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E A Beck

Publications and source records attributed to E A Beck.

At least 19 recordsLinked to original sources
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↗
PubMed1979

A modified assay for studies of cultured granulocyte precursors: cryopreservation of stimulating mononuclear cells.

Mononuclear cells (MNC), isolated from peripheral blood of healthy donors, were cryoprotected by dimethyl sulfoxide and stored in liquid nitrogen. Colony-stimulating factor (CSF), produced by cryopreserved MNC, was compared with that of nonfrozen controls in a double-layer agar system with human bone marrow as target cells. Our results indicate that cryopreserved MNC retain their ability to stimulate myelopoiesis-committed stem cells after freezing. In addition, evidence was obtained that CSF of feeder layers changes, depending on the duration of preincubation and cell concentration. In a system where either stimulating or target cells are cryopreserved the dynamics of interactions between normal or abnormal cell lines can thus be studied.

Blood Preservation↗
PubMed1977

Effects of amidination and chemical cross-linking on human factor VIII (antihemophilic factor).

The bifunctional reagent dimethyl suberimidate, reacting with primary amino groups of proteins, was used to cross-link highly purified human factor VIII. Reaction products were reduced with beta-mercaptoethanol or treated with Rhizopus arrhizus triglyceride lipase. The proportions of the dissociated subunits and their oligomers were calculated from the relative staining intensities of individual bands following polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. Low concentrations of dimethyl suberimidate (up to 0.5 mM) produced covalently linked dimers which retained full functional (coagulant and von Willebrand factor) activities. Treatment with increasing concentrations of dimethyl suberimidate resulted in an almost simultaneous appearance of both trimeric and tetrameric species, suggesting the existence of specific intra-dimer contacts. A parallel decrease of functional activities was observed at higher concentrations of dimethyl suberimidate. A monofunctional reagent (ethyl acetimidate), reacting similarly with primary amino groups, amidinated factor VIII at rates similar to dimethyl suberimidate. Up to 40% amidinated factor VIII retained full biological activities. We conclude that the most reactive lysine residues are not involved in the active sites responsible for either coagulant or von Willebrand activity.

Blood Coagulation↗
PubMed1977

Pharmacokinetics of heparin after a single intravenous or subcutaneous injection.

In 2 groups of 4 probands the pharmacokinetics of heparin were investigated by a physiologic method (factor Xa inhibition) and an isotope method (35S-heparin) following a single intravenous injection of 5000 IU heparin and a single subcutaneous injection of 10000 IU heparin respectively. Following intravenous administration the anticoagulant effect and radioactivity fall exponentially. The half-life is about 50 min and the distribution volume 3000 ml (factor Xa inhibition) and 3800 ml (radioactivity). With subcutaneous injection peak concentrations above 0.2 IR heparin/ml were measured by both methods 2-4 h after administration; 9 h after administration, no further factor Xa inhibition was detectable in 2 probands. In the light of these and previously published results, some practical aspects of the conduct of therapy and prophylaxis of thromboembolism with heparin are discussed.

Antithrombin III↗