bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of the Fertilase®-Laser System versus the Conventional Mechanical Method to Immobilize Spermatozoa for Intracytoplasmic Sperm Injection
Authors: Debrock Sophie , Spiessens Carl , Afschrift Hilde , van der Auwera Ingrid , D’Hooghe Thomas M.
Published: August 2003

A randomized controlled trial was set up to test the hypothesis that the fertilization rate of oocytes after intracytoplasmic sperm injection (ICSI) is higher after immobilization of the spermatozoa with the Fertilase®-laser system technology than after immobilization of the spermatozoa with the conventional mechanical method. Metaphase II oocytes were injected with spermatozoa that were immobilized with the conventional mechanical method (group A, n = 177) or with spermatozoa that were immobilized with the Fertilase-laser system technology (group B, n = 179). The fertilization rate per successfully injected oocyte was comparable in group A (62.6%; 92/147) and in group B (56.3%; 89/158)(p = 0.3). No difference could be observed in fertilization rates of oocytes injected with spermatozoa that were immobilized with the Fertilase-laser system technology compared to spermatozoa immobilized with the conventional mechanical method.
Human Granulosa Cells after Ovulation Induction Show Caspase-Independent Cell Death
Authors: De Neubourg Diane , Gerris Jan , Knaapen Michiel , Kockx Mark
Published: August 2003

It has become clear that apoptosis is an essential part of normal folliculogenesis and that granulosa cells in particular demonstrate intense cellular activity as well as programmed cell death. Although the entire mechanism of apoptosis appears to be conserved through many species, it now becomes clear that different cells may use different pathways within this system. We examined human granulosa cells after ovulation induction during an in vitro fertilization procedure to study apoptosis in this particular situation. We demonstrated a loss of cytokeratin staining as granulosa cells differentiate. We also detected that granulosa cells with apoptotic morphology did not stain for terminal deoxy-UTP nick end labeling and we showed the absence of immunoreactivity for caspase-cleaved cytokeratin, caspase-cleaved poly-ADP-ribose polymerase and caspase-cleaved caspase-3 in apoptotic granulosa cells. These data provide strong arguments for a caspase-independent cell death mechanism in human granulosa-lutein cells.
Anti-heavy-chain monoclonal antibodies directed to the acidic regions of the factor VIII molecule inhibit the binding of factor VIII to phospholipids and von Willebrand factor
Authors: Villard Sylvie , Grailly Sabrina , Gilles Jean-Guy , Granier Claude , Saint-Remy Jean-Marie , Barrowcliffe Trevor , Raut Sanjeev
Published: September 2003

Recent studies have shown that inhibitors develop against acidic regions of the FVIII molecule, which contain important functional sites. However, their mechanisms of inhibition are not well understood. In this study, two anti-human FVIII mouse monoclonal antibodies (MAbs), directed towards the exposed acidic regions of the FVIII molecule, were developed, characterised and their mechanisms of inhibition investigated. The two MAbs, F7B4 and F26F6, had inhibitory titres of 32 and 944 BU/mg respectively, had high affinities for the FVIII molecule (KD ~ nM range) and recognised sequences V357-F360 on the acidic a1 region and E724-L731 on the acidic a2 region of the FVIII heavy-chain (HC), respectively. F7B4 inhibited the rate of FXa generation by activated FVIII, whilst both antibodies inhibited FVIII activation by thrombin and blocked thrombin cleavage of FVIII. Furthermore, F7B4 and F26F6 inhibited FVIII binding to (a) phospholipids (IC50: 77 nM and 40 nM respectively), and (b) VWF (IC50: 93 nM and 267 nM respectively), despite both having HC specificity. Experiments with F(ab’)2 fragments confirmed the above findings. Taken together these data represent novel findings in that anti-acidic HC antibodies can inhibit FVIII function by a variety of mechanisms, in particular by interfering with the binding of FVIII to phospholipids & VWF.
Activated thrombin-activatable fibrinolysis inhibitor attenuates spontaneous fibrinolysis of batroxobin-induced fibrin deposition in rat lungs
Authors: Wu Chengliang , Dong Ningzheng , da Cunha Valdeci , Martin-McNulty Baby , Tran Katherine , Nagashima Mariko , Wu Qingyu , Morser John , Wang Yi-Xin
Published: September 2003

Studies have shown that inhibition of TAFI by small peptides enhances pharmacological effects of tPA in animal models of thrombosis, suggesting that TAFI modulates the fibrinolytic system. In this study, we investigated the effect of activated human TAFI (TAFIa) on endogenous fibrinolysis in a rat model of intravascular fibrin deposition. 125I-labeled fibrinogen was injected intravenously followed by a bolus injection of batroxo-bin, a thrombin-like enzyme. Batroxobin cleaved fibrinogen to form insoluble fibrin that was deposited in tissues, including the lungs. This was shown by a decrease of radioactivity in the blood as a result of consumption of 125I-labeled fibrinogen and an elevation of radioactivity in the lungs 5 min following batroxobin administration. Endogenous fibrinolysis was detected by a gradual increase in radioactivity in the blood and a decrease in radioactivity in the lungs at 30 min, an indication of radio-labeled fibrin degradation products (FDPs) being released into the circulation from the tissues. Intravenous administration of human TAFIa dose-dependently attenuated the later phase reduction of radioactivity in the lungs. When the dose of TAFIa was 218 μg/kg, giving a peak plasma level of TAFIa 0.9 ± 0.05 μg/ml, the spontaneous fibrinolysis was completely prevented. These results provide direct evidence that an increase in circulating TAFIa impairs endogenous clot lysis in a rat model of fibrin deposition.
Drotrecogin alfa (activated) (recombinant human activated protein C) reduces host coagulopathy response in patients with severe sepsis
Authors: Yan S. , Margolis Benjamin , Lorente José , Russell James , Freebairn Ross , Spapen Herbert , Riess Hanno , Basson Bruce , Johnson III Gerald , Kinasewitz Gary , Dhainaut Jean-François ,   for the PROWESS Sepsis Study Group
Published: September 2003

Drotrecogin alfa (activated) improved survival in patients with severe sepsis in PROWESS, a double-blind, study of 1690 adult patients randomized to drotrecogin alfa (activated) at 24 μg/kg/h (N=850) or placebo (N=840) infused for 96 hours. Pharmacodynamic effects of drotrecogin alfa (activated) were assessed with 15 prospectively defined systemic biomarkers of hemostasis, inflammation and endothelial injury. The last-observation-carried-forward (LOCF) method of imputation for missing observations was the prospectively defined statistical method. The results were also analyzed with only the observed values without imputation for missing data (repeated measures analysis). With both statistical methods, drotrecogin alfa (activated)-treated patients demonstrated antithrombotic (reduced markers of thrombin generation and accelerated normalization of anticoagulant factor, protein C and fibrinolytic factors) and anticoagulant (prolonged PT and APTT) effects compared with placebo. A profibrinolytic (reduction in plasminogen activator inhibitor-1) effect was significant only with the LOCF imputation method in observed case and percent change from baseline analyses. An anti-inflammatory (reduction in interleukin-6) effect was significant only with the LOCF imputation method in change from baseline and percent change from baseline analyses. Drotrecogin alfa (activated) is a new and promising agent for treatment of patients with severe sepsis. The extensive analysis of systemic biomarkers confirms the previously published antithrombotic effects. However, the present results using different statistical methods do not provide a strong basis for systemic anti-inflammatory or pro-fibrinolytic effects. These latter two effects may occur at the local or cellular level. The systemic biomarkers reported here might not be the most appropriate approach to demonstrate these potential effects of drotrecogin alfa (activated).
Shedding of active tissue factor by aortic smooth muscle cells (SMCs) undergoing apoptosis
Authors: Brisset Anne-Cécile , Terrisse Anne-Dominique , Dupouy Dominique , Tellier Lise , Pech Stéphane , Navarro Chantal , Sié Pierre
Published: September 2003

As apoptosis of neo-intimal SMCs is a feature of advanced atherosclerotic plaques, the procoagulant properties of SMCs of synthetic phenotype undergoing apoptosis were investigated. SMCs isolated from rat aorta obtained 10 days after balloon injury, previously found to up-regulate Tissue Factor (TF) and Tissue Factor Inhibitor (TFPI) and to release large amounts of TFPI (Ghrib et al. Thromb Haemost 2002;87:1043-50), were sensitive to the apoptosis induced by Fas-ligand. During this process, surface TF activity rose by a factor 10 over 6 hours, in parallel with a proportional increase in prothrombinase, while TF protein expressed at the membrane significantly decreased. The microparticles (MPs) produced during SMC death bore intact and functional TF, but the release of TFPI did not change, so that the balance shifted to a procoagulant state during apoptosis. Shed MPs enhanced thrombus formation in flowing whole blood over collagen coated-glass slides. Apoptotic SMCs in atherosclerotic plaques represent a reservoir of highly thrombogenic material, released into the blood stream in case of spontaneous or mechanical plaque disruption.
Patterns of use of heparins in ACS
Authors: Kraxner Wilfried , Hödl Ronald , Gabriel Steg Philippe , Budaj Andrzej , Gulba Dietrich , Sadiq Immad , Van de Werf Frans , White Kami , Fox Keith , Klein Werner ,   for the GRACE Investigators
Published: September 2003

A systematic study that compares the patterns of use of unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) in patients with acute coronary syndromes (ACS) has, to date, not been carried out in the “real-world” setting. The aim of this report is to identify patterns of use of UFH and LMWH and to report their correlates and outcomes in a broad spectrum of ACS patients enrolled in the observational Global Registry of Acute Coronary Events (GRACE).

The use of LMWH and UFH was analysed in 13,231 ACS patients according to patient history, concomitant treatment and invasive procedures in US and non-US sites. Frequency of use in hospitals with and without facilities for percutaneous coronary interventions (PCI) was investigated, and outcomes were analysed.

Results show that younger patients (<60 years), those receiving antiplatelet therapies, thrombolytics, beta-blockers, angiotensin-converting enzyme inhibitors, patients admitted to hospitals with PCI facilities, and patients undergoing invasive procedures were more likely to receive UFH, or both UFH and LMWH than LMWH alone (80.1% enoxaparin, 19.9% other LMWH). LMWH was used less often in US than non-US sites. After adjusting for confounding variables, patients receiving LMWH had significantly lower rates of hospital mortality (P=0.009) and major bleeding (P<0.0001). Similar results were observed in patients with ST-segment elevation myocardial infarction and non-ST-segment elevation myocardial infarction or unstable angina.

We can conclude that UFH tends to be used more frequently than LMWH, but hospital outcomes appeared to be better with LMWH after adjusting for covariables.
The antiaggregating activity of clopidogrel is not affected by N-acetyl L-cysteine
Authors: Lalé Alain , Savi Pierre , Herbert Jean-Marc
Published: September 2003

N-acetyl L-cysteine (NAC) is widely used to treat obstructive bronchopulmonary diseases. It has thiol reactive properties, accounting for its mucolytic activity. Clopidogrel is a potent antithrombotic compound, metabolised by the liver which generates an active metabolite containing a thiol reactive group, responsible for an irreversible interaction with the platelet P2Y12ADP receptor.

The aim of this study was to determine if NAC interferes with the antiaggregating activity of clopidogrel. For this purpose, NAC (100 µM) was incubated with platelets from rats treated or not with clopidogrel (5 mg/kg, PO, -2 h). Clopidogrel treatment strongly inhibited aggregation but this effect was not modified by NAC. In another experiment, a low concentration of the active metabolite of clopidogrel (0.3 µg/ml) was incubated with platelets from men or rats, in the absence or presence of NAC (100 µM). When stimulated by ADP (2.5 µM), platelet aggregation was inhibited by the active metabolite when incubated alone. In the presence of NAC, the inhibition by the active metabolite was not modified, therefore clearly indicating that NAC cannot reduce the thiol reactive part of the active metabolite of clopidogrel and does not interfere with its anti-aggregating activity. Moreover, in rats treated for 5 days with NAC (150 mg/kg), the activity of clopidogrel (5 or 10 mg/kg) against ADP-induced platelet aggregation was neither inhibited nor increased. This demonstrates that the generation of the active metabolite of clopidogrel is not affected by NAC.

In conclusion, we have found that NAC does not restore the “normal” properties of P2Y12on platelets from clopidogrel-treated animals, it does not interfere with the antiaggregating activity of the active metabolite of clopidogrel, and does not interfere with the generation of the active metabolite.
Comparison of PFA-100 testing and bleeding time for detecting platelet hypofunction and von Willebrand disease in clinical practice
Authors: Posan Emoke , McBane Robert , Grill Diane , Motsko Cheri , Nichols William
Published: September 2003

The PFA-100 instrument (Platelet Function Analyzer, Dade Behring) has been reported to be superior to the bleeding time (BT) as a screening test of primary hemostasis. However evaluation of this device has been principally limited to selected populations.

The study’s aim was to determine testing performance in clinical practice, by comparing the PFA-100 to the BT for the identification of von Willebrand disease (VWD) and intrinsic platelet hypofunction.

From 1998-2000, PFA-100 closure time (CT) for epinephrine-collagen (EPI) and ADP-collagen (ADP) cartridges and modified Ivy BTs were performed on outpatients referred for testing for suspected or known hemorrhagic diathesis (n=346). Evaluation included assays of von Willebrand factor and platelet aggregometry in addition to platelet flow cytometry and electron microscopy when indicated. The normal distribution of PFA-100 CTs was determined using blood samples from 61 normal donors studied on 155 occasions.

Results show that thirty-four patients met the diagnostic criteria for VWD and 31 patients were diagnosed with congenital or acquired intrinsic platelet hypofunction. The sensitivity of the PFA-100 for identification of VWD was significantly better (p<0.01) than the BT with similar specificity. In contrast, the PFA-100 was comparable, but not superior to the BT for detecting platelet hypofunction.

We conclude that the PFA-100 performance compares favor-ably to the BT for the identification of intrinsic platelet hypofunction in clinical practice with superior sensitivity for detecting VWD.Therefore, the PFA-100 could replace the BT for purposes of screening for VWD and intrinsic platelet hypofunction. When clinical suspicion is strong, testing should be supplemented with assays of von Willebrand factor and platelet aggregometry.
Regulation of human factor IX expression using doxycycline-inducible gene expression system
Authors: Wang Xiaomin , Siemetzki Ulrike , Albert Thilo , Oldenburg Johannes , Hanfland Peter , Poller Wolfgang , Brackmann Hans-Hermann , Srour Mahmoud , Fechner Henry , Schwaab Rainer
Published: September 2003

Following substitution therapy with human factor IX (hFIX) concentrate, therapy of haemophilia B by viral gene transfer has become an attractive alternative therapy in recent years. However, high doses of expressed hFIX, which can already be achieved in animal studies, may cause thrombosis in humans (van Hylckama Vlieg et al., 2000). Thus, it should be possible to maintain transgene expression within the therapeutic range. Therefore, we inserted elements of the tetracycline (Tet)-dependent Tet-On gene regulatory system into replication deficient adenovectors. The new system consists of two adenovec-tors: a response vector expressing hFIX (Ad5. TRE.hFIX), and a regulator vector expressing a second generation reverse tetracycline transactivator controlled by a CMV- (Ad5. CMV.rtTA) or human alpha1-antitrypsin-promoter (Ad5.hAAT.rtTA). Expression studies in four human cell lines showed high expression of hFIX from Ad5. TRE.hFIX in all cell lines in combination with Ad5. CMV.rtTA regulator vector, but only high specific expression in HepG2-cells in combination with Ad5.hAAT.rtTA regulator vector. Additionally, up- and down-regulation of hFIX expression could be demonstrated in vitro with the Ad5. TRE.hFIX/Ad5. CMV.rtTA combination and modulating doxycycline concentrations. When SCID-mice were infected with the Ad5. TRE.hFIX/Ad5. CMV.rtTA combination, up- and down-regulation of hFIX expression was achieved by oral doses of doxycycline for a period of at least two months. Replacement of the Ad5. CMV.rtTA vector by the Ad5.hAAT.rtTA vector showed minimal expression of hFIX in vivo. Although hFIX expression showed a slow and gradual decrease over time in vivo with the Ad5. CMV.rtTA vector, it remained within the therapeutic range. To date, regulation of hFIX has not been described in this way.
Thrombin generation in severe haemophilia A and B: the endogenous thrombin potential in platelet-rich plasma
Authors: Siegemund Thomas , Siegemund Annelie , Scholz Ute , Engelmann Lothar , Petros Sirak
Published: September 2003

Thrombin generation was investigated in platelet-rich plasma (PRP) from 11 healthy controls, 17 patients with severe haemophilia A and 7 patients with severe haemophilia B. Mean endogenous thrombin potential (ETP) in arbitrary fluorescence units (FU) was 226.9 ± 44.6, 186.4 ± 22.5, 154.2 ± 41.3 in controls, haemophilia A and B, respectively, all at a platelet count of 200 × 109/l (p = 0.004 for controls vs.haemophilia A, p = 0.003 for controls vs. haemophilia B, no significant difference between haemophilia A and B). The contribution of FVIII to thrombin generation in haemophilia A was 1.31 ± 0.16 FU/% of FVIII:C activity , while for FIX in haemophilia B this was 0.80 ± 0.21 FU/% of FIX activity. There was an almost linear relationship between increasing platelet count and thrombin generation up to a mean platelet count of 100 × 109/l. Further increase in platelet count has only a marginal influence on thrombin generation. Platelets increase ETP in haemophilia A by 0.184 ± 0.022 FU/109platelets/l and in haemophilia B by 0.319 ±
0.085 FU/109platelets/l, and this was significantly different between the two groups (p = 0.0002). This influence of platelets diminishes with increasing concentration of either FVIII or FIX. In conclusion, there is a difference in thrombin generation between haemophilia A and B, and this may be attributed to the role of platelets in the assembly of the tenase complex on their surface.
Frequency of the TAFI –438 G/A and factor XIIIA Val34Leu polymorphisms in patients with objectively proven pulmonary embolism
Authors: Zidane Majida , de Visser Marieke , ten Wolde Marije , L.Vos Hans , de Monyé Wouter , Bertina Rogier , Huisman Menno ,   the Antelope study group
Published: September 2003

Deep vein thrombosis (DVT) and pulmonary embolism (PE) are considered to be two forms of the same disease, however it is not fully understood what determines their clinical presentation.

Proteins encoded by the FXIIIA and TAFI genes are involved in stabilizing the fibrin clot and in making it more lysis resistant. The FXIIIA 34Leu and TAFI –438A alleles might protect against DVT. Information on such an association with PE is either contradictory or missing. We hypothesized that both polymorphisms might influence the formation and fate of emboli and accordingly the risk of PE. We determined the frequencies of both polymorphisms in patients with objectively demonstrated PE.

The frequency of FXIIIA Leu34Leu in PE patients and non-PE patients was 4.5% and 8.8%, [OR 0.5 (95% CI: 0.1 to 1.9)], respectively. For –438 A/A TAFI genotype the frequency was 1.5% and 8.1% [OR 0.1 (95% CI: 0.02 to 1.1)], respectively.
Induction of plasminogen activator inhibitor 1 by the PPARα ligand, Wy-14,643, is dependent on ERK1/2 signaling pathway
Authors: Banfi Cristina , Auwerx Johan , Poma Federica , Tremoli Elena , Mussoni Luciana
Published: September 2003

Impairment of the fibrinolytic system, mostly due to elevated plasma levels of plasminogen activator inhibitor 1 (PAI-1), is often associated with metabolic disorders such as diabetes mellitus and insulin-resistance syndrome. Moreover, insulin, as we have previously shown, directly stimulates PAI-1 production with a mechanism underlying a complex signaling network which ultimately leads to ERK activation.

In this study we have analyzed the effects of agonists of the per-oxisome proliferator-activated receptor (PPAR) alpha and gamma on PAI-1 biosynthesis in HepG2 cells in the presence or absence of insulin. The high affinity PPARα agonist, Wy-14,643, increased basal and insulin-stimulated PAI-1 antigen release with a mechanism involving gene transcription. We then investigated whether the MAP kinase pathway also plays a role in the stimulatory properties of Wy-L4,643. Wy-L4,643 increases phosphorylation of ERK and p38 in a time-dependent manner without affecting that of SAPK/JNK or ERK5. Moreover, the MEK (ERK kinase) inhibitors, PD98059 and UO126, completely prevented PAI-1 induction by Wy-14,643 without inhibiting the activation of a reporter gene carrying the PPRE element. Interestingly, the addition of p38 inhibitor followed by insulin and Wy-14,643 resulted in a greater than additive stimulation of PAI-1 secretion acting through ERK1/2 phosphorylation.

In contrast, the synthetic PPARγ agonist, rosiglitazone, did not change PAI-1 level, although this compound induced transcription from the PPRE-driven luciferase reporter construct.

In conclusion, Wy-14,643 induces PAI-1 gene expression, in the presence or absence of insulin, with a mechanism which is independent on PPARα activation and requires signaling through the ERK1/2 signaling pathway.
High prevalence of elevated clotting factor VIII in chronic thromboembolic pulmonary hypertension
Authors: Bonderman Diana , Turecek Peter , Jakowitsch Johannes , Weltermann Ansgar , Adlbrecht Christopher , Schneider Barbara , Kneussl Meinhard , Rubin Lewis , Kyrle Paul , Klepetko Walter , Maurer Gerald , Lang Irene
Published: September 2003

Chronic thromboembolic pulmonary hypertension (CTEPH) is an enigmatic disorder lacking signs, symptoms and classical risk factors for venous thromboembolism.

The objective of the prospective case controlled study, carried out at the Pulmonary Hypertension Unit, University Hospital Vienna, Austria, was to investigate whether plasma FVIII is elevated in CTEPH patients.

The study examined 122 consecutive patients diagnosed with CTEPH. Plasma FVIII was measured and compared with plasma FVIII of healthy controls (n=82) and of patients with non-thromboembolic pulmonary arterial hypertension (PAH, n=88).

Results show that CTEPH patients had higher FVIII levels than controls (233±83IU/dl versus 123±40IU/dl, p<0.0001) and PAH patients (158±61IU/dl, p<0.0001). Plasma FVIII one year after surgery (212±94IU/dl) was statistically unchanged compared with preoperative values (FVIII: 226±88IU/dl, n=25). FVIII>230IU/dl was more prevalent in CTEPH patients (41%) than in controls (5%, p<0.0001) and PAH patients (22%, p=0.022).

We can conclude that elevated plasma FVIII is the first prothrombotic factor identified in a large proportion of CTEPH patients.
Platelet factor 4 localization in carotid atherosclerotic plaques: correlation with clinical parameters
Authors: Pitsilos Stephanie , Hunt Jennifer , Mohler Emile , Prabhakar Anand , Poncz Mortimer , Dawicki Jennine , Khalapyan Tigran , L.Wolfe Megan , Fairman Ronald , Mitchell Marc , Carpenter Jeffrey , Golden Michael , Cines Douglas , Sachais Bruce
Published: September 2003

Emerging evidence supports a role for platelets in the progression of atherosclerosis in addition to an involvement in thrombotic vascular occlusion. Platelet Factor 4 (PF4), a chemokine released by activated platelets, stimulates several pro-atherogenic processes. Therefore, we examined the localization of PF4 and the homologous protein, Neutrophil Activating Protein-2 (NAP-2) in lesions representing the evolution of human atherosclerotic plaques. Carotid plaques from 132 patients with critical carotid stenosis and 6 autopsy specimens were studied. Clinical, histologic and immunohistochemical data were analyzed using a χ2-test. PF4 was detected in the cytoplasm of luminal and neovascular endothelium, in macrophages and in regions of plaque calcification. The presence of PF4 in macrophages and neovascular endothelium correlated with lesion grade (p = 0.004; p = 0.044). Staining of macrophages for PF4 correlated with the presence of symptomatic atherosclerotic disease (p = 0.028). In early lesions, PF4 was commonly found in macrophages of early lesions (Grade I/II), whereas NAP-2 was rarely present.

In conclusion, correlation between PF4 deposition, lesion severity and symptomatic atherosclerosis suggests that persistent platelet activation may contribute to the evolution of athero-sclerotic vascular lesions. These studies support the rationale for the chronic use of anti-platelet therapy in patients at risk for developing symptomatic atherosclerosis.
Matrix metalloproteinase-9 expression in post-hypoxic human brain capillary endothelial cells: H2O2 as a trigger and NF-κB as a signal transducer
Authors: Kolev Krasimir , Skopál Judit , Simon László , Csonka Éva , Nagy Zoltán , Machovich Raymund
Published: September 2003

The haemorrhagic transformation in ischemic stroke involves disruption of the integrity of the microvascular beds, partially based on the action of matrix metalloproteinases (MMPs). The objective of the present study was to evaluate the contribution of microvascular endothelial cells from human brain (HBECs) to MMPs’ expression and regulation under conditions relevant to brain ischemia. MMPs and their inhibitors were examined with zymography, Western-blotting, ELISA and MMP-activity assay in cultured HBECs. Four-hour hypoxia (pO2=60 mmHg) elevated the level of MMP-9 in the supernatant of the HBECs and this early response required collagen-matrix. Active oxygen species sustained the increased MMP-9 activity for at least 24 h. In the post-hypoxic period 20 μmol/L H2O2 caused a 6-fold increase in the specific activity of MMP-9 over the nor-moxic cells and a comparable effect was exerted by thrombin (50 nmol/L) and leukocyte elastase (10 nmol/L). The role of NF-κB, a redox-state sensitive transcription factor, was evaluated with immunofluorescence confocal microscopy and immunoblotting of nuclear and cytoplasmic extracts. The oxidative stress-dependent MMP-9 induction was accompanied by a significant increase in the NF-κB localized in the nuclei and these responses were blunted with a proteasome inhibitor (MG132). Consequently, according to our in vitro data HBECs are a source of MMP-9, which is under the control of triggers relevant to the ischemic/reperfused brain (reactive oxygen species, thrombus and inflammation related proteases) and this regulation is partially based on NF-κB activation. The reported regulation of endothelium-derived MMP-9 supports its potential involvement in the post-hypoxic disturbances of the cerebral micro-circulation.
A tetrameric glycoprotein Ib-binding protein, agglucetin, from Formosan pit viper: structure and interaction with human platelets
Authors: Wang Wen-Jeng , Ling Qing-Dong , Liau Ming-Yi , Huang Tur-Fu
Published: September 2003

Agglucetin, a tetrameric agglutination inducer from the Formosan pit viper, has been identified as a platelet membrane glycoprotein (GP) Ib agonist and directly agglutinated fixed-platelets in the absence of von Willebrand factor (vWf). Here, we resolved the complete cDNA sequences of agglucetin subunits (α1, α2, β1 and β2) by molecular cloning. Each cloned cDNA encoding the leader peptide (23 residues) and the mature subunit (131/135/123/126 residues) shares a high degree of homology to each other and the C-type lectin-like GP Ib-binding proteins (BPs). Furthermore, agglucetin specifically caused platelet agglutination and surface exposure of integrin αIIbβ3 with a GPIb-dependent manner in washed platelets, based on the observation that the enhanced expression of functional αIIbβ3 was suppressed by a GPIb-cleaving metalloproteinase, crotalin. Pretreating platelets with staurosporine or BAPTA-AM also completely blocked the exposure of functional αIIbβ3, suggesting that the activation of protein kinase C and intracellular calcium mobilization are involved in the GPIb-dependent signaling. In human platelet-rich plasma (PRP), agglucetin elicited sequential biphasic responses of platelet agglutination and aggregation in a GPIb- and αIIbβ3-dependent manner, respectively, implying that other cofactors may amplify platelet activation to trigger aggregation.

The nucleotide sequences reported in Figs. 1 and 2 have deposited in the GenBank, EMBL and DDBJ Nucleotide Sequence Databases under the accession numbers: AF540645 for agglucetin-α1 subunit, AF540646 for agglucetin-α2 subunit, AF540647 for agglucetin-β1 subunit and AF540648 for agglucetin-β2 subunit.
Optimal low-molecular-weight heparin regimen in major orthopaedic surgery
Authors: Laporte Silvy , Quenet Sara , Molliex Serge , Auboyer Christian , Decousus Hervé , Mismetti Patrick , Zufferey Paul
Published: September 2003

Low-molecular-weight heparins (LMWH) are routinely used for thromboprophylaxis in major lower limb orthopaedic surgery. However the optimal LMWH regimen, offering the greatest efficacy with an acceptable risk of bleeding, has not been clearly established with regard to dose and timing of treatment initiation. We performed a meta-analysis of all available randomised trials comparing LMWH to placebo. Relative risks (RR) and corresponding 95% confidence intervals (CI) were calculated. By means of subgroup analysis, we evaluated the consistency of the results according to the timing of treatment initiation (preoperative versus postoperative) and dose of LMWH used (low doses, i.e. 4000 anti-Xa IU or below versus high doses). The possibility of a dose-effect relationship of LMWH was also evaluated by meta-regression. Thirteen studies were included (1925 patients). In four studies, LMWH treatment was started postoperatively. Daily LMWH doses ranged from 3000 anti-Xa IU to over 6000 anti-Xa IU. Compared to placebo, LMWH significantly reduced the risk of asymptomatic deep-vein thrombosis (DVT) (RR=0.51, 95% CI=[0.45-0.59], p<0.001) without significantly increasing the risk of major haemorrhage (RR=0.80 [0.36-1.79], p=0.58). We found no convincing evidence that starting prophylaxis preoperatively was associated with a significantly reduced risk of asymptomatic DVT relative to starting postoperatively. Our results showed a strong correlation between the risk of DVT and LMWH dose (meta-regression, test of slope p=0.03).These findings are tentative because the comparisons are across trials, but nevertheless suggest that the different LMWH regimens currently recommended are effective and safe.
PML/RARα plays a role for basal activity and retinoid-induced repression of the tissue factor promoter in acute promyelocytic leukemia cells
Authors: Öberg Fredrik , Mackman Nigel , Nilsson Kenneth , Siegbahn Agneta , Tenno Taavo
Published: September 2003

Constitutive expression of tissue factor (TF) by acute promyelocytic leukemia (APL) cells may contribute to thrombotic complications. In this study we examined the transcriptional mechanisms of all-transretinoic acid (ATRA)-induced down-regulation of TF in the APL cell line NB4, by analysis of stable clones expressing the luciferase gene under the control of 5’ flanking regions of the TF gene. We show that the TF promoter is constitutively active in NB4 cells, and that ATRA induces rapid suppression of the promoter. Basal activity and ATRA-induced suppression of TF promoter is determined by the proximal -383 to +121 bp of the promoter. Electrophoretic mobility shift assays demonstrate the binding of Fos/Jun complexes to two TF promoter AP-1 sites in this region. Both complexes were suppressed by ATRA treatment. The ectopic expression of the APL-specific PML/RARα oncoprotein in U-937 cells results in induction of TF mRNA and promoter activity. Interestingly, this PML/RARα-mediated increase in TF promoter activity is sensitive to ATRA treatment. These data indicate that TF expression in APL cells is exacerbated by the presence of the PML/RARα fusion protein, and implicates the loss of Fos/Jun binding to the TF promoter in ATRA-induced suppression of TF.
Homozygous and double heterozygous Factor V Leiden and Factor II G20210A genotypes predispose infants to thromboembolism but are not associated with an increase of foetal loss
Authors: Hundsdoerfer Patrick , Vetter Barbara , Stöver Brigitte , Bassir Christian , Scholz Tristess , Grimmer Ingrid , Mönch Eberhard , Ziemer Sabine , Rossi Rainer , Kulozik Andreas
Published: September 2003

Prospective and controlled data about the individual risk profile in asymptomatic children with homozygous or double heterozygous risk genotypes for Factor V Leiden (FVL) and factor II (FII) G20210A are currently unavailable. The systematic and prospective observational study presented here was designed to determine the impact of the homozygous and double heterozygous FVL and FII G20210A genotypes on the prenatal and postnatal risk profiles of affected children. Risk infants and heterozygous controls were identified by screening of 85,304 neonates. Follow-up included the comparison of prenatal and postnatal development, ultrasonography of brain and kidneys, and a panel of independent determinants of thrombophilia. The numbers of identified or expected FVL homozygotes and double heterozygotes did not differ significantly (FVL: 116 ver-sus 91, p=0.08; FVL/FII: 94 versus 76, p=0.17), indicating the absence of a prenatal disadvantage. A prenatal advantage was suggested in FII homozygotes, whose identified number far exceeded the expected (19 versus 4, p=0.002). Clinical and/or imaging abnormalities indicated spontaneous thromboembolic events in 4 of 129 risk infants (3%) but in none of the 178 controls (p=0.02). Physical and neurological development was normal in both groups during the first 2 years of life. The risk genotypes appear to confer a significant predisposition for spontaneous thromboembolic events in infancy without impeding development within the first two years of life. Foetal risk genotypes do not cause an increased foetal loss rate. Moreover, homozygous FII G20210A appears to be associated with a prenatal advantage.
Inhibition of tumor cell-induced platelet aggregation and lung metastasis by the oral GpIIb/IIIa antagonist XV454
Authors: Mousa Shaker , Amaya Mildred , Blaydes Susan , Desai Hina , Meyer Todd , Francis John , Amirkhosravi Ali
Published: September 2003

Platelets are known to play a role in blood borne metastasis. Previous experimental studies have suggested that platelet GpIIb/IIIa may be a therapeutic target. However, the need for intravenous administration limits the potential application of current GpIIb/IIIa inhibitors to cancer therapy. The aim of the present study was to assess the efficacy of a novel, non-peptide oral GpIIb/IIIa antagonist (XV454) on tumor cell-induced platelet aggregation in vivo and on experimental metastasis. A Lewis lung carcinoma (LL2) mouse model of experimental metastasis was used in this study. XV454 (100 μg) was administered intravenously (via tail vein) or orally (gavages) to 20 g mice. To determine the effect of XV454 on platelet aggregation, blood samples were collected by cardiac puncture 10 minutes after intravenous and 1-24 hrs after oral XV454, and platelet function was assessed by aggregometry, thrombelastography and the Platelet Function Analyzer (PFA100®). The effect of XV454 on tumor cell-induced thrombocytopenia was determined 10 minutes after intravenous and 3 hrs after oral XV454 administration. Tumor cells (2 x 106) were injected intravenously and 15 minutes after cell injection, platelet count was measured and compared to baseline (pre-injection) counts. To assess the effect on metastasis, XV454 was administered intravenous or orally 10 minutes and 3 hrs before tumor cell injection, respectively. Eighteen days later, surface lung tumor nodules were counted and the total lung tumor burden assessed. In a fourth group, in addition to the initial oral dose (before tumor cell injection), oral XV454 was given daily for the first week and three times in the second week. Administration of XV454 (5 mg/kg) completely inhibited platelet aggregation and this effect persisted for at least 24 hrs after oral delivery. Both intravenous and oral XV454 significantly inhibited tumor cell-induced thrombocytopenia (P<0.01), the number of surface lung tumor nodules (80-85%; P<0.001) and total tumor burden (83% for intravenous group; 50% oral [single treatment] group; 91% oral [multiple treatment] group, P<0.001). Overall, these data provide further evidence for the effect of oral and intravenous GpIIb/IIIa antagonism on tumor cell-platelet interaction and metastasis.
Potent inhibition of angiogenesis by D,L-peptides derived from vascular endothelial growth factor receptor 2
Authors: Piossek Christine , Thierauch Karl-Heinz , Schneider-Mergener Jens , Volkmer-Engert Rudolf , Bachmann Martin , Korff Thomas , Augustin Hellmut , Germeroth Lothar
Published: September 2003

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and plays a central role in angiogenesis and vasculogenesis. Therefore, VEGF and its receptors VEGFR-1 and VEGFR-2 are prime targets for anti-angiogenic intervention which is thought to be one of the most promising approaches in cancer therapy. Recently, we have discovered a VEGFR-2-derived peptide (247RTELNVGIDFNWEYP261) representing a potential binding site to VEGF. Using the spot synthesis technique, systematic D-amino acid substitutional analyses of this peptide were conducted and the resulting D, L-peptides inhibit VEGF binding to VEGFR-2 at half maximal concentration of 30 nM.The serum-stable D, L-peptides further inhibited auto-phosphorylation of the VEGFR-2 at nanomolar concentrations. Testing of the peptides in a spheroid-based angiogenesis assay demonstrated a potent anti-angiogenic effect in vitro. The rational design of potent and stable anti-angiogenic peptide inhibitors from their parent receptors provides a feasible route to develop novel leads for anti-angiogenic medicines.
Platelet hyperactivity after statin treatment discontinuation
Authors: Laura Pasqui Anna , Pastorelli Marcello , Bova Giovanni , Di Renzo Michela , Leo Alessandro , Cercignani Michela , Palazzuoli Alberto , Auteri Alberto , Bruni Fulvio , Puccetti Luca
Published: September 2003

Hydroxymethyl-glutaryl-CoA-reductase inhibitors (statins) reduce cardiovascular events by cholesterol lowering as well as by non-lipid related actions. Among them, the modulation of platelet activity could play a relevant role in vascular protection. Furthermore withdrawal of statins has been associated with increased cardiovascular event rate. The aim of our study was to evaluate platelet activity after cerivastatin discontinuation in eighteen subjects that did not accept other drugs and in sixteen subjects continuing treatment with simvastatin. Fourteen subjects at the end of the discontinuation period decided to receive other drugs (simvastatin) and they were evaluted six weeks later. We measured complete lipid profile by the chromogenic method (LDL-C was calculated); oxidized-LDL (ox-LDL; ELISA), platelet P-selectin (P-sel) expression (flow cytometry detection), platelet aggregation (% change of transmitted light), intracellular citrullin production (iCit; HPLC) as an indicator of intracellular NO synthase activity at baseline and 7, 14, 28, 60 days after statin discontinuation. P-sel expression and platelet aggregation were increased at 14 days (p< 0.001 and p< 0.05) in association with raised ox-LDL (r= 0.30, p< 0.05) and decreased iCit (r= 0.53, p< 0.01). Increased LDL-C was related to P-sel and platelet aggregation at 28 days (r= 0.30, p< 0.05). Subjects continuing statin treatment had no significant changes of P-sel at 28 (p= 0.221) and 60 days (p=0.238). Subjects treated with simvastatin after 60 days of diet showed a significant reduction of P-sel and platelet aggregation after six weeks of treatment (p< 0.01).

Our data suggest a platelet hyperactivation state in the second week after statin discontinuation which is partially related to raised LDL-C. Such a finding could participate in the increased cardiovascular event rate after statin discontinuation.
Antithrombotic and thrombolytic efficacy of YM-254890, a Gq/11 inhibitor, in a rat model of arterial thrombosis
Authors: Taniguchi Masatoshi , Moritani Yumiko , Hayashi Kazumi , Saito Tetsu , Takasaki Jun , Nagai Koji , Inagaki Osamu , Shikama Hisataka , Kawasaki Tomihisa
Published: September 2003

We examined the antithrombotic and thrombolytic effects of the Gq/11 inhibitor YM-254890 in an electrically-induced carotid artery thrombosis model in rats. YM-254890 dose-dependently inhibited ex vivo ADP-induced platelet aggregation after i.v. bolus injection. In the thrombosis study, YM-254890 dose-dependently prolonged time to occlusion at doses of 3 and 10 μg/kg i.v. and decreased occlusion rate at 10 μg/kg i.v. In the thrombolysis study, YM-254890 at 30 μg/kg i.v. shortened the time to reperfusion and prevented reocclusion after thrombolysis with a modified tissue-type plasminogen activator. YM-254890, at 10 μg/kg and more, significantly improved carotid patency status after thrombolysis. However, at 30 μg/kg and more, YM-254890 decreased systemic blood pressure. These results suggest that YM-254890 may be effective for treating Gq-mediated diseases, and that YM-254890 is a useful tool for investigating the biological roles of Gq/11.
Factor V Leiden and the risk of stillbirth in a German population
Authors: Grimm Rita , Robinson Daniel , Robinson Constanze , Kohlmann Thomas , Schuster Gudrun , Alte Dietrich , Herrmann Falko , John Ulrich , Völzke Henry
Published: September 2003

An association between the factor V Leiden variant and an increased risk of pregnancy loss has been reported. Most previous studies were performed with clinically recruited patients and controls. This approach may cause selection bias. The present analysis was performed with the aim to investigate the association between the factor V Leiden mutation and the risk of stillbirth in a population-based sample.

The Study of Health in Pomerania (SHIP) is a survey that was carried out in North East Germany. A random sample from the population aged 20 to 79 years was taken. The total SHIP population comprised 4,310 participants. The presence of the factor V Leiden variant was determined by PCR and Mnl I digestion. The presence of the factor V Leiden variant was neither associated with the number of pregnancies nor with the number of children per women. Data from 1,768 females who had at least one pregnancy with known outcome was available for the present analysis. Seventy-three women (4.1%) reported at least one stillbirth. Women with and without the factor V Leiden mutation did not differ with respect to the number of women with at least one stillbirth (OR for factor V Leiden variant 1.57; 95%-CI 0.76 – 3.25). Furthermore, the number of women with two or more stillbirths, the number of stillbirths per affected woman and the number of stillbirths per number of pregnancies per woman was similar between both genotype groups.

In conclusion, there is no association between the factor V Leiden mutation and the risk of stillbirth in a representative population sample.
Duration of exposure to high fluid shear stress is critical in shear-induced platelet activation-aggregation
Authors: Jian-ning Zhang , Bergeron Angela , Yu Qinghua , Sun Carol , McBride Latresha , Bray Paul , Dong Jing-fei
Published: September 2003

Platelet functions are increasingly measured under flow conditions to account for blood hydrodynamic effects. Typically, these studies involve exposing platelets to high shear stress for periods significantly longer than would occur in vivo. In the current study, we demonstrate that the platelet response to high shear depends on the duration of shear exposure. In response to a 100 dyn/cm2 shear stress for periods less than 10-20 sec, platelets in PRP or washed platelets were aggregated, but minimally activated as demonstrated by P-selectin expression and binding of the activation-dependent αIIbβ3 antibody PAC-1 to sheared platelets. Furthermore, platelet aggregation under such short pulses of high shear was subjected to rapid disaggregation. The disaggregated platelets could be re-aggregated by ADP in a pattern similar to unsheared platelets. In comparison, platelets that are exposed to high shear for longer than 20 sec are activated and aggregated irreversibly. In contrast, platelet activation and aggregation were significantly greater in whole blood with significantly less disaggregation. The enhancement is likely via increased collision frequency of platelet-platelet interaction and duration of platelet-platelet association due to high cell density. It may also be attributed to the ADP release from other cells such as red blood cells because increased platelet aggregation in whole blood was partially inhibited by ADP blockage. These studies demonstrate that platelets have a higher threshold for shear stress than previously believed. In a pathologically relevant timeframe, high shear alone is likely to be insufficient in inducing platelet activation and aggregation, but acts synergistically with other stimuli.
Antibodies directed to protein S in patients with systemic lupus erythematosus: prevalence and clinical significance
Authors: Bertolaccini Maria , Sanna Giovanni , Ralhan Shvaita , Gennari Laura , Merrill Joan , Hughes Graham , Khamashta Munther
Published: September 2003

Antibodies directed against protein S (anti-ProtS) may be involved in the development of thrombosis in patients with the antiphospholipid syndrome. We assessed the prevalence and clinical significance of anti-ProtS and evaluated their immunological characteristics in 184 patients with SLE and 99 healthy donors. All patients were tested for IgG anti-ProtS by an in-house ELISA. Plasma levels and functional activity of protein S were also tested.

Anti-ProtS were found in 57 patients (31%) and 4 healthy controls (4%). Patients with thrombosis had anti-ProtS more frequently than controls (29% vs 4%, OR 9.5 [95% CI 3.07-29.3], p<0.0001). Anti-ProtS were more frequent in patients with venous thrombosis and in those with arterial thrombosis, than in controls (41% vs. 4%, OR 16.5 [95% CI 5-54], p< 0.0001 and 23% vs. 4%, OR 7 [95%CI 2.1-23.5], p=0.0008, respective-ly). Patients with prematurity, preeclampsia and intra-uterine growth restriction had anti-ProtS more frequently than the control group (36%, 47% and 44% vs. 4%; OR 13.6 [95% CI 2.8-66], p=0.003, OR 21 [95% CI 5-86], p<0.0001 and OR 19 [95% CI 4-99], p=0.0014, respectively). Plasma levels of free protein S were not statistically different between patients with and without anti-ProtS and controls (77.9% [20.7-100] vs. 83.7% [52.7-100] vs. 89% [62-101], respectively). Free protein S functional activity was no different between subgroups (105% [48-230] in anti-ProtS positive vs. 123% [95-283] in anti-ProtS negative vs. 136% [60-174] in controls).

Anti-ProtS are frequent in SLE patients with thrombosis and pregnancy morbidity. These antibodies do not interfere with free protein S in plasma since its level and/or functional activity are not impaired.