bluuebunny/crossref_metadata_2025 huggingface dataset:
Lyapunov–Krasovskii‐type criteria on ISS and iISS for impulsive time‐varying delayed systems
Authors: Peng Shiguo
Published: March 2018

This study investigates the problem of input‐to‐state stability (ISS) and integral input‐to‐state stability (iISS) of impulsive time‐varying delayed systems. Some Lyapunov–Krasovskii‐type criteria on ISS and iISS which are effective for destabilising impulses and stabilising impulses are derived from advanced linear dissipation inequalities under average impulsive interval constraints. The conditions which require the coefficients of the linear dissipation inequalities on the Lyapunov–Krasovskii functionals to be constants in the existing results on ISS/iISS of impulsive delayed systems are weakened. In this study, with the aid of the notions of uniformly exponentially stable function and average impulsive interval, the results allow the coefficients of the developed linear dissipation inequalities on the Lyapunov–Krasovskii functionals to be time‐varying functions which can take both positive and negative values, and the impulsive intervals of an impulsive sequence are allowed to have arbitrarily small lower bound and enough big upper bound simultaneously. Two examples are presented to illustrate the effectiveness of the results.
Stochastic optimal control for sampled‐data system under stochastic sampling
Authors: Sun Haoyuan , Chen Jie , Sun Jian
Published: March 2018

In this study, the problem of designing a stochastic optimal controller for sampled‐data systems whose sampling interval is subjected to a certain probability distribution is addressed. To design the controller, the Kronecker product operation and the Vandermonde matrix were introduced. A design method of the stochastic optimal controller is proposed. It is shown that the controller guarantee that the closed‐loop system has exponentially mean square stability. Finally, the simulation results illustrate the effectiveness and practicability of the proposed method.
Hierarchical thermal network analysis of axial‐flux permanent‐magnet synchronous machine for electric motorcycle
Authors: Chen Qixu , Liang Deliang , Gao Lin , Wang Qingshan , Liu Yibin
Published: March 2018

A novel 24 kW 12‐slots/10‐poles axial‐flux permanent‐magnet synchronous machine (AFPMSM) with external rotor motor applied to the large electric motorcycle is introduced in this study, which has the characteristics of the yokeless and segmented armature for power density improvement. The hierarchical method is used for calculation of stator iron core loss based on the analytical solution of air‐gap flux density, which cuts the prototype into many equivalent linear motor slices in the circumferential direction. A major advantage of hierarchical methods over traditional iron core loss calculation is its higher accuracy and rapidity. Then a lumped parameter T‐type thermal network model is presented for AFPMSM temperature rise calculation. A 3D liquid–solid coupling model is compared with the 2D T‐type lumped parameter thermal network. Finally, a temperature rise measurement platform is established to verify the above‐mentioned methods. Testing result shows that the hierarchical method and T‐type thermal network have a higher accuracy to predict each part temperature.
Effect of damper winding on accuracy of wound‐rotor resolver under static‐, dynamic‐, and mixed‐eccentricities
Authors: Totoonchian Farid
Published: March 2018

Most of the resolvers used in high precision servomechanism are two‐pole, wound‐rotor ones. The configuration of their stator winding is based on variable‐turn, on‐tooth method. However, there is different opportunity for their rotor: using distributed winding or on‐tooth one. Furthermore, based on the operating principle of the resolver its rotor needs a single phase winding. While, in many practical cases a two‐phase winding that one of them is short‐circuited (damper winding) is employed for rotor. In this study, the effect of different configurations for rotor winding on the resolver performance in terms of the average of absolute position error, maximum position error, and total harmonic distortion of induced voltages envelopes is investigated. Then, the effect of damper winding on the performance of the resolver under static‐, dynamic‐, and mixed‐eccentricities are studied. In this study, all simulations are performed using three‐dimensional time stepping finite‐element method and finally, the experimental tests on the studied sensor using a precision test setup are employed to approve the simulation results.
Die dentolabiale Ästhetik
Authors: Mansour Sonia , Pospiech Peter
Published: March 2018

In einer Welt, in der Ästhetik und äußeres Aussehen immer bedeutender werden, spielt insbesondere die Schönheit der Zähne in den Vorstellungen der Patienten eine dominante Rolle, was sich als Leistungsangebot und auch wesentlicher Bestandteil der meisten Praxishomepages widerspiegelt. Gutes Aussehen steht in direktem Zusammenhang mit einem attraktiven Lächeln und schönen Zähnen und damit einer gesteigerten Lebensqualität.
Synthesis, Structure, and Biological Activity of 2,6-Disubstituted 2,3a,4a,6,7a,8a-Hexaazaperhydrocyclopenta[def]fluorene-4-thioxo­-8-ones
Authors: Barsegyan Yana , Baranov Vladimir , Kravchenko Angelina , Strelenko Yuri , Anikina Lada , Karnoukhova Valentina , Kolotyrkina Natalya
Published: March 2018

A two-step one-pot condensation for the synthesis of previously inaccessible 2,6-disubstituted 2,3a,4a,6,7a,8a-hexaazaperhydrocyclopenta[def]fluorene-4-thioxo-8-ones from simple reagents [5-thioxohexahydroimidazo[4,5-d]imidazol-2(1H)-one, paraformaldehyde and amines] was developed. The structure, antimicrobial, and anticancer activity of a number of synthesized compounds were studied.
Latent Brønsted Base Solvent-Assisted Amide Formation from Amines and Acid Chlorides
Authors: Otsuka Rikuto , Maruhashi Kazuo , Ohwada Tomohiko
Published: March 2018

Weakly basic amines, including even neutral amines such as nitroaniline and aminocarboxylic acids, react with acid chlorides very efficiently in N,N-dimethylacetamide (DMAC), without addition of a base, to give the corresponding amides in high yields. The role of DMAC and related solvents as latent Brønsted bases was studied in these amidation reactions. Less basic amines, such as aromatic amines, reacted with benzoyl chloride faster than more basic aliphatic amines.
Effects of (Pre-)analytical Variables on Activated Protein C Resistance Determined Via a Thrombin Generation-based Assay
Authors: Curvers Joyce , Christella M. , Thomassen G. D. L. , de Ronde Hans , Bertina Rogier , Rosendaal Frits , Tans Guido , Rosing Jan
Published: March 2018

The normalized activated protein C sensitivity ratio (nAPC-sr) determined with an assay that quantifies the effect of APC on thrombin formation initiated via the extrinsic coagulation pathway identifies hereditary and acquired defects of the protein C system. We investigated the influence of assay conditions (analytical variables) and plasma handling (pre-analytical variables) on nAPC-sr obtained with this APC resistance test. The effect of the analytical variables (CaCl2, phospholipid and APC concentrations and the concentration and source of tissue factor) was determined in pooled normal plasma. Inhibition of thrombin formation by APC was dependent on the APC concentration and was also affected by the tissue factor, Ca2+ and phospholipid concentrations. Thus, strict standardization of reactant concentrations is required to obtain reproducible nAPC-sr. Three different tissue factor preparations were compared by determining nAPCsr in plasma samples obtained from 90 healthy individuals. nAPC-sr were similar for all three tissue factor preparations although, compared with the noncommercially available tissue factor used in earlier studies, values determined with commercial tissue factor preparations showed larger variation. Pre-analytical variables, investigated in plasma of nine volunteers (3 normal individuals and 6 individuals with an APCresistant phenotype) were: concentration of anticoagulant (3.2% vs. 3.8% trisodiumcitrate), time before processing of blood (0, 4 and 24 h), centrifugation speed, storage temperature of plasma (–20° C vs. –80° C) and sample thawing. Multiple linear regression analysis showed that only the citrate concentration affected the nAPC-sr, which was higher in samples collected in 3.2% trisodiumcitrate than in samples collected in 3.8% trisodiumcitrate.
A Prospective Evaluation of the Heat Shock Protein 70 Gene Polymorphisms and the Risk of Stroke
Authors: Bates David , Ridker Paul , Zee Robert
Published: March 2018

Genetic polymorphisms of heat shock protein 70-kD (HSP70) gene family have recently been hypothesized to be risk factors for cerebral ischemia. However, no prospective epidemiological data evaluating this gene family are available. The present investigation was conducted to examine the possible associations between the HSP70-1 nucleotide 190, HSP70-2 nucleotide 1267, and HSP70-hom nucleotide 2437 polymorphisms and the incidence of stroke in a large cohort of initially healthy men. 14916 apparently healthy men were followed over a 12-year period for incident stroke. Employing a nested case-control study design, 338 study participants who developed stroke (cases) and 338 ageand smoking-matched study participants who remained healthy during follow-up (controls) were evaluated. All observed genotype frequencies were in Hardy-Weinberg equilibrium. The allele and genotype distributions of the polymorphisms tested were similar among cases and controls, such that the relative risk of future stroke was 0.89 for HSP70-1 nucleotide 190 (95%CI = 0.70–1.12; p = 0.31), 1.13 for HSP70-2 nucleotide 1267 (95%CI = 0.90–1.42; p = 0.29); and 0.89 for HSP70-hom nucleotide 2437 (95%CI = 0.65–1.21; p = 0.45), assuming an additive model. No evidence of association was observed assuming dominant or recessive mode of inheritance. In this large, prospective study, genetic polymorphisms in the HSP70 genes were not associated with risks of future stroke. Screening for these polymorphisms is unlikely to be a useful tool for risk assessment.
Fibrinogen Assembly and Crosslinking on a Fibrin Fragment E Template
Authors: Siebenlist Kevin , Hernandez Irene , Wall Joseph , Hainfeld James , Mosesson Michael
Published: March 2018

There is an ongoing controversy concerning whether crosslinked γ chains in fibrin are oriented “transversely” between fibril strands or “end-to-end” along fibril strands. From the latter viewpoint, Veklich et al. [Proc Natl Acad Sci (USA) 95: 1438, 1998] observed that fibrinogen fibrils that had been assembled on a fibrin fragment E template, crosslinked with factor XIIIa, and then dissociated in acetic acid solution, were aligned end-to-end. This led to the conclusion that crosslinked γ chains in fibrin under physiological conditions were also aligned endto-end. To assess its validity we studied the assembly and organization of fibrinogen molecules on a des AB-fibrin fragment E (E-des AB) or a des A-fibrin fragment E (E-des A) template.

We evaluated the roles of E polymerization sites EA and EB, and D association sites γXL, Da, Db, βC, and αC in this process. EA:Da interactions caused fibrinogen: E “DED” complexes to form, and markedly enhanced the γ chain crosslinking rates of fibrinogen or des αC-fibrinogen. Fibrinogen crosslinking without added fibrin E was slower, and that of des αC-fibrinogen was still slower. These events showed that although αC domains promote fibrinogen fibril assembly and crosslinking, they contribute little to increasing the EA:Da-dependent crosslinking rate. Electron microscopic (STEM) images of E-des AB and fibrinogen plus factor XIIIa showed single-, double-, and multistranded fibrils with interstrand DED complexes aligned side-to-side. This alignment was due to βC:βC contacts resulting from D subdomain rearrangements initiated by the EB:Db interactions, and also occurred in mixtures of des αC-fibrinogen with E-des AB.

In contrast, a mixture of fibrinogen and E-des A plus XIIIa revealed double-stranded fibrils with interstrand DED complexes in a halfstaggered arrangement, an alignment that we attribute to crosslinking of γXL sites bridging between fibrils strands. These and other features of E-des A-based fibrinogen fibrils, including interstrand γ chain bridges and early and extensive lateral fibril strand associations concomitant with accelerated γ chain crosslinking, indicate that crosslinking of fibrin fibril strands takes place preferentially on transversely positioned γ chains.
Long-term Persistence of Anti-factor VIII Antibody-secreting Cells in Hemophilic Mice after Treatment with Human Factor VIII
Authors: Hausl Christina , Maier Elisabeth , Ahmad Rafi , Turecek Peter , Dorner Friedrich , Reipert Birgit , Schwarz Hans
Published: March 2018

The analysis of anti-factor VIII (FVIII) antibody-secreting cells (ASC) at different anatomic sites provides valuable information about the nature of the anti-FVIII immune response in hemophilic mice after treatment with human FVIII. An Elispot system is described that is suitable for analyzing frequencies and IgG subclasses of anti-FVIII ASC at the single-cell level. Hemophilic mice were treated with four doses of FVIII. Anti-FVIII antibodies in blood as well as anti-FVIII ASC in spleen and bone marrow were analyzed after each dose of FVIII and subsequently up to 22 weeks after termination of the FVIII treatment. Anti-FVIII ASC first appeared in the spleen where they were detectable after two intravenous doses of FVIII. Their appearance correlated with that of anti-FVIII antibodies in blood plasma. Anti- FVIII ASC in bone marrow were detectable after three doses of FVIII and were probably cells that initially formed in the spleen and subsequently migrated to the bone marrow. Whereas the frequency of anti- FVIII ASC in the spleen increased up to the fourth dose of FVIII and declined thereafter, in the bone marrow it remained constant for up to at least 22 weeks after the termination of the FVIII treatment. Titers of anti-FVIII antibodies in blood plasma increased up to the fourth dose of FVIII, then remained high constantly for 14 weeks and decreased but the antibodies were still detectable for up to at least 22 weeks after the fourth dose of FVIII. The IgG-subclass distribution of anti-FVIII ASC was similar in spleen and bone marrow and matched the subclasses of anti-FVIII antibodies in blood plasma indicating that both organs contribute to circulating antibodies in the blood.
Differential Regulation of Human Platelet Responses by cGMP Inhibited and Stimulated cAMP Phosphodiesterases
Authors: Manns J. M. , Brennan K. J. , Sheth S. B. , Colman R. W.
Published: March 2018

Platelets contain two cAMP phosphodiesterases (PDEs) which regulate intracellular cAMP levels, cGMP-inhibited cAMP PDE (PDE3A) and cGMP-stimulated PDE (PDE2A). Using the PDE3 inhibitor, milrinone and the PDE2 inhibitor, erythro-9-(2-hydroxyl-3-nonyl)adenine (EHNA), we have explored the contribution of each PDE to the regulation of platelet function. Inhibition of PDE2 resulted in higher levels of intracellular cAMP than inhibition of PDE3A suggesting this PDE may be the more important regulator of cAMP in human platelets. However, a concentration-dependent inhibition of agonist-induced aggregation was observed with milrinone while little effect was seen with EHNA. In addition, we observed a concentration-dependent inhibition in the increase of intracellular Ca2+ with PDE3 inhibition and significantly less with PDE2 inhibition. PDE3 inhibition also resulted in a concentration-dependent increase in cAMP-mediated phosphorylation of the vasodilator-stimulated phospho-protein (VASP) whereas there was no significant increase with PDE2 inhibition. In each of these experiments, synergism was noted with the combination of milrinone and EHNA. These results suggest that cAMP pools may be localized and the various PDEs regulate specific pools. These data also suggest that inhibitors of PDE3A may be more effective antiplatelet agents.
A Performance Evaluation of Commercial Fibrinogen Reference Preparations and Assays for Clauss and PT-derived Fibrinogen
Authors: Lawrie A. S. , Kitchen S. , Gaffney P. J. , Howarth D. , Lowe G. D. O. , Martin J. , Purdy G. , Rigsby P. , Rumley A. , Mackie I. J.
Published: March 2018

The wide availability of fibrinogen estimations based on the prothrombin time (PT-Fg) has caused concern about the variability and clinical utility of fibrinogen assays. In a multi-centre study, we investigated fibrinogen assays using various reagents and analysers. Clauss assays generally gave good agreement, although one reagent gave 15–30% higher values in DIC and thrombolysis. Two commercial reference preparations had much lower potencies than the manufacturers declared, and plasma turbidity influenced parallelism in some Clauss assays. PT-Fg assays gave higher values than Clauss and showed calibrant dependent effects, the degree of disparity correlating with calibrant and test sample turbidity. Analyser and thromboplastin dependent differences were noted. The relationship between Clauss and PT-Fg assays was sigmoid, and the plateau of maximal PT-Fg differed by about 2 g/l between reagents. ELISA and immunonephelometric assays correlated well, but with a high degree of scatter. Antigen levels were higher than Clauss, but slightly lower than PT-Fg assays, which appeared to be influenced by degraded fibrinogen. Clauss assays are generally reproducible between centres, analysers and reagents, but PT-Fg assays are not reliable in clinical settings.
Enzyme immunoassay detection of platelet-derived microparticles and RANTES in acute coronary syndrome
Authors: Uehata Shigeru , Saito Sonoko , Osumi Kazuoki , Ozeki Yasushi , Kimura Yukio , Nomura Shosaku
Published: March 2018

Platelet-derived microparticles (PDMPs) are produced by platelet activation or physical stimulation under various conditions. To evaluate changes in platelet and chemokine function in patients undergoing percutaneous transluminal coronary angioplasty (PTCA), we measured and compared levels of PDMPs and a C-C chemokine, regulated on activation normally T-cell express and secreted (RANTES), by ELISA. Levels of PDMP and RANTES in patients with acute coronary syndrome were significantly higher than those in the control groups (PDMP: 20.1 ± 2.9 vs 80.4 ± 7.3 U/ml, p < 0.001; RANTES: 18.6 ± 3.7 vs 52.1 ± 4.6 ng/ml, p < 0.01), but did not differ between the control groups and patients with stable angina. PDMP levels were higher in patients with acute myocardial infarction (AMI) than in patients with unstable angina (PDMP:115.0 ± 7.1 vs 63.9 ± 6.2 U/ml, p < 0.001). There was no difference in the RANTES levels, however, between patients with AMI and patients with unstable angina. PDMP and RANTES levels were significantly decreased after PTCA (PDMP, p < 0.001; RANTES, p < 0.05), but without differences between the two groups. In addition, the level of PDMP was significantly correlated with that of RANTES or soluble CD40 ligand. These findings suggest that PTCA may prevent the development of AMI-associated complications in which activated platelets and RANTES play roles. Our ELISA method appears to be sufficient for monitoring PDMP and RANTES levels after PTCA in patients with acute coronary syndrome.
Association between TAFI antigen and Ala147Thr polymorphism of the TAFI gene and the angina pectoris incidence
Authors: Morange Pierre , Scarabin Pierre , Alessi Marie , Luc Gérald , Arveiler Dominique , Ferrieres Jean , Amouyel Philippe , Evans Alun , Ducimetiere Pierre , Juhan-Vague Irène ,   on behalf of the PRIME Study group
Published: March 2018

Thrombin activatable fibrinolysis inhibitor (TAFI), a recently described inhibitor of fibrinolysis, has been hypothesized as playing a role in atherothrombosis. However, the evidence from retrospective studies, which have evaluated the role of TAFI in vascular risk, is conflicting.

In a prospective cohort (the PRIME Study) of nearly 10 000 apparently healthy men recruited in France (Lille, Strasbourg, Toulouse) and Northern Ireland (Belfast), we measured baseline plasma concentration of TAFI antigen among 143 participants (81 from France and 62 from Ireland) who subsequently developed angina pectoris and among 286 age-matched participants who remained free of disease during the 5 years of follow-up. Genotyping of the Ala147Thr polymorphism located in the TAFI gene was performed using an allele specific PCR. In France, mean levels of TAFI were significantly higher at baseline among men who subsequently developed angina pectoris compared with their control subjects (119 versus 107 %; p = 0.02). The risk of future angina pectoris increased with increasing tertiles of TAFI (p = 0.02), such that men in the highest tertile at study entry had a 5-fold higher relative risk than those in the lowest tertile (95% confidence interval, 1.38 to 18.58) after controlling for the conventional cardiovascular risk factors. No such difference was observed in Northern Ireland. In France, Thr/Thr carriers of the Ala147Thr polymorphism were significantly more frequent in cases than in controls (p = 0.01) leading to a relative risk of angina pectoris of 2.7 (95%CI 1.2-5.8).

Increase in plasma TAFI antigen levels is a risk factor for angina pectoris in France. Genotyping for the Ala147Thr polymorphism seems to be a reliable tool to assess the risk mediated by TAFI.
Fibrin-based biomaterials to deliver human growth factors
Authors: Wong Catalina , Inman Elisabeth , Spaethe Reiner , Helgerson Sam
Published: March 2018

Fibrin-based biomaterial preparations can be used as provisional growth matrices for cells important in tissue repair during wound healing in vivo. Their efficacy can be enhanced by including bioactive agents that promote specific cellular responses. This study examined the controlled delivery of the angiogenic growth factors bFGF, VEGF165, and VEGF121 using biomatrix preparations prepared from Fibrin Sealant product components. The growth factors were added prior to formation of the Fibrin Sealant clots, and the release kinetics of the proteins from the clots measured. The results indicated that the proteins were released from the clots more slowly in the order bFGF << VEGF165 < VEGF121. The biologic activity of the growth factors delivered from Fibrin Sealant clots was established by assaying growth stimulation of human microvascular endothelial cells (HMVEC) and angiogenesis in the chicken embryo chorioallantoic membrane (CAM) model of neovascularization. In the latter assay, clots containing bFGF, VEGF165, or V EGF121 all displayed angiogenic activity. However, delivery of either bFGF, VEGF165, or VEGF121 alone resulted in a significant percentage of clots becoming filled with blood, indicating that the newly developing vessels invading the clots were leaky and immature. In contrast, this hemorrhaging behavior did not occur with delivery of combinations, e.g., (VEGF165 + VEGF121) or (VEGF165 + bFGF), indicating that the vessels were more mature than those produced in response to single growth factors. Thus, delivering a combination of growth factors constituted an improvement over the delivery of individual growth factors for enhancing neovascularization.

Part of this research was presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society held in Munich, Germany.
Inhibition of platelet activation in congestive heart failure by aldosterone receptor antagonism and ACE inhibition
Authors: Schäfer Andreas , Fraccarollo Daniela , Hildemann Steven , Christ Michael , Eigenthaler Martin , Kobsar Anna , Walter Ulrich , Bauersachs Johann
Published: March 2018

An increased risk of thrombembolic events in congestive heart failure (CHF) has been attributed to a hypercoagulable state including vascular endothelial dysfunction and platelet activation.

After experimental myocardial infarction, male Wistar rats were treated with placebo, the ACE inhibitor trandolapril, the selective aldosterone receptor antagonist eplerenone or the combination of both, for 10 weeks. Platelet-bound fibrinogen and surface-expressed P-selectin were not modulated in rats without CHF compared with sham-operated animals, but were significantly increased in CHF rats (LVEDP>15mmHg). In CHF rats, ACE inhibition significantly reduced platelet P-selectin expression while bound fibrinogen was not modulated. Eplerenone reduced P-selectin expression to a comparable extent, while platelet-bound fibrinogen was normalised. Combination therapy with eplerenone and trandolapril completely abolished both the increased P-selectin expression as well as fibrinogen binding. Phosphorylation of platelet vasodila-tor-stimulated phosphoprotein (VASP) at both Ser157 and Ser239, which reflects the activity of platelet inhibitors including nitric oxide, was significantly reduced in platelets from placebo-treated CHF rats, and was completely normalised by combination treatment, but only marginally increased by either mono-therapy.

The results show that platelet activation was evident only in CHF rats. Monotherapy with ACE inhibition or eplerenone partially reduced this increased platelet activation, which was completely rescued to basal levels by combination therapy. Increased nitric oxide bioavailability can only partially explain the reduced platelet activation by eplerenone and ACE inhibition.
TNF 41-62 and TNF 78-96 Have Distinct Effects on LPS-induced Tissue Factor Activity and the Production of Cytokines in Human Blood Cells
Authors: Rekda Ø. , Svendsen J.S. , Østerud B. , Eliassen L.T.
Published: March 2018

Biological activities of peptides representing two different regions in the TNF molecule were investigated. We have earlier reported that one of the peptides studied, TNF 36-62, induced chemotaxis in granulocytes and monocytes. TNF 41-62, a shorter analog of TNF 36-62, possessed similar chemotactic effects. Both peptides caused a weak enhancement of LPS -induced IL-6 production and tissue factor activity by monocytes in whole blood. The third peptide studied, TNF 78-96, was selected from a region located on the opposite side of the β-sheet sandwich structure of the TNF molecule, and includes the loop 84-88 that has been shown to be involved in TNF receptor interaction. TNF 78-96 possessed properties quite different from TNF 36-62 and TNF 41-62. It amplified several fold PMA-induced secretion of elastase, and enhanced significantly PMA-induced secretion of cathepsin G from the neutrophils, activities which were effectively abolished by an anti-human TNF antibody. The TNF 78-96 peptide also inhibited LPS-induced TF activity in monocytes of whole blood, and it abolished the TNF enhancing effect of LPS-induced TF activty in a dose dependent manner. This suggests that the TNF 78-96 peptide may bind to the TNF receptor(s), without potentiating the same signals as native TNF. It may thereby prevent binding of the native TNF and the resultant activation effect of TNF. It also, at high concentrations, inhibited LPS-induced IL-6 production whereas it caused a doubling of LPS-induced IL-8 in monocytes and granulocytes in whole blood. These results clearly show that distinct TNF activities can be induced by peptide sequences taken from different regions of TNF. The TNF 78-96 peptide might be useful in downregulation of LPS-induced monocyte activations in vivo.
Thrombopoietin Increases Platelet Sensitivity to α-Thrombin via Activation of the ERK2-cPLA2 Pathway
Authors: Gorter Gertie , Akkerman Jan-Willem , van Willigen Gijsbert
Published: March 2018

Thrombopoietin (TPO) regulates stem cell proliferation and maturation of megakaryocytes by activating the c-Mpl-receptor, a member of the hematopoietic cytokine family. As human platelets possess c-Mplreceptors and supraphysiological concentrations of TPO trigger platelet aggregation and secretion, we searched for the signalling pathways through which the c-Mpl-receptor might activate platelets. A physiological concentration of TPO (20 ng/mL) did not trigger platelet functions, but increased their sensitivity to α-thrombin resulting in a 4-fold faster dense granule secretion. The effect of TPO was abolished by indomethacin and caused by synergism with signal generation by α-thrombin at the level of the cytosolic phospholipase A2 (cPLA2) pathway resulting in more arachidonate release, cPLA2 phosphorylation and thromboxane A2 formation. A similar synergism was seen at the level of extracellular signal-regulated kinase 2 (ERK2 or p42-MAPK). These data suggest, that TPO increases the sensitivity of platelets to α-thrombin by enhancing cPLA2 activation via the ERK2-cPLA2 pathway.
Humanized Severe Combined Immunodeficient Mice as a Potential Model for the Study of Tolerance to Factor VIII
Authors: Vanzieleghem Beatrijs , Gilles Jean , Desqueper Benoît , Vermylen Jos , Saint-Remy Jean-Marie
Published: March 2018

A Severe Combined Immunodeficient (SCID) mouse model has been established to evaluate experimental conditions leading to the production of factor VIII (FVIII) autoantibodies. To this end, we humanized 10 groups of 7 mice with peripheral blood mononuclear cells of 10 unrelated healthy blood donors (15 X 106 cells/mouse). Mice were injected with saline or immunized i.p. with 50 IU of a plasma derived human FVIII 24 h after reconstitution. Further immunization was made with 25 IU of FVIII every fortnight during 6 weeks and animals were sacrificed after 8 weeks. All reconstituted mice showed a spontaneous production of anti-FVIII antibodies in the absence of immunization with the corresponding antigen. However, no differences were observed regarding the quantity or the quality of these antibodies produced in the immunized or the saline group, indicating that tolerance to FVIII had been transferred with cell reconstitution. Affinity purified FVIII specific antibodies were capable of inhibiting FVIII activity and preventing the binding of FVIII to phospholipids in a dose-dependent manner. Immunoprecipitation experiments showed that the antibodies recognized only the C1 and C2 light chain domains. Since antibodies of interest can be found in the SCID mouse model and, moreover, since they are qualitatively comparable with the source donor’s antibodies, this model provides a tool to study the regulation of tolerance against self antigens in normal subjects and in acquired haemophilia patients.
Reduced Expression of Endothelial Cell Markers after Long-term Transdermal Hormone Replacement Therapy in Women with Coronary Artery Disease
Authors: Arnesen Harald , Hofstad Ann-Elin , Os Ingrid , Seljeflot Ingebjørg
Published: March 2018

The effects of 12 months hormone replacement therapy (HRT) on biochemical markers associated with endothelial function were studied in 98 postmenopausal women with CAD, who were randomized to transdermal HRT or a control group.

A significant reduction in the levels of von Willebrand factor in the HRT-group compared to controls was seen after 3 months, maintained after 12 months (p <0.001). Significant reduction in the HRT-group compared to controls was also seen in VCAM-1 after 3 months, sustained after 12 months (p = 0.013 and p = 0.045, respectively), and E-selectin was reduced by about 20% after 3 months on HRT, the reduction being statistically significant after 12 months (p <0.001). Significantly reduced levels of ICAM-1 were also seen after 12 months (p = 0.048). No effects could be observed on tPA-antigen or thrombomodulin.

The reduction in procoagulant and proinflammatory markers of endothelial function after long-term transdermal HRT could indicate a beneficial effect on the endothelium and thus a potentially modulating effect on the progression of atherosclerosis in women with CAD.
A Randomized Cross-over Study on the Effects of Levonorgestrel- and Desogestrel-containing Oral Contraceptives on the Anticoagulant Pathways
Authors: Tans Guido , Curvers Joyce , Middeldorp Saskia , Thomassen M. Christella , Prins Martin , Bouma Bonno , Büller Harry , Meijers Joost , Rosing Jan
Published: March 2018

The use of oral contraceptives (OC) causes disturbances of the procoagulant, anticoagulant and fibrinolytic pathways of blood coagulation which may contribute to the increased risk of venous thrombosis associated with OC therapy. Here we report the results of a cyclecontrolled randomized cross-over study, in which we determined the effects of so-called second and third generation OC’s on a number of anticoagulant parameters. In this study, 28 non-OC using women were randomly prescribed either a second generation (150 µg levonorgestrel/30 µg ethinylestradiol) or a third generation OC (150 µg desogestrel/30 µg ethinylestradiol) and who switched to the other OC after a two month wash out period. The anticoagulant parameters determined were: antithrombin (AT), α2-macroglobulin (α2-M), α1-antitrypsin, protein C inhibitor (PCI), protein C, total and free protein S and activated protein C sensitivity ratios (APC-sr) measured with two functional APC resistance tests which quantify the effect of APC on either the activated partial thromboplastin time (aPTT) or on the endogenous thrombin potential (ETP). During the use of desogestrel-containing OC the plasma levels of αc2-M, α1-antitrypsin, PCI and protein C significantly increased, whereas AT and protein S significantly decreased. Similar trends were observed with levonorgestrel-containing OC, although on this kind of OC the changes in AT, PCI and protein S (which was even slightly increased) did not reach significance. Compared with levonorgestrel, desogestrel-containing OC caused a significant decrease of total (p <0.005) as well as free protein S (p <0.0001) and more pronounced APC resistance in both the aPTT (p = 0.02) and ETPbased (p <0.0001) APC resistance tests. These observations indicate that the activity of the anticoagulant pathways in plasma from users of desogestrel-containing OC is more extensively impaired than in plasma from users of levonorgestrel-containing OC.
Plasminogen Activator Inhibitor-1 (PAI-1) 4G/5G Polymorphism, Coronary Thrombosis, and Myocardial Infarction in Middle-aged Finnish Men who Died Suddenly
Authors: Perola Markus , Wartiovaara Ulla , Peltonen Leena , Palotie Aarno , Penttilä Antti , Karhunen Pekka , Mikkelsson Jussi
Published: March 2018

High plasminogen activator inhibitor-1 (PAI-1) plasma levels increase future risk of myocardial infarction (MI). The 4G allele of the 4G/5G polymorphism of the PAI-1 gene has been associated with increased plasma levels of PAI-1. The association of the PAI-1 polymorphism with coronary narrowings, coronary thrombosis and myocardial infarction (MI) was studied in a prospective autopsy series of 300 middle-aged Caucasian Finnish men (33 to 69 yrs) suffering sudden out-of-hospital death (Helsinki Sudden Death Study). The 4G allele was found in 76.8% of men with sudden cardiac death (SCD) compared to 67.5% in men who died accidentally and 63.2% in men who died of other diseases (p = 0.08 and p = 0.055, respectively). Men possessing the 4G allele had more often acute MI (OR 3.5; p <0.05) and coronary thrombosis (OR 5.5; p = 0.01) compared to 5G homozygotes. 5G homozygotes, comprising one third of the men in our study, seem to be at a decreased risk of thrombosis, whereas carriers of the common 4G allele have an increased risk of thrombosis, AMI and possibly SCD compared to 5G homozygotes.
Identification of Eight Novel Single-Nucleotide Polymorphisms at Human Tissue-type Plasminogen Activator (t-PA) Locus: Association with Vascular t-PA Release In Vivo
Authors: Ladenvall Per , Wall Ulrika , Jern Sverker , Jern Christina
Published: March 2018

Recently, we reported that an Alu insertion polymorphism of the tissue-type plasminogen activator (t-PA) gene is associated with vascular t-PA release rates in man. In the current study we searched the t-PA gene for putative functional genetic variants in linkage disequilibrium (LD) with this polymorphism. Healthy individuals with different Alu genotypes and contrasting t-PA release rates were studied. Regulatory and coding regions of the t-PA gene were sequenced. Eight singlenucleotide polymorphisms (SNPs) were identified. Three of these were in significant LD with the Alu polymorphism and consequently associated with t-PA release rates; one in the far upstream enhancer, one in exon 6, and one in intron 10. The enhancer SNP resides within a GC box. Electrophoretic mobility shift assay (EMSA) revealed a reduced binding affinity of Sp1 to the T allele, which is the allele associated with a low t-PA release rate. Variations in exon 6 and intron 10 were silent and without apparent effect on splicing, respectively.
A New T-287C Polymorphism in the 5’ Regulatory Region of the Tissue Factor Pathway Inhibitor Gene
Authors: Moatti Didier , Haidar Bassam , Fumeron Frédéric , Gauci Laurent , Boudvillain Olivier , Seknadji Patrick , Ollivier Véronique , Aumont Marie , de Prost Dominique
Published: March 2018

Tissue factor pathway inhibitor (TFPI) is an important regulator of the extrinsic blood coagulation pathway. We screened the untranslated 5’ region of the TFPI gene for polymorphisms and investigated their possible involvement in arterial thrombosis. The allele frequencies of a new polymorphism, located 287 base pairs upstream of the transcription start site (T-287C), and that of the previously described C-399T polymorphism, were similar in cases and controls. In controls, the -287C allele was associated with significantly higher levels of total TFPI antigen, arguing for an effect of this polymorphism on TFPI gene expression. In controls, the C-399T polymorphism did not alter TFPI levels. In the cases, however, decreased total and post-heparin free TFPI levels and increased F1+2 levels were significantly associated with the -399T allele. These findings suggest that the T-287C and C-399T polymorphisms are not associated with an increased risk of coronary heart disease, a result which should be confirmed by a larger study. However, their influence on outcome, or a link with subtypes of acute coronary syndromes, cannot be excluded.
Molecular Characterisation and Three-Dimensional Structural Analysis of Mutations in 21 Unrelated Families with Inherited Factor VII Deficiency
Authors: Peyvandi Flora , Jenkins Vincent , Mannucci Pier , Billio Atto , Zeinali Sirous , Perkins Stephen , Perry David
Published: March 2018

Factor VII (FVII) is a four-domain glycoprotein that plays a critical role in the initiation of blood coagulation. Hereditary deficiencies of this plasma protein results in a bleeding diathesis that varies in severity amongst affected patients. We have analysed the FVII gene in 27 patients with FVII deficiency from 21 unrelated families predominantly of Middle-Eastern extraction. A total of 19 different mutations were identified, of which 12 were novel and 7 had been previously reported. Nine of the 12 novel mutations were missense mutations located in the Gla domain (Ser23Pro), the second epidermal growth factor domain (Cys135Arg) and the catalytic serine protease domain (Arg247Cys, Arg277Cys, Ser282Arg, Pro303Thr, Ser363Ile, Trp364Cys, Trp364Phe), of which five are homozygous. Three novel splice mutations were identified in intron 1a (IVS1a+5), intron 2 (IVS2+1) and intron 6 (IVS6+1). Of the seven previously reported mutations, five were missense mutations of which three are homozygous (Gln100Arg, Arg152Gln, Arg304Gln, Cys310Phe and Thr359Met), one was a 17 bp deletion (10585del17bp) and one was a splice site mutation within intron 7 (IVS7+7). This study has significantly extended the current database of FVII mutations, including the number of known homozygous mutations. Conformational analyses of crystal structures for FVIIa and the FVIIa-tissue factor complex provided likely explanations for the effect of the missense mutations on FVIIa secretion or function. In particular, since 23 missense mutations were located to the serine protease domain, mostly to the region between the catalytic triad and the contact surface with tissue factor, this showed that the orientation of the serine protease domain relative to bound tissue factor in the complex is crucial for functional activity.
Discrimination of von Willebrands Disease (VWD) Subtypes: Direct Comparison of von Willebrand Factor:Collagen Binding Assay (VWF:CBA) with Monoclonal Antibody (MAB) Based VWF-capture Systems
Authors: Henniker Anthony , Facey David , Hertzberg Mark , Favaloro Emmanuel
Published: March 2018

Discrimination of von Willebrand’s Disease (VWD) subtypes is important since it influences management. Qualitative [ie Type 2A, 2B, 2M] defects exhibit von Willebrand factor (VWF) discordance and give high VWF:Ag to VWF:’activity’ ratios. Classically, VWF:’activity’ is assessed using the VWF:RCof assay. The VWF:CBA is an ELISAbased VWF-functional adhesive assay which has consistently proved to be superior to VWF:RCof. A commercially available monoclonal antibody (MAB) based ELISA assay system claimed to mimic a VWF:RCof-like activity has also been recently described (‘SE’), as has the production and characterisation of a large number [n = 10] of locally generated anti-VWF MAB. In the current study, we have adapted these MAB to in-house ELISA assays to assess their utility for VWD diagnosis and subtype discrimination, and to compare them with other assay systems. Thus, the VWF:CBA, VWF:RCof by agglutination, the SE assay, and in-house MAB based assays have been directly compared for their ability to discriminate Type 1 [n = 9] from Type 2 VWD samples [phenotypes 2A and 2B; n = 11]. In summary, MAB-based systems can be used to measure VWF and confirm a diagnosis of VWD, as well as exhibiting some VWD-subtype-discriminatory capabilities. However, better evidence of VWF-discordance was usually achieved using the VWF:RCof (agglutination) assay, while the greatest degree of VWFdiscordance was consistently observed using the VWF:CBA assay. In conclusion, the VWF:CBA assay proved to offer the best diagnostic predictive tool for a Type 2 VWD defect, while MAB-based systems appear to be less effective in this regard.

Abbreviations: HMW: High Molecular Weight [VWF], MAB: Monoclonal antibody (/antibodies), PNP: Pooled Normal Plasma, RIPA: Ristocetin induced platelet aggregation, VWD: von Willebrands disease (/disorder), VWF: von Willebrand Factor, VWF:Ag: von Willebrand Factor Antigen (assay), VWF:CBA: Collagen Binding [Activity] Assay for VWF, VWF:RCof: Ristocetin Cofactor Assay for VWF
Sequence Alignment between vWF and Peptides Inhibiting the vWF-collagen Interaction Does not Result in the Identification of a Collagen-binding Site in vWF
Authors: van der Plas R. M. , Vandecasteele G. , Vauterin S. , Huizinga E. G. , Sixma J. J. , Deckmyn H. , Vanhoorelbeke K.
Published: March 2018

We previously found that two peptides (N- and Q-peptide) selected by phage display for binding to an anti-vWF antibody, were able to inhibit vWF-binding to collagen (1). The sequence of those peptides could be aligned with the sequence in vWF at position 1129-1136 just outside the A3-domain. As the peptides represent an epitope or mimotope of vWF for binding to collagen we next wanted to study whether the alignment resulted in the identification of a new collagen binding site in vWF. We mutated the 1129-1136 VWTLPDQC sequence in vWF to VATAPAAC. Expressing this mutant vWF (7.8-vWF) in a fur-BHK cell line resulted in well processed 7.8-vWF containing a normal distribution of molecular weight multimers. However, binding studies of this mutant vWF to rat tail, human and calf skin collagens type I, to human collagen types III and VI, revealed no decrease in vWF-binding to any of these collagens. Thus, although the N-and Q-peptides did inhibit the vWF-collagen interaction, the resulting alignment with the vWF sequence did not identify a collagen binding site, pointing out that alignments (although with a high percentage of identity) do not always result in identification of binding epitopes. However, suprisingly removal of the A3-domain or changing the vWF sequence at position 1129-1136 resulted in an increase of vWF-binding to human collagen type VI and to rat tail collagen type I, implying that these changes result in a different conformation of vWF with an increased binding to these collagens as a consequence.
Levels of Activated FXII in Survivors of Myocardial Infarction – Association with Circulating Risk Factors and Extent of Coronary Artery Disease
Authors: Kohler Hans , Carter Angela , Stickland Max , Grant Peter
Published: March 2018

Little data is available regarding the activated form of factor XIIa (FXIIa) in survivors of myocardial infarction. 292 Caucasian patients characterised for extent of coronary atheroma by angiography and for a past history of myocardial infarction and 77 healthy controls were included in the study. To investigate the relationship between coronary artery disease, activated factor XII and other circulating factors, we studied levels of FXIIa, cholesterol, triglycerides, fasting insulin, fibrinogen, FVII:C, t-PA antigen and PAI-1 antigen. Factor XIIa levels were higher in all patients [2.5 (2.3-2.6) ng/ml] and in patients with a history of MI [2.6 (2.4-2.9) ng/ml] than in controls [1.9 (1.7-2.1) ng/ml], p <0.0001. In patients, FXIIa levels positively correlated with FVII:C, BMI, cholesterol, insulin, PAI-1 antigen, t-PA antigen and triglycerides. In controls FXIIa levels only correlated with PAI-1 antigen and triglycerides. FXIIa levels were strongly associated with extent of coronary stenosis: 2.8 (2.6-3.1) ng/ml and 2.6 (2.3-2.9 ng/ml) in those with 2 and 3 vessels stenosed compared to 2.1 (1.9-2.3) ng/ml in those with 0 vessel stenosed (p = 0.0004). Activated FXII relates to both extent of coronary atheroma and to a past history of myocardial infarction and clusters with features of insulin resistance.
Attenuation of Thrombolysis-induced Increase of Plasminogen Activator Inhibitor-1 by Intravenous Enalaprilat
Authors: Wagner Andreas , Gwechenberger Marianne , Herkner Harald , Müllner Marcus , Woisetschläger Christian , Laggner Anton , Hirschl Michael
Published: March 2018

We examined the effect of intravenous enalaprilat on the course of PAI-1 plasma levels in 23 patients with acute myocardial infarction undergoing thrombolytic therapy. All patients received 100 mg aspirin, 1000 IU/h heparin, thrombolysis with 100 mg rt-PA within 90 min, and betablockers. Eleven out of 23 patients received 5 mg enalaprilat intravenously prior to thrombolysis. Blood samples for determination of PAI-1 plasma levels were collected on admission, 2, 4, 6, 12, and 24 h after thrombolysis. PAI-1 plasma levels in patients receiving enalaprilat were similar to those of the control patients before thrombolysis (5 ng/ml, 95% confidence interval: 2-10 vs. 7 ng/ml, 95% confidence interval: 2-10; p = 0.5). The PAI-1AUC was 9 ng/ml/h (95% confidence interval: 5-10) in the enalaprilat group and 19 ng/ml/h (95% confidence interval: 13-26) in the control group (p = 0.0006). The maximum difference was observed 6 h after thrombolysis (enalaprilat: 13 ng/ml, 95% confidence interval: 5-25, control: 42 ng/ml, 95% confidence interval: 18-55; p = 0.003).

Our study clearly demonstrates that application of intravenous enalaprilat prior to thrombolysis attenuates the thrombolysis-related increase of PAI-1. This finding may suggest a possible therapeutic approach to influence the fibrinolytic system in patients with acute myocardial infarction after thrombolysis.