bluuebunny/crossref_metadata_2025 huggingface dataset:
Mise à l’essai d’un dispositif renouvelé d’enseignement de l’orthographe auprès d’élèves d’un lycée professionnel en France
Authors: Maynard Catherine , Brissaud Catherine , Armand Françoise
Published: July 2018

L’apprentissage de l’orthographe française constitue une pierre d’achoppement importante chez les élèves (Manesse et Cogis, 2007). Les difficultés qu’ils rencontrent sont notamment dues à la polyvalence graphique du français et à sa morphologie silencieuse, ce qui se traduit par la présence de nombreuses lettres muettes (Fayol et Jaffré, 2008). Dans ce contexte, certains dispositifs, tels que les ateliers de négociation graphique (Haas et Maurel, 2009) et les dictées métacognitives (Nadeau et Fisher, 2014a), et certaines approches, telles qu’une approche intégrée d’enseignement de l’orthographe (Allal et al., 2001), semblent avoir un effet positif sur le développement de la compétence orthographique des élèves. Dans notre recherche, nous proposons de concevoir et de mettre à l’essai, auprès d’élèves d’un lycée professionnel en France, un dispositif d’intervention s’inspirant de ces interventions et adapté au profil des élèves de notre échantillon. Les effets de la mise en oeuvre du dispositif sur la compétence orthographique des élèves sont mesurés au moyen d’une dictée et d’une production écrite. Nous constatons ainsi que le dispositif expérimenté tend à favoriser le développement de la compétence orthographique des élèves, notamment pour ce qui est des accords adjectivaux et verbaux et de l’orthographe des suffixes adverbiaux en –ment. Des pistes d’adaptation du dispositif mis à l’essai sont suggérées.
Molecular Diagnostics in Pediatric Brain Tumors: Impact on Diagnosis and Clinical Decision-Making — A Selected Case Series
Authors: Bächli Heidi , Ecker Jonas , van Tilburg Cornelis , Sturm Dominik , Selt Florian , Sahm Felix , Koelsche Christian , Grund Kerstin , Sutter Christian , Pietsch Torsten , Witt Hendrik , Herold-Mende Christel , von Deimling Andreas , Jones David , Pfister Stefan , Witt Olaf , Milde Till
Published: July 2018

Central nervous system (CNS) tumors account for the highest mortality among pediatric malignancies. Accurate diagnosis is essential for optimal clinical management. The increasing use of molecular diagnostics has opened up novel possibilities for more precise classification of CNS tumors. We here report a single-institutional collection of pediatric CNS tumor cases that underwent a refinement or a change of diagnosis after completion of molecular analysis that affected clinical decision-making including the application of molecularly informed targeted therapies. 13 pediatric CNS tumors were analyzed by conventional histology, immunohistochemistry, and molecular diagnostics including DNA methylation profiling in 12 cases, DNA sequencing in 8 cases and RNA sequencing in 3 cases. 3 tumors had a refinement of diagnosis upon molecular testing, and 6 tumors underwent a change of diagnosis. Targeted therapy was initiated in 5 cases. An underlying cancer predisposition syndrome was detected in 5 cases. Although this case series, retrospective and not population based, has its limitations, insight can be gained regarding precision of diagnosis and clinical management of the patients in selected cases. Accuracy of diagnosis was improved in the cases presented here by the addition of molecular diagnostics, impacting clinical management of affected patients, both in the first-line as well as in the follow-up setting. This additional information may support the clinical decision making in the treatment of challenging pediatric CNS tumors. Prospective testing of the clinical value of molecular diagnostics is currently underway.
Endoscopic Ultrasound-Guided Drainage and Treatment of Symptomatic Pancreatic Fluid Collection following Acute or Acute-on-Chronic Pancreatitis – A Single Center Case Series
Authors: Kaczmarek Dominik , Nattermann Jacob , Strassburg Christian , Weismüller Tobias
Published: July 2018

Abstracts

Introduction Pancreatic fluid collection (PFC) is a common complication of acute pancreatitis. Endoscopic ultrasound (EUS)-guided drainage, which is often followed by direct endoscopic necrosectomy (DEN), has become the primary approach to treat PFC, including pancreatic pseudocysts (PP) and walled-off necrosis (WON). We aimed to determine retrospectively the short- and long-term results of patients treated in our endoscopy unit and to identify parameters that are associated with treatment efficacy and outcome.

Methods The data of 41 consecutive patients with post-pancreatitic PFC, who underwent endoscopic transmural intervention between 2014 and 2016, were analyzed retrospectively. After an initial EUS-guided puncture, one or more plastic stents were placed and DEN was performed if necrotic tissue remained.

Results The mean diameter of the PFC was 74.0 ± 4.8 mm. Of the PFCs, 29.3% were classified as PP and 70.7% as WON. Altogether, 196 transmural endoscopic procedures were performed, including 73 endoscopic necrosectomies in a subgroup of 21 patients (20 WON, 1 PP). Initial technical success was achieved in 97.6% of patients and the short-term clinical success rate was 90.2%. The long-term clinical success rate was 82.9%, since four patients died from septic shock and/or multiple organ failure and three patients developed recurrent PFC some months after the initial discharge from endoscopic treatment. Procedural complications were registered in 9 patients during 10 of 196 endoscopic procedures (5.1%): bleeding (6), cardiorespiratory insufficiency (2), perforation with pneumoperitoneum (1), aspiration with respiratory insufficiency (1), and non-perforating superficial damage of the gastric wall (1). Neither the size of the PFC nor the initial value of C-reactive protein (CRP) or other biochemical markers were correlated with efficacy or outcome of treatment. Only the cumulative number of days with CRP > 50 mg/L significantly correlated with the number of follow-up endoscopic sessions and DEN. Fungal colonization of PFC correlated significantly (p < 0.05) with the risk of mortality (44% vs. 0%), need for intensive care treatment (66.7% vs. 25%), and sepsis (55.6% vs. 12.5%).

Conclusions We confirm that EUS-guided drainage followed by DEN in patients with solid necrotic material is an effective and relatively safe therapeutic approach. Prolonged elevation of CRP and fungal colonisation of the PFC are associated with a worse course of the disease.
New Hirsutinolide-Type Sesquiterpenoids from Vernonia cinerea Inhibit Nitric Oxide Production in LPS-Stimulated RAW264.7 Cells
Authors: Kuo Li-Ming , Tseng Pei-Yi , Lin Yu-Chi , Liaw Chia-Ching , Zhang Li-Jie , Tsai Keng-Chang , Lin Zhi-Hu , Ho Hsiu-O , Kuo Yao-Haur
Published: July 2018

Phytochemical investigation of ethanol extracts from two Taiwanese collections of Vernonia cinerea resulted in the isolation of eighteen hirsutinolide-type sesquiterpenoids, including seven new ones designated as vernolides E – K (1–7). All structures were determined by a combination of detailed spectroscopic analyses (NMR and MS) and comparison with reported data. In an in vitro anti-inflammatory assay, compounds 3, 7, 9, 11, and 14 exhibited strong inhibitory activities toward NO production by LPS-induced RAW264.7 macrophages, with IC50 values of 1.18, 0.85, 0.66, 0.71 and 0.45 µM, respectively, without affecting cellular viability at 40 µM. Preliminary structure-activity relationships indicate that the ester groups at C-8 and C-13 may enhance inhibition of NO production.
Markers of Hypercoagulability in Patients with Hemophilia B Given Repeated, Large Doses of Factor IX Concentrates during and after Surgery
Authors: Santagostino E , Mannucci P M , Gringeri A , Tagariello G , Baudo F , Bauer K A , Rosenberg R D
Published: July 2018

Purer factor IX (FIX) concentrates have been produced for the treatment of hemophilia B in the attempt to reduce the risk of thrombotic complications associated with the use of prothrombin complex concentrates. To evaluate ex vivo whether or not FIX concentrates activate the coagulation system in conditions associated with a high risk for thrombosis, we measured markers of hypercoagulability in 10 patients with hemophilia B who underwent surgery, mainly orthopedic procedures, covered by multiple concentrate infusions (40-80 U/kg/day). Postinfusion plasma levels of prothrombin fragment 1+2 and factor X activation peptide did not differ significantly from the presurgical levels, neither before nor after each concentrate dose. Therefore, it appears that prolonged treatment of patients with hemophilia B undergoing high risk surgical procedures with high doses of FIX concentrate does not cause systemic activation of coagulation. This suggests that purified FIX concentrates are preferable to prothrombin complex concentrates for conditions associated with an increased risk of thrombosis.
Studies on the Fibrinolytic System in Human Plasma: Quantitative Determination of Plasminogen Activators and Proactivators
Authors: Kluft C
Published: July 2018

Effects due to plasma plasminogen activators and proactivators are usually studied in assay systems where inhibitors influence the activity and where the degree of activation of proactivators is unknown. Quantitative information on activator and proactivator levels in plasma is therefore not available

Studies on the precipitating and activating properties of dextran sulphate in euglobulin fractionation presented in this paper resulted in the preparation of a fraction in which there was optimal recovery and optimal activation of a number of plasminogen activators and proactivators from human plasma. The quantitative assay of these activators on plasminogen-rich fibrin plates required the addition of flufenamate to eliminate inhibitors. The response on the fibrin plates (lysed zones) could be coverted to arbitrary blood activator units (BAU). Consequently, a new activator assay which enables one to quantitatively determine the plasma level of plasminogen activators and proactivators together is introduced.

Two different contributions could be distinguished: an activity originating from extrinsic activator and one originating from intrinsic proactivators. The former could be assayed separately by means of its resistance to inhibition by Cl-inactivator. Considering the relative concentrations of extrinsic and intrinsic activators, an impression of the pattern of activator content in plasma was gained. In morning plasma with baseline levels of fibrinolysis, the amount of extrinsic activator was negligible as compared to the level of potentially active intrinsic activators. Consequently, the new assay nearly exclusively determines the level of intrinsic activators in morning plasma. A pilot study gave a fairly stable level of 100 ± 15 BAU/ml (n = 50). When fibrinolysis was stimulated by venous occlusion (15 min), the amount of extrinsic activator was greatly increased, reaching a total activator level of 249 ± 27 BAU/ml (n = 7).
The Effects of Compound 48/80 on the Fibrinolytic Activity of Dogs
Authors: Eyüboğlu Hughette , Akcasu Alâeddin
Published: July 2018

It has been demonstrated that histamine by itself, did play some role in the activation mechanism of fibrinolysis. In this particular work, it was demonstrated that histamine at doses not affecting blood pressure levels, did not exert any direct immediate effect on fibrinolytic activity.

Compound 48/80, a powerful mast cell depleter and histamine liberator, by itself, has no direct molecular effect on fibrinolytic activity, but rather, through the release of activators and/or inhibitors form mast cells, will exert either an activating or inhibiting action, according to the dose used. Low doses, such as 0.2 mg/kg i. v. will achieve a higher degree of activator release, while doses of 1.0 mg/kg will result in higher inhibitors release from mast cells.
Prevention of Intravascular Thrombus Formation on Plastic Catheters with Heparin-Benzalkonium Complex: in Vivo and in Vitro Studies
Authors: Evans Roger W , Pasmantier Mark W , Coleman Morton , Wagner Franklin S , Flair Robert C
Published: July 2018

A complex of heparin and a quaternary ammonium compound dissolved in an organic solvent was investigated for its potential ability to prevent clotting on the surface of plastic catheters. Despite the complexing of heparin to ammonium, anticoagulant activity remained on in vitro testing. Both the heparin complex and its organic solvent alone partially prevented catheter thrombus formation when tested in an in vivo animal model system. Neither, however, was totally effective in preventing thrombosis.
A in Vivo Assay for Standardizing Heparin Preparations
Authors: Pomeroy A R
Published: July 2018

The limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.

The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.

The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.
Correlation of Specific Platelet Activation Markers with Carotid Arterial Wall Thickness
Authors: Ghaddar Habib M , Cortes Jorge , Salomaa Veikko , Kark Jeremy D , Davis C Edward , Folsom Aaron R , Heiss Gerardo , Stinson Valarie , Wu Kenneth K ,   For the Atherosclerosis Risk in Communities Study Investigators
Published: July 2018

Many studies have shown inereased platelet activation in patients with coronary artery, cerebrovascular and peripheral vascular diseases. However, the temporal relationship between platelet activation and arterial atherosclerosis is unclear. To answer this basic question, we measured the plasma concentrations of two specific platelet activation markers, β-thromboglobulin (βTG) and platelet factor 4 (PF4) in 459 cases with increased carotid arterial wall thickness and 459 age-, sex- and race-matched controls selected from a cohort of 15,800 men and women, aged 45-64 who participated in the Atherosclerosis Risk in Communities Study. These participants had no acute vascular symptoms or known cardiovascular disease. The mean values of βTG and PF4 were significantly higher in cases than in controls. However, when analyzed by quartiles using conditional logistic regression, only βTG exhibited a significant association with carotid wall thickness, while PF4 did not. The odds ratio (OR) determined by multivariate logistic regression analysis was significantly higher for the uppermost quartile of βTG (OR=1.7, 95% Cl 1.1-2.5) compared to the lower 3 quartiles. This OR was 2.3 in white men (95% Cl 1.2-4.2), 1.4 in white women (95% Cl 0.6-3.0) and 1.0 in blacks (95% Cl 0.4-2.5).

This study indicates that plasma βTG may be useful as a marker for early atherosclerosis in middle aged adults, particularly in white men. It also suggests that platelet activation has an independent role in the pathogenesis of atherosclerosis, although the possibility that this may be a response to carotid atherosclerosis cannot be excluded.
Prolonged Expression of Procoagulant Activity of Human Platelets Degranulated by Thrombin
Authors: Kinlough-Rathbone Raelene L , Perry Dennis W
Published: July 2018

Platelets are exposed to thrombin when they take part in arterial thrombus formation, and they may return to the circulation when they are freed by fibrinolysis and dislodged by flowing blood. Thrombin causes the expression of procoagulant activity on platelets, and if this activity persists, the recirculating platelets may contribute to subsequent thrombosis. We have developed techniques to degranulate human platelets by treatment with thrombin, and recover them as single, discrete platelets that aggregate in response to both weak and strong agonists. In the present study we examined the duration of procoagulant activity on the surface of thrombin-degranulated platelets by two methods: a prothrombinase assay, and the binding of 125I-labeled annexin. Control platelets generated 0.9 ± 0.4 U thrombin per 107 platelets in 15 min. Suspensions of thrombin-degranulated platelets formed 5.4 ± 0.1 U thrombin per 107 platelets in this time. Binding of 125I-annexin V was also greater with thrombin-treated platelets than with control platelets (controls: 1.7 ±0.1 ng annexin/107 platelets; thrombin-degranulated platelets: 6.8 ± 0.2 ng annexin/107 platelets). With thrombin-degranulated platelets, increased procoagulant activity and annexin binding persisted for at least 4 h after degranulation and resuspension, indicating that the catalytic activity for the prothrombinase complex is not reversed during this time. These platelets maintained their ability to aggregate for 4 h, even in response to the weak agonist, ADP. Thus, platelets that have taken part in thrombus formation and returned to the circulation may contribute to the promotion of further thrombotic events because of the persistence of procoagulant activity on their surface.
Sod and GSH Inhibit the High Glucose-Induced Oxidative Damage and the PDGF Increased Secretion in Cultured Human Endothelial Cells
Authors: Curcio Francesco , Pegoraro Isabella , Russo Patrizia Dello , Falleti Edmondo , Perrella Giusepplna , Ceriello Antonio
Published: July 2018

Poor control of blood glucose has been established as a key pathogenetic mechanism in the vascular complications of diabetes. It has been reported that glucose may autooxidize generating free radicals which have been suggested to delay proliferation, to modify mobility, to influence platelet-derived growth factor and other secretory protein production in a variety of cell systems. Platelet-derived growth factor, in turn, may induce proliferation and migration of vascular smooth muscle cells and thus play a role in atherogenesis. In the present study the effects of antioxidants on the high glucose-dependent oxidative cell damage and increased platelet-derived growth factor secretion have been investigated using cultured human endothelial cells. Our findings show that rising the glucose concentration in the culture medium from 5 mM to 20 mM, increased the production of free radicals cell damage markers, such as malondialdehyde and conjugated dienes, as well as the production of platelet-derived growth factor. The addition of superoxide dismutase or glutathione prevents both such effects. These results suggest that antioxidants may be a helpful therapeutic adjuvant to reduce the vascular complications of diabetes.
Thrombin Prevents the Expression of Inducible Nitric Oxide Synthase in Vascular Smooth Muscle Cells by a Proteolytically-Activated Thrombin Receptor
Authors: Schini-Kerth Valérie B , Fißithaler Beate , Andersen Thomas T , Fenton John W , Vanhoutte Paul M , Busse Rudi
Published: July 2018

Proteolytically active forms of thrombin (α- and γ-thrombin) and thrombin receptor peptides inhibited the release of nitrite, a stable endproduct of nitric oxide, evoked by interleukin-1 β(IL-1 β) in cultured vascular smooth muscle cells while proteolytically inactive forms [D-Phe-Pro-Arg chloromethyl ketone-α-thrombin (PPACK-α- thrombin) and diisopropylphosphoryl-α-thrombin (DIP-α-thrombin)] had either no or only minimal inhibitory effects. Under bioassay conditions, perfusates from columns containing IL-1 β-activated vascular smooth muscle cells or cells treated with IL-1βplus PPACK-α-thrombin relaxed detector blood vessels. These relaxations were abolished by the inhibitor of nitric oxide synthesis, NG-nitro-L arginine. No relaxations were obtained with untreated cells or IL-1 β-treated cells in the presence of α-thrombin. The expression of inducible nitric oxide synthase mRNA and protein in vascular smooth muscle cells by IL-1 β was impaired by α-thrombin. These results demonstrate that thrombin regulates the expression of the inducible nitric oxide synthase at a transcriptional level via the proteolytic activation of the thrombin receptor in vascular smooth muscle cells
Sulfated Polysaccharide Anticoagulants Suppress Natural Killer Cell Activity In Vitro
Authors: Johann Sabine , Zoller Christine , Haas Sylvia , Blümel Günther , Lipp Martin , Förster Reinhold
Published: July 2018

In contrast to the well studied anticoagulant activity of sulfated polysaccharides (SPS), little is known about their influence on immune competent cells. Using two naturally derived SPSs (unfractionated heparin and low molecular weight heparin), one semi-synthetic SPS (pentosanpolysulfate), and one synthetic SPS (lactobionic acid) as well as the polypeptide hirudin we investigated the effect of these drugs on natural killer cell activity in vitro. We demonstrate that all SPSs tested significantly suppress the activity of natural killer cells at clinically relevant dosages. At all concentrations and at all effector:target ratios tested pentosanpolysulfate was the most potent natural killer cell inhibitor. In contrast, hirudin had no effect on natural killer cell function. Furthermore, scanning electron microscopy revealed that reduced natural killer cell activity is paralleled by decreased lymphocyte cell size and altered cell surface structures. Our results indicate that defined therapeutically applied SPSs can interfere with the tumor cell killing process.
Decreased Plasma Tissue Factor Pathway Inhibitor Activity in Ischemic Stroke Patients
Authors: Abumiya Takeo , Yamaguchi Takenori , Terasaki Tadashi , Kokawa Toshinori , Kario Kazuomi , Kato Hisao
Published: July 2018

Although tissue factor pathway inhibitor (TFPI) plays an essential role in the regulation of blood coagulation, the quantitative changes in its levels in thrombotic disease are still undefined. We compared TFPI activity in ischemic stroke patients and control subjects matched for age and cholesterol level to determine whether TFPI activity is changed in the disease. TFPI activity was significantly lower in the stroke patients (1.01 ± 0.24 U/ml) than in the control subjects (1.10 ± 0.16 U/ml). In relation to clinical subtypes of stroke, TFPI activity in atherothrombotic infarction (0.93 ± 0.19 U/ml) and lacunar infarction (0.99 ± 0.23 U/ml) was significantly lower than in the control subjects, whereas the level in cardioembolic infarction (1.16 ± 0.31 U/ml) was not. No relationship could be established between TFPI activity and other haemostatic parameters reflecting the production of thrombin/fibrin and the activation of fibrinolysis. These results may suggest that the moderately lower TFPI activity in stroke patients could be due to atherosclerotic changes rather than to consumptive coagulopathy.
The Effect of Desmopressin on Reducing Blood Loss in Cardiac Surgery – A Meta-Analysis of Double-Blind, Placebo-Controlled Trials
Authors: Cattaneo Marco , Harris Alan S , Strömberg Ulf , Mannucci Pier Mannuccio
Published: July 2018

The effect of desmopressin (DDAVP) on reducing postoperative blood loss after cardiac surgery has been studied in several randomized clinical trials, with conflicting outcomes. Since most trials had insufficient statistical power to detect true differences in blood loss, we performed a meta-analysis of data from relevant studies. Seventeen randomized, double-blind, placebo-controlled trials were analyzed, which included 1171 patients undergoing cardiac surgery for various indications; 579 of them were treated with desmopressin and 592 with placebo. Efficacy parameters were blood loss volumes and transfusion requirements. Desmopressin significantly reduced postoperative blood loss by 9%, but had no statistically significant effect on transfusion requirements. A subanalysis revealed that desmopressin had no protective effects in trials in which the mean blood loss in placebo-treated patients fell in the lower and middle thirds of distribution of blood losses (687-1108 ml/24 h). In contrast, in trials in which the mean blood loss in placebo-treated patients fell in the upper third of distribution (>1109 ml/24 h), desmopressin significantly decreased postoperative blood loss by 34%. Insufficient data were available to perform a sub-analysis on transfusion requirements. Therefore, desmopressin significantly reduces blood loss only in cardiac operations which induce excessive blood loss. Further studies are called to validate the results of this meta-analysis and to identify predictors of excessive blood loss after cardiac surgery.
Inhibition of Thrombin by Peptides Containing Lysyl-α-Keto Carbonyl Derivatives
Authors: Lewis Sidney D , Ng Assunta S , Lyle Elizabeth A , Mellott Michael J , Appleby Sandra D , Brady Stephen F , Stauffer Kenneth J , Sisko John T , Mao Shi-Shan , Veber Daniel F , Nutt Ruth F , Lynch Joseph J , Cook Jacquelynn J , Gardll Stephen J , Shafer Jules A
Published: July 2018

Several H-N-Me-D-Phe-Pro-Lysyl-α-keto carbonyl derivatives were shown to be potent thrombin inhibitors (Ki 0.2 to 27 nM). The inhibitory potencies of these compounds toward tissue plasminogen activator, plasmin and factor Xa were minimal; however, substantial cross-reactivity versus trypsin was observed (Ki values from 0.5 to 1500 nM). Inhibition of thrombin by α-keto carbonyl compounds appeared to occur via a one-step reversible reaction. The α-keto carbonyl inhibitors bound thrombin with a second order rate constant (k, 1–4 μM-1s-1) that was 10–100-fold slower than that expected for a diffusion-controlled reaction. Certain α-kelo earbonyl inhibitors were as potent (on a weight basis) as hirudin when evaluated in a rat arterial thrombosis model. The modest oral bioavailability (10–19%) in rats demonstrated for three of the α-keto carbonyl thrombin inhibitors suggests the possibility that α-keto amide containing thrombin inhibitors may have utility as orally-active antithrombotic agents.
Development of an ELISA for Autoantibodies to Prothrombin Showing their Prevalence in Patients with Lupus Anticoagulants
Authors: Arvieux J , Darnige L , Caron C , Reber G , Bensa J C , Colomb M G
Published: July 2018

Some lupus anticoagulants (LA) have been shown to be directed against phospholipid-bound prothrombin. While developing an ELISA to detect anti-prothrombin autoantibodies in patient serum or plasma, no or very low signal was observed using human prothrombin immobilized on plain polystyrene plates. In contrast, the same LA-positive samples bound specifically to prothrombin coated on γ-irradiated plates, depending on the radiation dose, in the absence of added calcium and phospholipid. Optimization of the assay required the addition of 0.1% Tween 20 to the buffers. Antibody specificity for immobilized prothrombin was ascertained by competition using liposome-bound prothrombin, since fluid-phase prothrombin competed poorly. Seventy-seven of 139 patients (55.4%) with LA related to a variety of underlying diseases possessed anti-prothrombin antibodies (27 IgG, 35 IgM and 15 both isotypes), either isolated or more often associated with anti-(β2 glycoprotein I (β2GPI) antibodies. These included 67-71% of the patients with systemic lupus erythematosus and related disorders, primary antiphospholipid antibody syndrome or drug-induced LA (autoimmune groups), but only 19-20% of those with infection or malignancy (p <0.001). As previously shown for anti-β2GPI antibodies, IgG2 was the predominant IgG subclass reactive with prothrombin. Thus, autoimmune patients with LA have a high incidence of antibodies to β2GPI and prothrombin, the binding of which could similarly require high antigen density and/or exposure of cryptic epitopes resulting from protein interaction with an irradiated (i. e. more anionic) polystyrene surface.
Ultrastructural Detection of Surface Exposed Phosphatidylserine on Activated Blood Platelets
Authors: Stuart M C A , Bevers E M , Comfurius P , Zwaal R F A , Reutelingsperger C P M , Frederik P M
Published: July 2018

Phosphatidylserine (PS) is normally restricted to the inner leaflet of the plasma membrane of cells (including blood platelets). Upon cell activation PS may become exposed to the outer surface of the cell. Cell membranes with surface exposed PS at the outside form a catalytic surface for coagulation reactions. When platelets are activated with ionophore or with thrombin in combination with thapsigargi, calcium induced scrambling of phospholipids takes place, resulting in PS exposure. Concomitant with PS exposition structural changes take place. On resting and activated platelets we combined the immunocytochemical detection of surface exposed PS with (ultra)structural information. Blood platelets were activated in the presence of annexin V, a protein which binds to PS in the presence of Ca2+. Annexin V was found to bind to lipid bilayers containing more than 5 mole % PS as estimated by binding of fluorescent-labelled annexin V to liposomes with varying PS concentrations. After vitrification, freeze-substitution and embedding of the platelets, annexin V was located on ultra thin sections, as detected by an anti-annexin V antibody and gold labelled protein A. Upon activation, the platelets show two different forms; irregular platelets with unchanged cytoplasm and round cells with apparently diluted cytoplasm. Activation with ionophore initially resulted in both forms, but after ten minutes only round platelets with diluted cytoplasm were observed. Both forms of these platelets as well as the microvesicles were found to be annexin V positive. However upon activation with thrombin in combination with thapsigargin, only the round cells with diluted cytoplasm and microvesicles were annexin V positive, whereas platelets with unchanged cytoplasm, even when microvesicles are present, are negative for annexin V.
Effects of Heparin and Hirudin on Thrombin Generation and Platelet Aggregation after Intrinsic Activation of Platelet Rich Plasma
Authors: Gallistl Siegfried , Muntean Wolfgang , Leis Hans Jörg
Published: July 2018

The effects of unfractionated heparin (UH) and recombinant hirudin (rH) on prothrombin activation, free thrombin generation, and platelet aggregation induced by endogenously generated thrombin after intrinsic activation of platelet rich plasma were compared. Free thrombin generation and platelet aggregation were assessed simultaneously by delaying fibrinogen polymerisation with GPRP.

UH more effectively inhibited prothrombin activation and free thrombin generation than rH. Increasing concentrations of rH had hardly any effect on the peak amount of free thrombin, while in the presence of 400 nM UH only traces of free thrombin were detected. Comparison of TAT and THC (thrombin-hirudin complex) generated until the onset of platelet aggregation on a molar basis showed that much more thrombin was inactivated in the presence of rH than in plasma containing UH. The explosive generation of free thrombin in hirudinized plasmas was accompanied by a markedly steeper aggregation curve as compared to heparinized plasmas. The generation of thromboxane B2 was markedly delayed in the presence of UH but not influenced in the presence of rH.

Our results suggest that UH is more effective than rH in inhibiting prothrombin activation after intrinsic activation of platelet rich plasma, while rH prevents clotting more by direct inactivation of already generated thrombin. The inability of even high concentrations of rH to prevent the explosive generation of free thrombin might contribute to the observed inefficiency of rH to inhibit platelet aggregation.
A Novel Nonsense Mutation Associated with an Exon Skipping in a Patient with Hereditary Protein S Deficiency Type I
Authors: Okamoto Yoshihiro , Yamazaki Tomio , Katsumi Akira , Kojima Tetsuhito , Takamatsu Junki , Nishida Mikio , Saito Hidehiko
Published: July 2018

The genetic defect in a patient with hereditary type I protein S (PS) deficiency was investigated. All the exons and intron-exon junctions of the patient’s PS gene were amplified by PCR and subjected to heteroduplex screening. Only the PCR product of exon 4 revealed heteroduplex bands. A novel nonsense mutation, Ser62 (TCA) to Stop (TGA) was found in exon 4. RT-PCR detected the aberrant mRNA in the patient’s platelets, which was markedly reduced in amount and lacked the region of exon 4, suggesting that the nonsense mutation affected the mutated mRNA metabolism and induced exon skipping. The skipping of exon 4 causes an in-frame deletion of 29 amino acids which just construct the thrombin-sensitive region of the PS molecule. The loss of such an important domain as well as the quantitative decrease in the mutated mRNA appear to be responsible for the type I PS deficiency in this patient.
Risk Factors for Bleeding during Treatment of Acute Venous Thromboembolism
Authors: Wester Jos P J , de Valk Harold W , Nieuwenhuis Karel H , Brouwer Catherine B , van der Graaf Yolanda , Meuwissen Otger J A Th , Hart Herman C , Sixma Jan J , Dirk Banga Jan
Published: July 2018

Objective: Identification of risk factors for bleeding and prospective evaluation of two bleeding risk scores in the treatment of acute venous thromboembolism.

Design: Secondary analysis of a prospective, randomized, assessor-blind, multicenter clinical trial.

Setting: One university and 2 regional teaching hospitals.

Patients: 188 patients treated with heparin or danaparoid for acute venous thromboembolism.

Measurements: The presenting clinical features, the doses of the drugs, and the anticoagulant responses were analyzed using univariate and multivariate logistic regression analysis in order to evaluate prognostic factors for bleeding. In addition, the recently developed Utrecht bleeding risk score and Landefeld bleeding risk index were evaluated prospectively.

Results: Major bleeding occurred in 4 patients (2.1%) and minor bleeding in 101 patients (53.7%). For all (major and minor combined) bleeding, body surface area ≤2 m2 (odds ratio 2.3, 95% Cl 1.2-4.4; p = 0.01), and malignancy (odds ratio 2.4, 95% Cl 1.1-4.9; p = 0.02) were confirmed to be independent risk factors. An increased treatment-related risk of bleeding was observed in patients treated with high doses of heparin, independent of the concomitant activated partial thromboplastin time ratios. Both bleeding risk scores had low diagnostic value for bleeding in this sample of mainly minor bleeders.

Conclusions: A small body surface area and malignancy were associated with a higher frequency of bleeding. The bleeding risk scores merely offer the clinician a general estimation of the risk of bleeding. In patients with a small body surface area or in patients with malignancy, it may be of interest to study whether limited dose reduction of the anticoagulant drug may cause less bleeding without affecting efficacy.
More Effective Suppression of Hemostatic System Activation in Patients Undergoing Cardiac Surgery by Heparin Dosing Based on Heparin Blood Concentrations rather than ACT
Authors: Despotis George J , Heinrich Joist J , Hogue Charles W , Alsoufiev Alexander , Joiner-Maier D , Santoro Samuel A , Spitznagel Edward , Weitz Jeffrey I , Goodnough Lawrence T
Published: July 2018

This study was designed to determine whether the maintenance of higher than usual patient-specific heparin concentrations during cardiopulmonary bypass (CPB) was associated with more effective suppression of hemostasis system activation.

Thirty-one patients scheduled for repeat cardiac sugery or combined procedures (i.e., coronary revascularization + valve repair/replacement) were consented and enrolled in this study. All patients received porcine heparin and protamine and were randomly assigned to monitoring of anticoagulation by either celite ACT alone (Control, n = 16) or by kaolin ACT combined with on-site measurements of whole blood heparin concentration (Intervention, n = 15). Blood specimens collected before administration of heparin, before weaning from CPB and after administration of protamine were analyzed with a battery of coagulation assays.

Patients in the intervention cohort received appreciably greater heparin doses than control patients, resulting in higher anti-Xa heparin levels at the end of CPB. Fibrinopeptide A and D-dimer levels were higher in the control group before discontinuation of CPB. Percent decrease during CPB were greater in the control group for factors V and VIII, fibrinogen and antithrombin III. Percent decrease in complement 3 was greater in the control group after protamine and bleeding times measured in the Intensive Care Unit were significantly more prolonged in this group.

Maintenance of higher patient-specific heparin concentrations during CPB more effectively suppresses excessive hemostatic system activation than do standard heparin doses chosen based on measurement of ACT. These findings may explain, at least in part, the significant reduction in perioperative blood loss and blood product use when higher heparin concentrations are maintained.
Pathogenetic Analysis of Five Cases with a Platelet Disorder Characterized by the Absence of Thromboxane A2(TXA2)-Induced Platelet Aggregation in Spite of Normal TXA2 Binding Activity
Authors: Fuse Ichiro , Hattori Akira , Mito Masao , Higuchi Wataru , Yahata Kazuaki , Shibata Akira , Aizawa Yoshifusa
Published: July 2018

Five patients with mild bleeding tendencies characterized by defective thromboxane A2 (TXA2)-induced platelet aggregation are reported. The platelets of all the patients had the ability to bind exogenous TXA2. Bleeding time was markedly prolonged in one patient. In three of the five patients, synthetic TXA2 mimetic (STA2)-induced platelet responses, including IP3 formation, Ca2+ mobilization, phosphatidic acid formation and GTPase activities were selectively defective, suggesting impaired coupling between the TXA2 receptor and phospholipase C activation. However, in the remaining two patients, these responses were all within normal limits. This suggests that the defective site of this type of platelet disorder is heterogenous and that signaling mechanisms other than the TXA2 receptor-phospholipase C pathway are also involved in TXA2-induced platelet aggregation.
Autoimmune Antiphospholipid Antibodies Are Directed against a Cryptic Epitope Expressed when β2-Glycoprotein I Is Bound to a Suitable Surface
Authors: Pengo Vittorio , Biasiolo Alessandra , Fior Maria Grazia
Published: July 2018

The antiphospholipid antibodies (aPL) present in autoimmune disorders are associated with thromboembolic episodes, and their binding to phospholipids (PL) is mediated by a plasma cofactor, β2-glycoprotein I (β2GPI). Both PL and β2GPI seem necessary for binding, thus indicating that the two components comprise the epitope against which aPL are directed. Using an anti-β2GPI antibody ELISA with the antigen adsorbed onto polyvinylchloride (PVC) plates, we detected high antibody titres in 12 out of 12 plasmas from patients with the antiphospholipid syndrome. No or very low positivity was obtained when the same ELISA was carried out in polystyrene (PST) plates, while an increasing positivity was found when processed (i.e. more hydrophilic) or COOH-surface PST plates were used. When (β2GPI dependent IgG-aPL were purified using agarose-immobilized cardio- lipin, 4 out of 4 preparations were highly positive in anti-β2GPI antibody ELISA using PVC plates, while β2GPI was not fully recognized by aPL-IgG when adsorbed onto PST plates. These findings demonstrate that aPL are, in fact, anti-β2GPI antibodies directed against a cryptic epitope which is expressed when β2GPI is bound to anionic phospholipid, or another suitable surface.
Tissue Factor Activity of Syncytiotrophoblast Plasma Membranes and Tumoral Trophoblast Cells in Culture
Authors: Reverdiau P , Jarousseau A C , Thibault G , Khalfoun B , Watier H , Lebranchu Y , Bardos P , Gruel Y
Published: July 2018

During pregnancy, important modifications of hemostasis occur resulting in mothers in hypercoagulability and the role of placental cells such as trophoblast cells has been hypothesized. In this study, we first showed that syncytiotrophoblast plasma membranes, isolated from normal human placenta, expressed a strong tissue factor (TF) activity. We then studied TF activity of two continuous trophoblast cell lines (JEG-3 and BeWo) in comparison to human umbilical vein endothelial cells (HUVEC) and transformed human endothelial cells (ECV-304). TF assays were performed on intact detached confluent cells. Unstimulated JEG-3 and BeWo cells exhibited a very high TF activity which slightly increased after 2 to 4 h TNF-α stimulation. In contrast, HUVEC and ECV-304 had a lower basal TF activity which was mainly inducible by TNF-a, with a maximum effect after 4 to 6 h stimulation. For both cell types, TF activity was decreased to basal value after 16-hour TNF-α stimulation. These results support that trophoblast cells are able to express TF but the involvement of this property in the hemostatic physiological changes observed during pregnancy, remains to be demonstrated.
The APTT Monitoring of Heparin - The ISTH/ICSH Collaborative Study
Authors: Poller L , Van der Velde E A
Published: July 2018

In an international collaborative study three candidate reference and the local APTT reagent have been tested with lyophilized plasmas containing heparin added in vitro and fresh plasmas from heparinized patients to determine whether it was possible to standardize APTT heparin monitoring.

The APTT responses of patients with thrombosis were much less to the same concentrations of heparin so only these plasmas were used to calibrate the reagents.

A calibration constant (b) was determined for each local APTT system from the orthogonal regression slope of the patients’ and normals’ plasmas. Although the calibration was based on relatively limited numbers of patients’ plasmas the results gave no consistent indication that this method of analysis with the reference preparations was incorrect. Two of the three candidate reference reagents performed almost equally well and one of these reagents is recommended as the APTT reference preparation. Use of a single b value for a brand of reagent for all laboratories is not advised. Each laboratory must perform its own local calibration. The adoption of a reference APTT reagent together with the use of orthogonal regression analysis would be a useful step to initiate APTT heparin monitoring standardization and to develop safe and effective local therapeutic ranges.
Desmin 370, a Low Molecular Weight Dermatan Sulfate, Reduces the Weight of Preformed Thrombi in Rats Made Afibrinogenemic by Ancrod
Authors: Barbanti Miriam , Calanni Fiorella , Marchi Egidio , Semeraro Nicola , Colucci Mario
Published: July 2018

Desmin 370 (D370), a low molecular weight dermatan sulfate, has been shown to induce a marked reduction of the weight of preformed venous thrombi in rats and rabbits by mechanisms that appeared largely independent of inhibition of thrombus accretion. In order to provide further support for such a mechanism, we exploited the defibrinating capacity of ancrod to obtain a thrombosis model characterized by the lack of thrombus growth and thus sensitive only to agents promoting thrombus lysis. Thrombus formation in anesthetized rats was induced by vena cava ligature. Injection of ancrod (5 U/kg) 5 h after induction of venous stasis caused a more than 95% reduction in plasma fibrinogen and prevented thrombus accretion as indicated by the lack of thrombus weight increase during the 3h experimental period (12.2 ° 0.6 vs 14.5 ° 1 as compared to 12.6 ° 0.6 vs 19.6 ° 0.8, p <0.01, in control rats) and by the almost complete (>90%) inhibition of125I- fibrin(ogen) binding to thrombi. Moreover, when ancrod was given 1 h before vena cava ligature, no thrombi were formed within 2 h whereas at the same time interval visible thrombi were present in all control rats. Administration of D370 (10 mg/kg) to thrombus bearing rats, 1 h after induction of afibrinogenemia, resulted in a significant reduction in thrombus weight (43% after 2h, p <0.01) which was only slightly lower than that recorded in normofibrinogenemic rats (54%). Enhancement of plasma fibrinolytic activity by ancrod had no influence on thrombus lysis and was not at all affected by administration of D370. These data provide additional and more direct evidence that D370 may promote thrombus lysis independently of inhibition of thrombus accretion.
Fibrin Polymerization Defect in HIV-infected Patients-Evidence for a Critical Role of Albumin in the Prolongation of Thrombin and Reptilase Clotting Times
Authors: Toulon Pierre , Frere Elyane , Bachmeyer Claude , Candia Nathalie , Blanche Philippe , Sereni Daniel , Sicard Didier
Published: July 2018

Thrombin clotting time (TCT) and reptilase clotting time (RCT) were found significantly prolonged in a series of 72 HIV-infected patients drawn for routine coagulation testing. Both TCT and RCT were highly significantly correlated with albumin (r = -0.64, and r = -0.73 respectively, p<0.0001). TCT and RCT were significantly higher (p<0.0001) in a series of 30 other HIV-infected patients selected on their albumin level below 30.0 g/l (group l) than in 30 HIV-infected patients with albumin level above 40.0 g/l or in 30 HIV-negative controls; the two latter groups were not different. In vitro supplementation of plasma from group 1 patients with purified human albumin up to 45.0 g/l (final concentration) lead to a dramatic shortening effect on both TCT and RCT, which reached normal values. The TCT and RCT of the purified fibrinogen solutions (2.0 g/l final concentration) were not different in the three groups, and normal polymerization curves were obtained in all cases. This further ruled out the presence of any dysfibrinogenemia in the plasma from group 1 patients. Using purified proteins, highly significant correlations were demonstrated between the albumin concentration and the prolongations of both TCT and RCT, which were of the same magnitude order than those found in the patients plasma. These results suggest that hypo-albuminemia is responsible for the acquired fibrin polymerization defect reported in HIV-infected patients. The pathophysiological implication of the low albumin levels was suggested by the finding of decreased albumin levels (associated with prolonged TCT and RCT) in a small series of the eight HIV-infected patients who developed thrombotic complications.
One Month Follow-up of Haemostatic Variables in Patients Undergoing Aortocoronary Bypass Surgery
Authors: Mannucci L , Gerometta P S , Mussoni L , Antona C , Parolari A , Salvi L , Biglioli P , Tremoli E
Published: July 2018

It is already known that activation of the coagulation and fibrinolytic system occurs in patients undergoing cardiopulmonary bypass (CPB). We have thus studied twenty patients (10 treated with aprotinin during CPB and 10 untreated) both during the intraoperative period and during thirty days follow up. In untreated patients D-dimer levels increased 4-fold during CPB and the levels were above baseline for the whole follow up (p<0.0001). D-dimer levels were reduced in aprotinin treated patients in comparison to untreated patients (p = 0.0172); levels then gradually increased to the values of the untreated patients over the following 24 h later and remained higher during the thirty day follow up. The behavior of haemostatic variables in the 24 h after CPB did not vary between untreated and aprotinin treated patients. In particular, five minutes after protamine sulphate administration, levels of F1 + 2 and TAT rose significantly (p = 0.0054, p = 0.0022 respectively), whereas fibrinogen significantly decreased (p<0.0001) and PAI-1 antigen levels were reduced. Two days after CPB the concentrations of F1 + 2 and TAT lowered, whereas fibrinogen and PAI-1 antigen levels increased. On the 5th, 8th and 30th days after CPB, F1 + 2 and TAT levels remained higher than those reported at baseline in both groups of patients, whereas fibrinogen levels increased over basal levels in aprotinin treated patients only.

Thus, in addition to the activation of the coagulation and fibrinolytic system occurring during the intraoperative period, in patients undergoing CPB, there are alterations of haemostatic variables up to thirty days from surgery.