrid II were found to be heterozygous for an unknown substitution preventing thrombin cleavage at AαArg16. A case of dysfibrinogenaemia (fibrinogen Ashford) with a normal fibrinopeptide release stoichiometry was confirmed to have a primary polymerization abnormality using purified fibrin monomers. Similarly, a case of hypodysfibrinogenaemia (fibrinogen London V) had normal fibrinopeptides and a fibrin polymerization abnormality. In one case of hypofibrinogenaemia and two cases of afibrinogenaemia, no fibrinopeptide or functional abnormalities could be definitely established. This rapid and simple method of fibrinopeptide analysis is recommended for screening of plasma samples taken from patients suspected of having abnormalities of fibrinogen synthesis.

POSSIBLE CELL RECRUITMENT IN HUMAN LEUKÆMIA

Abstract Nuclear material prepared from peripheral-blood leucocytes and marrow cells of eleven patients with acute leukaemia either stimulated or repressed D.N.A. synthesis in cells from the same person. Such responses did not occur with cells from patients without leukaemia. It is suggested that the cells which respond are immunocompetent lymphocytes or stem cells and the observed differences in reactivity are probably related to different stages of the disease process. Marrow nuclei invariably stimulated D.N.A. synthesis by marrow cells, and a similar process of recruitment may be responsible for leukaemic cell proliferation in vivo. Factors released by fragmenting leukaemic blast-cells may cause other normal leucocytes to develop leukaemic characteristics.

Experimental evidence for renal catabolism of fibrin fragment β15–42

The elimination of labelled human fibrin fragment beta 15-42 from the circulation has been studied in an experimental rat model. When 125I beta 15-42 was injected as a bolus into a control group of rats, its elimination from the circulation could be fitted to a two compartment exponential model. A fast initial elimination had a t1/2 of less than 2 min and this was followed by a slower component with a calculated t1/2 of 135 min. The fast component was caused by equilibration of peptide throughout the intra and extra vascular spaces and the slow component reflected the action of catabolic processes once equilibration had been attained. Rats that had undergone bilateral nephrectomy eliminated significantly less of the labelled peptide than the control animals at a given time and the t1/2 of the slow component was prolonged, but not significantly, to 159 min. Rats with ligated ureters had statistically indistinguishable elimination curves from control rats. Examination of the heterogeneity of labelled peptide in plasma samples taken during the experiments by immunoprecipitation and by gel filtration revealed progressive extensive degradation in the control and ureteral ligated rats, but less degradation in nephrectomized rats. These results suggest that beta 15-42 is eliminated, in part, from the circulation by uptake and catabolism by the kidney. It is concluded that impaired renal function may results in elevated plasma levels of beta 15-42 antigen in human renal failure without the need for an increased rate of production of the peptide.