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Biomedical subjects

E A Beck

Publications and source records attributed to E A Beck.

At least 55 records · Page 3Linked to original sources
PubMed1981

Fractionation of individual, biologically active factor VIII multimers.

We have designed an electrophoretic system for the fractionation of individual, biologically active multimers of factor VIII. Human factor VIII, purified by gel filtration on Sepharose CL-2B from plasma cryoprecipitate, was submitted to electrophoresis without SDS on 2.0% polyacrylamide gels in 0.04 M Tris/0.06 M Tes buffer, pH 7.5. Staining with Coomassie blue revealed a series of protein bands. Measurement of electrophoretic mobility showed constant size intervals between adjacent bands. Electrophoresis in a second dimension, in the presence of SDS, resulted in an identical order of mobilities, suggesting that the different migration rates of factor VIII proteins in the first electrophoretic system were size- and not charge-dependent. After electrophoresis in the absence of SDS both factor VIII coagulant and ristocetin cofactor activities as well as factor VIII-related antigen were recovered by elution from gel slices. The distribution of activity peaks resembled that of Coomassie-stained factor VIII proteins found in control gels. We thus demonstrate that an electrophoretic fractionation of factor VIII multimers is possible even at neutral pH where factor VIII activities are retained.

Antigens↗
PubMed1981

Binding of bovine factor VIII-coated colloidal gold particles to receptors on platelet membranes.

Bovine factor VIII/platelet aggregating factor was adsorbed into gold granules and the protein-gold complex added to either formalin-fixed or fresh washed human platelets. Following aggregation, binding of gold granules to the platelets was measured by monitoring the optical density of colloidal gold remaining in the supernatant. Scatchard analysis of binding data indicated that multiple classes of binding sites were present. The number of high affinity binding sites per formalin-fixed platelet depended on the concentration of ristocetin: 420 gold granules were calculated to bind at 1.4 mg/ml of ristocetin, 610 at 0.6 mg/ml of ristocetin and 875 when no ristocetin was added. Fresh washed platelets bound 1350 granules per cell in the absence of ristocetin. We conclude that during platelet aggregation, induced by bovine factor VIII, the binding sites on the platelet surface are only partially occupied.

Animals↗
PubMed1981

Binding of colloidal gold granules, coated with bovine factor VIII, to human platelet membranes.

Platelet aggregating factor (PAF) activity is preferentially associated with the larger molecular forms of bovine factor VIII and diminishes considerably after dissociation into smaller oligomers by mild disulphide reduction. PAF activity was regained by the binding of small factor VIII oligomers to colloidal gold granules with a mean diameter of 23 nm. In contrast, adsorption of large factor VIII polymers onto gold particles resulted in partial loss of PAF activity. Thus, an optimum multimeric size of bovine factor VIII appears to be required for the maximal expression of PAF activity. Gold granules, coated with reduced factor VIII, can be used as an electron-dense label. Their interaction with surface receptors for bovine factor VIII on either viable or formalin-fixed human platelets was demonstrated by transmission and scanning electron microscopy.

Animals↗
PubMed1981

Factor VIII activity in chronic renal disease.

F VIII coagulant, F VIII-related antigen and F VIII ristocetin cofactor activity were significantly increased in 68 patients with various chronic renal diseases. All three F VIII functions correlated generally well with each other. A striking relationship between some F VIII activities and serum creatinine was detectable in patients with glomerulonephritis and kidney transplants, with mild or moderate renal insufficiency. This correlation was no longer present in terminal renal failure. The results suggest that in initial stages of renal disease elevated F VIII levels may be attributable to glomerular endothelial damage. In terminal renal failure, however, increased F VIII concentrations seem to result from nonspecific causes related to uremia such as acute phase reactions.

Adolescent↗
PubMed1980

[Increased von Willebrand activity of low molecular-weight factor VIII following binding to gold granules].

High molecular weight factor VIII was partially reduced with beta-mercaptoethanol. Disulfide reduction resulted in progressive dissociation of the multimeric protein with a concomitant decrease in von Willebrand activity. The binding of reduced small factor VIII oligomers to gold particles with an average diameter of 20--25 nm led to marked "activation" of their von Willebrand activity. A similar increase in activity was also observed following adsorption of native small molecular weight forms of factor VIII to gold granules. Binding of initially highly active high molecular weight factor VIII to gold granules resulted in inhibition of von Willebrand activity, probably by formation of superaggregates. The platelet aggregating function of factor VIII in the circulation appears to depend largely on the size of this multimeric protein, which must be in the range of several millions for its maximum expression.

Adsorption↗
PubMed1980

Cryoprotection of human bone marrow committed stem cells (CFU-c) by dextran, glycerol and dimethyl sulfoxide.

Dextran, glycerol and dimethyl sulfoxide (DMSO), alone or in combination, were used for cryoprotection of human bone marrow cells. The viability of cryopreserved cells was assessed by culture of myelopoiesis-committed stem cells (CFU-c) in vitro. A significantly better protection against freezing injury was obtained by 9% dextran in combination with 3 or 5% DMSO, and also with 5 or 10% DMSO alone, than with either 15% glycerol or 9% dextran with 1% DMSO.

Animals↗
PubMed1980

[Normal reference values in frequently carried out laboratory analyses hematologic cytology and blood coagulation)].

In 310 individuals (165 men and 145 women) significant pathological processes were exluded by reference to the patient's history, by physical investigation and by detailed laboratory tests (clinical chemistry, hematological cytology and most important coagulation analyses). Hematological laboratory values were then evaluated for age and sex differences, and in women according to menstruation versus post-menopause an oral contraception. Only the well known sex-specific differences in hematocrit values, hemoglobin and red cell counts were statistically highly significant (Z > 10). All other hematological values were pooled after exclusion of methodological errors. According to the non-symmetrical distribution of hematological laboratory values, percentiles were calculated. The 2.5 to 97.5 percentiles thus established now serve as "normal hematological reference values" for our laboratory.

Adult↗
PubMed1980

Studies on factor VIII-related protein. IV. Interaction of galactose-specific lectins with human factor VIII/Von Willebrand factor.

Factor VIII of human cryoprecipitate was purified and fractionated on Sepharose CL-2B into three fractions of progressively decreasing multimer size and ristocetin cofactor activity. Following complete disulfide reduction, the resulting subunits were electrophoresed on 3% polyacrylamide gels and subsequently stained with two galactose-specific, fluorescein-labelled lectins from Ricinus communis (RCAI and RCAII). Measurements of fluorescence indicated that the reduced chains, derived from the largest factor VIII multimers, have stronger binding affinity for both lectins than those obtained after reduction of smaller factor VIII species. Ristocetin cofactor activity of purified factor VIII was competitively inhibited by both Ricinus lectins and by concanavalin A. RCAI-lectin was found to be a considerably more efficient inhibitor than RCAII or concanavalin A. Following removal of sialic acid from factor VIII, the inhibiting effect of RCAII-lectin was markedly potentiated, probably by exposing additional galactose residues, some of which must be located close to the ristocetin cofactor 'active site' of factor VIII. Ristocetin cofactor activity was also strongly inhibited with RCAII-lectin for binding sites which are located on the surface factor VIII multimers. Our results suggest that RCAI-lectin, which contains galactose-specific binding sites per molecule, and anti-factor VIII antibodies inhibit ristocetin cofactor activity by crosslinking and aggregation of factor VIII multimers.

Antibodies↗
PubMed1980

Effects of low-dose 'factor VIII inhibitor bypassing activity (FEIBA)' in resistant haemophilia.

Possible effects and side-effects of 'factor VIII inhibitor bypassing activity (FEIBA)' were tested on 7 haemophiliacs with high-titre antibody to factor VIII (resistant haemophilia). FEIBA was administered both to hospitalised patients and as part of a home therapy programme. Serious side-effects, attributable to FEIBA, were one episode of hypersensitivity and, possibly, hepatitis. Our preliminary data suggest that early injection of FEIBA, using considerably lower doses than suggested by the manufacturer, may shorten the duration of immobilisation of haemophiliacs with inhibitor against factor VIII in comparison with infusion of factor VIII and/or supportive care alone, at least following musculoskeletal bleeding. A decrease or complete disappearance of the inhibitor was observed in all patients receiving FEIBA alone.

Adult↗
PubMed1980

Idiopathic thrombocytopenia with giant platelets.

Long-lasting, possibly constitutional thrombocytopenia, characterized by the presence of giant platelets, was found in a young woman. Conventional methods underestimated the number of circulating platelets and yielded erroneously elevated white cell counts. Platelet dysfunction was suggested by mild bleeding, prolongation of the Ivy bleeding time, and impaired platelet aggregation by collagen, adenosine diphosphate and adrenaline. The reported data suggest that this patient has a unique abnormality of platelet production.

Adult↗
PubMed1979

[Is there a correlation between sugar content, thrombocyte aggregating activity and molecular size of factor VIII?].

Human and bovine factor VIII, isolated from cryoprecipitates of fresh plasma by gel filtration on Sepharose CL-2B, gave similar elution patterns and showed comparable distribution of oligomers on SDS agarose electrophoretic gels. The carbohydrate content of individual factor VIII bands, measured by reaction with dansyl hydrazine or binding of glucose/mannose specific concanavalin A, was not directly related to the size or von Willebrand activity of factor VIII oligomers. Staining of disulfide-reduced factor VIII subunits, in polyacrylamide gels, with galactose-specific fluorescein-labelled Ricinus communis lectins, showed an increased binding affinity with increasing size and von Willebrand activity of the parent factor VIII. The von Willebrand activity was strongly inhibited by reaction with Ricinus RCAI lectin, whereas concanavalin A inhibited platelet aggregation only at concentrations above 1 mg/ml. These results suggest that galactose residues are involved in the aggregation of platelets by factor VIII.

Animals↗
PubMed1979

Studies on factor VIII-related protein. III. Size distribution and carbohydrate content of human and bovine factor VIII.

Human and bovine factor VIII were isolated from cryoprecipitate of fresh frozen plasma by gel filtration on Sepharose CL-2B. The elution diagrams and SDS-agarose electrophoretic analysis of eluted fractions show no significant differences in the size-distribution of factor VIII aggregates between the two species. Agarose gels were stained for carbohydrate by two methods: (1) the dansyl hydrazine reaction following oxidation with periodic acid and (2) staining with fluorescein-labeled concanavalin A. Results of both procedures indicate that in human factor VIII neither the size distribution nor its ristocetin cofactor activity are related to carbohydrate content. Bovine factor VIII contains slightly less sugar than the human preparation as judged from the relative dansyl hydrazine staining intensities. In contrast to human factor VIII, the binding affinity for concanavalin A of bovine factor VIII was gradually decreased with increasing aggregate size. This finding suggests an impaired accessibility of reactive sugar residues in large aggregates of bovine factor VIII.

Animals↗
PubMed1979

A thiocholine ester of cinnamic acid inhibits crosslinking of fibrin without specific binding to donor lysines.

In the presence of activated factor XIII, 2-diethylbenzyl-aminoethylthiol-14C-transcinnamate bromide completely inhibited the crosslinking of fibrin. However, all three fibrin chains bound the cinnamic acid, and, in the alpha- and gamma-chains, the binding of label was not restricted to the crosslinking donor sites as might be expected. Furthermore, even in the absence of activated factor XIII, fibrinogen and fibrin incorporated cinnamic acid. Thus, as well as reacting with the functional thiol group of factor XIII, the thiocholine ester of cinnamic acid is incorporated non-specifically throughout the fibrinogen and fibrin subunit chains. The thiocholine ester used differs in this respect from dansyl cadaverine which is incorporated enzymatically and exclusively to the (acceptor) sites involved in crosslinking. Thiocholine ester of cinnamic acid cannot be used as a label for localization of specific crosslinking donor sites.

Chemical Phenomena↗
PubMed1979

Studies on factor VIII-related protein. I. Ultrastructural and electrophoretic heterogeneity of human factor VIII-related protein.

Human factor VIII-related protein precipitates with specific heterologous anti-bodies directed against purified factor VIII and supports ristocetin-induced aggregation of washed platelets. We purified human factor VIII from cryoprecipitate by subsequent gel filtration on crosslinked large-pore agarose. Factor VIII-related protein appeared as a large aggregate following electrophoresis on 3% polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS). The same material was separated into multiple bands (molecular weight in excess of several millions) following electrophoresis on SDS-1% agarose gels. After complete disulfide reduction of factor VIII-related protein and electrophoresis on SDS-5% polyacrylamide gels a single subunit chain (Mr approximately equal to 200 000) was revealed. Analysis of this protein, in its non-reduced state, by negative contrast electron microscopy showed filaments of markedly variable size. The calculated molecular weight of such filaments ranged from about 0.6.10(6) to 20.10(6). We conclude that size heterogeneity is an essential feature of human factor VIII-related protein.

Disulfides↗
PubMed1979

Studies on factor VIII-related protein. II. Estimation of molecular size differences between factor VIII oligomers.

Human factor VIII-related protein was isolated from cryoprecipitate by agarose (Sepharose CL-2B) gel filtration. Electrophoresis on SDS-2% polyacrylamide-0.5% agarose gels revealed size heterogeneity of factor VIII-related protein which was similar to that shown by SDS-1% agarose gel electrophoresis and electron microscopy. The apparent molecular weights were compared with those of crosslinked IgM oligomers and corresponded to values of up to 20 . 10(6) for factor VIII eluting close to the void volume of our gel filtration column. Measurement of mobility intervals on electrophoretic gels suggested a constant size difference between adjacent bands. Smaller aggregates were found in later eluates from Sepharose columns as well as following partial reduction of factor VIII with cysteine. In order to compare the size difference between small and large aggregates of factor VIII-related protein we calibrated the SDS-2% polyacrylamide-0.5% agarose gels with factor VIII which had been crosslinked with dimethyl suberimidate and subsequently disulfied-reduced with 2-metcaptoethanol. By combination of calibration ranges, constant intervals were measured for large and smaller factor VIII aggregates. The interval between any neighboring protein bands, which were immunologically identified as factor VIII-related protein, was equal to the dimer of the basic factor VIII subunit chain. We conclude that factor VIII aggregates correspond to multimers of a dimeric molecule, i.e. pairs of the basic subunit chain.

Chromatography, Gel↗
PubMed1979

[Principal symptoms in hemostasis disorders].

In a retrospective study the results of laboratory investigations were correlated with actual or previous bleeding symptoms of 40 patients. In 22 patients, a defect of hemostasis was documented in the laboratory, whereas the bleeding disorder, suggested by severe hemorrhage, could not be classified in 3 additional patients. In the remaining 15 individuals, no abnormality could be detected by the available laboratory methods. In the group with a documented bleeding disorder, 13 of 22 patients had prolonged bleeding following dental extraction or other surgery of the oral cavity. Other bleeding symptoms were equally distributed among patients with and without a documented defect. 3 patients had a positive family history, i.e. relatives with a hemorrhagic tendency. In 6 patients, a correlation between bleeding and the ingestion of acetylsalicylic acid could be established, while in 4 it was probable. Hemorrhagic complications following oral surgery, particularly in combination with drugs known to interfere with platelet function and a positive family history, strongly suggest an abnormality of hemostasis. Among the 22 patients with a documented defect of hemostasis, only 6 were found who did not present at least one of these leading symptoms.

Blood Coagulation Disorders↗
PubMed1979

Preparation of factor VIII-deficient plasma by immunoadsorption.

An immunoadsorbent was prepared by coupling rabbit and human antibodies against human factor VIII to Sepharose CL-2B. The resulting insoluble antibodies completely removed factor VIII and fibrinogen from normal human citrate plasma. Other coagulation factors were satisfactorily recovered in the eluted plasma. Following addition of fibrinogen, the factor VIII-deficient plasma was used for calibration of the one-stage factor VIII assay and compared with the hemophilic plasma. Parallel straight lines were obtained in the log-log plot against VIII:C, indicating that the artificial reagent can be used as a substitute for hemophilic plasma in determination of factor VIII procoagulant activity. The immunoadsorbent can be regenerated and repeatedly used for affinity chromatography binding of factor VIII.

Animals↗