bluuebunny/crossref_metadata_2025 huggingface dataset:
HVDC grid protection algorithm design in phase and modal domains
Authors: Leterme Willem , Pirooz Azad Sahar , Van Hertem Dirk
Published: June 2018

To meet the required operation speed for protection of meshed voltage source converter (VSC) high‐voltage direct current (HVDC) grids, travelling wave‐based algorithms operating in the submillisecond time‐frame can be used. The domain in which these algorithms operate, i.e. modal or phase, determines their performance in fault discrimination, fault type classification, and faulted pole selection. In the recent literature, high‐speed algorithms have been proposed for various VSC HVDC grid configurations and transmission line types; yet, the choice of domain has received insufficient attention. This study offers recommendations for the choice of domain for protection algorithm design of HVDC overhead line or cable systems in symmetric monopolar and bipolar configurations. The theoretical analysis of this study, which is based on fundamental wave propagation theory, indicates that the preferred domain for protection algorithms for cable and overhead line systems are the phase and modal, respectively. Furthermore, the study provides comprehensive guidelines to construct detection functions for both configurations and discusses the errors introduced by approximations. Finally, study results from a bipolar overhead line test system demonstrate the advantages of modal over phase domain for fast fault discrimination and classification and illustrate practical problems associated with non‐ideal detection functions.
Biological pest control using a model‐based robust feedback
Authors: Puebla Hector , Roy Priti Kumar , Velasco‐Perez Alejandra , Gonzalez‐Brambila Margarita M.
Published: June 2018

Biological control is the artificial manipulation of natural enemies of a pest for its regulation to densities below a threshold for economic damage. The authors address the biological control of a class of pest population models using a model‐based robust feedback approach. The proposed control framework is based on a recursive cascade control scheme exploiting the chained form of pest population models and the use of virtual inputs. The robust feedback is formulated considering the non‐linear model uncertainties via a simple and intuitive control design. Numerical results on three pest biological control problems show that the proposed model‐based robust feedback can regulate the pest population at the desired reference via the manipulation of a biological control action despite model uncertainties.
Grundlagen der kontrastverstärkten Sonografie (CEUS)
Authors: Strunk Holger , Marinova Milka
Published: June 2018

Die kontrastverstärkte Sonografie (CEUS) wird für viele Fragestellungen und in zahlreichen Fachdisziplinen eingesetzt, wenn auch mit unterschiedlichen Indikationen und Schwerpunkten. Dabei spielt die Erfahrung des Untersuchers sowohl für die Befunderhebung als auch für die Befundbeurteilung eine wesentliche Rolle. Im Folgenden werden die Grundlagen und die wichtigsten Einsatzgebiete der CEUS dargestellt.
Bauchaortenaneurysma – Diagnostik und interventionell-radiologische Therapie
Authors: Anton Susanne , Stahlberg Erik , Goltz Jan
Published: June 2018

Das Bauchaortenaneurysma (BAA) als eine fokale Erweiterung aller Wandschichten der Aorta abdominalis ist ein komplexes und lebensbedrohliches Krankheitsbild – Albert Einstein und Charles de Gaulle sind an einer Ruptur verblutet. Eine endovaskuläre Aortenreparatur (EVAR) oder offen-chirurgische Therapie haben das Ziel, das Rupturrisiko niedrig zu halten bzw. zu eliminieren und die Lebenserwartung des Patienten zu verbessern.
„The Big Five“ – Kataraktchirurgie bei multiplen okulären Komorbiditäten: individuelle Vorgehensweise zur Komplikationsvermeidung
Authors: Borkenstein Andreas , Borkenstein Eva-Maria
Published: June 2018

Das Auftreten von 5 Komorbiditäten (Cataracta senilis, Pseudoexfoliatio lentis, Fuchs-Endotheldystrophie, altersbedingte Makuladegeneration, Keratoconjunctivitis sicca) kann zu einer massiven Einschränkung des Sehvermögens führen. Engmaschige Verlaufskontrollen und die gezielte Therapie jeder einzelnen Erkrankung sind notwendig, um mit den entsprechenden Vorsichtsmaßnahmen die erfolgreiche Operation des grauen Stars durchführen zu können. Diese Fallserie zeigt, dass die Kataraktoperation trotz infauster Prognose zu visueller Rehabilitation führen kann.
Childhood Craniopharyngioma: A 22-Year Challenging Follow-Up in a Single Center
Authors: Tosta-Hernandez Patrícia , Siviero-Miachon Adriana , da Silva Nasjla , Cappellano Andrea , Pinheiro Marcelo , Spinola-Castro Angela
Published: June 2018

Craniopharyngioma is a sellar/suprasellar benign tumor whose aggressiveness may imply in endocrine disturbances (hypothalamic obesity and hormone deficiencies). Fifty-seven patients were evaluated according to clinical characteristics, hypothalamic involvement, type of treatment, anthropometric variables, adiposity indexes (body mass index Z score category at diagnosis and post-treatment, total body fat, visceral adipose tissue, and metabolic syndrome components) and analyzed through multiple regression and logistic models. Patients were stratified according to growth hormone deficiency and recombinant human growth hormone use. Mean ages at diagnosis and at study evaluation were 9.6 and 16.6 years old, respectively. A set of 43/57 (75.4%) patients presented with important hypothalamic involvement, 24/57 (42.1%) received surgical treatment and cranial radiotherapy, and 8/57 (14%) interferon-α exclusively. Fifty-five patients (96.5%) were considered growth hormone deficient, and 26/57 (45.6%) grew despite no recombinant human growth hormone replacement therapy. At diagnosis, 12/57 (21%) patients were obese, and 33/57 (57.9%) at study evaluation, and after 3.2 years (median) post first therapy. There was no influence of height Z score on body mass index Z score. Body mass index Z score at diagnosis positively influenced body mass index Z score, total body fat, waist circumference and the presence of the metabolic syndrome post-treatment. Replacement of recombinant human growth hormone decreased total body fat and visceral adipose tissue. Craniopharyngioma patients worsened body mass index Z score category 3.2 years (median) after first treatment. Body mass index Z score increased due to real weight gain, without height decrease. Replacement of recombinant human growth hormone had beneficial effect on adiposity.
Palliativmedizinisches Denken und Handeln im Notarztdienst – eine Befragung zur Strukturqualität
Authors: Tiesmeier Jens , Salomon Fred , Lehning Brigitte , Tomaszczyk Matylda , Henzler Dietrich , Jakob Thomas
Published: June 2018

Fragestellung Je nach untersuchter Region beträgt der Anteil von Patienten mit einer palliativen Grunderkrankung 3 – 10 % (ca. 1 % in einer Terminalphase) der Gesamteinsätze im Rettungsdienst. Dabei ist ein breites Spektrum zu bewältigen. Es reicht von Symptomkrisen und psychosozialen Belastungen des Umfelds, über erkrankungsunabhängige Notfälle bis hin zur Frage der Durchführung einer Reanimation. Hierbei besteht ein Spannungsfeld zwischen dem kurativen Ansatz der Notfallrettung und dem Ansatz einer palliativen, symptomkontrollierenden Therapie vor Ort. Unter Zeitdruck müssen die Gesamtsituation erfasst und medizinische, juristische und ethische Aspekte im Sinne des Patienten gewürdigt werden. Es wurde der Frage nachgegangen, welche Strukturen und Hilfsmittel dem Notarzt für ein palliativmedizinisches Vorgehen und Denken im Einsatz zur Verfügung stehen.

Material und Methoden Datenerhebung zur Strukturqualität bei der Versorgung von Palliativpatienten mittels Fragebogen bei den Ärztlichen Leitern Rettungsdienst der Region Ostwestfalen-Lippe und der Stadt Münster.

Ergebnisse Rücklaufquote 8/8. Gesamtzahl der regelmäßig aktiven Notärzte pro Gebietskörperschaft 71 – 80; hiervon mit Zusatzweiterbildung Palliativmedizin 0 – 10. Fragenauswahl (in Klammern Anzahl der Nennungen), Strukturdaten Leitstelle: Kontaktdaten des ambulanten Palliativmediziners (z. B. Mobilfunknummer) für Disponenten zur Verfügung: ja (7), nein (1). Spezielle Schulungen der Disponenten: ja (1), nein (7). Strukturdaten zum Advance-Care Planning: Qualitätszirkel/Treffen Rettungsdienst/Palliativnetzwerk: ja (2), nein (6), geplant (1). Regional standardisierte Notfallausweise o. Ä.: ja (4), nein (4), geplant (1). Entscheidungshilfen im Einsatz: Standard operating Procedure (SOP) als Entscheidungspfad vorhanden: ja (3), nein (5), geplant (1). Ausstattung der Rettungsmittel: Port-Nadeln: ja (7), nein (1); MAD-Systeme (Intranasal Mucosal Atomization Device): ja (8), Gesamtmenge Morphinsulfat vor Ort ≥ 40 mg: ja (7), nein (1).

Diskussion und Schlussfolgerung Optionen, die ein palliativmedizinisches Vorgehen für die Notfallmedizin bieten können, wurden bislang uneinheitlich implementiert. Dies gilt für die Weiterbildung in medizinischen, rechtlichen und ethischen Fragen, die Ausstattung und Hilfsmittel auf den Rettungsmitteln sowie für die Kooperation mit den ambulanten Partnern. Mögliche Konflikt- und Schnittmengen sollten lokal und überregional erkannt und besprochen werden. Ziel ist ein souveränes und differenziertes Vorgehen zwischen kurativer Behandlungsoption und palliativer Symptomkontrolle, um im Sinne des Patienten handeln zu können.
Reaction of 1-Trimethylsilyl-1,2-epoxy-3-alkanols with Alkynes and Application to the Synthesis of 18-HEPE
Authors: Kobayashi Yuichi , Nanba Yutaro , Morita Masao
Published: June 2018

A reaction of epoxy alcohols (anti and/or syn isomers) ­derived from (E)-TMS-CH=CHCH(OH)R with TMS-C≡CLi in THF/HMPA s­tereoselectively afforded (E)-TMS-C≡C-CH=CHCH(OH)R. The (E) stereo­chemistry was independent of the anti/syn stereochemistry, but the syn isomers showed higher reactivity than the anti isomers. The reaction was applied to the synthesis of (18R)- and (18S)-HEPE.
Manganese-Catalyzed Kumada Cross-Coupling Reactions of Aliphatic Grignard Reagents with N-Heterocyclic Chlorides
Authors: Matson Ellen , Petel Brittney , Purak Merjema
Published: June 2018

Herein we report the use of manganese(II) chloride for the catalytic generation of C(sp2)–C(sp3) bonds via Kumada cross-coupling. Rapid and selective formation of 2-alkylated N-heterocyclic complexes were observed in high yields with use of 3 mol% MnCl2THF1.6 and under ambient reaction conditions (21 °C, 15 min to 20 h). Manganese-catalyzed cross-coupling is tolerant toward both electron-donating and electron-withdrawing functional groups in the 5-position of the pyridine ring, with the latter resulting in an increased reaction rate and a decrease in the amount of nucleophile required. The use of this biologically and environmentally benign metal salt as a catalyst for C–C bond formation highlights its potential as a catalyst for the late-stage functionalization of pharmaceutically active N-heterocyclic molecules (e.g., pyridine, pyrazine).
Fibrinolysis and Fibrinogenolysis in Disseminated Intravascular Coagulation
Authors: Takahashi Hoyu , Tatewaki Wataru , Wada Ken , Niwano Hiroe , Shibata Akira
Published: June 2018

In order to assess precisely the fibrinolytic state in disseminated intravascular coagulation (DIC), plasma levels of fibrinogenolysis products (FgDP), fibrinolysis products (FbDP) and fibrinogenolysis plus fibrinolysis products (TDP) were measured with newly developed enzyme-linked immunosorbent assays based on monoclonal antibodies in 72 patients with DIC at presentation. Not only FbDP and TDP but also FgDP were markedly elevated in patients with DIC. When analyzed according to the underlying disease categories, the relative proportion of FgDP to TDP was high in patients with acute promyelocytic leukemia and vascular diseases, and it was the lowest in patients with sepsis. Correlation analysis revealed that plasma levels of FgDP correlated negatively with alpha 2-antiplasmin and positively with plasmin-alpha 2-antiplasmin complex (PAP) and a ratio of PAP to thrombin-antithrombin III complex (TAT). These findings indicate that besides fibrinolysis, fibrinogenolysis is markedly accelerated in the majority of the patients with DIC.
Hereditary Dysfunctional Protein C Molecules (Type II): Assay Characterization and Proposed Classification
Authors: Marlar Richard A , Adcock Dorothy M , Madden Renée M
Published: June 2018

Protein C (PC) deficiency is among the increasing number of recognized causes of hereditary thrombotic disease. Two types of PC deficiency have been described: 1) Type I, which is characterized by a concomitant decrease in PC activity and antigen, and 2) Type II. characterized by disproportionately low activity compared to antigen (i.e. a dysfunctional molecule). To date, only a small number of Type II patients have been described. This study was undertaken to evaluate a number of dysfunctional PC molecules by comparing PC clotting and amidolytic activities with antigen levels. For these studies, an automated PTT-based clotting PC assay was developed. This assay was sensitive to 1% of a normal plasma pool, specific, accurate, and reproducible (± 12%). A good correlation (r = 0.918) of the clotting activity to antigen was found in normal individuals and Type I heterozygous and homozygous patients. To classify Type II PC deficient patients, the antigen, amidolytic and clotting PC levels were compared in ten affected families. The clotting activities were decreased in all affected members, whereas the antigen levels were within the normal limits. In four of the 10 families, the amidolytic activity was normal and similar to the antigen levels. This suggests that in certain families, defects in the PC molecule occur in regions not associated with amidolytic functions. From these studies, the molecular basis of Type II PC deficiency is varied and complex, involving different functional domains of the PC molecule. Therefore, we have suggested a nomenclature algorithm for Type II PC deficiency based on the location of the defect within the specific domains of the PC molecule.
Diagnosis of Protein C Deficiency in Patients on Oral Anticoagulant Treatment: Comparison of Three Different Functional Protein C Assays
Authors: Pabinger I , Kyrle P A , Speiser W , Stoffels U , Jung M , Lechner K
Published: June 2018

The efficacy of three different protein C activity assays and of protein C antigen determination for the diagnosis of protein C deficiency was studied in 13 protein C deficient patients (11 with type I, 2 with type II deficiency) and in 51 presumably nondeficient patients (control group), both groups being on oral anticoagulant (OAC) treatment. For protein C activity measurement (1) the assay according to Francis (slightly modified) with thrombin activation and measurement of activated protein C in the aPTT system, (2) an assay using Protac activation and chromogenic substrate (Protac-CS) and (3) an assay using Protac activation and the aPTT system (Protac-PTT) were used. Protein C antigen was determined by Laurcll immunoelec-trophoresis. The three activity assays gave different results, with the highest values obtained by the Protac-CS assay and the lowest values by the Protac-PTT assay. The Francis assay gave intermediate results.

Protein C activity and antigen values were significantly lower in protein C deficient patients compared to the control group. Protein C activity tests had a higher discriminative power than the antigen determination. After taking into account the intensity of treatment, by the Francis assay all deficient and non-deficient patients were correctly classified, by the Protac-CS and the Protac-PTT assay 2 and 4 patients, respectively, were misclas-sified and by the antigen assay 8 patients were misclassified. Calculation of the ratios of protein C activity to factor II activity was of high discriminative power.

We conclude that for diagnosis of protein C deficiency protein C activity tests are superior to antigen determination not only in type II but also in type I deficient patients. Certain statistical procedures can further improve the discrimination between deficient and non-deficient patients.
The Antithrombotic Properties of Human Prothrombin Fragment 1.2 in Mice
Authors: Chung Amy , Ofosu Frederick A , Blajchman Morris A
Published: June 2018

We have investigated the antithrombotic properties of prothrombin fragment 1.2 (F1.2) in this study. To do this, we established the minimum concentration of human placental tissue factor or human a-thrombin that was lethal in mice within 5 min after intravenous injection. Prothrombin F1.2 protected the mice from the lethal effect of tissue factor or α-thrombin in a dose dependent manner, with 500 μg (14 nmoles) of prothrombin F1.2 per mouse being the minimum amount required to protect all mice from the lethal effect of either thrombogenic stimulus. The minimum dose of heparin which protected mice from the lethal effect of thrombin or tissue factor was 6 units or ∼3.3 nmoles. The observation that prothrombin F1.2 has antithrombotic properties suggests prothrombin F1.2 can modulate coagulation in vivo, as it has previously been shown to do in vitro.
Characterization of a Panel of Monoclonal Antibodies to Human Coagulation Factor XI and Detection of Factor XI in Hep G2 Cell Conditioned Medium
Authors: Ohkubo Y , O’Brien D P , Kanehiro T , Fukui H , Tuddenham E G D
Published: June 2018

We have produced a panel of ten monoclonal antibodies specific to coagulation factor XI. Western blot analysis demonstrates that 9 of these antibodies react with the heavy chain of factor XI and one with the light chain. Seven of these antibodies inhibit factor XI and factor XIa activity.

We have used immobilised monoclonal antibody for the production of factor XI deficient plasma and to purify factor XI to homogeneity with high yield in a simple two-step procedure. These monoclonal antibodies were used to develop highly sensitive immunoassays capable of detecting less than 0.01 u factor XI antigen ml−1. A strong correlation was found between antigen and activity levels in 11 patients with hereditary deficiency indicating that none was cross-reacting material positive. Cultured Hep G2 cells were found to synthesize small amounts of factor XI antigen and this could also be detected by functional assay and by western blot analysis.
The Dependence of the International Sensitivity Index on the Coagulometer Used to Perform the Prothrombin Time
Authors: Ray M J , Smith I R
Published: June 2018

This study was designed to detect any effect that different types of coagulation instrument may have on the International Sensitivity Index (ISI) of a thromboplastin.

Manufacturers of commercial thromboplastins now calibrate their reagents against the World Health Organization international reference preparation to assign them an IST. This enables the prothrombin time (PT) estimated with that reagent to be expressed as an International Normalised Ratio (INR).

One batch of Thromborel S was calibrated against the Australasian Reference Thromboplastin (ART). The Thromborel S was used on three photo-optical instruments, the Automated Coagulation Laboratory (ACL) (Instrumentation Laboratory), the Cobas Fibro (Roche), and the Coag-a-Pet (General Diagnostics). PTs using ART were performed manually using the reference method.

The ISIs calibrated in our laboratory when the ACL and Cobas Fibro were used were not significantly different at the 95% level, being 1.102 ± 0.018 and 1.134 ± 0.022 respectively. The ISI with the Coag-a-Pet of 1.223 ± 0.023 was significantly different to that of the ACL and the Cobas Fibro at the 95% level.

The flowcharts for a computer program to perform the necessary calculations are provided. The program allows for the entry and editing of data from the calibration procedure, and provides a mean normal PT and normal range, the ISI and 95% confidence limits of the calibration, and a chart for the conversion of the test PTs to INRs.

The authors have made available an IBM compatible program for the calibration of thromboplastins.
Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel
Authors: Kuyas C , Haeberli A , Walder P , Straub P W
Published: June 2018

With an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the α-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (<5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogcn) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.
The Fibrinolytic Potential of the Normal Primate following the Generation of Thrombin In Vivo
Authors: Giles Alan R , Nesheim Michael E , Herring Steven W , Hoogendoorn Hugh , Stump David C , Heldebrant Charles M
Published: June 2018

Parameters of the fibrinolytic system were studied in a primate model where the generation of thrombin was promoted in vivo. The procoagulant stimulus used was a combination of human factor Xa in combination with phosphatidylcholine/phos-phatidylserine lipid vesicles (PCPS) as the source of coagulant active phospholipid. The dosage of each component was formulated to provide a gradation of thrombin generating potential assessed prior to in vivo study in an in vitro clotting assay. These ranged from 25.25 - 36.60 pMole/kg (factor Xa) and 18.85 - 56.30 nMole/kg (PCPS). In each case, the ratio of the dose of factor Xa/PCPS was maintained at 0.65 (pMole factor Xa/ nMole PCPS). Individual dosage combinations producing recalcification clotting times in vitro of 15, 20, 25 and 30 s were used in detailed in vivo studies. Previous studies in dogs had confirmed the thrombin generating potential of factor Xa/PCPS infusions and demonstrated an associated activation ot protein C and increased fibrinolytic activity. This has now been extensively characterized in the chimpanzee as follows: 10 min after the infusion of the highest dose (36.6 pMole factor Xa/56.3 nMole PCPS kg bodyweight), the level of circulating t-PA had risen to 900 ng/ml (antigen), 885 IU/ml (functional). Dosage was observed with the lowest dose of 12.25 pMole factor Xa and 18.85 nMole PCPS being associated with relatively minor increases in circulating t-PA activity. There were no changes in u-PA at any dosage during the full time course of the experimental period (90 min). Plasminogen activation was also apparent with alpha-2 antiplasmin levels falling to 30 - 40% of pre-infusion levels at the highest dosages. There was also a significant consumption of fibrinogen and evidence of active fibrinolysis manifested by major increases in the levels of FDP, D-dimer and B-beta 1-42. The data strongly suggested that this was predominantly fibrinolysis rather than fibrinogenolysis and that the fibrinolytic response observed resulted from a major release of t-PA from available stores consequent to thrombin generation and presumably subsequent fibrin generation. These data illustrate the enormous fibrinolytic potential of the intact normal primate and may provide a model for study of the mechanism(s) by which the regulation of t-PA availability can be up- or down-regulated in health and disease.
Physico-Chemical Properties of Recombinant Desulphatohirudin
Authors: Electricwala A , Irons L , Wait R , Carr R J G , Ling R J , Grossenbacher H , Atkinson T
Published: June 2018

Physico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. (13) for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies (17)
Ethanol Inhibition of Thrombus Formation on Collagen-Coated Glass
Authors: Owens Charlene K , Mclntire Larry V , Lasslo Andrew
Published: June 2018

Epi-fluorescent video microscopy was used to evaluate the effect of ethanol on platelet mural thrombus formation. Whole blood, treated with ethanol, was perfused over collagen coated glass in a parallel-plate flow chamber at a shear rate of 1,000/s. Digital image processing and photodiode measurements were used to analyze the dynamics of thrombus growth on this surface. Ethanol concentrations as low as 0.02% v/v were found to inhibit 45 + 33% (± S.D.) of normal platelet accumulation on the slide while 0.2% v/v ethanol effected an 82 ± 15% inhibition of mural thrombus formation. While platelet adhesion to the collagen surface appeared unaffected by ethanol concentrations up to 0.1% v/v, 0.2% v/v ethanol had an effect on adhesion as well as aggregation. These results imply that low ethanol concentrations inhibit the formation of mural thrombi in a model of a damaged blood vessel at physiological shear rates. This inhibition would not be detected in systems which measure bulk aggregation, e.g. in aggregometric determinations.
Proteolytic and Immunologic Comparison of Human and Bovine von Willebrand Factor
Authors: Bakhshi Meenakshi R , Kirby Edward P
Published: June 2018

The structures of bovine and human vWF were compared by proteolysis with Staphylococcus aureus V8 protease and rattlesnake venom Protease I. Fragments were analyzed for chain composition, heparin binding, collagen binding, platelet agglutinating activity and recognition by a panel of monoclonal antibodies which reacted with both bovine and human vWF. Similar large fragments from the C-terminal domain of vWF were seen in each case. The N-terminal domain resulting from cleavage of bovine vWF was much smaller than that seen upon digestion of human vWF with V8 protease. Protease I destroyed the heparin binding domain in human vWF. Bovine vWF was much less sensitive to proteolysis than was human vWF.
Selective Staining of Platelet Glycoproteins Using Two-Dimensional O’Farrell Gel Electrophoresis and Avidin-Biotin-Conjugated Lectins
Authors: Kehrel Beate , Kokott Renate , Balleisen Leopold , Stenzinger Werner , Clemetson Kenneth J , van de Loo Jürgen
Published: June 2018

A time-saving and sensitive high resolution method lor the analysis and identification of platelet glycoproteins using non-radioactive compounds has been developed. Platelet proteins (50 μg) of normal subjects were separated by isoelectric focusing and SDS-PAGE. Proteins were either stained with silver or electroblotted onto nitrocellulose. Nitrocellulose blots were treated with the following biotinylated lectins : Abrus precatorius lectin, concanavalin A, Lens culinaris lectin, or wheat germ agglutinin. Staining was achieved by avidin-biotin-coupled peroxidase using 4-chloro-l-naphthol as the substrate. A rapid overview of platelet glycoproteins may be obtained by applying all the lectins to a single blot.
Hypothyroidism Leads to More Small-Sized Platelets in Circulation
Authors: van Doormaal J J , van der Meer J , Oosten H R , Halie M R , Doorenbos H
Published: June 2018

The effect of induced hypothyroidism on platelet count and platelet volume distribution was studied in twelve athyreotic patients, After a two weeks withdrawal of triiodothyronine supplementation, platelet count and the ratio between platelet and red cell count were increased in all patients. Furthermore, mean platelet volume was declined and platelet distribution width was risen. Thus, hypothyroidism appears to increase the number of circulating platelets, especially the smaller ones.
Two Genes Homologous with Human Protein S cDNA Are Located on Chromosome 3
Authors: Amstel J K Ploos van , van der Zander A L , Bakker E , Reitsma P H , Bertina R M
Published: June 2018

A cDNA coding for the carboxy-terminal region of human protein S and containing a complete 3’-untranslated region, was isolated by a combination of antibody screening of a λgt11 human liver cDNA expression library and in situ hybridization of a pUC9 human liver cDNA library.

Hybridization analysis of human total DNA with radiolabelled non-overlapping cDNA restriction fragments revealed the existence of two genes per haploid genome homologous with the protein S cDNA. Both genes were mapped to chromosome 3 using human-rodent cell hybrids. Neither of the genes showed polymorphism for sixteen different enzymes upon hybridization with the protein S cDNA.
Congenital Deficiency of Alpha-2-Adrenoceptors on Human Platelets : Description of Two Cases
Authors: Tamponi Giacomo , Pannocchia Antonella , Arduino Carlo , Bazzan Mario , Dora Nadine Della , Schinco Piercarla , Buraglio Mauro , Eandi Mario
Published: June 2018

The biochemistry and functionality of platelets from two related subjects (mother and son) with alpha-2-adrenoceptor-deficient platelets has been evaluated. Radioligand binding experimentes with the specific alpha-2-adrenergic-receptor antagonist, 3H-yohimbine, showed a drastic reduction of alpha-2-adrenoceptors in platelets from both subjects in comparison with the control values. Electron microscopy studies revealed a normal morphology and a normal number of alpha granules and dense bodies. Levels of adenine nucleotides; 5-hydroxytryptamine; B-thromboglobulin; platelet-factor-4 and thromboxane A2 production were within normal limits.

Platelet aggregation and 5-hydroxytryplamine production in response to adrenalin (at concentrations up to 50 μM) were absent, whereas ADP, AA, PAF, collagen and thrombin-induced aggregation, secretion, Ca++ flux and thromboxane A2 production were normal.

The inhibitory effect caused by different concentrations of prostacyclin on Ca++ flux, aggregation, secretion and thromboxane A2 production of platelet functionally lacking of alpha-2-adrenoceptor was not distinguishable from control platelets and platelets preincubated with yohimbine.
Immunoaffinity Purification of HTC Rat Hepatoma Cell Plasminogen Activator-Inhibitor-1
Authors: Zeheb Ron , Rafferty Usha M , Rodriguez Miguel A , Andreasen Peter , Gelehrter Thomas D
Published: June 2018

Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits tissue-type plasminogen activator activity by inducing a specific plasminogen activator-inhibitor (PAI-1). Using immobilized polyclonal antibodies raised against HT-1080 human fibrosarcoma PAI-1, we have purified HTC PAI-1 from serum-free medium conditioned by dexamethasone-treated HTC hepatoma cells and shown it to be antigenically related to human PAI-1. Greater than 100-fold purification with greater than 75% yield was achieved in a single step. The purified PAI-1 migrates on SDS-polyacrylamide gels as a single major band of 49 kDa with a minor band of 46 kDa. Digestion of PAI-1 with endoglycosidase F causes a shift toward faster migrating species which retain inhibitory activity. The purified PAI-1 was stable at pH 2.5, lost 50% of its activity after 15 min at 45° C, and showed marked activation after treatment with SDS or guanidine-HCl. Purified PAI-1 rapidly inhibited and formed complexes with both tissue-type and urokinase-type plasminogen activators. Polyclonal rabbit antirat PAI-1 antibodies were raised which immunoprecipitate both free and complexed PAI-1.
Fibrinolytic Response and Fibrin Fragment D-Dimer Levels in Patients with Deep Vein Thrombosis
Authors: Declerck P J , Mombaerts P , Holvoet P , De Mol M , Collen D
Published: June 2018

An enzyme-linked immunosorbent assay for fragment D-dimer was developed with the use of two monoclonal antibodies directed against specific non-overlapping antigenic determinants, present in fragment D-dimer of crosslinked fibrin but not in fragment D of non crosslinked fibrin or of fibrinogen, The lower limit of sensitivity of the assay when applied to human plasma, is 25 ng/ml. Concentration of fragment D-dimer in plasma from healthy individuals was 177 ,± 83 ng/ml (mean ± SD). In plasma of 11 out of 12 patients with phlebographically confirmed acute deep vein thrombosis, fragment D-dimer levels were significantly increased. Fragment D-dimer was not increased in 9 out of 10 patients with recurrent idiopathic deep vein thrombosis during clinically silent episodes.

Total t-PA antigen and free t-PA antigen concentrations were measured using previously developed ELISAs. Nine of the 12 patients with acute deep vein thrombosis showed a significant increase of total t-PA antigen (from 8.6 ± 6.9 ng/ml to 21 ± 16 ng/ml) after venous occlusion but in 3 of these free t-PA remained undetectable. Five of the 10 patients with recurrent deep vein thrombosis responded to venous occlusion with a significant increase of total t-PA antigen (from 6.7±3.2 ng/ml to 14 ± 7.9 ng/ml) but, in all patients, free t-PA antigen remained undetectable.

It is concluded that the combined assays of total and free t-PA antigen and of fragment D-dimer may be useful for the evaluation of the dynamics of the fibrinolytic system in physiological and pathological conditions.
Antithrombin III Binding to Surface Immobilized Heparin and Its Relation to F Xa Inhibition
Authors: Kodama K , Pasche B , Olsson P , Swedenborg J , Adolfsson L , Larm O , Riesenfeld J
Published: June 2018

The mode of F Xa inhibition was investigated on a thromboresistant surface with end-point attached partially depoly-merized heparin of an approximate molecular weight of 8000. Affinity chromatography revealed that one fourth of the heparin used in surface coating had high affinity for antithrombin III (AT). The heparin surface adsorbed AT from both human plasma and solutions of purified AT. By increasing the ionic strength in the AT solution the existence of high and low affinity sites could be shown. The uptake of AT was measured and the density of available high and low affinity sites was found to be in the range of 5 HTid 11 pic.omoles/cmf, respectively Thus the estimated density of biologically active high and low ailmity heparm respectively would be 40 and 90 ng/cm2 The heparin coating did not take up or exert F Xa inhibition by itself. With AT adsorbed on both high and low affinity heparin the surface had the capacity to inhibit several consecutive aliquots of F Xa exposed to the surface. When mainly high affinity sites were saturated with AT the inhibition capacity was considerably lower. Tt was demonstrated that the density of AT on both high and low affinity heparin determines the F Xa inhibition capacity whereas the amount of AT on high affinity sites limits the rate of the reaction. This implies that during the inhibition of F Xa there is a continuous surface-diffusion of AT from sites of a lower class to the high affinity sites where the F Xa/AT complex is formed and leaves the surface. The ability of the immobilized heparin to catalyze inhibition of F Xa is likely to be an important component for the thromboresistant properties of a heparin coating with non-compromized AT binding sequences.
Sources of Variability in Coagulation Factor Assays
Authors: Thompson S G , Martin J C , Meade T W
Published: June 2018

Estimates of the components of variation of a number of coagulation factor assays are presented and are compared with those for other haematological and biochemical variables. They have been derived from a study in which fourteen volunteers gave blood samples approximately bi-monthly for three years. The systematic between-batch variation w7as generally only a small component of the total variation. However, the proportion of the total variation due to differences between people ranged from about 15% in the case of two measures of antithrombin III to about 85% for cholesterol and haemoglobin. For factor VII activity and fibrinogen, the figure was just over 70%. These estimates of the components of variation are not generally available in the literature. In view of the increasing interest in the role of coagulation factors in the pathogenesis of arterial disease, estimates of this kind are needed both for the interpretation of observed relationships as well as for planning sample sizes for future studies.
Identification of mRNA Coding for Factor VII Protein in Human Alveolar Macrophages - Coagulant Expression May Be Limited Due to Deficient Postribosomal Processing
Authors: McGee Maria P , Wallin Reidar , Devlin Robert , Rothberger Henry
Published: June 2018

Clotting factors synthesized by monocytes and macrophages may initiate coagulation reactions during inflammation. Functional vitamin K-dependent coagulation factors have been found to be associated with human monocytes/macrophages, but there are no reports identifying mRNA coding for vitamin K-dependent proteins in these cells. In the present studies, factor VII mRNA was found in total RNA extracted from freshly isolated human alveolar macrophages using hybridization with a complementary DNA probe. On the other hand, vitamin K-dependent carboxylase activity which is required for postribosomal modification of the protein, was not detectable in the macrophages before or after culture, and human blood mononuclear leukocytes also lacked this enzyme activity. Control human and rat hepatoma cells exhibited high levels of carboxylase activity within the same experiments. Using sensitive kinetic assays, no increase in factor VII activity was detected during culture of alveolar macrophages under conditions promoting 1.78 ± .24 (n = 8) fold increases of tissue factor activity. These findings with freshly isolated cells demonstrate that alveolar macrophages synthesize factor VII mRNA in vivo. However, the mRNA was found in the absence of evidence for γ-carboxylase activity or processing of the factor into a functional clotting enzyme. The results imply that functional expression of any synthesized coagulation factor VII in alveolar macrophages may be limited or prevented due to a cellular deficiency at the level of postribosomal processing.
Risks to the Fetus of Anticoagulant Therapy During Pregnancy
Authors: Ginsberg Jeffrey S , Hirsh Jack , Turner D Christoper , Levine Mark N , Burrows Robert
Published: June 2018

The use of anticoagulants during pregnancy is problematic because of the potential adverse effects to the mother and the fetus. Heparin does not cross the placenta, and thus, it was surprising that a recent report concluded that heparin therapy during pregnancy was as risky as oral anticoagulant therapy. Therefore, we performed a literature review of fetal/infant outcomes following anticoagulant therapy during pregnancy. We examined 186 reports which described fetal/infant outcomes in 1,325 pregnancies associated with anticoagulant therapy. The rates of adverse fetal/infant outcomes including death, prematurity and congenital malformations following treatment with heparin, oral anticoagulants, or both were calculated. The previously described high rate of adverse feta/infant outcomes with heparin- treated patients, could be accounted for by the frequent use of heparin in pregnancies with comorbid conditions independently associated with adverse outcomes and by reports of uncomplicated prematurity. After excluding such pregnancies, outcomes in heparin-treated patients are similar to the normal population.
The Relative Antithrombotic Effectiveness of Heparin, a Low Molecular Weight Heparin, and a Pentasaccharide Fragment in an Animal Model
Authors: Thomas D P , Merton R E , Gray E , Barrowcliffe T W
Published: June 2018

The antithrombotic efficacy of unfractionated heparin (UFH), a low molecular weight heparin (LMWH) and a synthetic pentasaccharide (PENTA) has been compared in an animal model for stasis thrombosis. We have also compared the relative ability of these three agents to impair thrombin generation in vitro and in vivo, and measured their effects on anti-Xa activity and thrombin clotting times. UFH, LMWH and PENTA all had the capacity to impair thrombogenesis, although there were marked differences in their relative effectiveness. Reduction of thrombin generation to 20% of control values was closely correlated with the prevention of thrombosis after 20 minutes’ stasis, but this was only achieved with UFH. The same dry weight dose of LMWH or PENTA reduced thrombin generation to about half control values, and neither significantly impaired thrombus formation after 20 minutes’ stasis. Impaired thrombin generation correlated better than anti-Xa activity with prevention of stasis thrombosis. In this model, UFH was clearly superior to LMWH and PENTA as an antithrombotic agent.
A Rapid and Simple Method for the Separation of Four Molecular Forms of Human Plasminogen
Authors: Nieuwenhuizen W , Traas D W
Published: June 2018

At least four molecular forms of plasminogen are known. Two of those forms have glutamic acid at their amino-terminal end, and are designated as glu-plasminogen. The other two have lysine, methionine and/or valine as amino-terminal amino acid and are collectively designated as lys-plasminogen. Two subforms (I and II) each of glu- and lys-plasminogen exist. The I-forms are glycosylated at asn-288 and thr-345, whereas the II-forms are only glycosylated at thr-345. In a previous publication (Thromb Haemostas 1984; 52: 347-349) we have described the separation of the I- and II-forms of plasminogen in lysine-Sepharose in phosphate buffers. Now we have combined those findings with the differential affinity of glu- and lys-plasminogen for aminohexyl-Sepharose through their aminohexyl-sites, recently described by Christensen (Biochem J 1984; 223: 431-421). Acid/urea electrophoresis, end-group determination and carbohydrate analysis show that the combination of affinity chromatography on lysine-Sepharose in phosphate buffers, and on aminohexyl-Sepharose provides an efficient procedure to separate the four molecular forms of plasminogen.
Affinity Purification of Plasma Proteins: Characterization of Six Affinity Matrices and their Application for the Isolation of Human Factor VIII
Authors: te Booy M P W M , Riethors W , Faber A , Over J , König B W
Published: June 2018

For the purification of coagulation factor VIII, (1,1’-carbonyldiimida zole [CDI] -activated) Sepharose CL-48 was functionalized with two aminoalkyl and four aminoalkyl-carbamylalkyl ligand- spacer combinations. The affinity matrices were contacted with human plasma. All affinity matrices showed complete adsorption of factor VIII (>90%) and three aminoalkyl-carbamylalkyl Sepharoses gave factor-VIII recoveries of 50-65% and a factor-VIII preparation with a specific activity of 1–2 U factor VIII/mg of protein. Furthermore, no fibrinogen, immunoglobulin G and albumin could be detected in the isolated factor VIII. Optimal results were obtained using the di-methyl-aminopropyl-carbamylpentyl-Sepharose affinity matrix.
In Vitro Carboxylation of a Blood Coagulation Factor IX Precursor Produced by Recombinant-DNA Technology
Authors: Soute Berry A M , Balland Alain , Faure Thérèse , de la Salle Henri , Vermeer Cees
Published: June 2018

Blood coagulation factor IX (Christmas factor) is a plasma protein which is required for normal haemostasis. A functional deficiency of factor IX results in haemophilia B, a bleeding disorder which is generally treated by infusions of factor IX concentrates prepared from pooled human plasma. The use of human blood products is connected with the risk of transmitting viral agents responsible for diseases such as hepatitis B and AIDS. Recombinant DNA techniques may provide the means to produce the required proteins without exposing the patients to these risks and at lower costs. One of the problems which has to be overcome before recombinant factor IX can be used for therapeutical purposes is related to the vitamin K-dependent carboxylation of its 12 NH2-terminal glutamate residues. In cell cultures this carboxylation, which is required to render the protein its procoagulant activity, is far from complete, especially at high expression levels. In this paper we describe the in vitro carboxylation of non and/or partly carboxylated recombinant factor IX produced by transformed Chinese hamster ovary cells. The identity of the newly formed Gla residues was verified and it could be demonstrated that all carboxyl groups had been incorporated into the recombinant factor IX.
A Prospective Study of Haemostatic Tests at 28 Weeks Gestation as Predictors of Pre-Eclampsia and Growth Retardation
Authors: Thornton J G , Molloy B J , Vinall P S , Philips P R , Hughes R , Davies J A
Published: June 2018

A panel of haemostatic tests was perfomed on 400 primiparous women at 28 weeks to test whether one or more could predict the development of pregnancy complications. Fifteen women subsequently developed pre-eclampsia with significant proteinuria and 13 delivered growth retarded infants. There were no significant differences between mothers in the pre-eclampsia group and 22 randomly selected controls. A stepwise logistic discriminant analysis of the data did not produce a significant model. In the growth retarded group only beta thromboglobulin levels were significantly lower than in the controls (p <0.05), although in the logistic discriminant analysis the inclusion of both beta thromboglobulin and fibrin degradation products led to a borderline significant improvement in fit of the model. We conclude that the haemostatic variables studied are not significantly changed at 28 weeks nor clinically useful predictors of either pre-eclampsia or fetal growth retardation.
Immunogold Electron-Microscopic Localization of Calpain I in Human Erythrocytes
Authors: Samis John A , Elce John S
Published: June 2018

With a view to understanding the function of calpain I (EC 3.4.22.17) in vivo, the localization of the enzyme was studied by immunoelectron microscopy in human erythrocytes. Thin sections of the cells embedded in Spurr’s resin were exposed to solutions of monoclonal anti-calpain I or control antibodies, biotin antimouse IgG antibodies, and streptavidin-gold. Most of the calpain I (93%) was found to be distributed throughout the cytoplasm, and only 7% of the gold particles were associated with the erythrocyte membrane. Erythrocytes were Ca2+-loaded by means of the calcium ionophore A23187, and the rise in intracellular [Ca2+] was demonstrated both by crenation of the cells, and by activation of calpain which was detected by immunoblotting. The proportions of cytosolic and membrane-bound gold labelling were, however not altered by Ca2+-loading. These results are not consistent with the hypothesis that activation of calpain requires membrane-binding.