bluuebunny/crossref_metadata_2025 huggingface dataset:
Engineering biofunctional in vitro vessel models using a multilayer bioprinting technique
Authors: Schöneberg Jan , De Lorenzi Federica , Theek Benjamin , Blaeser Andreas , Rommel Dirk , Kuehne Alexander J. C. , Kießling Fabian , Fischer Horst
Published: July 2018

Recent advances in the field of bioprinting have led to the development of perfusable complex structures. However, most of the existing printed vascular channels lack the composition or key structural and physiological features of natural blood vessels or they make use of more easily printable but less biocompatible hydrogels. Here, we use a drop-on-demand bioprinting technique to generate in vitro blood vessel models, consisting of a continuous endothelium imitating the tunica intima, an elastic smooth muscle cell layer mimicking the tunica media, and a surrounding fibrous and collagenous matrix of fibroblasts mimicking the tunica adventitia. These vessel models with a wall thickness of up to 425 µm and a diameter of about 1 mm were dynamically cultivated in fluidic bioreactors for up to three weeks under physiological flow conditions. High cell viability (>83%) after printing and the expression of VE-Cadherin, smooth muscle actin, and collagen IV were observed throughout the cultivation period. It can be concluded that the proposed novel technique is suitable to achieve perfusable vessel models with a biofunctional multilayer wall composition. Such structures hold potential for the creation of more physiologically relevant in vitro disease models suitable especially as platforms for the pre-screening of drugs.
Repeated social defeat stress impairs attentional set shifting irrespective of social avoidance and increases female preference associated with heightened anxiety
Authors: Higashida Shu , Nagai Hirotaka , Nakayama Kazuki , Shinohara Ryota , Taniguchi Masayuki , Nagai Midori , Hikida Takatoshi , Yawata Satoshi , Ago Yukio , Kitaoka Shiho , Narumiya Shuh , Furuyashiki Tomoyuki
Published: July 2018

Repeated social defeat stress (R-SDS) induces multiple behavioral changes in mice. However, the relationships between these behavioral changes were not fully understood. In the first experiment, to examine how the social avoidance is related to R-SDS-impaired behavioral flexibility, 10-week-old male C57BL/6N mice received R-SDS followed by the social interaction test and the attentional set shifting task. R-SDS impaired attentional set shifting irrespective of the development of social avoidance. In the second experiment, to examine whether R-SDS affects sexual preference and how this behavioral change is related to the social avoidance and R-SDS-heightened anxiety, another group of 10-week-old male C57BL/6N mice were subjected to R-SDS followed by the social interaction test, the female encounter test and the elevated plus maze test. The anxiety was heightened in the defeated mice without social avoidance, but not in those which showed social avoidance. Furthermore, female preference was increased specifically in the defeated mice which showed heightened anxiety, but was not related to the level of social avoidance. Together, these results showed that attentional set shifting is more sensitive to R-SDS than social interaction, and that female preference is affected by R-SDS in association with heightened anxiety rather than the social avoidance.
Case study: patient-derived clear cell adenocarcinoma xenograft model longitudinally predicts treatment response
Authors: Vargas Roberto , Gopal Priyanka , Kuzmishin Gwendolyn B. , DeBernardo Robert , Koyfman Shlomo A. , Jha Babal K. , Mian Omar Y. , Scott Jacob , Adams Drew J. , Peacock Craig D. , Abazeed Mohamed E.
Published: July 2018

There has been little progress in the use of patient-derived xenografts (PDX) to guide individual therapeutic strategies. In part, this can be attributed to the operational challenges of effecting successful engraftment and testing multiple candidate drugs in a clinically workable timeframe. It also remains unclear whether the ancestral tumor will evolve along similar evolutionary trajectories in its human and rodent hosts in response to similar selective pressures (i.e., drugs). Herein, we combine a metastatic clear cell adenocarcinoma PDX with a timely 3 mouse x 1 drug experimental design, followed by a co-clinical trial to longitudinally guide a patient’s care. Using this approach, we accurately predict response to first- and second-line therapies in so far as tumor response in mice correlated with the patient’s clinical response to first-line therapy (gemcitabine/nivolumab), development of resistance and response to second-line therapy (paclitaxel/neratinib) before these events were observed in the patient. Treatment resistance to first-line therapy in the PDX is coincident with biologically relevant changes in gene and gene set expression, including upregulation of phase I/II drug metabolism (CYP2C18, UGT2A, and ATP2A1) and DNA interstrand cross-link repair (i.e., XPA, FANCE, FANCG, and FANCL) genes. A total of 5.3% of our engrafted PDX collection is established within 2 weeks of implantation, suggesting our experimental designs can be broadened to other cancers. These findings could have significant implications for PDX-based avatars of aggressive human cancers.
Halo-methylphenyl substituted neutral tripodal receptors for cation-assisted encapsulation of anionic guests of varied dimensionality
Authors: Manna Utsab , Das Gopal
Published: July 2018

Two neutral tris([(halo-methylphenyl)amino]ethyl)-urea receptors with asymmetric electronic aryl-functionalization have been developed for encapsulation of anionic guests of variable size.
Sub-molecular spectroscopy and temporary molecular charging of Ni-phthalocyanine on graphene with STM
Authors: Zhao Mali , Almarzouqi Faisal , Duverger Eric , Sonnet Philippe , Dujardin Gérald , Mayne Andrew J.
Published: July 2018

In this study, the self-assembled molecular network and electronic properties of Ni-phthalocyanine (NiPc) molecules on monolayer graphene (MLG)/6H-SiC(0001) were studied by room temperature Scanning Tunnelling Microscopy (STM) and Density Functional Theory (DFT) calculations.
Principal component analysis for automatic extraction of solid-state kinetics from combined in situ experiments
Authors: Guccione Pietro , Palin Luca , Belviso Benny Danilo , Milanesio Marco , Caliandro Rocco
Published: July 2018

A new algorithm to extract in an automatic way kinetic parameters from a set of measurements from in situ experiments is presented and applied to X-ray powder diffraction and Raman spectroscopy.
Unveiling the multifunctional roles of hitherto known capping ligand oleic acid as blue emitter and sensitizer in tuning the emission colour to white in red-emitting phosphors
Authors: Sekar S. , Muller J. George , Karthikeyan J. , Murugan P. , Lakshminarasimhan N.
Published: July 2018

Multifunctional roles of oleic acid as capping ligand, blue emitter and sensitizer are revealed in obtaining white light emission from Eu3+-based red phosphors.
Correction: Activating molecular oxygen by Au/ZnO to selectively oxidize glycerol to dihydroxyacetone
Authors: Meng Ye , Zou Shihui , Zhou Yuheng , Yi Wuzhong , Yan Yang , Ye Bin , Xiao Liping , Liu Juanjuan , Kobayashi Hisayoshi , Fan Jie
Published: July 2018

Correction for ‘Activating molecular oxygen by Au/ZnO to selectively oxidize glycerol to dihydroxyacetone’ by Ye Meng et al., Catal. Sci. Technol., 2018, 8, 2524–2528.
Retention and multiphase transformation of selenium oxyanions during the formation of magnetite via iron(ii) hydroxide and green rust
Authors: Börsig Nicolas , Scheinost Andreas C. , Shaw Samuel , Schild Dieter , Neumann Thomas
Published: July 2018

This article provides new insights into selenite and selenate reduction chemistry and immobilization mechanisms in the presence of Fe(ii) and O2.
Ruthenium–indolizinone complexes as a new class of metalated heterocyclic compounds: insight into unconventional alkyne activation pathways, revelation of unexpected electronic properties and exploration of medicinal application
Authors: Chung Lai-Hon , Ng Sze-Wing , Yeung Chi-Fung , Shek Hau-Lam , Tse Sheung-Ying , Lo Hoi-Shing , Chan Siu-Chung , Tse Man-Kit , Yiu Shek-Man , Wong Chun-Yuen
Published: July 2018

The first examples of metal–indolizinone complexes prepared by Ru-assisted activation of pyridine-tethered alkynes exhibit moderate cytotoxicity against several human cancer cell lines.
The promise of antireflective gold electrodes for optically monitoring the electro-deposition of single silver nanoparticles
Authors: Lemineur Jean-François , Noël Jean-Marc , Combellas Catherine , Ausserré Dominique , Kanoufi Frédéric
Published: July 2018

Combined to electrochemical actuation, it allows the dynamic in situ visualization of the electrochemical growth and dissolution of individual Ag nanoparticles.
Mannan-oligosaccharide modulates the obesity and gut microbiota in high-fat diet-fed mice
Authors: Wang Hongshan , Zhang Xiaojuan , Wang Shanshan , Li Heng , Lu Zhenming , Shi Jinsong , Xu Zhenghong
Published: July 2018

MOS attenuates HFD induced obesity, affects MS parameters, modulates the gut microbiota and reverses species-level microbial changes caused by HFD.
Recent advances in synthetic methodologies for transition metal-free Ullmann condensation reactions
Authors: Pant Preeti Lalit , Shankarling Ganapati S.
Published: July 2018

Different methodologies for transition metal-free Ullmann condensation reactions.
Stabilisation of small mono- and bimetallic gold–silver nanoparticles using calix[8]arene derivatives
Authors: Ray Priyanka , Clément Marie , Martini Cyril , Abdellah Ibrahim , Beaunier Patricia , Rodriguez-Lopez José-Luis , Huc Vincent , Remita Hynd , Lampre Isabelle
Published: July 2018

We report a facile approach to synthesise small Au–Ag alloyed nanoparticles using a new cali[8]arene derivative as a stabiliser.
Unraveling the discrepancies in size dependence of hardness and thermal stability in crystalline/amorphous nanostructured multilayers: Cu/Cu–Ti vs. Cu/HfO2
Authors: Yue T. , Wang Y. Q. , Zhang J. Y. , Wu K. , Li G. , Kuang J. , Liu G. , Sun J.
Published: July 2018

Compared with the as-deposited crystalline/amorphous Cu/X (X = Cu-Ti, HfO2) multilayers, the Cu/Cu-Ti samples manifest annealing hardening, while the Cu/HfO2 samples manifest annealing softening.
Phase-selective synthesis of self-supported RuP films for efficient hydrogen evolution electrocatalysis in alkaline media
Authors: Ge Ruixiang , Wang Shuo , Su Jianwei , Dong Yan , Lin Yichao , Zhang Qiuju , Chen Liang
Published: July 2018

Phase-selective synthesis of RuP/CC enables superior HER activity with an overpotential of 13 mV at 10 mA cm−2 in 1.0 M KOH.
Efficient syntheses and anti-cancer activity of xenortides A–D including ent/epi-stereoisomers
Authors: Esmati N. , Maddirala A. R. , Hussein N. , Amawi H. , Tiwari A. K. , Andreana P. R.
Published: July 2018

One-pot, two-step, total synthesis of naturally occurring xenortides A, B, C and D (Xens A–D).
Cyclotrimerization of phenylacetylene catalyzed by a cobalt half-sandwich complex embedded in an engineered variant of transmembrane protein FhuA
Authors: Thiel A. , Sauer D. F. , Mertens M. A. S. , Polen T. , Chen H.-H. , Schwaneberg U. , Okuda J.
Published: July 2018

An (η5-cyclopentadienyl)cobalt(i) complex was covalently incorporated in an engineered variant of the β-barrel protein FhuA. The new biohydrid catalyst cyclotrimerized phenylacetylene to give regioisomeric triphenylbenzenes.
Cationic and hydrolysable branched polymers by RAFT for complexation and controlled release of dsRNA
Authors: Cook Alexander B. , Peltier Raoul , Hartlieb Matthias , Whitfield Richard , Moriceau Guillaume , Burns James A. , Haddleton David M. , Perrier Sébastien
Published: July 2018

The complexation and sustained release of dsRNA from highly branched polymers prepared via RAFT polymerisation and copolymerisation of the monomers DMAEA, DMAPA, and DMAEMA, is reported.
Using 3D printed physical models to monitor knowledge integration in biochemistry
Authors: Babilonia-Rosa Melissa A. , Kuo H. Kenny , Oliver-Hoyo Maria T.
Published: July 2018

Noncovalent interactions determine the three-dimensional structure of macromolecules and the binding interactions between molecules. Students struggle to understand noncovalent interactions and how they relate to structure–function relationships. Additionally, students’ difficulties translating from two-dimensional representations to three-dimensional representations add another layer of complexity found in macromolecules. Therefore, we developed instructional resources that use 3D physical models to target student understanding of noncovalent interactions of small molecules and macromolecules. To this effect, we monitored indicators of knowledge integration as evidenced in student-generated drawings. Analysis of the drawings revealed that students were able to incorporate relevant conceptual features into their drawings from different sources as well as present their understanding from different perspectives.
Prediction of disulfide dihedral angles using chemical shifts
Authors: Armstrong David A. , Kaas Quentin , Rosengren K. Johan
Published: July 2018

Chemical shifts can be used to predict the conformation of disulfide bonds, greatly improving resolution of solution NMR structures.
Definition, Classification and Diagnosis of Diabetes Mellitus
Authors: Petersmann Astrid , Nauck Matthias , Müller-Wieland Dirk , Kerner Wolfgang , Müller Ulrich , Landgraf Rüdiger , Freckmann Guido , Heinemann Lutz
Published: July 2018

Aim of recommendations like this one issued by the German Diabetes Association is to provide the GP and diabetologist and his team with information he needs for his daily practice. These recommendations are updated annually. They are written by a group of experts, but they are not evidence based guidelines. This specific recommendation for diabetes diagnosis briefly describes the diabetes types and the different options for diagnosis. Also the caveats and the practical procedure are presented.
Use of Ultrasound in the Diagnostic Work-Up of Adult Intussusception – A Multicenter Retrospective Analysis
Authors: Klinger Christoph , Riecken Bettina , Dietrich Christoph Frank , Dirks Klaus , Caca Karel , Fröhlich Eckhart
Published: July 2018

Purpose To evaluate the use of ultrasound (US) in the diagnostic work-up of adult intussusception (AI).

Methods This multicenter study includes 26 consecutive patients diagnosed with AI between January 2010 and November 2017. A retrospective chart analysis was conducted with a focus on abdominal US findings and diagnostic accuracy of different imaging modalities (ultrasound, computed tomography (CT), magnetic resonance imaging). If available, surgical and pathological findings served as the gold standard (76.9 %). US examiners certified according to DEGUM grade 2 or 3 were classified as experts. Otherwise, they were regarded to have basic skills.

Results During diagnostic work-up, 92.3 % underwent abdominal US. US was the first-line imaging modality in 88.5 % of cases. The accuracy regarding the detection of AI (85 %), correct localization (95 %) and detection of complications (100 %) was excellent and comparable with CT (81 %, 90.5 %, and 91.7 %) when performed by experts. 72.7 % of tumorous lead points were detected by experienced examiners. In contrast, AI was detected in only 45.5 % of cases by examiners with basic skills. AI was diagnosed prior to surgery in all patients.

Conclusion US is reliable in the diagnostic work-up of AI when performed by experienced examiners with high-quality equipment. US, CT and MRI should be used in a complementary fashion since combination provides excellent sensitivity regarding the detection and correct localization of AI as well as the detection of complications. The impact of real-time imaging is illustrated by supplementary videos.
A Novel Study Design to Systematically Explore the Impact of Trial Methodology on Psychopharmacological Treatment Outcome in Patients with Depression
Authors: Severus Emanuel , Sauer Cathrin , Bauer Michael , Ostacher Michael , Anghelescu Ion-George
Published: July 2018

Introduction Randomized, double-blind, placebo-controlled trials were developed to draw rather unbiased conclusions regarding the efficacy of antidepressants in the treatment of a major depressive episode (internal validity), mostly with the purpose of formal approval of new compounds in this indication. However, at the same time, data suggest that the very process of randomization and blinded administrations of placebo will have a significant impact on the efficacy of the antidepressant tested and therefore may limit the external validity of results obtained from this type of studies. Therefore, there is an urgent need to systematically study the impact of randomization/placebo control/blinding on patient population, efficacy, tolerability, and external validity in the psychopharmacological treatment of patients with a major depressive episode.

Methods To develop a study design that allows the systematic exploration of the impact of trial design on characteristics of included patient population and outcome.

Results We propose a study design including sample size calculation and statistical analysis in which patients with a major depressive episode are randomized to 3 distinct study designs that differ with regard to control, randomization, and blindness.

Discussion The results of the proposed study design may have substantial consequences when it comes to how to best interpret the results of traditional randomized, double-blind, placebo-controlled trials in the acute treatment of major depressive disorder. Furthermore, they may lead to the implementation of new study designs that may be more suitable for assessing the effectiveness of new antidepressant compounds in everyday clinical practice.
Palladium-Catalyzed Asymmetric 1,4-Addition of Diarylphosphines to α,β-Unsaturated Sulfonamides
Authors: Xiao Fanhua , Duan Wei-Liang , Dai Guo-Fa , Song Yu-Chuan
Published: July 2018

A pincer palladium-catalyzed asymmetric 1,4-addition of diarylphosphines to α,β-unsaturated sulfonamides was realized for the synthesis of chiral sulfonamide phosphines with up to 98% ee under mild conditions.
An Expedient Synthesis of Ketocalix[6]arene Hexamethyl Ether
Authors: Biali Silvio , Itzhak Norbert
Published: July 2018

p-tert-Butylcalix[6]arene hexamethyl ether was oxidized in one step to the corresponding ketocalix[6]arene via photochemical bromination (100 W spot lamp) with NBS in a chloroform/water mixture. A pentamethyl ketocalix[6]arene derivative was obtained by reaction of the ketocalix[6]arene hexamethyl ether with boron tribromide dimethyl sulfide complex, and characterized by X-ray crystallography. In the presence of base, the compound undergoes an intramolecular nucleophilic aromatic substitution reaction.
A Comparative Analysis of D-Dimer Assays in Patients with Clinically Suspected Pulmonary Embolism
Authors: van Beek Edwin J R , van den Ende Bram , Berckmans René J , van der Heide Yvonne T , Brandjes Dees P M , Sturk Augueste , ten Cate Jan W
Published: July 2018

To avoid angiography in patients with clinically suspected pulmonary embolism and non-diagnostic lung scan results, the use of D-dimer has been advocated. We assessed plasma samples of 151 consecutive patients with clinically suspected pulmonary embolism. Lung scan results were: normal (43), high probability (48) and non-diagnostic (60; angiography performed in 43; 12 pulmonary emboli). Reproducibility, cut-off values, specificity, and percentage of patients in whom angiography could be avoided (with sensitivity 100%) were determined for two latex and four ELISA assays.

The latex methods (cut-off 500 μg/1) agreed with corresponding ELISA tests in 83% (15% normal latex, abnormal ELISA) and 81% (7% normal latex, abnormal ELISA). ELISA methods showed considerable within- (2–17%) and between-assay Variation (12–26%). Cut-off values were 25 μg/l (Behring), 50 μg/l (Agen), 300 μg/l (Stago) and 550 μg/l (Organon). Specificity was 14–38%; in 4–15% of patients angiography could be avoided.

We conclude that latex D-dimer assays appear not useful, whereas ELISA methods may be of limited value in the exclusion of pulmonary embolism.
Molecular Conversions of Recombinant Staphylokinase During Plasminogen Activation in Purified Systems and in Human Plasma
Authors: Ueshima S , Silence K , Collen D , Lijnen H R
Published: July 2018

Recombinant staphylokinase (STAR) is produced as a 136 amino acid protein with NH2-terminal sequence Ser-Ser-Ser (mature STAR, HMW-STAR), which may be converted to lower molecular weight forms (LMW-STAR) by removal of the first six residues (yielding STAR-Δ6 with NH2-terminal Gly-Lys-Tyr-) or the first ten residues (yielding STAR-Δ10 with NH2-terminal Lys-Gly-Asp-). In the present study the occurrence and effects of these conversions during plasminogen activation by HMW-STAR were studied in purified systems and in human plasma.

In stoichiometric mixtures of HMW-STAR and native human plasminogen (Glu-plasminogen), rapid and quantitative conversion of HMW-STAR to LMW-STAR occurred, concomitant with exposure of the active site in the plasmin-STAR complex. NH2-terminal amino acid sequence analysis revealed the sequence Lys-Gly-Asp- in addition to the known sequences of the Lys-plasmin chains, identifying STAR-Δ10 as the derivative generated from HMW-STAR. In mixtures of catalytic amount of HMW-STAR and human plasminogen, plasmin generation occurred progressively, following an initial lag phase, during which HMW-STAR was converted to LMW-STAR. Plasmin-mediated conversion of HMW-STAR to LMW-STAR obeyed Michaelis-Menten kinetics with K
m = 3.6 μM and k
2 = 0.38 s−1. The specific clot lysis activities of HMW-STAR (122,000 ± 8,000 units/mg) and LMW-STAR (129,000 ± 8,000 units/mg) were indistinguishable.

In an in vitro system consisting of a 60 μl plasma clot submerged in 250 μl plasma, 80% clot lysis within 1 h was obtained with 70 nM HMW-STAR. This was associated with fibrinogen depletion and conversion of 20% of the HMW-STAR to LMW-STAR. Addition of 100 nM HMW-STAR to human plasma in the absence of a clot did not induce significant fibrinogen breakdown (≥ 90% residual fibrinogen after 2 h), and was not associated with significant coversion to LMW-STAR (≤10% within 2 h). With 400 nM HMW-STAR, fibrinogen depletion in plasma occurred within 1 h, and 80% conversion to LMW-STAR was observed. Thus, at fibrinolytically active concentrations which do not cause fibrinogen breakdown, no significant conversion of HMW-STAR to LMW-STAR occurs in human plasma in the absence of a clot.

These findings indicate that the conversion of HMW-STAR to LMW-STAR may occur in association with clot lysis, but is not required to induce it.
Influence of Variability of Interpretation of Contrast Venography for Screening of Postoperative Deep Venous Thrombosis on the Results of a Thromboprophylactic Study
Authors: Couson Frédéric , Bounameaux Colette , Didier Cominique , Geiser Daniel , Meyerovitz Michael F , Schmitt Hans-Erich , Schneider Pierre-Alain , Bounameaux Henri
Published: July 2018

To assess interobserver variability of venography for screening for postoperative deep venous thrombosis (DVT), we used 185 bilateral ascending contrast venograms (366 lower limbs) which were performed using the long-leg film technique, in the frame of a trial of the efficacy of two low-molecular-weight-heparin fractions (initial evaluation). These venograms were submitted in a multicenter setting to three further readers who performed a serial scoring as DVT, no DVT or non-evaluable. DVTs were diagnosed in 78, 55 and 59/366 limbs (initial evaluation: 58). The proximal locations of DVTs were 16, 9 and 16 (initial evaluation: 15) and the non evaluable limbs 3, 5 and 18 (initial evaluation: 0). Apparent pairwise agreement between the three readers ranged from 87 to 90% (true coefficient of agreement Kappa 0.63-0.70). It ranged from 89 to 93% (Kappa 0.63-0.74) for the comparison between the readers and the initial evaluation. This considerable degree of disagreement among experienced readers should be taken into account in calculating sample sizes in prospective thromboprophylactic studies. Indeed, one of the two prophylactic regimens proved to be significantly (p = 0.012, p = 0.031, p = 0.049) or non-significantly (p = 0.073) superior to the other one depending upon the reading of venograms.
ARIC Hemostasis Study - III. Ouality Control
Authors: Chambless Lloyd E , McMahon Robert , Finch Andrea , Sorlie Paul , Heiss Gerardo , Lyles Robert , Wu Kenneth K
Published: July 2018

Methods and results from the quality assurance program of the Atherosclerosis Risk in Communities (ARIC) Study regarding hemostasis variables are presented, following up previous reports in this journal on standardized procedures for blood collection and processing (7) and an organized plan for the performance of those procedures (8). Efforts were made to control for and assess all sources of variability, from venipuncture to laboratory analysis, including also local field center processing and sample shipping. The quality control program included (a) a standardized protocol for blood collection and processing; (b) training, certification, and annual recertification of field center personnel for blood collection and processing; (c) monitoring of fasting times, phlebotomy times, processing times, and shipping problems; (d) hemostatic laboratory internal quality control; (e) a replicate blood sample program; (f) an intraindividual variability study; and (g) continual monitoring of quality control and study participants’ data. This paper focused on items (c), (d), and (e). Measures of Variation, generally Standard deviations and coefficients of Variation, are estimated for replicate blood sampling and internal quality control data, for activated partial thromboplastin time, fibrinogen, factor VII and VIII activity, von Willebrand factor, antithrombin-III, and protein C. The results demonstrate that it is possible to reliably measure these hemostatic variables in a large multicenter study.
Antibodies to β2-Glycoprotein I Inhibit Phospholipid Dependent Coagulation Reactions
Authors: Brandt John T
Published: July 2018

Lupus anticoagulants are antibodies that inhibit phospholipid dependent coagulation reactions in vitro. These antibodies are of clinical interest because of their association with a variety of clinical manifestations characterized by microvascular thrombosis. Although these antibodies were originally thought to be directed at negatively charged phospholipid, recent studies have suggested that they may be directed at phospholipid-protein complexes. The effect of antibodies directed against β2-glycoprotein I (β2-GP I, apolipoprotein H) on phospholipid-dependent coagulation reactions has been studied. Polyclonal and monoclonal antibodies to β2-GP I were found to inhibit thrombin generation in a dose dependent manner. Inhibition of thrombin formation was due to specific interaction with β2-GP I. There was no evidence that inhibition was due to crossreactivity with other proteins involved in the prothrombinase complex. These findings document that antibodies directed against β2-GP I can have anticoagulant activity analogous to lupus anticoagulant activity and are consistent with the recent observation of such activity in lupus anticoagulant patient samples.
A Quantitative Thrombin Time for Determining Levels of Hirudin and Hirulog™
Authors: Reid Thomas J , Alving Barbara M
Published: July 2018

The anticoagulant effect of recombinant hirudin (rHir) and HirulogTM has been monitored in patients with the activated partial thromboplastin time. Accurate monitoring with this test cannot be achieved if plasmas contain heparin, lupus anticoagulants, low concentrations of fibrinogen or other factors, or elevated fibrinogen-fibrin degradation products (FDP). We have therefore developed a simple, rapid, sensitive clot-based method, the quantitative thrombin time (QTT), to measure levels of rHir and HirulogTM in patient plasma (or whole blood). The QTT is performed by mixing a 1:10 dilution of patient plasma (50 μl) with human fibrinogen (50 μl, 128 mg/dl) at 37° C; the clotting time is initiated by adding human thrombin (50 μl, 5-7.5 U/ml). The concentration of HirulogTM or rHir in plasma can be determined by comparing the QTT in patient plasma with a Standard curve that is generated by adding different concentrations of anticoagulant to pooled normal plasma. Studies with whole blood using the same procedure yield similar results. In the absence of HirulogTM or rHir, the baseline QTT is the same in normal and abnormal plasmas (fibrinogen <150 mg/dl and FDP as high as 1024 μg/ml, elevated FDP alone, lupus anticoagulant, or heparin <0.9 U/ml). When known concentrations of either rHir or HirulogTM are added to abnormal plasmas, the mean observed concentrations as determined by the QTT deviate from the expected values by less than 10% (range 0-19%). The data indicate that the QTT is a simple, rapid, and accurate test for the determination of levels of rHir and HirulogTM in plasma or whole blood.
Continuous Registration of Thrombin Generation in Plasma, Its Use for the Determination of the Thrombin Potential
Authors: Hemker H C , Wielders S , Kessels H , Béguin S
Published: July 2018

A method is described by which the time-course of thrombin generation in plasma can be obtained from a continuous optical density recording of p-nitroaniline (pNA) production in a 2:3 diluted plasma. A chromogenic substrate, methylmalonyl-methylanalyl-arginyl-pNA (SQ68), is used that is specifically split by thrombin but at a low rate. The thrombin that appears and disappears in the plasma does not split more than 5% of the substrate added, so the rate of substrate conversion is in good approximation proportional to the amidolytic activity in the plasma over the entire period of thrombin generation. The course of the enzyme concentration can be calculated from the amidolytic activity curve. It is shown that the thrombin generation curves obtained in this way are essentially identical to those obtained via the classical subsampling method.

The presence of SQ 68 influences the amount of free thrombin that appears in plasma because it competitively inhibits the inactivation of thrombin by AT III and α2 macroglobulin. The inhibition of the thrombin peak by heparin, relative to an uninhibited control, remains unaltered by the presence of the substrate.

From the course of thrombin activity and the prevailing decay constants, the course of prothrombin conversion velocity can be calculated. Prothrombin conversion was seen to be inhibited at high (>500 μM) substrate concentrations only, and experimental conditions are found under which the inhibition of the clotting process by the substrate is negligible

The amidolytic activity is the sum of the activities of free thrombin and of the α2 macroglobulin-thrombin complex formed. Via a mathematical procedure the amount of SQ 68 that has been split by thrombin alone and not by the a2 macroglobulin-thrombin complex, can be derived from the course of the optical density.

The total amount of SQ 68 eventually split by thrombin alone is proportional to the surface under the thrombin generation curve, i. e. to the time-integral of free thrombin. This value, that we call the thrombin potential (TP), directly indicates how much of any physiological substrate can potentially be split by the thrombin being generated in the plasma.
Correlation Between Platelet Aggregation and Dephosphorylation of a 68 kDa Protein Revealed through the Use of Putative PKC Inhibitors
Authors: Turini Marco E , Gaudette Douglas C , Holub Bruce J , Kirkland James B
Published: July 2018

The efficacy of two structurally and functionally unrelated protein kinase C (PKC) inhibitors, chelerythrine and calphostin C, was assessed in intact human platelets by studying platelet aggregation in response to Stimulation with phorbol 12-myristate 13-acetate (PMA) or the thromboxane-A2 mimetic, U46619. Surprisingly, both inhibitors increased aggregation in response to PMA, but decreased aggregation in response to U46619. To further explore this phenomenon, gel electrophoresis of 32P-labelled proteins from PMA- or U46619-stimulated platelets in the presence and absence of the two putative PKC inhibitors was performed. Although neither chelerythrine nor calphostin C proved to be effective PKC inhibitors in intact human platelets, a strong correlation between the dephosphorylation of a 68 kDa protein and the rate of platelet aggregation was observed. From these results, the indiscriminate use of PKC inhibitors in whole platelets is questioned and attention is drawn to the role of protein dephosphorylation in platelet activation. The 68 kDa protein was the major phosphorylated substrate in resting platelets. Okadaic acid increased phosphorylation of this band, indicating active phosphate group turnover under resting conditions.
The Difference Between Platelet and Plasma FXIII Used to Study the Mechanism of Platelet Microvesicle Formation
Authors: Holme Pål André , Brosstad Frank , Solum Nils Olav
Published: July 2018

The formation of microvesicles from platelets was induced either by activation of the complement system by a monoclonal antibody to CD9, or by incubation of platelets with the calcium ionophore A23187. A filter technique to isolate the microvesicles without plasma contamination is described. The microvesicles contained FXIIIa2 from the platelet cytoplasm which shows that these particles contain significant amounts of intracellular material. This was shown by the use of crossed immunoelectrophoresis with rabbit antibodies to total human platelet proteins in the second dimension gel and polyclonal antibodies against the a- and b-subunit of FXIII in the intermediate gel. The FXIIIa2 in the microvesicle was found to be functional as an enzyme. To prove this, it was shown that FXIII in its immunoprecipitate arc could catalyze the incorporation of monodansylcadaverine into casein as identified by fluorescence of this arc in ultraviolet light. The observation that the plasma form of FXIII (FXIIIa2b2) was absent from the microvesicles collected by the filtration technique, whereas it was present in platelet fragments obtained by mechanical disruption by ultrasonication, indicates that the activation-dependent microvesicles are formed by a true budding process with the inclusion of intracellular, but not extracellular material.
Autopsy Findings in Three Patients with von Willebrand Disease Type IIB and Type III: Presence of Atherosclerotic Lesions without Occlusive Arterial Thrombi
Authors: Federici A B , Mannucci P M , Fogato E , Ghidoni P , Matturri L
Published: July 2018

It has been reported that pigs with severe von Willebrand disease (vWd) are protected from spontaneous and diet-induced atherosclerosis, but there are very few studies in human patients. Autopsies were carried out on three patients with vWd (one case with the type IIB variant and two cases with type III, a variant similar to that protecting pigs from atherosclerosis), aged 73, 44 and 52 years at the time of death. Hemorrhagic diathesis was an important cause of death for these patients. Atherosclerosis lesions were found in all patients, though of different extent and localization: severe lesions in all vascular areas in the patient with type IIB, few and slighter in all areas in the two patients with type III, except that in one of them there were moderately severe lesions in the coronary arteries. There was no clinical or pathologic evidence of thrombosis in the coronary arteries or other arteries. These data indicate that severe vWd did not protect completely against the development of atherosclerosis, although it is possible that intensive lifelong treatment with plasma concentrates had a modifying influence in these patients.
Inhibitory Effect of Staphylokinase on Platelet Aggregation
Authors: Suehiro Akira , Oura Yoshio , Ueda Motoo , Kakishita Eizo
Published: July 2018

We investigated the effect of staphylokinase (SAK), which has specific thrombolytic properties, on human platelet aggregation. Platelet aggregation induced with collagen was observed following preincubation of platelets in platelet-rich plasma (PRP) or washed platelet suspension (WP) with SAK at 37° C for 30 min. SAK inhibited platelet aggregation in PRP only at the highest examined concentration (1 x 10-4 g/ml). Although SAK did not inhibit platelet aggregation in WP which contained fibrinogen, it did when the platelets had been preincubated with SAK and plasminogen. The most effective concentration in WP was 1 x 10-6 g/ml. The effect could be inhibited by adding aprotinin or α2-antiplasmin. The highest generation of plasmin in the same preincubation fluid was detected at 1 x 10-6 g/ml SAK. We concluded that SAK can inhibit platelet aggregation in WP by generating plasmin and/or fibrinogen degradation products, but is only partially effective in PRP because of the existence of α2-antiplasmin.
Multicentre Evaluation of Commercial Kit Methods: Plasminogen Activator Inhibitor Activity
Authors: Gram J , Declerck P J , Sidelmann J , Jespersen J , Kluft C
Published: July 2018

In order to study the analytical performance of different commercial kits for determination of plasminogen activator inhibitor (PAI) activity we distributed eight selected split samples to 11 European laboratories experienced with haemostasis testing. Three different laboratories were involved in the production of data from each of the commercial kits tested. A considerable variation of PAI activity results reported from the laboratories testing the same commercial kits was observed. The range of reported results could in individual samples exceed the median value indicating an interlaboratory variation of more than 100%.

When we harmonized the results reported from different kits in different laboratories by means of an international standard from National Institute for Biological Standards and Control (NIBSC) we still observed that the results produced by some kits deviated systematically from results produced by other kits. Also, the harmonized results were used to estimate the overall coefficient of variation (CV) of PAI activity determined in various laboratories by different kits. We observed an inverse correlation between the PAI activity level and the CV with a CV of about 10Q% for low PAI activity levels and a CV of about 16% for high PAI activity levels. The high imprecision of the kits in the low concentration range of PAI activity indicates that unspecific factors in plasma may interfere with determination of active PAI. This was confirmed by the evaluation of the results from one of the plasma samples, which was PAI-1 depleted. The laboratories involved in the testing reported for this sample a mean value of 6.1 IU/ml.

In conclusion, the currently available kits for determination of PAI activity are not accurate for measurement of PAI-1 in plasma, and the kits give imprecise results particularly at low activity levels.
Human Spleen Insoluble Fibrinolytic Proteinase Acting at Neutral pH: Its Partial Purification and Characterization
Authors: Okamoto Utako , Nagamatsu Yoko , Amemiya Takehiko
Published: July 2018

An insoluble fibrinolytic enzyme with a molecular weight of approximately 30000, was purified from the human spleen. A single protein band possessing fibrinolytic activity was obtained on polyacrylamide gel disk electrophoresis at pH 4.5.

The enzyme, tentatively termed spleen fibrinolytic proteinase (SFP), degraded fibrinogen at neutral pH following Michaelis-Menten kinetics. This fibrinogenolytic activity was not inhibited by t-AMCHA, a specific plasmin inhibitor. SFP barely degraded certain synthetic ester or polypeptide substrates for trypsin, chymotrypsin, plasmin, Xa, elastase and collagenase. These results indicate a different nature for SFP compared to other enzymes examined. SFP was found to digest no elastin and its fibrinogenolytic activity was strongly inhibited by STI, indicating that it was not an elastase. SFP required neither Zn++ nor Ca+ + for its fibrinogenolytic activity, indicating that it differed from metal-dependent proteinases such as collagenase. SFP was inhibited by DFP but not by TLCK, suggesting that it contains an active serine residue, but no trypsin type histidine at its active center. These results appear to show that SFP is a unique proteinase in the spleen, which is capable of degrading fibrin and fibrinogen at neutral pH.
A Method to Determine the Optimal Intensity of Oral Anticoagulant Therapy
Authors: Rosendaal F R , Cannegieter S C , van der Meer F J M , Briët E
Published: July 2018

Oral anticoagulant therapy has been shown to be effective for several indications. The optimal intensity of anticoagulation for each indication, however, is largely unknown. To determine this optimal intensity, randomised clinical trials are conducted in which two target levels of anticoagulation are compared. This approach is inefficient, since the choice of the target levels will be arbitrary. Moreover, the achieved intensity is not taken into account.

We propose a method to determine the optimal achieved intensity of anticoagulation. This method can be applied within a clinical trial as an “efficacy-analysis”, but also on data gathered in day-to-day patient care.

In this method, INR-specific incidence rates of events, either thromboembolic or hemorrhagic, are calculated. The numerator of the incidence rate is based on data on the INR at the time of the event. The denominator consists of the person-time at each INR value, summed over all patients, and is calculated from all INR measurements of all patients during the follow-up interval. This INR-specific person-time is calculated with the assumption of a linear increase or decrease between two consecutive INR determinations. Since the incidence rates may be substratified on covariates, efficient assessment of the effects of other factors (e.g. age, sex, comedication) by multivariate regression analysis becomes possible.

This method allows the determination of the optimal pharmacological effects of anticoagulation, which can form a rational starting point for choosing the target levels in subsequent clinical trials.
Herpes Simplex Virus Decreases Endothelial Cell Plasminogen Activator Inhibitor
Authors: Bok Robert A , Jacob Harry S , Balla Jozsef , Juckett Margaret , Stelle Theresa , Shatos Marie A , Vercellotti Gregory M
Published: July 2018

Herpes simplex virus (HSV) infection is histopathologically associated with vascular injury, fibrinoid necrosis and inflammatory cell infiltrates. We have previously shown in vitro that HSV infection of human umbilical vein endothelial cells (HUVEC) promotes a procoagulant phenotype manifest by the induction of tissue factor, the loss of thrombomodulin, and an increase in platelet adhesion. In these studies we examined the effects of HSV infection on HUVEC plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator (t-PA). HSV infection caused the loss of PAI-1 in the extracellular matrix (ECM) and that released into the supernatant of HUVEC. Both activity and antigen levels of the Serpin inhibitor are diminished as a result of HSV infection. The loss of inhibitor is not secondary to diminished vitronectin (Vn), the primary binding protein of PAI-1 in the ECM, but appears to be secondary to decreased synthesis at the RNA level. Tissue plasminogen activator (t-PA). synthesis is also decreased in endothelial HSV infection. PAI-1 loss may further promote a procoagulant phenotype in HSV infection in vivo.
The Role of Cyclic Nucleotides in the Release of Tissue-Type Plasminogen Activator and von Willebrand Factor
Authors: Tranquille N , Emeis J J
Published: July 2018

The modulation of the induced acute release of tissue-type plasminogen activator (t-PA) and of von Willebrand factor (vWF) by compounds affecting cyclic nucleotide levels was studied, using an isolated rat hindleg perfusion system. Platelet-activating factor (PAF; 5 nM) or bradykinin (0.8 (μM) were used to induce release of t-PA and vWF.

The guanylate cyclase activators sodium nitroprusside and atrial natriuretic factor reduced the induced release of t-PA and vWF. Release was not affected by inhibiting nitric oxide production with NG-nitro-L-arginine. The effects of nitroprusside and atrial natriuretic factor could not be reproduced by infusion of 8-bromo-cGMP.

The adenylate cyclase activator forskolin had no effect on bradykinin-induced release of t-PA and vWF, reduced PAF-induced t-PA release, but potentiated PAF-induced vWF release. These modulatory effects were only partially mimicked by infusion of 8-bromo-cAMP.

None of the compounds tested was able to induce the release of t-PA or of vWF in the absence of stimulation by bradykinin or platelet-activating factor. Cyclic nucleotides can thus modulate, but not induce, the acute release of t-PA and vWF from perfused rat hindlegs.
The Course of Preexistent Immune Abnormalities in HIV Negative Haemophiliacs Treated for Two Years with a Monoclonal Purified Factor VIII Concentrate
Authors: Smid W M , van der Meer J , Smit J W , Halie M R
Published: July 2018

The administration of factor VIII concentrates has been associated with immune abnormalities in patients with severe haemophilia A, even in the absence of HIV infection.

The effects of a monoclonal purified factor VIII concentrate, Hemofil M (Baxter), on preexistent immune abnormalities were assessed over a 2 year period in 22 HIV negative haemophiliacs. They were treated previously with other concentrates, and received Hemofil T from 1983 to 1988. No HIV infection was demonstrated. No serologic evidence for other viral (re)-infections was seen. A decrease of HLA-DR expression on non-B lymphocytes in the first year (P = 0.026), and a decrease of T4-T8 ratio over the 2 years were found (P = 0.0016). Skin tests were non-contributive. The decrease in HLA-DR expression is suggestive for an improved immune function, possibly due to a reduced content of protein or virus in this concentrate.
Problems of Oral Anticoagulation in an Adult with Homozygous Protein C Deficiency and Late Onset of Thrombosis
Authors: Pescatore P , Horellou H M , Conard J , Piffoux M , Van Dreden P , Ruskone-Fourmestraux A , Samama M
Published: July 2018

We describe a 57-year-old woman with homozygous protein C deficiency and mild thrombotic manifestations consisting of three spontaneous distal deep vein thromboses occurring after the age of 45. Previous surgery and pregnancies had been uneventful. Low but detectable protein C antigen and activity levels (both 20%) were discovered on the occasion of skin necrosis induced by oral anticoagulation. This therapy was interrupted because of skin necrosis and several episodes of disseminated intravascular coagulation (DIC) at the initiation of treatment despite a cautious protocol. No recurrent thromboembolic event has occurred in our patient using prophylactic doses of low molecular weight heparin for 24 months. New therapeutic approaches might be the administration of low molecular weight heparin or oral anticoagulation associated with protein C replacement in the induction period. This case reflects the variability of expression of protein C deficiency as well as the potential hazards of antivitamin K anticoagulation in this disorder.