bluuebunny/crossref_metadata_2025 huggingface dataset:
Na-K-ATPase in rat cerebellar granule cells is redox sensitive
Authors: Petrushanko I. , Bogdanov N. , Bulygina E. , Grenacher B. , Leinsoo T. , Boldyrev A. , Gassmann M. , Bogdanova A.
Published: November 2005

Redox-induced regulation of the Na-K-ATPase was studied in dispersed rat cerebellar granule cells. Intracellular thiol redox state was modulated using glutathione (GSH)-conjugating agents and membrane-permeable ethyl ester of GSH (et-GSH) and Na-K-ATPase transport and hydrolytic activity monitored as a function of intracellular reduced thiol concentration. Depletion of cytosolic and mitochondrial GSH pools caused an increase in free radical production in mitochondria and rapid ATP deprivation with a subsequent decrease in transport but not hydrolytic activity of the Na-K-ATPase. Selective conjugation of cytosolic GSH did not affect free radical production and Na-K-ATPase function. Unexpectedly, overloading of cerebellar granule cells with GSH triggered global free radical burst originating most probably from GSH autooxidation. The latter was not followed by ATP depletion but resulted in suppression of active K+influx and a modest increase in mortality. Suppression of transport activity of the Na-K-ATPase was observed in granule cells exposed to both permeable et-GSH and impermeable GSH, with inhibitory effects of external and cytosolic GSH being additive. The obtained data indicate that redox state is a potent regulator of the Na-K-ATPase function. Shifts from an “optimal redox potential range” to higher or lower levels cause suppression of the Na-K pump activity.
Upregulation of apical NHE3 in renal OK cells overexpressing the rodent α1-subunit of the Na+pump
Authors: Gomes Pedro , Soares-da-Silva Patrício
Published: November 2005

Vectorial Na+reabsorption across the proximal tubule is mediated by apical entry of Na+, primarily via Na+/H+exchanger isoform 3 (NHE3), and basolateral extrusion via the Na+pump (Na+-K+-ATPase). We hypothesized that regulation of Na+reabsorption should involve not only the activity of the basolateral Na+-K+-ATPase, but also the apical NHE3, in a concerted manner. To generate a cell line that overexpresses Na+-K+-ATPase, opossum kidney (OK) cells were transfected with the rodent Na+-K+-ATPase α1-subunit (pCMV ouabain vector), and native cells were used as a control. The existence of distinct functional classes of Na+-K+-ATPase in wild-type and transfected cells was confirmed by the inhibition profile of Na+-K+-ATPase activity by ouabain. In contrast to wild-type cells, transfected cells exhibited two IC50values for ouabain: the first value was similar to the IC50of control cells, and the second value was 2 log units greater than the first, consistent with the presence of rat and opossum α1-isozymes. It is shown that transfection of OK cells with Na+-K+-ATPase increased Na+-K+-ATPase and NHE3 activities. This was associated with overexpression of the Na+-K+-ATPase α1-subunit and NHE3 in transfected OK cells. The abundance of the Na+-K+-ATPase β1-subunit was slightly lower in transfected OK cells. In conclusion, the increase in expression and function of Na+-K+-ATPase in cells transfected with the rodent Na+pump α1-subunit cDNA is expected to stimulate apical Na+influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.
Pyrogenicity of CpG-DNA in mice: role of interleukin-6, cyclooxygenases, and nuclear factor-κB
Authors: Kozak Wieslaw , Wrotek Sylwia , Kozak Anna
Published: November 2005

Bacterial DNA containing unmethylated cytosine-phosphate-guanosine motif (CpG-DNA) has been identified as a pathogen-associated molecular pattern, which is recognized by Toll-like receptors and activates immune cells to produce cytokines. The aim of the study was to characterize the ability of CpG-DNA to induce fever in mice. Intravenous administration of unmethylated CpG-DNA 1826 triggered an elevation of body temperature (Tb) lasting several hours. The magnitude of Tbelevation increased with an increase of dose of the oligonucleotide (administered in a range from 0.01 mg/kg to 1.0 mg/kg). A fever-like increase of Tbin mice was partially dependent on IL-6, as IL-6 deficient mice responded with reduced fever to the CpG-DNA 1826. Meloxicam and sulindac sulfide, inhibitors of cyclooxygenases, reduced fever in mice challenged with CpG-DNA 1826, indicating that the process may also depend on prostaglandins. In fact, plasma levels of prostaglandin E2, as well as IL-6, increased at 4 h postinjection of CpG-DNA 1826 into mice. These data demonstrate that the pathophysiological mechanism of the increase of Tbinduced by CpG-DNA 1826 is similar to fever induced by LPS. Both LPS and CpG-DNA 1826 failed to produce elevation of Tbin mice deficient for a nuclear factor-κB (NF-κB) gene, further supporting the hypothesis that the two pyrogens provoke fever, using the same components of the cellular signaling metabolism. However, parthenolide, an inhibitor of I-κB kinase reduced fever due to CpG-DNA 1826, and did not affect fever to LPS, suggesting that the two structurally dissimilar pyrogens may affect different intracellular pathways leading to the upregulation of NF-κB. In support of this hypothesis, we demonstrate that C3H/HeJ mice, known to exhibit a mutation in the Toll-like receptor-4 gene, do not respond with fever to LPS. They respond, however, with fever after injection of CpG-DNA 1826. We conclude that bacterial DNA shares with components of the bacterial wall the capacity to elicit fever and may, consequently, be part of a novel class of exogenous pyrogens.
The effect of lung inflation on the inspiratory action of the canine parasternal intercostals
Authors: Leduc Dimitri , Troyer André De
Published: November 2005

Inflation induces a marked decrease in the lung-expanding ability of the diaphragm, but its effect on the parasternal intercostal muscles is uncertain. To assess this effect, the phrenic nerves and the external intercostals were severed in anesthetized, vagotomized dogs, such that the parasternal intercostals were the only muscles active during inspiration, and the endotracheal tube was occluded at different lung volumes. Although the inspiratory electromyographic activity recorded from the muscles was constant, the change in airway opening pressure decreased with inflation from −7.2 ± 0.6 cmH2O at functional residual capacity to −2.2 ± 0.2 cmH2O at 20-cmH2O transrespiratory pressure ( P < 0.001). The inspiratory cranial displacement of the ribs remained virtually unchanged, and the inspiratory caudal displacement of the sternum decreased moderately. However, the inspiratory outward rib displacement decreased markedly and continuously; at 20 cmH2O, this displacement was only 23 ± 2% of the value at functional residual capacity. Calculations based on this alteration yielded substantial decreases in the change in airway opening pressure. It is concluded that, in the dog, 1) inflation affects adversely the lung-expanding actions of both the parasternal intercostals and the diaphragm; and 2) the adverse effect of inflation on the parasternal intercostals is primarily related to the alteration in the kinematics of the ribs. As a corollary, it is likely that hyperinflation also has a negative impact on the parasternal intercostals in patients with chronic obstructive pulmonary disease.
Music during Bronchoscopic Examination: the Physiological Effects
Authors: Triller Nadja , Eržen Damjan , Duh Štefan , Petrinec Primožič Marija , Košnik Martina
Published: November 2005

Background: Patients scheduled for bronchoscopic procedures are often anxious and frightened. Reduction in the state of anxiety during an invasive procedure may prevent some possible complications. Music has been proposed as a safe nonpharmacological antianxiety intervention. Objective: We followed up physiological indicators of anxiety (blood pressure, heart rate) during bronchoscopic examination to determine the effect of music on the level of anxiety. Methods: Two hundred adult patients were included in the study. Blood pressure, heart rate, procedures performed during bronchoscopy and duration of examination were monitored. Patients’ overall feelings during the procedure were rated from 0 (without unpleasant feelings) to 10 (unbearable). All patients used the Visual Analogue Scale (VAS). Results: Two hundred adult patients referred for bronchoscopy were included in the study: 93 patients received music during the procedure and 107 served as control. Therewere no significant differences between the two groups in terms of age, sex, indications for bronchoscopy, procedures performed during bronchoscopy, duration of the examination and patients’ subjective perception of the procedure. The mean hart rate, systolic and diastolic blood pressures were significantly lower in the music group compared to the control group. Conclusions: Our findings suggest that the application of music reduces anxiety during bronchoscopic examination as physiological indicators of anxiety, the mean heart rate, systolic and diastolic blood pressures, were significantly lower in the music group.
Influence of Nasal Resistance on Initial Acceptance of Continuous Positive Airway Pressure in Treatment for Obstructive Sleep Apnea Syndrome
Authors: Sugiura Tatsuki , Noda Akiko , Nakata Seiichi , Yasuda Yoshinari , Soga Taro , Miyata Seiko , Nakai Sigeru , Koike Yasuo
Published: November 2005

Background: Continuous positive airway pressure (CPAP) is considered as the standard therapy for obstructive sleep apnea syndrome (OSAS), but some patients with OSAS are unable to accept CPAP due to nasal obstruction and poor nasal airflow. Objectives: We assessed the influence of nasal resistance before beginning CPAP treatment on the initial acceptance of CPAP in OSAS patients. Methods: The study subjects comprised 77 patients (74 males, 3 females) with primary OSAS, all of whom received CPAP treatment with nasal masks. Before trials, all subjects underwent overnight polysomnography, and nasal resistance was measured with active anterior rhinomanometry in the seated position on the first day of CPAP trial. Results: The CPAP treatment was accepted by 56 patients after the initial trials with overnight polysomnography. Body mass index, the number of apnea/hypopnea episodes per hour (apnea/hypopnea index; AHI), and the number of episodes per hour with an oxygen desaturation of >3% (oxygen desaturation index) were significantly higher (p < 0.01) and nasal resistance was lower (p = 0.003) in patients who accepted CPAP than in those who did not. Logistic regression analysis, with patient age, body mass index, Epworth sleepiness scale score, AHI, oxygen desaturation index, and nasal resistance before CPAP treatment as explanatory variables, showed that nasal resistance (OR + 0.1 Pa/cm3/s: 1.48; p = 0.002) and AHI (OR + 1 event/h: 0.93; p = 0.003) were significant factors for CPAP non-acceptance. Conclusions: Nasal resistance before the beginning of CPAP treatment has a significant effect on the acceptance of CPAP in OSAS patients, and hence, could be a predictive parameter for the initial acceptance of CPAP.
An important role for the activation peptide domain in controlling factor IX levels in the blood of haemophilia B mice
Authors: Begbie Megan E. , Mamdani Asif , Gataiance Sharon , Eltringham-Smith Louise J. , Bhakta Varsha , Hortelano Gonzalo , Sheffield William P.
Published: November 2005

The factors responsible for the removal of injected factor IX (fIX) from the blood of individuals with haemophilia B are only partly understood, and may include binding to endothelial or subendothelial sites, passive extravasation related to size or charge, or interactions requiring fIX activation. To investigate these issues, we have produced and characterised recombinant fIX proteins with amino acid changes: Δ155–177, an internal deletion which removes most of the activation peptide while retaining the activation cleavage sites; S365A, which inactivates the serine protease activity of fIXa; and K5A, previously shown to eliminate fIX binding of endothelial/subendothelial collagen IV. All proteins were expressed in stably transfected HEK 293 cells, purified by immunoaffinity chromatography, and compared to the wild type HEK 293-derived protein (fIX (WT)). Mutant fIX proteins K5A and Δ155–177 exhibited 72 and 202% of the specific activity of fIX (WT), respectively; S365A was without activity. Following intravenous injection in haemophilia B (fIX knockout) mice, recoveries did not differ for fIX (WT) and Δ155–177, but were higher for K5A and S365A. The terminal catabolic halflife of Δ155–177, alone among the mutants, was increased, by 45% versus fIX (WT). Nine hours post-injection, the observed areas under the clearance curve (AUCs) of Δ155–177 and K5, but not S365A, were elevated 2-fold. Δ155–177 was equally effective as fIX (WT) in reducing blood loss following tail vein transection in haemophilia B mice. Our results suggest that deletion of the multiple sites of fIX post-translational modification found within the activation peptide eliminated important fIX clearance motifs.
Platelet adhesion: Structural and functional diversity of short dystrophin and utrophins in the formation of dystrophinassociated-protein complexes related to actin dynamics
Authors: Cerecedo Doris , Martínez-Rojas Dalila , Chávez Oscar , Martínez-Pérez Francisco , García-Sierra Francisco , Rendon Álvaro , Mornet Dominique , Mondragón Ricardo
Published: November 2005

Platelets are dynamic cell fragments that modify their shape during activation. Utrophin and dystrophins are minor actin-binding proteins present in muscle and non-muscle cytoskeleton. In the present study, we characterised the pattern of Dp71 isoforms and utrophin gene products by immunoblot in human platelets. Two new dystrophin isoforms were found, Dp71f and Dp71d, as well as the Up71 isoform and the dystrophin-associated proteins, α and β-dystrobrevins. Distribution of Dp71d/Dp71Δ110
m, Up400/Up71 and dystrophin-associated proteins in relation to the actin cytoskeleton was evaluated by confocal microscopy in both resting and platelets adhered on glass. Formation of two dystrophin-associated protein complexes (Dp71d/Dp71Δ110
m ~DAPC and Up400/Up71~DAPC) was demonstrated by co-immunoprecipitation and their distribution in relation to the actin cytoskeleton was characterised during platelet adhesion. The Dp71d/Dp71Δ110
m ~DAPC is maintained mainly at the granulomere and is associated with dynamic structures during activation by adhesion to thrombin-coated surfaces. Participation of both Dp71d/Dp71Δ110
m ~DAPC and Up400/Up71~DAPC in the biological roles of the platelets is discussed.
Inverse relationship between plasminogen activator inhibitor-1 activity and adiponectin in overweight and obese women
Authors: Mertens Ilse , Ballaux Dominique , Funahashi Tohru , Matsuzawa Yuji , Planken Marc Van der , Verrijken An , Ruige Johannes B. , Van Gaal Luc F.
Published: November 2005

Adipose tissue is an active endocrine organ secreting different adipokines such as plasminogen activator inhibitor-1 (PAI-1) and adiponectin, among many others. In this study, we investigated the association between PAI-1 activity and serum adiponectin levels in a group of 444 overweight and obese women and assessed the interrelationship with visceral adipose tissue (VAT; CT-scan L4-L5), insulin resistance (HOMA-IR), HDL cholesterol (HDL-chol) and inflammation (hs-CRP). PAI-1 was inversely related to adiponectin (r=-0.25, p<0.001; adjusted for age and BMI).After adjustment for age, VAT, HOMA-IR and hs-CRP, the relationship remained significant (r=-0.15; p=0.001), but disappeared after additional adjustment for HDL-chol (r=-0.09; p=0.067). Subjects were divided in two groups according to the median levels of adiponectin or PAI-1 levels. PAI-1 activity (19.1±11.4 vs. 15.8±8.6 AU/ml; p=0.003) and adiponectin levels (9.8±4.6 vs. 8.4±4.0 μg/ml; p<0.001) were significantly higher in the low adiponectin/PAI-1 groups. The difference in PAI-1 remained significant after adjustment for age and BMI (p=0.001), became borderline significant after adjustment for age and VAT (p=0.052), and disappeared after adjustment for age and HOMA-IR (p=0.116) or age and HDL-chol (p=0.443).The difference in adiponectin levels remained significant after adjustment for age, VAT, HOMA-IR and hs-CRP (p=0.006), but disappeared after additional adjustment for HDL-chol (p=0.089). Further analyses suggest a contribution of HOMA-IR and/or HDL-chol in the relationship between PAI-1 and adiponectin. HDL-chol was found to be the only factor independently determining both factors. In conclusion, in overweight and obese women, PAI-1 activity was inversely related to serum adiponectin, independent of visceral adipose tissue.
The effects of supra-normal protein C levels on markers of coagulation, fibrinolysis and inflammation in a human model of endotoxemia
Authors: Spiel Alexander O. , Firbas Christa , Mayr Florian B. , Leitner Judith M. , Schmidt Bily , Knöbl Paul , Varadi Katalin , Jilma Bernd
Published: November 2005

The protein C pathway serves as a modulating system with both anti-inflammatory and anticoagulant properties and is intimately involved in the pathophysiology of inflammation and sepsis. Treatment with recombinant human activated protein C (rhAPC) can reduce the mortality of severe sepsis. We investigated whether an elevation of plasma protein C levels to supranormal levels by infusion of a protein C zymogen concentrate has an effect on coagulation, protein C activation or inflammation in a human endotoxemia model. Eleven healthy male volunteers were enrolled in a double-blind, placebo-controlled two-way cross-over trial. Ten minutes after infusion of 2ng/kg endotoxin each volunteer received either placebo or a plasmaderived protein C zymogen concentrate (Ceprotin®, Baxter) (150 U/kg as a slow bolus infusion followed by 30 U/kg/h continuous infusion until 4 hours after LPS-infusion). Protein C antigen and activity increased 4– to 5-fold after infusion of the concentrate. APC was generated during endotoxin-induced inflammation in the placebo (1.6 fold increase) and the protein C period (4.0-fold increase).The increase of APC levels correlated with the TNF-α and IL-6 release in both periods (r=0.65–0.68; p<0.05) and paralleled the protein C antigen and activity levels in the period with supranormal protein C levels. Supra normal protein C levels resulted in slightly, although non-significant, lower tissue factor mRNA expression and thrombin generation (TAT, F1+2). Systemic inflammation (TNF-α, IL-6) was not influenced by protein C zymogen concentrate administration. Infusion of protein C zymogen was safe and no adverse effects occurred. The increase of protein C levels several fold above the normal range resulted in a proportional increase of the APC levels, but had no major anticoagulant, anti-inflammatory or profibrinolytic effects. Low grade endotoxemia itself induces significant protein C activation, which correlates with the TNF release.
Endurance training reduces circulating asymmetric dimethylarginine and myeloperoxidase levels in persons at risk of coronary events
Authors: Penka Martina , Grdić Marija , Steiner Sabine , Strasser Barbara , Ziegler Sophie , Zorn Gerlinde , Maurer Gerald , Simeon-Rudolf Vera , Wojta Johann , Huber Kurt , Richter Bernhard , Niessner Alexander
Published: November 2005

Asymmetric dimethylarginine (ADMA), myeloperoxidase (MPO) and paraoxonase 1 (PON1) are directly involved in the pathogenesis of atherosclerosis by modulation of oxidative stress and/or NO bioavailability. We aimed to assess whether endurance exercise which is known to be cardioprotective could beneficially affect these novel risk markers. Thirty-two subjects (31–68 yrs, 56% males) with elevated cardiovascular risk including ten patients with coronary artery disease volunteered for a supervised 12-week endurance training (196 ± 15 min/week).Their fitness evaluated by 2 km test runs improved significantly after training (pre: 17.3±0.8 vs. post: 15.7±0.9min, p<0.001). ADMA (pre: 0.94±0.03 vs. post: 0.75±0.04 μmol l−1) and MPO (pre:296.8±22.2 vs.post:185.7±19.5 ng ml−1) serum levels decreased significantly by 17.6±4.6% and 28.5±7.5%, respectively, after training (both p<0.001).Their down-regulation was inversely correlated (ADMA: r=-0.609, p<0.001, MPO: r=-0.437, p=0.014) with the up-regulation of plasma cGMP levels (Cyclic-guanosine 3`,5`-monophosphate; pre: 1.6±0.12 vs. post: 2.21±0.2 μmol ml−1, p=0.001) reflecting NO production. PON1 activity towards phenylacetate was not significantly influenced by training (pre: 133±6 vs. post: 130±5 μmol ml−1 min−1, p=0.375). In a matched inactive control group (n=16) ADMA, MPO, cGMP levels and PON1 activity did not change over time. ADMA, MPO and cGMP changes were significantly different between participants and controls (all p<0.05). Regular endurance exercise was successful in reducing the circulating levels of two promising cardiovascular risk markers, ADMA and MPO, in persons prone to cardiac events. These changes may result in numerous antiatherosclerotic effects such as improvement of NO bioavailability, reduction of oxidative stress and lipid peroxidation.
Functional heterogeneity of alloantibodies against the human platelet antigen (HPA)-1a
Authors: Kroll Hartmut , Penke Gabriele , Santoso Sentot
Published: November 2005

The integrin αIIbβ3 is the major fibrinogen receptor on the platelet membrane and plays a crucial role for platelet aggregation. The β3-subunit carries the human platelet alloantigen (HPA)-1a, which is the main target for alloantibodies (alloabs) responsible for foetal and neonatal alloimmune thrombocytopenia (FNAIT) and post-transfusion purpura (PTP).Whereas PTP is almost invariably associated with severe bleeding, the clinical presentation of FNAIT ranges from mild thrombocytopenia to severe haemorrhagic diathesis. However, this clinical heterogeneity is not fully understood as it is not explained solely by the variability of the platelet count. Here, we examined the ability of HPA-1a alloabs from mothers with FNAIT (n = 43) and PTP patients (n = 8) to inhibit cell adhesion to fibrinogen and asked if this inhibition was correlated with the heterogeneity of the clinical picture. Stably transfected cells expressing HPA-1a (β3-Leu33) and –1b (β3-Pro33) isoforms were incubated with sera containing HPA-1a alloabs and were allowed to adhere to immobilised fibrinogen. The inhibitory activity was measured as percentage of cell adhesion in the presence of patient sera versus normalAB serum. Only two FNAIT sera specifically inhibited the adhesion of HPA-1a, but not HPA-1b cells. Two other FNAIT sera blocked the adhesion of HPA-1a as well as HPA-1b cells. Interestingly, all four neonates with inhibitory HPA-1a alloabs (9% of all sera) suffered severe bleeding. In comparison, the majority of PTP sera (75%) inhibited cell binding to fibrinogen, four PTP sera selectively inhibited the adhesion of HPA-1a cells whilst 2 sera impaired the binding of both allotypes. Our observations indicate that 1) HPA-1a alloabs are heterogeneous in their ability to interfere with fibrinogen binding, and 2) inhibition of the αIIbβ3 fibrinogen receptor by HPA-1a alloabs may contribute to pronounced bleeding in patients with alloimmune thrombocytopenia.
Urokinase plasminogen activator released by alveolar epithelial cells modulates alveolar epithelial repair in vitro
Authors: Leer Coretta Van , Stutz Monika , Haeberli André , Geiser Thomas
Published: November 2005

Intra-alveolar fibrin is formed following lung injury and inflammation and may contribute to the development of pulmonary fibrosis. Fibrin turnover is altered in patients with pulmonary fibrosis, resulting in intra-alveolar fibrin accumulation, mainly due to decreased fibrinolysis. Alveolar type II epithelial cells (AEC) repair the injured alveolar epithelium by migrating over the provisional fibrin matrix. We hypothesized that repairing alveolar epithelial cells modulate the underlying fibrin matrix by release of fibrinolytic activity, and that the degree of fibrinolysis modulates alveolar epithelial repair on fibrin. To test this hypothesis we studied alveolar epithelial wound repair in vitro using a modified epithelial wound repair model with human A549 alveolar epithelial cells cultured on a fibrin matrix. In presence of the inflammatory cytokine interleukin-1β, wounds increase by 800% in 24 hours mainly due to detachment of the cells, whereas in serum-free medium wound areas decreases by 22.4 ± 5.2 % (p<0.01). Increased levels of D-dimer, FDP and uPA in the cell supernatant of IL-1β-stimulated A549 epithelial cells indicate activation of fibrinolysis by activation of the plasmin system. In presence of low concentrations of fibrinolysis inhibitors, including specific blocking anti-uPA antibodies, alveolar epithelial repair in vitro was improved, whereas in presence of high concentrations of fibrinolysis inhibitors, a decrease was observed mainly due to decreased spreading and migration of cells. These findings suggest the existence of a fibrinolytic optimum at which alveolar epithelial repair in vitro is most efficient. In conclusion, uPA released by AEC alters alveolar epithelial repair in vitro by modulating the underlying fibrin matrix.
Dose-finding and pharmacokinetics of therapeutic doses of tinzaparin in pediatric patients with thromboembolic events
Authors: Kuhle Stefan , Massicotte Patti , Dinyari Maria , Vegh Patsy , Mitchell Debra , Marzinotto Velma , Chan Antony , Pieniaszek Hank , Mitchell Lesley G.
Published: November 2005

In children, there is an increasing off-label use of low molecular weight heparin (LMWH). However, there is an absence of information on dosing and pharmacokinetics of LMWH over all age groups. The objectives of the current study were to determine i) the once daily dose required to achieve anti-Xa levels of 0.5–1.0 IU/mL, ii) the pharmacokinetics and iii) preliminary safety data using tinzaparin. The study took the form of a single centre open-label Phase II study performed in 35 children requiring anticoagulation for treatment of thromboembolism. Age groups studied were: 0-<2 months; 2 months-<1 year; 1-<5 years; 5-<10 years; 10–16 years. Both population pharmacokinetic analysis using nonlinear mixed-effect modeling techniques and model-independent pharmacokinetic methods were employed. Results showed a relationship of age and dose requirements, clearance, time to peak anti-Xa level and volume of distribution. Younger children required an increased dose, cleared tinzaparin more rapidly, had anti-Xa levels peak earlier and had an increased volume of distribution. Younger children were more likely to be below target range than older children, with up to 75% of children <1 year being below the target anti-Xa level. Four recurrences and one major bleed occurred. In conclusion, there is an inverse relationship of age on dose requirements related to volume of distribution, clearance and time to peak anti- Xa. Children <5 years likely require dose adjustment samples to be drawn 2–3 hours post injection. Infants require anti-Xa levels to be monitored at least twice monthly.
The first assessment of soluble CD146 in women with unexplained pregnancy loss
Authors: Bardin Nathalie , Martin Luc De Saint , Le Martelot Marie Thérèse , Bohec Caroline , Roche Sylvie , Mottier Dominique , Dignat-George Françoise , Pasquier Elisabeth
Published: November 2005

The mechanisms responsible for pregnancy loss have not all been elucidated. CD146 is a cell adhesion molecule involved in the control of both endothelium integrity and intermediate trophoblast invasiveness, two potential key features in the pathogenesis of pregnancy loss. As CD146 is detectable as a soluble form in the plasma (sCD146), we investigated sCD146 plasma levels in women with a history of pregnancy loss. We conducted a paired case-control study to compare sCD146 plasma levels in 100 women with unexplained pregnancy losses (2 or more consecutive losses at or before 21 weeks of gestation, or at least one later loss) and in 100 age-matched control women (no pregnancy loss and at least one living child). The sCD146 concentrations were determined at least 2 months after the last obstetrical event. Patients and controls were comparable regarding thrombophilia. Among the patients, 83 women experienced early pregnancy losses (average of 3 losses, mean gestation of 6.6 weeks) and 22 women suffered at least one late pregnancy loss. We found significantly higher sCD146 plasma levels in the 100 patients compared to age matched control women (p<0.001). The sCD146 plasma levels did not correlate with the number of pregnancy losses nor with the mean gestation time. Alterations in sCD146 plasma levels could be related to endothelial dysfunction associated to defective endovascular trophoblast invasiveness. Additional studies should explore whether sCD146 assessment could provide diagnostic and prognostic information with a view to screening and thus managing women with unexplained pregnancy loss.
Is factor V Leiden a risk factor for thrombotic microangiopathies without severe ADAMTS13 deficiency?
Authors: Krieg Soraya , Studt Jan-Dirk , Sulzer Irmela , Lämmle Bernhard , Hovinga Johanna A. Kremer
Published: November 2005

About 60% of patients diagnosed with acute thrombotic thrombocytopenic purpura (TTP) display a severe ADAMTS13 deficiency. Recently, Raife et al. concluded from a small case series, that factor V Leiden (FVL) might constitute a risk factor for acute thrombotic microangiopathy (TMA) without severe ADAMTS13 deficiency. Therefore, we determined ADAMTS13 activity and FVL carrier-ship in 256 consecutive patients presenting with various forms of acute TMA, including patients diagnosed with TTP or hemolytic-uremic syndrome (HUS). The overall prevalence of FVL was 8.2% (6.25% among patients diagnosed with TTP, and 9% among those with HUS) concordant with the FVL prevalence reported in Europe. FVL was present in 9.9% of patients with ADAMTS13 activity <10% and in 9.7% of those with normal ADAMTS13 activity (>50%). We conclude that FVL is not more prevalent inTMA patients without as compared to those with severe ADAMTS13 deficiency. The prevalence of FVL carriers in certain HUS subgroups (HUS with ADAMTS13 activity >50%) reaching 12.3% suggests that a contributory role of FVL in the pathogenesis of defined forms of HUS needs further study.
Molecular weight determines the frequency of delayed type hypersensitivity reactions to heparin and synthetic oligosaccharides
Authors: Alban Susanne , Kaufmann Roland , Lindhoff-Last Edelgard , Boehncke Wolf-Henning , Ludwig Ralf J. , Schindewolf Marc
Published: November 2005

Eczematous lesions, resulting from type IV sensitizations are well-known and relatively frequent cutaneous adverse effects of s.c. heparin therapy. If anticoagulation is further required intravenous heparin, heparinoids or lepirudin may be used as a substitute. However, these alternatives are not optimal in terms of practicability and/or safety-profiles. As molecular weight of different heparin preparations has repetitively been implied to determine the frequency of sensitization, we hypothesized, that due to its low molecular weight the pentasaccharide fondaparinux may provide a practicable and safe anticoagulant therapy in patients with delayed type hypersensitivity reactions (DTH) to heparin and other oligosaccharides. To test this concept, patients referred for diagnosis of cutaneous reactions after s.c. anticoagulant treatment underwent a series of in vivo skin allergyand challenge-tests with unfractionated heparin, a series of low molecular weight heparins (nadroparin, dalteparin, tinzaparin, enoxaparin and certoparin), the heparinoid danaparoid and the synthetic pentasaccharide fondaparinux. In total, data from twelve patients was evaluated. In accordance with previously published data, we report a high crossreactivity among heparins and heparinoids. In contrast – and in support of our initial hypothesis – sensitization towards the synthetic pentasaccharide fondaparinux was rarely observed. Plotting the cumulative incidence against the determined molecular weight of the individual anticoagulant preparations, shows that molecular weight generally is a key determinant of sensitization towards heparins and other oligosaccharides (r2=0.842, p=0.009). Hence, fondaparinux may be used as a therapeutic alternative in patients with cutaneous DTH relations towards heparin and other polysaccharides.
Pharmacodynamic markers in the early clinical assessment of otamixaban, a direct factor Xa inhibitor
Authors: Ozoux Marie-Laure , Chu Valeria , Simcox Kelly , Marks Vanessa , Freyburger Geneviève , Sibille Michel , Paccaly Anne , Shukla Umesh
Published: November 2005

This manuscript reports the assessment of pharmacodynamic (PD) markers of anti-coagulation in the first-in-man study with the novel direct Factor Xa (FXa) inhibitor, otamixaban, with a brief description of safety and pharmacokinetic (PK) findings. The study comprised ten consecutive parallel groups of healthy male subjects (6 active, 2 placebo per group). Eight groups received escalating intravenous doses of otamixaban as 6-hour infusions (1.7 to 183 μg/kg/h) and two groups received a bolus dose (30 or 120 μg/kg) with a 6-hour infusion (60 or 140 μg/ kg/h, respectively). PD markers included anti-FXa activity and clotting time measurements, i.e. activated Thromboplastin Time (aPTT), ProthrombinTime (PT), Heptest® ClottingTime (HCT), and Russell’s Viper Venom-induced clotting Time (RVVT). In addition, Endogenous Thrombin Potential (ETP) was assessed in the bolus-plus-infusion dose groups. Otamixaban was well tolerated. Otamixaban plasma concentrations increased with escalating dose, were maximal at the end-of-infusion (Ceoi), and decreased rapidly as the infusion was stopped. Anti-FXa activity coincided with otamixaban plasma concentrations and clotting time measurements followed the same pattern. Maximal changes from baseline at Ceoi were 1.9 ± 0.2 for aPTT, 2.0 ± 0.2 for PT, 5.1 ± 0.6 for HCT, and 4.5 ± 1.2 for RVVT. Otamixaban inhibited thrombin generation (24% decrease in ETP) and a delay in thrombin generation was noticed in vitro at high concentrations.
Point-of-care testing of the international normalized ratio in patients with antiphospholipid antibodies
Authors: Samsa Gregory P. , Ortel Thomas L. , Perry Stephanie L.
Published: November 2005

Antiphospholipid antibodies can influence the results of clotting tests in a subset of patients, which can be a major obstacle in monitoring warfarin. The aim was to determine if point-of-care testing of the International Normalized Ratio (INR) is influenced by antiphospholipid antibodies. We compared 59 patients receiving warfarin for a diagnosis of antiphosphoipid antibody syndrome (APS) to 49 patients receiving warfarin for atrial fibrillation to evaluate the consistency between INR results obtained by different methods. INR results obtained by finger stick (capillary whole-blood) and venipuncture (non-citrated and citrated whole-blood) were compared with our laboratory plasma-based prothrombin time assay. Five patients (8%) with APS and both elevated anti-β2glycoprotein I levels and positive lupus anticoagulants had non-measurable ProTime® INR results and generally higher Hemochron® Signature INR results than the plasma-based method, but the corresponding chromogenic factor X results were not supratherapeutic. For the remaining patients, differences between the plasma-based INR and the point-of-care INR results ranged from 0.2±0.2 to 0.4±0.3. The differences were similar for patients with APS and atrial fibrillation for all INR comparisons with the exception of the plasma-based method compared with the ProTime, which showed a mean absolute difference of 0.4±0.3 for APS patients and of 0.2±0.2 for atrial fibrillation patients (p=0.02). In a subset ofAPS patients, the ProTime® system will not yield an INR result and the HEMochron Signature (citrate and non-citrate whole-blood) INR results will exhibit elevated INR results. For this subset of APS patients, we suggest using an alternative method to monitor warfarin.
D-dimer, P-selectin, and microparticles: Novel markers to predict deep venous thrombosis
Authors: Myers Daniel D. , Hawley Angela E. , Longo Christopher , Henke Peter K. , Guire Kenneth E. , Schmaier Alvin H. , Wakefield Thomas W. , Rectenwald John E.
Published: November 2005

Current plasma markers for diagnosis of deep venous thrombosis (DVT) allow for exclusion of the diagnosis, but lack adequate specificity to establish the diagnosis. Thus, a prospective study was performed to determine the sensitivity and specificity of plasma assays for D-dimer, soluble P-selectin (P-selectin), and total microparticles in patients with documented DVT by duplex ultrasound. Three groups of individuals were examined: 30 normals; 22 positive for DVT on duplex ultrasound (Group 2); and 21 symptomatic, but negative on duplex ultrasound for DVT (Group 3). Group 1 individuals had D-dimer values of 1.53±0.12 mg/l and P-selectin values of 0.34±0.05 ng/mg total protein. Group 2 vs. Group 3 individuals had D-dimer values of 7.57±2.03 vs. 3.19±0.79 mg/l, p=0.02; P-selectin values of 0.98±0.11 vs. 0.55±0.08 ng/mg total protein, p<0.01; and microparticle values of 129±17% vs. 99±12% of control, p=ns. Using a logistic regression model with dichotomous variables, we determined a sensitivity of 73%, specificity of 81%, and accuracy of 77% when combining D-dimer, soluble P-selectin, and total microparticles to differentiate Group 2 from Group 3 patients. Logistic regression using continuous variables yielded similar results (p=0.05).This study demonstrates that plasma markers for DVT can be developed and achieve moderate sensitivity and specificity in diagnosing DVT. However for clinical applicability, the sensitivity/specificity will need to be improved. These studies also suggest the importance of soluble P-selectin in assessing DVT in humans.
Synergistic association between hypercholesterolemia and the C46T factor XII polymorphism for developing premature myocardial infarction
Authors: Roldán Vanessa , Corral Javier , Marín Francisco , Pineda Javier , Vicente Vicente , González-Conejero Rocío
Published: November 2005

Factor XII (FXII) plays a key role in both coagulation and fibrinolysis, thus its role in thrombotic processes is uncertain. Both genetic and environmental factors determine FXII plasma levels. A common C46T polymorphism in the Kozak region of F12 gene disturbs the translation of the protein leading to a significant reduction of FXII levels although its clinical significance is conflictive. We studied the F12 C46T polymorphism in 281 patients who had suffered from an acute myocardial infarction (MI) before 45-year-old and 550 control subjects from the same area. Serum levels of cholesterol, HDL, LDL, triglycerides and C reactive protein (CRP) were assayed in the MI group. The 46T allele slightly increased the risk to suffer from premature MI (OR:1.64; 95%CI: 1.14–2.37; p= 0.008). Moreover, patients carrying the 46T allele showed increased levels of CRP (p= 0.002). Interestingly, we found that the simultaneous presence of the 46T allele and hypercholesterolemia increases the risk to develop premature MI 2.26 times. The F12 C46T polymorphism, associated with a reduction of plasma FXII levels, seems to play a deleterious effect, predisposing the development of premature MI, especially in hypercholesterolemic patients. This effect could be associated with an increased pro-inflammatory state, as the 46T allele associates with high levels of CRP.
Discrepancy between tissue factor activity and tissue factor expression in endotoxin-induced monocytes is associated with apoptosis and necrosis
Authors: Klingenberg Olav , Øvstebø Reidun , Joø Gun-Britt , Westvik Åse-Brit , Kierulf Peter , Henriksson Carola E.
Published: November 2005

Tissue factor (TF), the main initiator of blood coagulation, contributes to the manifestation of disseminated intravascular coagulation following septic shock in meningococcal infection. Since a direct relationship between disease severity and lipopolysaccharide (LPS) concentration in the circulation has been shown, we hypothesized that the procoagulant and cytotoxic effects of endotoxin also in vitro were related to its concentration. In vitro studies, however, have frequently used much higher LPS concentrations than those observed in clinical samples. Using elutriation-purified human monocytes, we observed that LPS up to 1000 ng/ml exerted a concentration-dependent increase in TF activity (tenase activity, fibrin formation in plasma). Although there was a dose-dependent increase in TF activity, there was not a concomitant increase in TF expression at LPS concentrations above 1 ng/ml (flow cytometry, Western blotting, TF mRNA). Flow cytometry revealed that this discrepancy between TF activity and TF expression at endotoxin concentrations above 1 ng/ml, coincided with an LPS dose-dependent increase in cell surface phosphatidylserine (PS), considered to promote coagulation. The increased PS expression was associated with an increased number of 7-AAD-positive cells indicating cell death. We conclude that enhancement of monocyte procoagulant activity in vitro by high concentrations of LPS may result from increased PS exposure due to apoptosis and necrosis. Therefore, the LPS concentrations used to examine monocyte procoagulant activity in vitro, should be carefully chosen.
Presence of endothelial progenitor cells, distinct from mature endothelial cells, within human CD146+ blood cells
Authors: Delorme Bruno , Basire Agnès , Gentile Carla , Sabatier Florence , Monsonis Frédéric , Desouches Christophe , Blot-Chabaud Marcel , Uzan Georges , Sampol José , Dignat-George Françoise
Published: November 2005

CD146 is an adhesion molecule present on endothelial cells throughout the vascular tree. CD146 is also expressed by circulating endothelial cells (CECs) widely considered to be mature endothelial cells detached from injured vessels. The discovery of circulating endothelial progenitor cells (EPCs) originating from bone marrow prompted us to investigate whether CD146 circulating cells could also contains EPCs. We tested this hypothesis using an approach combining elimination of CECs by an adhesion step, followed by immunomagnetic sorting of remaining CD146+ cells from the non adherent fraction of cord blood mononuclear cells. When cultured under endothelial-promoting conditions, these cells differentiated as late outgrowth endothelial colonies: they grew as a cobblestone monolayer, were uniformly positive for endothelial markers and did not express leukocyte antigens. They highly proliferated and were expanded in long-term culture without alterations of their phenotypic and functional properties (DiI-ac-LDL uptake, wound repair, capillary-like network formation, and TNFα response). Moreover, these cells colonized a Matrigel plug in immunodeficient mice (NOD/SCID). Finally, using 4-color flow cytometry analysis of purified CD34+ cells, we clearly discriminated, CD146+ EPCs (CD146+ CD34+ CD45+ CD133+ or CD117+), and CD146+ CECs (CD146+ CD34+, CD45− CD133− or CD117−), both in cord and adult peripheral blood. The relative proportions of the two CD146+ subsets varied in patients with myocardial infarction as compared to healthy subjects. Our study establishes that, beside CECs, CD146+ circulating cells contain a subpopulation of EPCs with potential use in proangiogenic therapy. In addition, the dual measurement of CD146+ CECs and CD146+ EPCs offers a promising tool for monitoring vascular injury/regeneration processes in clinical situations.
Structure, function and biology of tissue factor pathway inhibitor-2
Authors: Chand Hitendra S. , Foster Donald C. , Kisiel Walter
Published: November 2005

Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa matrix-associated Kunitz-type serine proteinase inhibitor consisting of a short amino-terminal region, three tandem Kunitz-type domains and a positively charged carboxy-terminal tail. Human TFPI-2, previously designated as placental protein 5, inhibits a broad spectrum of serine proteinases almost exclusively through its first Kunitz-type domain, and is thought to play an important role in the regulation of extracellular matrix digestion and re-modeling. In this context, reduced synthesis of TFPI-2 has been related to numerous pathophysiological processes such as inflammation, angiogenesis, atherosclerosis, retinal degeneration and tumor growth/metastasis. In this review, we document current information regarding the expression of TFPI-2 by various tissues, its inhibitory activity and proteinase specificity in-vitro, and discuss possible physiological roles for this inhibitor based on in-vivo studies.
Transcriptional Profiles of Valvular and Vascular Endothelial Cells Reveal Phenotypic Differences
Authors: Butcher Jonathan T. , Tressel Sarah , Johnson Tiffany , Turner Debi , Sorescu George , Jo Hanjoong , Nerem Robert M.
Published: November 2005

Objective—

The similarities between valvular and vascular lesions suggest pathological initiation mediated through endothelium, but the role of hemodynamics in valvular endothelial biology is poorly understood.

Methods and Results—

Monolayers of porcine aortic endothelial cells (PAECs) or porcine aortic valve endothelial cells (PAVECs) were exposed to 20 dyne/cm
2
steady laminar shear stress for 48 hours, with static cultures serving as controls. Multiple microarray comparisons were made using RNA from sheared and control batches of both cell types. More than 400 genes were significantly differentially expressed in each comparison group. The resulting profiles were validated at the transcription and protein level and expression patterns confirmed in vivo by immunohistochemistry. PAVECs were found to be less intrinsically inflammatory than PAECs, but both cell types expressed similar antioxidant and antiinflammatory genes in response to shear stress. PAVECs expressed more genes associated with chondrogenesis, whereas PAECs expressed osteogenic genes, and shear stress had a protective effect against calcification.

Conclusions—

Transcriptional differences between PAVECs and PAECs highlight the valvular endothelial cell as a distinct organ system and suggest more attention needs to be given to valvular cells to further our understanding of similarities and differences between valvular and vascular pathology.
Expression Profiling Identifies Smooth Muscle Cell Diversity Within Human Intima and Plaque Fibrous Cap
Authors: Adams Lawrence D. , Geary Randolph L. , Li Jing , Rossini Anthony , Schwartz Stephen M.
Published: November 2005

Background—

The fibrous cap of the atherosclerotic lesion is believed to be critical to stability because disruption of the cap is the final event leading to plaque rupture. We have, therefore, used expression arrays to define the phenotype of the cap and other plaque components.

Methods and Results—

To identify unique expression programs able to distinguish the smooth muscle of the cap from other plaque smooth muscle cells, RNA profiles were determined in human carotid artery media, nonatherosclerotic adjacent intima, fibrous cap of advanced atherosclerotic plaques, and whole advanced plaque with cDNA arrays covering 21 000 or 26 000 Unigene clusters. The molecular signature of each tissue was dominated by a core gene-set with differential expression of <1% of clusters assayed.

Conclusions—

Both intima and cap expressed novel genes not previously associated with SMC pathology. If the cap is derived from a unique subpopulation, this pattern is the signature of that particular set of cells. The loss of RGS5 in the fibrous cap is of particular interest because of its role in vessel development and physiology.