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Biomedical subjects

D S Pepper

Publications and source records attributed to D S Pepper.

At least 19 recordsLinked to original sources
PubMed1992

Human vascular endothelial cells catabolise exogenous glycosaminoglycans by a novel route.

Heparin and other anticoagulant glycosaminoglycans were radiolabelled with 125I and their catabolism by human vascular endothelial cells in culture was studied. Heparin, heparan sulphate and pentosan polysulphate were associated with the cellular fraction and incorporated into the subendothelial matrix, but dermatan sulphate was not found in either fraction. High molecular weight, fully desulphated carbohydrate chains were major catabolic products of all those glycosaminoglycans which were taken up by the cells. Pentosan polysulphate was not degraded further, but the catabolism of heparan sulphate, and to a lesser extent that of heparin, also yielded small oligosaccharides. Thus the first step in catabolism of exogenous glycosaminoglycans by human vascular endothelial cells appears to be complete desulphation, which destroys their biological activity, followed by depolymerisation of the carbohydrate chain. This alternative to the sequential action of lysosomal exoenzymes is dependent upon binding to the cell; thus dermatan sulphate, which is not associated with the cellular fraction, is not catabolised.

Binding Sites↗
PubMed1992

Direct dye binding--a quantitative assay for solid-phase immobilized protein.

A direct dye-binding procedure was established for the quantification of protein after its immobilization on a solid phase, using IgG and BSA as model proteins. The assay, which in the range 0-5 mg protein/ml gel correlates well with indirect protein determination by A280 as well as determination of protein hydrolyzed from the gel, is based on a modified Bradford dye-binding assay. As the protein coupled to the gel binds the dye, a decrease in A465 of the supernatant is measured. Three solid supports commonly used for protein immobilization (Sepharose, Sephadex, Sephacryl) were found to be compatible with the dye-binding assay while nonspecific dye binding was found to HEMA gels. Protein was coupled to Sephacryl S-1000 using three different activation methods (aldehyde, hydrazine, and adipic acid dihydrazide). Artifactual dye-binding was not observed using any of the three different "linkers." The assay is easily carried out and represents a useful tool, e.g., when optimizing procedures for protein immobilization.

Coloring Agents↗
PubMed1992

Inhibition of thrombin generation by heparin and low molecular weight (LMW) heparins in the absence and presence of platelet factor 4 (PF4).

The ability of several low molecular weight (LMW) heparins and unfractionated heparin (UFH) to inhibit thrombin generation, and their anti-Xa and anti-IIa activities, were measured in the absence and presence of platelet factor 4 (PF4). The LMW heparins studied were 2-5 times less potent, on a weight basis, than UFH as inhibitors of thrombin generation in platelet-poor plasma; the inhibition of thrombin generation by LMW heparins correlated better with their anti-IIa activity (r = 0.98) than with their anti-Xa activity (r = 0.69). At low concentrations of PF4, the activity of LMW heparins in the thrombin generation test was neutralized less than that of UFH, but at higher PF4 concentrations all their activities could be neutralized except in anti-Xa assays. These observations support the hypothesis that anti-IIa activity is important for inhibition of thrombin generation by LMW heparins in vitro. However, when all the anti-IIa activity of LMW heparins was neutralized by PF4, considerable inhibitory activity remained in thrombin generation and anti-Xa assays, indicating that a portion of the anti-Xa activity of LMW heparins also contributes towards inhibition of thrombin generation.

Factor Xa Inhibitors↗
PubMed1992

Development, optimization and use of an enzyme linked immunosorbent assay (ELISA) to measure factor VIII antigen utilizing monoclonal antibodies.

An enzyme linked immunosorbent assay (ELISA) has been developed to measure VIII:Ag in plasma and concentrates. The assay utilizes two commercially available monoclonal antibodies to VIII:Ag and provides an alternative to the established immunoradiometric assay (IRMA). It has the advantage of not requiring the use of radioactive material and human antibodies. The assay sensitivity is 0.006 u/ml and the interassay coefficient of variation is 6.3%. Forty-eight samples with VIII:Ag levels ranging from 0.006 to 1.5 u/ml were assayed by both ELISA and IRMA. The coefficient of correlation between the two assays was 0.89. In addition to measuring human VIII:Ag, it is also possible to detect antigen in several animal plasma and sera.

Antibodies, Monoclonal↗
PubMed1978

Complete covalent structure of human beta-thromboglobulin.

The complete primary structure of the platelet-specific protein human beta-thromboglobulin has been determined. beta-Thromboglobulin consists of identical subunits of 81 amino acids, each with a molecular weight of 8851. The amino acid sequence of the beta-thromboglobulin subunit is: Gly-Lys-Glu-Glu-Ser-Leu-Asp-Ser-Asp-Leu-Tyr-Ala-Glu-Leu-Arg-Cys-Met-Cys-Ile-Lys-Thr-Thr-Ser-Gly-Ile-His-Pro-Lys-Asn-Ile-Gln-Ser-Leu-Glu-Val-Ile-Gly-Lys-Gly-Thr-His-Cys-Asn-Gln-Val-Glu-Val-Ile-Ala-Thr-Leu-Lys-Asp-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Pro-Asp-Ala-Pro-Arg-Ile-Lys-Lys-Ile-Val-Gln-Lys-Lys-Leu-Ala-Gly-Asp-Glu-Ser-Ala-Asp. Disulfide bridge-18 to half-cystine-58. The amino acid sequence of beta-thromboglobulin shows a marked homology with that of platelet factor 4. When the sequences are aligned for maximum homology, 42 of the 81 residues of beta-thromboglobulin are identical with those of platelet factor 4, including the position of the four half-cystines.

Amino Acid Sequence↗
PubMed1978

beta-thromboglobulin and deep vein thrombosis.

The measurement of plasma beta-thromboglobulin as a potential diagnostic test for venous thrombosis has been investigated in 16 normal volunteers, 24 patients presenting with deep vein thrombosis (DVT) or pulmonary embolism and 46 patients screened by 125I fibrinogen test (IFT) for post-operative DVT. The normal mean was 33 ng/ml (range 15-117 ng/ml). Of the 24 patients with clinical thrombotic disease 22 presented with DVT confirmed by phlebogram or IFT and 2 presented with embolism confirmed by lung scan. At the time of first presentation 12 out of 24 had betaTG values greater than 70 ng/ml. All except 3 of this group of 24 patients had values of greater than 70 ng/ml at some stage during a subsequent week of daily sampling. DVT was detected in 13 out of 46 screened post-operative patients. There was a rise om betaTG observed within 24 hr of the IFT becoming positive but the mean rise did not reach significance at the 5% level. An association between DVT and high betaTG values has been confirmed. However, its clinical value cannot yet be fully elucidated until factors, probably related to blood sampling and clearance, are further investigated.

Beta-Globulins↗
PubMed1977

The different forms of antithrombin II in serum.

Antithrombin III (AT III) complexes were isolated from human serum by affinity chromatography and gel filtration. In the first step of the preparation, using heparin-agarose chromatography, we observed that the complexed form of AT III bound less strongly to the gel than the free form and that about half of the AT III was free. With further purification a 2.5 X 10(5) molecular weight complex was isolated. Using 125I labelled human thrombin, this complex was radioactive indicating the presence of thrombin. Only in a synthetic thrombin-AT III system was a 9 X 10(4) molecular weight complex detected, but not in serum. These facts suggest that in serum AT III complexes may exist in a polymeric form. Also, an AT III antigen derived from the original AT III molecule, but not complexed, was isolated which may be a degradation product.

Antigens↗
PubMed1977

Spectrophotometric determination of factor Xa generation in factor IX concentrates.

Previous work from this department, concerned with testing the potential thrombogenicity of therapeutic factor IX concentrates, demonstrated that following recalcification of factor IX concentrates thrombin was generated within 3--30 minutes of incubation (Sas el al. 1975). The test developed (known as the TGt 50 test) is a two-stage assay and was thus found to be time consuming, tedious and tended to become inaccurate with long incubation periods and a large number of samples. A semiautomatic version of the test is reported in which the synthetic peptide Bz-ILE-GLU-GLY-ARG-pNA (S-2222) is added to recalcified, diluted factor IX concentrate in the micro-cuvette of a multiple sample recording spectrophotometer. Information can be obtained on (a) the amount of Xa (if any) present prior to recalcification (b) the initial amount of Xa formed and (c) the time taken to activate all factor =X to Xa. Direct graphical interpretation shows a number of qualitative differences between commercial preparations, but by either of the criteria (b) or (c) above, it is possible to place the different products into "activated" and "non activated" groups such that both the Xa generation times and TGt 50 tests identify the same two groups of products. This aggreement also indicates that the TGt 50 test is independent of the intrinsic factor V levels in the various concentrates.

Factor IX↗
PubMed1977

Half-life of platelet factor 4 (PF-4) in plasma and platelets from macaca mulatta.

Human platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipitation from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Animals↗
PubMed1977

Plasma beta-thromboglobulin in diabetes mellitus.

The plasma beta-thromboglobulin (betaTG) content was measured in 56 diabetic patients with known complications of this disease, including neuropathy, retinopathy, and ischemic skin lesions. Although two patients were found to have elevated levels beyond the normal range, there was no significant difference between the diabetic group as a whole and the group of 35 controls. The significance of these findings with regard to the proposed contribution of small-vessel platelet sequestration in the pathogenesis of late complications of diabetes mellitus is discussed.

Beta-Globulins↗