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Biomedical subjects

D S Pepper

Publications and source records attributed to D S Pepper.

At least 55 records · Page 3Linked to original sources
PubMed1984

The interaction of platelet factor 4 with heparins of different chain length.

The interaction of platelet factor 4 with heparin of varying chain lengths has been investigated by labelling the heparin with fluorescein isothiocyanate and monitoring the change in anisotropy of fluorescence when the protein is added to a solution of the polysaccharide. The shape of the titration curve depends on the Mr of the heparin and chains of Mr greater than 10 000 showed a definite break when the concentration of polysaccharide and protein became equimolar. Evidence is presented to show that most of the fluorescein label is linked to residual serines on the heparin. Similar break-points were observed if total fluorescence or light-scattering was used to monitor the interaction. Unlabelled heparin was used for the latter method. These results together with those obtained in buffer of high ionic strength lead us to propose a model where the heparin is wrapped around the tetrameric protein.

Fluorescein-5-isothiocyanate↗
PubMed1983

The interaction between collagens and factor VIII/von Willebrand factor: investigation of the structural requirements for interaction.

The blood protein Factor VIII/von Willebrand factor (FVIII/VWF) has been shown to bind to a variety of collagen polymers including (i), the native-type fibres (of collagens types I and III), (ii), segment-long-spacing (SLS) aggregates (of collagens types I, III, IV and V), (iii), the insoluble polymer obtained by random cross-linking of the type I monomer and (iv), the non-striated fibril (of type I) produced by alcohol precipitation. Relatively little binding of FVIII/VWF to the amorphous, non-fibrillar form of collagen (type I) produced by salt precipitation from acid solution was observed. No significant binding either to elastin or to the insoluble polymer derived by random cross-linking of bovine serum albumin was noted. The absorption of FVIII/VWF to collagens was affected by ionic concentration and FVIII/VWF was only totally bound at relatively low ionic strength. Binding of radiolabelled FVIII/VWF could be largely inhibited by an excess of the unlabelled protein. The interaction of FVIII/VWF with collagen fibres was inhibited in a concentration-dependent manner by monomeric collagen when present at relatively high concentrations. Gelatin did not appear to inhibit binding significantly. The structural requirements of collagen for binding to occur appear to resemble those required for collagen-induced platelet aggregation in which collagen quaternary structure rather than collagen type per se is the important factor. Loss of secondary or higher orders of structure of FVIII/VWF as a result of heat denaturation or reduction of disulphide bonds decreased or prevented binding. In accord with the association of biological activity with FVIII/VWF aggregates, optimal binding appeared to require the presence of aggregated FVIII/VWF.

Binding, Competitive↗
PubMed1983

A radioimmunoassay for thrombospondin, used in a comparative study of thrombospondin, beta-thromboglobulin and platelet factor 4 in healthy volunteers.

A radioimmunoassay was developed for the platelet alpha-granule protein thrombospondin; concentrations of thrombospondin as low as 3 ng ml-1 could be measured. There was no interference from other components of human biological fluids and no crossreactivity with beta-thromboglobulin (beta-TG) or platelet factor 4 (PF4). Plasma samples were stable when stored at -20 degrees C. Normal human plasma contained 105.0 +/- 31.0 ng thrombospondin ml-1 compared with beta-TG concentrations of 37.2 +/- 10.9 ng ml-1 and PF4 concentrations of 14.7 +/- 10.1 ng ml-1 when samples were carefully taken into a platelet inhibitor cocktail and processed at 0-4 degrees C. Release of thrombospondin during clotting of blood occurred at the same time as that of beta-TG and PF4 and resulted in a serum concentration of 17.5 +/- 5.5 micrograms ml-1. Assay of whole blood gave a platelet thrombospondin content of 89.1 +/- 28.3 ng/10(6) platelets. The concentration in normal urine fluctuated widely from 3 to 22.5 ng ml-1, and was unrelated to urine flow. The half-life of thrombospondin in vivo was about 9 h, much longer than that of either beta-TG or PF4. Unlike PF4, it was not released into the blood following an intravenous heparin injection. Bovine, ovine, canine and porcine sera contained thrombospondin which crossreacted immunologically with the human molecule; these species would be suitable animal models for the study of thrombospondin and its value as a platelet release marker.

Adult↗
PubMed1983

The immunoelectronmicroscopic localization of human platelet factor 4 in tissue mast cells.

Initial immunohistochemical localization of human platelet factor 4 (PF4) in tissue mast cells suggested that the protein was present in the mast cell granule. It was proposed that this could reflect binding of PF4 to heparin or heparan sulphate, known granule constituents. We report here the confirmation of granule localization by an immunoelectron microscopical method. The possible role of such binding is unknown, but the potential for cationic proteins of platelet origin interacting with vessel wall constituents is discussed.

Histocytochemistry↗
PubMed1983

Immunocytochemical localization of fibrinogen, platelet factor 4, and beta thromboglobulin in thin frozen sections of human blood platelets.

Affinity-purified monospecific antibodies against human fibrinogen and the platelet-specific proteins platelet factor 4 and beta thromboglobulin were used to localize these antigens in thin and ultra-thin frozen sections of mildly fixed, washed human blood platelets. By immunofluorescent double-labeling experiments the distribution of fibrinogen was compared to that of platelet factor 4 and beta thromboglobulin. All three antigens occurred in virtually all platelets and showed and identical, dotlike distribution. For immunoelectron microscopy we used protein A-colloidal gold on ultra-thin frozen sections to visualize the specific reaction indirectly. The staining for platelet factor 4, beta thromboglobulin, and fibrinogen localized exclusively over alpha-granules of washed platelets. Within the granules, platelet factor 4 was localized preferentially in the electron dense, alpha-granule nucleoid, whereas fibrinogen was more predominant in the electron-lucent granule periphery. Beta thromboglobulin localization did not show a preferential intragranular distribution.

Antigens↗
PubMed1982

A sensitive competitive binding assay for exogenous and endogenous heparins.

A new type of assay for heparins has been devised, in which the test material competes with 125I-labelled heparin for binding to protamine-Sepharose. The assay is very sensitive and will measure heparin concentrations down to 10 ng ml-1. It responds to both the degree of sulphation and the molecular weight of acidic polysaccharides, but is independent of their biological activities. It can be used to quantitate heparins in biological fluids after pretreatment of the samples with protease. In this way endogenous heparins were measured in normal human serum, plasma and urine. The assay is extremely versatile and has great potential for the investigation of endogenous and exogenous heparins.

Binding, Competitive↗
PubMed1982

The in vivo release of human platelet factor 4 by heparin.

Intravenous and subcutaneous injection of heparin or the heparin analogue SSHA into normal volunteers induced release of platelet factor 4 (PF4) but not beta-thromboglobulin (beta-TG). At low heparin doses the amount of PF4 released was related to the plasma heparin concentration achieved. The rise in plasma PF4 was coincident with, and appeared to be a response to, the increase in plasma heparin concentration rather than to the absolute heparin level. After the primary response, the system became refractory to further challenge by the same heparin dose; the full initial magnitude of the response was not regained until 144 h. after heparin was first injected. The maximum amount of PF4 released corresponded to only about 5% of that potentially available from platelets. Moreover, heparin did not stimulate PF4 release from whole blood in vitro. We have demonstrated the presence of PF4 on the vascular endothelium, and suggest that this is the immediate source of the PF4 released by heparin, though it is probably initially derived from platelets. The effect of such binding on the antithrombotic potential of the endothelial surface is discussed.

Animals↗
PubMed1982

Production of direct agglutinins by the combination of incomplete antibodies to protein A-bearing Staphylococcus aureus cells.

A novel method is described for the production of saline-reacting antibodies from "incomplete' blood grouping sera. IgG anti-D is combined to protein A-bearing Staphylococcus aureus (SA), forming a polyvalent anti-D assembly. The SA-anti-D has a high titre in saline and is stable on 4 degrees C storage and lyophilisation. The material was successful in Rh(D) typing cells sensitised in vitro with various antibodies. Saline-reacting SA-anti-Kell and SA-anti-C were also produced.

Agglutinins↗
PubMed1982

Immunological localisation of beta-thromboglobulin and platelet factor 4 in human megakaryocytes and platelets.

Beta-thromboglobulin (beta TG) and platelet factor 4 (PF4) have been localised in megakaryocytes and platelets using immunofluorescence and immunoperoxidase techniques. These studies support the concept of synthesis of the proteins by parent megakaryocytes. By immunoelectron microscopy both proteins have been visualised in the alpha granule of the platelet and megakaryocyte, supporting functional studies of the cytoplasmic localisation of these proteins. The light microscopic techniques may allow elucidation of the distribution and role of the megakaryocyte in the pulmonary circulation and, on a practical level, permit its identification and distinction from other multinucleate cells in extramedullary tissue.

Beta-Globulins↗
PubMed1981

Studies of a fibrinolytic enzyme from the larvae of Lonomia achelous (Cramer) using chromogenic peptide substrates.

A fibrinolytic agent purified from the haemolymph, hair secretion and whole body extract of Lonomia achelous (Cramer) cleaves various chromogenic peptide substrates. The best substrate were found to be pyro-Glu-Gly-Arg-pNA (S-2444) followed by D-Pro-Phe-Arg-pNA (S-2302) and Bz-Ile-Glu-(or) Gly-Arg-pNA (S-2222) designed for urokinase, plasma kallikrein and factor Xa, respectively. Using substrate S-2251 we also found a plasminogen activator.

Animals↗
PubMed1981

The transfer of platelet factor 4 from its proteoglycan carrier to natural and synthetic polymers.

1. Transfer of dansyl-platelet factor 4 complexed with a series of glycosaminoglycans to heparin has been detected and studied by measuring changes in the anisotropy of the dansyl fluorescence. The protein was most easily transferred from chondroitin sulphate and least easily from heparan sulphate. 2. The transfer of the dye-labelled protein from its biological chondroitin 4-sulphate proteoglycan carrier to natural and synthetic anionic polymers was similarily followed. The transfer to heparin and dermatan sulphate was shown to be the same whether 3 mM Ca2+ or 8 mM EGTA was present in the solution. 3. The shapes of the binding curves of the dansyl-factor to the polymers have been compared at I = 0.4M. 4. The observed changes in anisotropy of dye fluorescence have been correlated with the charge density and the stereochemistry of the charged groups of the natural polymers. Large complexes are observed with polymers of high negative charge/weight ratios. Less charged polymers containing disaccharide units of iduronic acid and glucosamine N-sulphate will also from large complexes at I = 0.15 M. 5. It is demonstrated that the release of a platelet factor 4 proteoglycan complex in vivo would result in the transfer of the protein to heparin, moderate quantities of either dermatan or heparan sulphates would not prevent this transfer.

Blood Coagulation Factors↗
PubMed1981

Production of cryoprecipitate of intermediate purity in a closed system thaw-siphon process.

The thaw-siphon procedure for routine production of cryoprecipitate is described briefly, and also its extension to a reproducible procedure for production of cryoprecipitates of intermediate purity. For the latter, the average yield of factor VIII is 60% of that in the prefrozen plasma and approximately 30% is present in the supernatant plasma. The product contains 0.53 units of VIII: C per mg of total protein (of which about 70% is clottable). The mean value of the ratio VIII: C/VIII: RAG is about 0.7. The yield and purity of the product compare favourably with those reported for intermediate purity factor VIII concentrates produced by large-scale plasma fractionation laboratories. The procedure offers an alternative pathway to the production of factor VIII concentrate of intermediate purity and can be operated in any blood bank with a minimum of equipment.

Antigens↗
PubMed1981

Changes in factor VIII complex activities during the production of a clinical intermediate purity factor VIII concentrate.

Samples taken at various stages of preparation of intermediate purity factor VIII concentrate were assayed for factor VIII coagulant activity (VIII:C), factor VIII coagulant antigen (VIIIC:Ag) and factor VIII related antigen (VIIIR:Ag). The antigen results were used to assess mechanical loss during fractionation as these markers are relatively stable. In contrast VIII:C is sensitive to both mechanical and inactivation losses. The major loss of factor VIII occurred during the cryoprecipitation and extraction step and was due to both mechanical loss and inactivation. Losses before and after this step were largely due to inactivation of the factor VIII. Assay of VIIIR:Ag in concentrates presented problems and a modified technique is suggested.

Antigens↗
PubMed1980

The multiple complexes formed by the interaction of platelet factor 4 with heparin.

The anisotropy of the fluorescence of dansyl (5-dimethylaminonaphthalene-1- sulphonyl) groups covalently attached to human platelet factor 4 was used to detect the macromolecular compounds formed when the factor was mixed with heparin. At low heparin/protein ratios a very-high-molecular-weight compound (1) was formed that dissociated to give a smaller compound (2) when excess heparin was added. 2. A large complex was also detected as a precipitate that formed at high protein concentrations in chloride buffer. It contained 15.7% (w/w) polysaccharide, equivalent to four or five heparin tetrasaccharide units per protein tetramer. In this complex, more than one molecule of protein binds to each heparin molecule of molecular weight greater than about 6 X 10(3).3. The stability of these complexes varied with pH, salt concentration and the chain length of the heparin. The limit complexes found in excess of the larger heparins consisted of only one heparin molecule per protein tetramer, and the failure to observe complexes with four heparin molecules/protein tetramer is discussed.

Blood Coagulation Factors↗