bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient approach for establishing the economic and operating reliability via optimal coordination of wind–PSH–solar‐storage hybrid plant in highly uncertain double auction competitive power market
Authors: Dawn Subhojit , Tiwari Prashant Kumar , Goswami Arup Kumar
Published: March 2018

This study presents a two‐stage competent and efficient approach for optimal operation of wind–pumped‐storage‐hydro (PSH)–solar–thermal‐storage hybrid power plant to get maximum system revenue and profit along with maintaining the grid frequency. The wind speed is predicted for a deregulated market and accordingly, the wind plants are committed to supplying the demand. The operation of PSH, battery and solar power are considered in order to minimise the adverse effect of imbalance cost which comes into the picture due to the mismatch between actual and predicted wind power. The proposed operating strategy for the complex hybrid plant helps to reduce the uncertainty of renewable power sources in an economical manner. Two new energy levels associated with pumped storage, i.e. PEopt and PElow and four energy levels associated with the battery, i.e. BEmax, BEopt, BElow and BEmin have been considered in this work to show the robustness of the proposed strategy. The proposed approach is implemented and compared using Mi‐Power, bat algorithm, particle swarm optimisation algorithm, genetic algorithm and cuckoo search algorithm. Modified IEEE 14‐bus system is used to validate the effectiveness of the proposed approach. The bilateral contracts with a double auction bidding model for the competitive power market are also considered for the implementation.
Aldazines in the Castagnoli–Cushman Reaction
Authors: Mikheyev Alexander , Kantin Grigory , Krasavin Mikhail
Published: March 2018

Aldazines were employed in the Castagnoli–Cushman reaction of homophthalic anhydride for the first time. The reaction proved to be distinctly diastereoselective when conducted at room temperature in acetonitrile, yielding predominantly the kinetic cis-configured adduct as a racemate. Thermodynamically more stable trans-configuration could be attained either via heating the cis-configured product in DMSO or via the action of a base (imidazole) in the course of CDI-promoted esterification or amidation of the carboxylic acid function in the initial adducts. Further manipulation of the remaining N-acylhydrazone moiety in the latter led to N-alkyl- or fully unprotected N-aminolactams.
A De Novo Synthetic Route to 1,2,3,4-Tetrahydroisoquinoline Derivatives
Authors: Fülöp Ferenc , Kiss Loránd , Ábrahámi Renáta , Fustero Santos
Published: March 2018

A novel synthetic approach was developed for the construction of the 1,2,3,4-tetrahydroisoquinoline framework possessing varied functions. The synthetic strategy was based on oxidative ring opening of some indene derivatives through their C=C bond, followed by double reductive amination of the dicarbonyl intermediates with various primary alkyl- or fluoroalkylamines.
Effect of New Synthetic Heparin Mimetics on Whole Blood Thrombin Generation In Vivo and In Vitro in Rats
Authors: Hérault J.P. , Bernat A. , Gaich C. , Herbert J.M.
Published: March 2018

The effect of new heparin mimetics (synthetic oligosaccharides) was studied in vitro with regard to thrombin generation (TG) in rat platelet rich plasma (PRP) and whole blood (WB) and in vivo on stasis-induced venous thrombosis in the rat.

TG in PRP and in WB was highly dependent on platelet count and strongly influenced by the haematocrit. The peak of TG appeared to be significantly higher in WB than in PRP whereas the endogenous thrombin potential (ETP) was not significantly different under either condition.

The effect of hirudin, the synthetic pentasaccharide SR90107/ Org31540 (SP) and heparin were measured on TG in PRP and WB. We then compared the effect of two new synthetic heparin mimetics (SR121903A and SanOrg123781) with potent and comparable antithrombin (AT) mediated activity against factor Xa and thrombin. These two compounds were made of a pentasaccharide with a high affinity to AT, prolonged at the non-reducing end by an oligosaccharide chain recognised by thrombin. In SR121903A, the charge density and charge distribution was analogous to that of heparin whereas in SanOrg123781 the charges were only located on the last 5 saccharides of the non-reducing end of the molecule. In PRP and in WB, SR121903A acted on the lag time and on the AUC whereas SanOrg123781 inhibited thrombin formation with no effect on the lag time. SanOrg123781 was more potent in inhibiting TG than SR121903A. This difference was due to the structures of the compounds that differed in their ability to be neutralised by platelet factor 4. The antithrombotic effect of the two compounds was examined in a venous thrombosis model in rats. We observed that SanOrg123781 was more active than SR121903A and heparin.

Taken together, these results indicate that the activity of oligosaccharides is greatly influenced by the global charge density of the molecule and show that SanOrg123781 is a potent and promising antithrombotic drug candidate.
Substitution of Gly-548 to Ala in the Substrate Binding Pocket of Prothrombin Perijá Leads to the Loss of Thrombin Proteolytic Activity
Authors: Sekine Osamu , Ebisawa Kazuyoshi , Umeyama Hideaki , Iwahana Hiroyuki , Ruiz-Sáez Arlette , de Bosch Norma , Matsuda Michio , Sugo Teruko
Published: March 2018

Prothrombin Perijá is a dysprothrombin derived from a homozygous patient that manifests low thrombin activity upon activation in a onestage assay. Purified prothrombin Perijá showed normal appearance on SDS-PAGE, and was cleaved normally to form α-thrombin by the prothrombinase complex. The activated form, thrombin Perijá, however, did not show any proteolytic activity towards native substrates, fibrinogen, protein C or various synthetic substrates for α-thrombin, but it was able to bind to antithrombin III, although the binding capacity was markedly reduced even in the presence of heparin. Thrombin Perijá showed full reactivity toward a small inhibitor, DFP, indicating that the molecular defect is in the substrate binding site in the thrombin molecule but not in the active site itself. By DNA sequence analysis of the patient prothrombin gene, we identified a G to C mutation at nucleotide 20016 in exon 14, which predicts a Gly-548 to Ala substitution in the prothrombin Perijá molecule. The structural modeling of thrombin Perijá suggests that Ala-548 is located close to the limb of the cavity wall of the substrate binding pocket, and that the methyl group blocks protrusion of the guanidino group of Arg into the cavity. This steric hindrance may well inhibit the access of Arg-containing substrates to the catalytic Ser-525 leading to the loss of proteolytic activity.
Molecular Modeling of the Seven Tandem Leucine-Rich Repeats within the Ligand-Binding Region of Platelet Glycoprotein Ibα
Authors: Whisstock James , Shen Yang , López José , Andrews Robert , Berndt Michael
Published: March 2018

Platelet glycoprotein (GP)Ib-IX-V mediates von Willebrand Factor (vWF)-dependent adhesion to vascular subendothelium at high shear in (patho)physiological thrombus formation. The ligand-binding domain of GPIb-IX-V is within the N-terminal 282 residues of GPIbα, that contains seven tandem leucine-rich repeats (Leu36–Ala200). Repeats 2–4 are critical for vWF binding. In this study, we have built molecular models of the seven leucine-rich repeats of human, canine and mouse GPIbα, providing novel insights into the species-specific interaction between human vWF and its receptor. Interestingly, a major difference between the models was a large negatively charged patch on the concave face of human, but not canine, repeats 2–4. In addition, five individual mutations within the leucine-rich repeats of GPIbα associated with the bleeding disorder Bernard-Soulier syndrome, that result in dysfunctional vWF binding, were mapped to the model of human GPIbα. This provides the basis for relating these genetic lesions to abnormal function of the receptor.
A Defect in Collagen Receptor-Ca2+ Signaling System in Platelets from Cattle with Chediak-Higashi Syndrome
Authors: Shiraishi Mitsuya , Kawashima Sachiko , Moroi Masaaki , Shin Yangchol , Morita Takashi , Horii Yoichiro , Ikeda Masahiro , Ito Katsuaki
Published: March 2018

Decreased platelet aggregation to collagen is a cause for bleeding diathesis of Chediak-Higashi syndrome (CHS). We investigated whether the collagen receptor-Ca2+ signaling system was impaired in platelets from cattle affected with CHS. A collagen-induced increase in cytosolic Ca2+ ([Ca2+]i) was depressed in CHS platelets, which was accompanied by a decrease in the production of inositol 1,4,5-trisphosphate. When the influences of endogenous arachidonic acid metabolites and ADP were excluded, convulxin or collagen-related peptide, which are specific agonists for the collagen receptor GPVI, increased [Ca2+]i in both normal and CHS platelets. In contrast, rhodocytin, which was thought to activate another collagen receptor GPIa/IIa, increased [Ca2+]i in CHS platelets to a lesser extent than in normal ones. Cytochalasin D, an actin polymerization inhibitor, depressed the response to collagen or rhodocytin but not the response to convulxin. Adhesion of CHS platelets to acid soluble type I collagen, which was mediated by GPIa/IIa, was similar to that of normal platelets. These results suggest that a defect in the rhodocytin-sensitive pathway is responsible for decreasing the response to collagen in CHS platelets. It remains to be determined which receptor is associated with the mechanism.
Protein C Global Assay in the Evaluation of Women with Idiopathic Pregnancy Loss
Authors: Sarig Galit , Lanir Naomi , Hoffman Ron , Brenner Benjamin
Published: March 2018

Since the majority of thrombophilic defects in women with pregnancy loss are in the protein C pathway, we have prospectively determined the diagnostic value of a protein C global assay in 60 consecutive women with pregnancy loss compared to 61 controls. Protein C activation time normalized ratio (PCAT-NR) in pregnancy loss women was significantly lower than controls (0.74 ± 0.16 vs. 0.99 ± 0.2; P <0.0001). PCAT-NR lower than cut off level of 0.8 were found in 42/60 (70%) of PL women compared to 7/61 (11%) of controls (OR = 18.0, 95% CI: 6.3-53.4, P < 0.0001).

Cut-off level of 0.8 successfully identified all pregnancy loss women with abnormality in the protein C pathway (12 factor V Leiden, 7 APC-Resistance without factor V Leiden, 15 low levels of protein S and 1 of protein C). Moreover, PCAT-NR below 0.8 was documented in 15/29 (52%) of PL women without thrombophilic risk factor compared to 3/55 (5%) of controls (OR = 18.6, 95% CI: 4.2-95.2, P <0.0001).

These results suggest that ProC Global may be useful as a screening test for protein C pathway abnormalities and may serve as a new thrombophilic risk factor in women with pregnancy loss.
Characterization of the Binding of Urokinase-Type Plasminogen Activator to the Asialoglycoprotein Receptor
Authors: van der Kaaden M. E. , Groeneveld E. , Barrett-Bergshoeff M. M. , van Berkel Th. J. C. , Kuiper J. , Rijken D. C.
Published: March 2018

In order to study the role of the asialoglycoprotein receptor (ASGPr) in the rapid plasma clearance of urokinase-type plasminogen activator (u-PA), a microtiter plate binding assay was developed using ASGPr purified from rat liver extracts. Urinary two-chain u-PA bound to immobilized ASGPr in a saturable manner with an EC50 of 0.2 µM. Binding was inhibited by rabbit antibodies against the ASGPr. In line with the known carbohydrate specificity of the ASGPr, GalNAc proved to be the most effective inhibitor from a series of monosaccharides, followed by Gal and Fuc, whereas GlcNAc was ineffective. The N-linked oligosaccharides of urinary u-PA do not terminate with the common Gal-GlcNAc element, but with a GalNAc-GlcNAc element which is partially sulfated. Sulfated forms of u-PA were separated from non-sulfated forms by using the lectin Wisteria floribunda agglutinin. Only the non-sulfated forms of u-PA (30% of the total) appeared to bind to the ASGPr. From different u-PA preparations used for thrombolytic therapy only urinary u-PA and u-PA produced by kidney cell cultures strongly bound to the ASGPr, whereas (recombinant) u-PA expressed in mouse myeloma cells, Chinese hamster ovary cells or E. coli scarcely bound to the receptor. It is concluded that u-PA bearing non-sulfated GalNAc-GlcNAc elements is specifically recognized by the ASGPr present on liver cells.
The −33T → C Polymorphism in Intron 7 of the TFPI Gene Influences the Risk of Venous Thromboembolism, Independently of the Factor V Leiden and Prothrombin Mutations
Authors: Borgel Delphine , Fumeron Frédéric , Moatti Didier , Alhenc-Gelas Martine , Grandchamp Bernard , Aiach Martine , Emmerich Joseph , Ameziane Nejma , Seguin Cendrine , de Prost Dominique
Published: March 2018

We have previously identified, in intron 7 of the TFPI gene, a T to C single-base polymorphism (−33T→C) which is strongly associated with total circulating TFPI antigen levels. Here we examined the influence of this polymorphism on the risk of venous thromboembolism. The polymorphism was identified in the PATHROS study population (330 cases with venous thromboembolism and 826 controls). The CC genotype was found in 6.4% of cases and 10.2% of controls (age-adjusted odds ratio 0.6; 95% CI 0.3–0.9; p = 0.03). This protective effect persisted after adjustment for oral contraception and the factor V Leiden and prothrombin gene polymorphisms. In 171 controls and 49 cases in whom blood was taken at least three months after the thrombotic event, the CC genotype was associated with significantly higher total TFPI levels than the TT genotype. These results suggest that the CC genotype of the TFPI intron 7 polymorphism is an independent protective factor for venous thromboembolism, an effect probably mediated by increased TFPI levels.
Effect of Homocysteine Reduction by B-Vitamin Supplementation on Markers of Clotting Activation
Authors: Klerk Mariska , Verhoef Petra , Verbruggen Bert , Schouten Evert , Blom Henk , Bos Gerard , den Heijer Martin
Published: March 2018

Homocysteine may have an effect on risk of cardiovascular disease by stimulating procoagulant factors and/or impair anti-coagulant mechanisms or fibrinolysis. However, data in humans of such effects are sparse. In this intervention study, we examined the effect of homocysteine lowering by B-vitamin supplementation on prothrombin fragments 1 and 2 (F1+2), thrombin-antithrombin complex (TAT), and fibrin degradation products (D-dimer). The study comprised 118 healthy volunteers, 50 with homocysteine > 16 µmol/L and 68 with homocysteine ≤ 16 µmol/L, who were randomized to placebo or highdose B-vitamin supplements (5 mg folic acid, 0.4 mg hydroxycobalamin, and 50 mg pyridoxine) daily for 8 weeks. Although homocysteine concentrations were 27.7% (p < 0.0001) reduced in the Bvitamin group compared to the placebo group, no effect on F1+2 and TAT concentrations was observed. A 10.4% reduction was observed for D-dimer (p = 0.08).

In conclusion, it appears that in healthy subjects homocysteine reduction by B-vitamin supplementation has a modest beneficial effect on clotting activation.
Reduced Activation of the Gla19Ala FX Variant via the Extrinsic Coagulation Pathway Results in Symptomatic CRMred FX Deficiency
Authors: Pinotti M. , Marchetti G. , Baroni M. , Cinotti F. , Morfini M. , Bernardi F.
Published: March 2018

We characterized a symptomatic CRMred factor X (FX) deficiency produced by the Glu19Ala mutation in the γ-carboxyglutamic-rich domain. FX activity levels in plasma were markedly reduced in prothrombin time assays (< 1-5%), whereas in activated partial thromboplastin assays (16%) and in RVV assays (17%) the reduction in activity mirrored that in antigen levels (17%). Activation of recombinant 19Ala-FX by factor IXa/factor VIIIa or RVV, and the activity in thrombin generation assays, were comparable to those of wild-type FX. Differently, complete activation of recombinant 19AlaFX required a factor VIIa/TF concentration 30-fold higher than that of wild-type FX. The recombinant FVIIa significantly reduced PT values in 19Ala-FX reconstituted plasma, thus suggesting an alternative approach for treatment of FX deficiencies characterized by defective FX activation.

The study of this FX deficiency provides an “in vivo” and “in vitro” model for the investigation of Gla domain interactions.
Activated Protein C Inhibits Lipopolysaccharide-Induced Tumor Necrosis Factor-α Production by Inhibiting Activation of both Nuclear Factor-κB and Activator Protein-1 in Human Monocytes
Authors: Yuksel Mehtap , Uchiba Mitsuhiro , Horiuchi Seikoh , Okabe Hiroaki , Okajima Kenji
Published: March 2018

Activated protein C (APC), an important natural anticoagulant, inhibits tumor necrosis factor-α (TNF-α) production and attenuates various deleterious events induced by lipopolysaccharide (LPS), contributing thereby to a significant reduction of mortality in patients with severe sepsis. In this study, we investigated the mechanism(s) by which APC inhibits TNF-α production by LPS-stimulated human monocytes in vitro. Although APC inhibited LPS-induced TNF-α production in a concentration-dependent fashion, diisopropyl fluorophosphate-treated APC, an active-site-blocked APC, had no effect. APC inhibited both the binding of nuclear factor-κB (NF-κB) to target sites and the degradation of IκBα. APC also inhibited both the binding of activator protein-1 (AP-1) to target sites and the activation of mitogen-activated protein kinase pathways. These observations strongly suggest that APC inhibited LPS-induced TNF-α production by inhibiting the activation of both NF-κB and AP-1 and that the inhibitory activity of APC might depend on its serine protease activity. These results would at least partly explain the mechanism(s) by which APC reduces the tissue injury seen in animal models of sepsis and in patients with sepsis.
Ciglitazone Inhibits Plasmin-Induced Proinflammatory Monocyte Activation via Modulation of p38 MAP Kinase Activity
Authors: Syrovets Tatiana , Schüle Almut , Jendrach Marina , Büchele Berthold , Simmet Thomas
Published: March 2018

Plasmin triggers chemotaxis and NF-κBand AP-1-mediated proinflammatory gene expression in human peripheral monocytes (PM). Compared with macrophages and dendritic cells, PM express mainly the peroxisome proliferator-activated receptor (PPAR) γ and traces of PPARα as detected by semiquantitative RT-PCR and immunoblotting. The PPARγ agonist ciglitazone, but not the PPARα agonist clofibric acid, concentration-dependently inhibited the plasmin-, but not the FMLP-induced PM chemotaxis. Similarly, release of interleukin (IL)-1α, IL-1β and tumor necrosis factor (TNF)-α from plasmin-stimulated PM was concentration-dependently inhibited by ciglitazone, but not by clofibric acid, while the LPS-induced TNF-α release remained unaffected by any of both PPAR agonists. Ciglitazone activates PPARγ as shown by a novel surface plasmon resonance analysis and inhibits the plasmin-induced activation of NF-κB and AP-1. It also inhibits p38 MAPK phosphorylation essential for the plasmin-induced PM chemotaxis and gene activation. Thus, activation of PPARγ by ciglitazone may allow controling of the plasmin-mediated recruitment and activation of PM at sites of inflammation.
Comparison of Outcomes after Hospitalization for Deep Venous Thrombosis or Pulmonary Embolism
Authors: Murin Susan , Romano Patrick , White Richard
Published: March 2018

Venous thrombosis and pulmonary embolism are commonly viewed as different manifestations of a single disease process, venous thromboembolism. Recent evidence suggests that there may be important differences between patients who manifest these two conditions. Using linked hospital discharge records we analyzed 71,250 patients hospitalized with a principal diagnosis of venous thrombosis alone or pulmonary embolism and analyzed predictors of rehospitalization within 6 months for venous thrombosis or pulmonary embolism. There were 51233 patients diagnosed with venous thrombosis alone and 21,625 diagnosed with pulmonary embolism. Comparing patients initially diagnosed with venous thrombosis alone to patients with pulmonary embolism, the relative risk of being rehospitalized with venous thrombosis within 6 months for venous thrombosis was 2.7. Conversely, when patients with pulmonary embolism were compared to patients with venous thrombosis alone, the relative risk of rehospitalization within 6 months with a diagnosis of pulmonary embolism was 4.2. In multivariate models the strongest predictor of recurrent thromboembolism manifest as pulmonary embolism was an initial diagnosis of pulmonary embolism and the strongest predictor of recurrence as venous thrombosis was an initial diagnosis of venous thrombosis. We conclude that the initial clinical manifestation of thromboembolism strongly predicts the manifestation of a recurrence. Venous thrombosis and pulmonary embolism appear to be distinct, albeit overlapping, clinical entities with different natural histories.

Presented at the International Society of Thrombosis and Haemostasis Meeting in Paris, France on July 9, 2001
Antithrombin ‘DREUX’ (Lys114Glu): A Variant with Complete Loss of Heparin Affinity
Authors: Huntington James , Conard Jacqueline , Carrell Robin , Mushunje Alec , Zhou Aiwu
Published: March 2018

Here we report the finding of a new natural antithrombin mutation that confirms the critical contribution of lysine 114 to the binding of the core heparin pentasaccharide, with the replacement of lysine 114 by glutamate causing a complete loss in affinity. The variant was identified in a father and son, the father having been investigated for an episode of cerebral ischaemia associated with hypercholesterolaemia. The variant forms SDS-stable complexes with activated factor X (fXa) and its thermal stability and rate of factor Xa inhibition in the absence of heparin are identical to those of normal antithrombin. Normal antithrombin binds to the high affinity heparin pentasaccharide with a Kd of 1nM, as detected by a 45% change in intrinsic fluorescence, resulting in a 230-fold increase in rate of factor Xa inhibition. However, no change in fluorescence was detected for the variant when titrated with heparin or the heparin pentasaccharide, nor was there detectable activation towards factor Xa, indicating a complete loss of heparin binding.
Leukocyte trans-endothelial migration: JAMs add new pieces to the puzzle
Authors: Preissner Klaus , Santoso Sentot , Chavakis Triantafyllos
Published: March 2018

The molecular details of leukocyte transmigration through the endothelial barrier (also called diapedesis), which is the final step of leukocyte extravasation from the circulation to a given site of inflammation, are by far not well understood. The present review will focus on the different mechanisms potentially involved in leukocyte trans-endothelial migration. Both homophilic and heterophilic interactions between leukocyte and endothelial cell receptors will be covered, with a particular focus on the growing gene family of junctional adhesion molecules (JAM). Deciphering their mechanisms of interaction will also allow to unravel novel strategies for therapeutic intervention in inflammatory or atherothrombotic diseases.
Orthopaedic surgery in persons with haemophilia
Authors: Rodriguez-Merchan E. C.
Published: March 2018

The orthopaedic problems of haemophilia and the surgical techniques most frequently performed by orthopaedic surgeons on haemophilia patients are summarised in this review article. Sometimes it can be recommendable to carry out two or three orthopaedic surgical procedures in a single operative session, with the aim of solving the functional problem in a more global way, which usually is polyarticular. There is no doubt that such an undertaking implies a greater anaesthetic risk, but also avoids the repetition of surgical procedures and saves factor comsumption. Close cooperation between haematologists, orthopaedic surgeons, rehabilitation physicians, paediatricians, psychologists, physiotherapists and nurses is paramount for the satisfactory result of any orthopaedic surgical procedure. There is no doubt that continuous prophylactic clotting factor replacement (prophylactic therapy) is the optimal way to avoid the orthopaedic problems of haemophilia that can still be seen today. Until such a goal can be reached, the orthopaedic surgeon will continue performing arthrocentheses, synoviortheses, synovectomies, tendon lengthenings, débridements, osteotomies, joint replacements, osteosynthesis of fractures and other less frequent surgical procedures on persons with haemophilia.
Localization of tissue factor pathway inhibitor to lipid rafts is not required for inhibition of factor VIIa/tissue factor activity
Authors: Jack Garnet , Page Keith , Tetzloff Tina , Hall Connie , Mast Alan , Dietzen Dennis
Published: March 2018

Tissue factor pathway inhibitor (TFPI) abrogates coagulation initiated by the factor VIIa/tissue factor catalytic complex. While the gene structure of TFPI suggests that it is a secreted protein, a large pool of TFPI is associated with the vascular endothelium through its affinity for a glycosylphosphatidylinositol (GPI)-linked membrane protein. Inhibition of tissue factor by TFPI coincides with the translocation of quaternary complexes containing tissue factor, factor VIIa, factor Xa, and TFPI to detergent-insoluble plasma membrane domains rich in cholesterol, sphingomyelin, and GPI-linked proteins known as lipid rafts and caveolae. It is not known if localization of TFPI to these membrane domains is required for its inhibition of tissue factor procoagulant activity. We generated chimeric TFPI molecules linked directly to the plasma membrane via a GPI anchor or hydrophobic transmembrane domain and expressed these in HEK293 cells that produce tissue factor but not endogenous TFPI. The GPI-anchored chimera was exclusively enriched in detergent-insoluble membrane fractions while the transmembrane molecule was not. Transfectants expressing equal levels of the GPI-linked or transmembrane TFPI displayed equal anticoagulant potency as assessed by tissue factor-mediated conversion of factor X to factor Xa. Disruption of lipid rafts with cyclodextrin likewise had no effect on the inhibitory activity of the transmembrane or GPI-linked TFPI chimeras in HEK293 cells, nor on endogenous TFPI expressed by ECV304 cells. Thus, we conclude that the GPI anchor and membrane localization to lipid rafts does not enhance inhibition of factor VIIa/ tissue factor by cell-surface associated TFPI.
Peripheral artery disease in type 2 diabetes: the role of fibrinolysis
Authors: Piarulli Francesco , Sartore Giovanni , Rossetti Ciro , Martano Luigi , Carraro Paolo , De Paoli Massimo , Fedele Domenico , Lapolla Annunziata
Published: March 2018

The aim of the present study is to verify the relationship between peripheral artery disease (PAD) and some coagulation/fibrinolysis parameters in type 2 diabetic patients.

Sixty-three type 2 diabetic patients, without PAD, were studied at baseline and after 4 years. Assessments included tissue-Plasminogen Activator (t-PA), Plasminogen Activator Inhibitor-1 antigen (PAI-1 Ag), Plasminogen Activator Inhibitor-1 activity (PAI-1 Act), Plasminogen (Pl), Fibrin peptide A (FPA), Fibrinogen (Fr), and the ankle/brachial pressure index (ABI).

We observed a significant difference between diabetic patients and controls as regards tPA (11.8 ± 5.4 vs. 6.6 ± 3.0 ng/ml; p <0.05 ) and PAI-1 Act (17.8 ± 9.2 vs. 11.7 ± 6.6 ng/dl; p <0.005). After 4 years 13 diabetic patients became vasculo-pathic and, at baseline, had significantly lower tPA (8.9 ± 4.8 vs. 12.5 ± 5.3; p <0.011), and higher PAI-1 Ag (50.8 ± 22.2 vs. 32 ± 22.2; p <0.006), and PAI-1 Act values (24.1 ± 9.5 vs. 16.1 ± 8.4; p <0.014), compared with 50 diabetic patients who did not develop PAD after 4 years.

These data show that the physiological equilibrium which exists between t-PA and PAI-1 moves towards higher levels in our diabetic patients compared with controls, at baseline, whereas diabetic patients who developed PAD showed a shift towards an antifibrinolytic pathway with diminished t-PA, increased PAI-1 Ag and PAI-1 Act and consequently procoagulant activity. Our study suggests that hypofibrinolysis may be involved in the future onset of PAD in type 2 diabetic patients.
Vascular endothelial growth factor (VEGF-A) plasma levels in non-small cell lung cancer: relationship with coagulation and platelet activation markers
Authors: Roselli Mario , Mineo Tommaso , Basili Stefania , Mariotti Sabrina , Martini Francesca , Bellotti Annamaria , Ambrogi Vincenzo , Spila Antonella , D’Alessandro Roberta , Gazzaniga Pier , Guadagni Fiorella , Ferroni Patrizia
Published: March 2018

Platelet activation, commonly found in lung cancer patients, may cause the release of angiogenic factors, such as vascular endothelial growth factor (VEGF-A). The present study was designed to investigate whether plasma VEGF-A levels were associated to different stages of non-small cell lung cancer (NSCLC). Moreover, sP-selectin, prothrombin fragment 1+2 (F1+2),thrombin-antithrombin III complex (TATc) and D-dimer levels were measured to test the hypothesis of an involvement of platelet and coagulation activation in tumor angiogenesis. VEGF-A, sP-selectin, F1+2, TATc and D-dimer levels were elevated in 65 patients with NSCLC, particularly in metastatic patients. sP-selectin (p <0.003) and F1+2 (p <0.005) levels were independently associated to VEGF-A. In addition, patients with positive levels of both sP-selectin and F1+2 had the highest levels of VEGF-A. In conclusion, our findings support the hypothesis that thrombin generation might induce platelet activation and VEGF-A release in NSCLC.
Elevation of FVIII: C in Venous Thromboembolism Is Persistent and Independent of the Acute Phase Response
Authors: Mumford Andrew , Manning Richard , Laffan Michael , O’Donnell James
Published: March 2018

Recent literature has suggested a role for elevated FVIII:C in venous thromboembolic disease (VTED). However since FVIII:C is known to rise in response to an acute phase reaction, it is difficult to determine whether the increased FVIII:C precedes the thrombosis or represents a secondary reactive phenomenon. In an attempt to address this question, we followed 35 patients with confirmed VTED, raised FVIII:C level (>1.5 iu/ml) and no other thrombotic tendency. Serial measurements of FVIII:C, vWF:Ag, C-reactive protein and fibrinogen were performed. We hypothesized that a persistent increase in FVIII:C in the absence of any other measures of ongoing acute phase response, would support the idea that elevation of FVIII:C is a constitutional phenomenon.

Of this initial group, 94% continued to have an elevated FVIII:C level throughout the period of follow up (median 8 months; range 3 to 39 months), with no significant difference between the FVIII:C levels determined at first estimation and those obtained during follow up (p = 0.58). Conversely, only 18% had evidence of an acute phase reaction when first assessed, and nonparametric ranking analysis demonstrated no correlation between FVIII:C and either C-reactive protein or fibrinogen (p = 0.315 and 0.425 respectively).We conclude that increased FVIII:C levels following VTED are persistent, independent of the acute phase reaction, and thus may represent a constitutional risk factor for VTED.
Factor II G20210A and Factor V G1691A Gene Mutations and Peripheral Arterial Occlusive Disease
Authors: Köppel Herwig , Brodmann Marianne , Pabst Edmund , Schallmoser Katharina , Toplak Hermann , Wascher Thomas , Pilger Ernst , Renner Wilfried
Published: March 2018

G to A mutations at positions 20210 of the prothrombin gene (F2) and 1691 of the factor V gene (F5) are established risk factors for venous thrombosis. Several factors associated with coagulation and/or fibrinolysis have been associated with arterial occlusive disease, but the role of F2 20210A and F5 1691A for arterial occlusive disease remains unclear.

To investigate if F2 20210A and F5 1691A are associated with peripheral arterial occlusive disease (PAOD).

We analyzed the prevalence of F2 20210A and F5 1691A alleles in 336 patients with documented PAOD at Fontaine stage II – IV and 300 controls without vascular disease. Allele frequencies in patients and controls were 0.013 and 0.022 for F2 20210A, and 0.042 and 0.045 for F5 1691, respectively, both differences being not statistically significant.

Our data suggest that mutations F2 G20210A and F5 G1691A are not associated with PAOD.
Purification and Characterization of Factor VII Inhibitor Found in a Patient with Life Threatening Bleeding
Authors: Miyamoto Seiji , Iwasa Atsushi , Ishii Masao , Okajima Kenji , Kamikubo Yu-ichi
Published: March 2018

We recently observed a patient with acquired inhibitor-induced F.VII deficiency whose plasma level of F.VII was < 1.0%. However, the biochemical nature of the inhibitor has not yet been clarified. In the present study, we purified the F.VII inhibitor from the patient’s plasma by using activated F.VII (F.VIIa)-conjugated gel and characterized the inhibitor. The results showed that the inhibitor comprised two kinds of antibodies: one was eluted with EDTA (antibody 1) and the other with glycine-HCl buffer (pH 2.3) (antibody 2) from the F.VIIa affinity gel. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis of these inhibitors demonstrated that both antibodies had features of immunoglobulin G1 (IgG1) with κ and λ-light chains. Antibody 1 bound to the immobilized F.VIIa with a high affinity in the presence of calcium ion, while antibody 2 bound to the F.VIIa very weakly and the binding was independent of calcium ion. Immunoblotting analysis demonstrated that antibody 1 bound to the light chain of F.VIIa after reduction with 2-mercaptoethanol, while it did not react with either the γ carboxyglutamic acid (Gla)-domainless light chain of F.VIIa or the heavy chain with the protease domain. Antibody 1 markedly inhibited the activity of tissue factor-F.VIIa complex. Based on these observations, it is suggested that F.VIIa autoantibody (antibody 1) recognizes the calcium-dependent conformation within or near the Gla domain and inhibits F.VIIa activity by interacting with the light chain.
Thirty-three Novel Mutations in the Protein C Gene
Authors: Gandrille Sophie , Aubry Marie-Laurence , Aiach Martine , Alhenc-Gelas Martine ,   deficiencies and the French INSERM network on molecular abnormalities responsible for protein C and protein S
Published: March 2018

We analyzed the protein C gene (PROC) with the denaturing gradient gel electrophoresis (DGGE) scanning strategy in a series of 129 patients with suspected protein C (PC) deficiency (93 with low plasma PC levels and 36 with borderline level). At least one sequence variation was found in 104 of the 129 patients. Thirty-nine sequence variations (found in 72 patients) were already reported detrimental mutations. Thirty-three were novel sequence variations, of which 19 (found in 25 patients) were probably detrimental. Five novel mutations (A1T, R9H, S11R, S12R and K193Q) were associated with qualitative plasma PC deficiency, suggesting or confirming the functional importance of amino acids at these positions. This strategy confirmed the diagnosis of inherited PC deficiency in 79/93 (84.9%) patients with low plasma PC levels and 14/36 (38.8%) patients with borderline values. In order to explain abnormal PC levels observed in patients who did not carry detrimental mutations, screening for the -1654C/T and -1641A/G PROC promoter polymorphisms known to influence plasma PC concentrations was performed. The frequency of the CG allele associated with lower PC concentrations was slightly but not significantly lower in 82 heterozygotes for detrimental PROC gene mutations than in 36 patients with no identified detrimental mutations.
Determination of the Putative Binding Sites for Thrombin Receptor Activating Peptide through a Hydropathic Complementary Approach
Authors: Suttnar Jiří , Dolečková Lucie , Dyr Jan
Published: March 2018

Putative binding sites in a platelet thrombin receptor (PAR-1) for the activating peptide SFLLRNPNDKYEPF (AP) and for the bradykinin analogue MKRPPGFSPFRSSRIG were revealed using a computer program for identifying complementary peptide segments. The program is based on the assumption that interactions of agonist’s peptides and protein’s receptors can be elucidated by complementary average hydropathies as much as possible equal by size and opposite by sign. Some of the computer-found putative binding sites were close to the supposed AP-PAR-1 contacts in the amino-terminal exodomain and in the second extracellulary loop of PAR-1. Peptides complementary to these binding sites were also computer-designed and were synthesized. They mostly inhibited the aggregation of gel filtered platelets by thrombin (0.025 U/mL) with IC50 in a high micromolar range of concentrations. The peptide complementary to site L258-Y269 of PAR-1 induced aggregation of gel filtered platelets with EC50 = 98 [µmol/L] related to thrombin (0.025 U/mL) aggregation response.
Heterogeneous Recognition of beta 2-glycoprotein I by Antibodies from Antiphospholipid Syndrome Patients
Authors: Guerin J. , Sim R. , Yu B-B. , Ferluga J. , Feighery C. , Jackson J.
Published: March 2018

Beta 2-glycoprotein I plays a pivotal role in the binding of antiphospholipid antibodies to phospholipid in patients with antiphospholipid syndrome. In this study the nature of the epitopes on beta 2-glycoprotein I (β2-GPI) recognised by sera from antiphospholipid syndrome (APS) patients (n = 15) was investigated and compared to rabbit polyclonal and mouse monoclonal anti-β2-GPI antibodies. β2-GPI was only recognised when bound to a high affinity binding support. The antigenic epitope on β2-GPI recognised by all APS patients was also dependent on disulphide bond integrity. Digestion of β2-GPI with elastase rapidly destroyed the epitope(s) on β2-GPI recognised by antibodies in 91% of APS patients. The main cleavage occurred at tryptophan316-lysine317 in the fifth domain. Digestion with staphylococcal V8 protease resulted in a 50% reduction in antibody binding in 81% of patients and the cleavage sites mainly involved the first domain of the molecule. There was considerable variability in the recognition of six different species of β2-GPI by serum from APS patients. The epitopes on β2-GPI bound by APS sera appear conformationally determined in all patients but are quite heterogeneous in the regions of β2-GPI that are recognised.
Multi-Cellular Activation In Vivo by Endotoxin in Humans – Limited Protection by Adenosine Infusion
Authors: Li Nailin , Soop Anne , Sollevi Alf , Hjemdahl Paul
Published: March 2018

The influence of adenosine infusion (40 µg/kg/min for 4 h) on inflammatory and hemostatic parameters was investigated in healthy males without (n = 10) or with (n = 11) intravenous endotoxin injection (4 ng/kg). Without endotoxin, adenosine elevated circulating leukocytes and circulating platelet-leukocyte aggregates. Endotoxin activated platelets and leukocytes in vivo. Platelet activation was seen as slightly increased platelet P-selectin expression, decreased platelet counts, and elevated plasma soluble P-selectin (from 39.6 ± 3.4 to 68.9 ± 6.6 ng/ml; P <0.01). Leukocyte activation was evidenced by increased CD11b expression (from MFI of 0.54 ± 0.02 to 2.21 ± 0.17; P <0.01) and plasma elastase levels (from 25.3 ± 2.5 to 169.3 ± 22.5 ng/ml; P <0.01). Endotoxin also enhanced platelet and leukocyte responsiveness to in vitro stimulation. Endotoxin induced von Willebrand factor secretion (from 92 ± 8 units to 265 ± 19 units at 4 h; P <0.001) and enhanced thrombin generation in vivo. Endotoxin induced leukocytosis and thus increased circulating platelet-leukocyte, mainly platelet-neutrophil, aggregates. Adenosine caused slight attenuation of platelet reactivity to agonist stimulation, enhanced the endotoxin-induced leukocytosis, and detained more platelet-leukocyte aggregates in circulation, but did not attenuate endotoxin-induced neutrophil elastase secretion, von Willebrand factor secretion, or thrombin generation. Thus, endotoxemia induces multi-cellular activation in vivo. Adenosine inhibits leukocyte adhesion and extravasation, and mildly attenuates platelet responsiveness and soluble P-selectin release. Adenosine has the potential of becoming a therapeutic antiinflammatory drug, but an optimal treatment strategy needs to be developed.
Functional Analyses of Patient-derived IgG Monoclonal Anticardiolipin Antibodies Using In Vivo Thrombosis and In Vivo Microcirculation Models
Authors: Liu Xiaowei , Espinola Ricardo , Olee Tsawei , Zhu Min , Harris Nigel , Chen Pojen , Pierangeli Silvia
Published: March 2018

Antiphospholipid antibodies (aPL) have been associated with thrombosis and pregnancy losses in patients diagnosed with antiphospholipid syndrome (APS) and enhance thrombus formation in vivo in mice, but the mechanism of thrombosis by aPL is not completely understood. It has been proposed that aPL may affect endothelial cell (EC) function and/or induce their activation, transforming their anticoagulant surface into procoagulant, thus predisposing to thrombosis. It has been proposed that aPL may affect EC cell function and/or induce their activation, transforming their anticoagulant surface into procoagulant, thus predisposing to thrombosis. This study proposes to test the hypotheses that some IgG anticardiolipins (IgG aCL) with thrombogenic properties in mice, exert their effects through activation of endothelium. We studied seven patient-derived monoclonal aCL for their thrombogenic properties in an in vivo pinch-induced thrombosis model, and their functional activities in activating EC by analyzing in vivo leukocyte adhesion to endothelium in microcirculation in venules in exposed murine cremaster muscle and in vitro adhesion molecule expression in cultured EC. The binding of the monoclonal aCL to EC was also tested. In addition to the previous identified thrombogenic IS2, four of the five new more IgG monoclonal aCL (from two patients) were found to be thrombogenic. Of these five thrombogenic aCL, three caused more in vivo leukocyte adhesion to EC in microcirculation, as compared to that induced by the H2 control human monoclonal IgG, and enhanced expression of adhesion molecules (particularly VCAM-1) on cultured EC. These data show that about 2/3 patientderived IgG monoclonal aCL are thrombogenic and suggest that some thrombogenic IgG aCL exert their effects through activating EC.

Abbreviations: aCL, anticardiolipin antibodies; aPL, antiphospholipid antibodies; APS, antiphospholipid syndrome; CL, cardiolipin; dRVVT, dilute Russell’s viper venom time; EC, endothelial cells; ELISA, enzyme-linked immunosorbent assay; GP, glycoprotein; h, hour; HUVEC, human umbilicalvein endothelial cells; ICAM, intercellular adhesion molecule; KCT, kaolin clotting time; LA, lupus anticoagulant; PBS, phosphate-buffered saline; PL, phospholipid; sq, square; TBS, Tris-buffered saline; VCAM, vascular cell adhesion molecule; PMBC, peripheral mononuclear blood cells
Protein S Thr103Asn Mutation Associated with Type II Deficiency Reproduced In Vitro and Functionally Characterised
Authors: Giri Tusar , de Frutos Pablo , Dahlbäck Björn
Published: March 2018

Protein S functions as a cofactor to activated protein C (APC) in the degradation of FVa and FVIIIa. In protein S, the thrombin sensitive region (TSR) and the first EGF-like domain are important for expression of the APC cofactor activity. A naturally occurring Thr103Asn (T103N) mutation in the first EGF-like domain of protein S has been associated with functional (type II) protein S deficiency. To elucidate the functional consequences of the T103N mutation, recombinant protein S mutant was expressed in mammalian cells and functionally characterised. The expression level of protein S T103N from transiently transfected COS 1 cells was equal to that of wild type protein S. The mutant protein S and wild type protein S were also expressed in 293 cells after stable transfection, and the recombinant proteins purified. In APTT-and PT-based coagulation assays, the mutant protein demonstrated approximately 50% lower anticoagulant activity as compared to wild type protein S. The functional defect was further investigated in FVa-and FVIIIa-degradation assays. The functional defect of mutant protein S was attenuated at increasing concentrations of APC. The results demonstrate the region around residue 103 of protein S to be of functional importance, possibly through a direct interaction with APC.
Single Founder Mutation (W380G) in Type II Protein C Deficiency in Finland
Authors: Kuismanen Kristiina , Holopainen Päivi , Vahtera Elina , Rasi Vesa , Krusius Tom , Levo Antti , Partanen Jukka
Published: March 2018

The present study investigated the genetic basis for type II protein C deficiency in Finland, where this form has an unusually high incidence. We demonstrated that, first, a single novel mutation W380G in the protein C gene (PROC) explained 25/26 index patients, estimated to represent two thirds of all families with type II deficiency in Finland. Second, extended chromosomal conservation, i. e. a specific haplotype, around the W380G mutation was indicated in unrelated patients. Third, a local geographical origin for the W380G mutation was suggested by genealogical data. These results are in contrast to the heterogeneity in type II protein C deficiency elsewhere, but closely parallel disorders of the Finnish disease heritage. The high frequency of the type II disease can be explained by founder effect and subsequent enrichment of a single mutation in Finland. The present study also provided a simple means for genetic diagnosis of this disease and the genetic test can be included in the routine screenings in this population.
Characterization of the Murine Coagulation Factor X Promoter
Authors: Wilberding Julie , Castellino Francis
Published: March 2018

Factor X (FX) is a vitamin K-dependent serine protease zymogen that functions in both the extrinsic and intrinsic pathways of blood coagulation. In this study, the 5’-flanking region of the murine FX gene was analyzed to determine those elements that govern its transcriptional activity and regulation. Consistent with other TATA-less promoters, murine FX contains two start sites of transcription, at bp −5 and −21 relative to the ATG translational initiation codon. The mRNA of FX was found in a number of tissues, including the liver, stomach, intestine, kidney, ovary, testes, spleen, skeletal muscle, and lung. Using DNase I footprinting, three areas of protection have been identified in the proximal 287 bp of the promoter, spanning bp −28 to −218. Further examination of this region revealed transcription factor binding sites for NF-Y, HNF-4, and a GATA factor. Electrophoretic mobility shift analysis (EMSA) confirmed the identities of NF-Y, HNF-4, and GATA-4, all of which were found by transient transfection analyses in HepG2 cells to influence the activity of the promoter. Ablation of the NF-Y site was most dramatic, reducing activity to 10% of that of the wild-type construct. Deletion of the HNF-4 site led to an activity of 25% of wild-type, and a GATA-4 mutation reduced activity to 63% of wild-type values. This investigation revealed the identity of the factors bound at the proximal promoter of the FX gene, and the relative importance of each. This is the first report of a member of the GATA family of transcription factors being important in the regulation of a coagulation-based gene.