bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple electrode aggregometry: A new device to measure platelet aggregation in whole blood
Authors: Calatzis Andreas , Penz Sandra , Losonczy Hajna , Siess Wolfgang , Tóth Orsolya
Published: December 2006

Several methods are used to analyse platelet function in whole blood. A new device to measure whole blood platelet aggregation has been developed, called multiple electrode platelet aggregometry (MEA). Our aim was to evaluate MEA in comparison with the single platelet counting (SPC) method for the measurement of platelet aggregation and platelet inhibition by aspirin or apyrase in diluted whole blood. Platelet aggregation induced by different concentrations of ADP, collagen and TRAP-6 and platelet inhibition by apyrase or aspirin were determined in citrateor hirudin-anticoagulated blood by MEA and SPC. MEA indicated that spontaneous platelet aggregation was lower, and stimulated platelet aggregation was higher in hirudin- than citrate-anticoagulated blood. In hirudin-anticoagulated, but not citrate-anticoagulated blood, spontaneous platelet aggregation measured by MEA was inhibited by apyrase. For MEA compared with SPC the dose response-curves of agonist-induced platelet aggregation in citrate- and hirudin-blood showed similar EC50 values for TRAP, and higher EC50 values for ADP (non-significant) and collagen (p<0.05). MEA and the SPC method gave similar results concerning platelet-inhibition by apyrase and aspirin. MEA was more sensitive than SPC to the inhibitory effect of aspirin in collagen-induced aggregation. In conclusion, MEA is an easy, reproducible and sensitive method for measuring spontaneous and stimulated platelet aggregation, and evaluating antiplatelet drugs in diluted whole blood. The use of hirudin as an anticoagulant is preferable to the use of citrate. MEA is a promising technique for experimental and clinical applications.
Review: Laboratory markers quantifying prothrombin activation and actions of thrombin in venous and arterial thrombosis do not accurately assess disease severity or the effectiveness of treatment
Authors: Ofosu Frederick
Published: December 2006

Thrombin is normally produced for hemostasis and physiological wound healing. Increased thrombin production in vivo, cell activation and inflammation mediated in part by thrombin are hallmarks of both arterial and venous thrombosis. Thrombin generates (pro) coagulant, mitogenic, inflammatory and anticoagulant responses by interacting witha variety of cells in vivo.Both direct and indirect thrombin inhibitors are effective drugs for preventing and treating the consequences of arterial and venous thrombosis. For these reasons, measurements of the production and activities of thrombin in vivo have the potential for gauging the extent of thromboembolism and the responses of patients to anticoagulant, antiplatelet and anti-inflammatory drugs. How-ever, a critical review of published information suggests that measurement of thrombin production and activity in patients at risk for and in patients with significant thrombosis generally does not provide information useful for clinical decision-making. This lack of clinical utility of levels of thrombin production in vivo may arise from two causes: the inability of the measurement to differentiate between physiological (hemostatic) and disease-related (pathological) sources and/or causes of thrombin production in vivo, and the inability of antithrombotic treatment modalities to permanently eliminate the stimuli that cause increased thrombin production evident in venous and arterial thrombosis.
A versatile strategy for preimplantation genetic diagnosis of haemophilia A based on F8-gene sequencing
Authors: Sánchez-García Jorge , Gallardo Dominique , Navarro Joaquima , Márquez Carmen , Gris Josep , Sánchez Maria , Altisent Carme , Vidal Francisco
Published: December 2006

Preimplantation genetic diagnosis (PGD) of hemophilia A (HA) and other X-linked diseases through sex selection implies that male embryos will be systematically discarded, even though 50% are unaffected. The objective of the present work was to develop a PGD protocol for direct mutation identification that could be applied to first polar bodies (1PBs) in several HA clinical cases. Single buccal cells from controls and patients, and 1PBs were subjected to primer extension preamplification (PEP) PCR followed by amplification of F8 gene coding and intronic flanking regions, and direct sequencing. Moreover, multiplex fluorescent amplification of four short tandem repeats was adapted to a single cell preamplification in order to rule out contamination and allele drop-out, and for confirmatory indirect diagnosis. A couple at risk of HA transmission, with a familial mutation characterized as a 41-bp duplication in exon 14 of the F8 gene, was selected for the first clinical study. After optimizing the protocol, the complete F8 gene coding sequence was obtained from single cells to demonstrate the sensitivity of our methodology although in any clinical case only the relevant region, not the whole gene, must be amplified. The woman enrolled in the first clinical case has completed the first in-vitro fertilization cycle, and seven oocytes were analyzed with concordant results by both linkage analysis and direct sequencing method. Only one oocyte, among those diagnosed as mutation free, developed to embryo at day 3. It was transferred but pregnancy was not achieved. This PGD procedure enables non-affected and noncarrier embryo selection in families with any point or smallrange mutation in the F8 gene, without the need for further custom-made modifications.
Rational humanization of the powerful antithrombotic anti-GPIbα antibody: 6B4
Authors: Fontayne Alexandre , Vanhoorelbeke Karen , Pareyn Inge , Van Rompaey Isabel , Meiring Muriel , Lamprecht Seb , Roodt Jan , Desmet Johan , Deckmyn Hans
Published: December 2006

Fab-fragments of the monoclonal antibody 6B4, raised against human glycoprotein Ibα (GPIbα), have a powerful antithrombotic effect in baboons by blocking the GPIbα binding site for von Willebrand factor (VWF), without significant prolongation of the skin bleeding time. In order to bring this antibody to the clinic,we here humanized for the first time an anti-human GPIbα by variable-domain resurfacing guided by computer modeling. First, the genes coding for the variable regions of the heavy and light chains of 6B4 were cloned and sequenced. Based on this,a three-dimensional structure of the Fv-fragment was constructed by using homology-based modeling, and with this and comparison with antibodies with known structure,”murine” putative immunogenic residues which are exposed, were changed for “human-like” residues. The humanized Fab-fragment, h6B4-Fab, was constructed in the pKaneo vector system, expressed and purified and showed in vitro an unaltered, even slightly higher binding affinity for its antigen than the murine form as determined by different ELISA set-ups and surface plasmon resonance. Finally, injection of doses of 0.1 to 1.5 mg/kg of h6B4-Fab in baboons showed that both pharmacokinetics and ex-vivo bio-activity of the molecule were to a large extent preserved.In conclusion, the method used here to humanize 6B4 by resurfacing resulted in a fully active derivative, which is now ready for further development.
Fucosylated chondroitin sulfate as a new oral antithrombotic agent
Authors: Fonseca Roberto , Mourão Paulo
Published: December 2006

Fucosylated chondroitin sulfate is a potent anticoagulant polysaccharide extracted from sea cucumber. Its anticoagulant activity is attributed to the presence of sulfated fucose branches. We have shown that intravascular injection of fucosylated chondroitin sulfate inhibits thrombus formation in a venous and an arterial shunt model in rats. Since this compound resists digestion by enzymes that cleave mammalian glycosaminoglycans, we investigated the possibility that fucosylated chondroitin sulfate might be absorbed after oral administration. In fact, after oral administration of fucosylated chondroitin sulfate to rats, we observed a dose-dependent increase in the plasma anticoagulant activity, as assessed by assays for activated partial thromboplastin time (aPTT) and thrombin time (TT) (about 3-and 5-fold, respectively) and by anti-IIa activity. Furthermore, animals receiving daily oral doses of this glycosaminoglycan showeda decrease in thrombus weight on experimental models of venous and arterial shunt thrombosis. This antithrombotic action clearly has a strong relationship with anticoagulant activity. Similar doses of heparin administered orally had no effect on the plasma anticoagulant activity or on the thrombus weight. Finally, we observed that fucosylated chondroitin sulfate given orally to rats did not modify the bleeding time. Overall, our results indicate that fucosylated chondroitin sulfate is absorbed after oral administration and could become a promising oral anticoagulant.
Plasminogen activator inhibitor-1 impairs plasminogen activationmediated vascular smooth muscle cell apoptosis
Authors: Rossignol Patrick , Anglès-Cano Eduardo , Lijnen Henri
Published: December 2006

The role of plasminogen activator inhibitor-1 (PAI-1) in vascular smooth muscle cell (VSMC) apoptosis mediated by plasminogen activation was studied with the use of aorticVSMC derived from mice with deficiency of PAI-1 (PAI-1-/-), tissue-type (t-PA-/-) or urokinase-type (u-PA-/-) plasminogen activator or from wildtype (WT) mice with corresponding genetic background. Plasminogen incubated with confluentVSMC was activated ina concentration-dependent and saturable manner for all four cell types, with maximal activation rates that were comparable for WT,u-PA-/and t-PA-/cells,but about two-fold higher for PAI-1-/cells. Plasminogen activation was impaired by addition of the lysine analogue 6-aminohexanoic acid, and by addition of t-PA and u-PA neutralizing antibodies, suggesting that it depends on binding to cell surface COOH-terminal lysine residues, and on plasminogen activator activity. Morphological alterations consistent with apoptosis were observed much earlier in PAI-1-/than in WT VSMC. Without addition of plasminogen, the apoptotic index was similar for all four cell types, whereas after incubation with physiological plasminogen concentrations, it was greater in PAI-1-/VSMC, as compared to WT, t-PA-/or u-PA-/VSMC. Furthermore, the apoptotic rate paralleled the release of plasmin. Thus, plasmin-mediated apoptosis of VSMC occurs via plasminogen activation by either t-PA or u-PA and is impaired by PAI-1.
Incidence of venous thromboembolism in first-degree relatives of patients with venous thromboembolism who have factor V Leiden
Authors: Kearon Clive , Leroyer Christophe , Mercier Bernard , Abgrall Jean , Le Gal Grégoire , Lacut Karine , Oger Emmanuel , Bressollette Luc , Ferec Claude , Lamure Michel , Mottier Dominique , Couturaud Francis ,   for the Groupe d’Etude de la Thrombose de Bretagne Occidentale (G.E.T.B.O)
Published: December 2006

The factor V Leiden (FVL) mutation, a genetic abnormality with an autosomal mode of inheritance, is associated with an increased risk of venous thromboembolism (VTE). We aimed to determine the annual incidence of VTE in first-degree relatives of patients with VTE and FVL and to identify factors in patients and the relatives that influence this incidence. In this retrospective and prospective cohort study, the incidence of objectively diagnosed first episodes of VTE was assessed in 553 first-degree relatives of 161 patients with acute VTE and FVL. The annual incidence of VTE was 0.43% (95% CI, 0.3 to 0.56) with FVL and 0.17 % (95% CI, 0.07 to 0.27) without FVL (relative risk of 2.5, 95% CI, 1.3 to 4.7). A majority (70%) of episodes of VTE were provoked, and this proportion was similar with and without FVL. A larger proportion of VTE was provoked in women (83%) that in men (33%), with the difference accounted for by pregnancy and use of oral contraceptives. The proportion of pregnancies complicated by VTE was 3.9% (95% CI, 2.0–5.8) with FVL and 1.4% (95% CI, 0.04–2.7) without FVL. FVL is associated with a two-to threefold increase in VTE in first-degree relatives of patients with VTE. No subgroup of relatives was identified who require more than routine prophylaxis because of a particularly high risk of VTE.
An investigation of the von Willebrand factor genotype in UK patients diagnosed to have type 1 von Willebrand disease
Authors: Grundy Pamela , Keeney Stephen , Lester William , Enayat Said , Guilliatt Andrea , Bowen Derrick , Pasi John , Keeling David , Hill Frank , Bolton-Maggs Paula , Hay Charles , Collins Peter , Cumming Anthony ,   on behalf of the UK Haemophilia Centre Doctors’ Organisation
Published: December 2006

Forty families diagnosed by UK centres to have type 1 VWD were recruited. Following review, six families were re-diagnosed to have type 2 VWD, one to have a platelet storage pool disorder, and one family was determined to be unaffected. Direct DNA sequencing of the promoter region and all exons and intronic boundaries of the VWF gene identified six mutations likely to be causative of VWD in index cases of nine of the 32 (28%) confirmed type 1 VWD families. These included R1205H (3614G>A) VWD Vicenza, P1648fsX45 (4944delT), D141G (422A>G) and three splice site mutations: 3108+5G>A, 7437+1G>A and 3379+1G>A. The Y1584C (4751A>G) polymorphism was present in eight additional families. No significant VWF gene mutation or polymorphism was identified in 15 of the32 type 1VWD index cases (47%). Haplotype studies were performed using a panel of VWF polymorphisms to investigate the segregation in families of VWD phenotype with the VWF gene. In 13 of the 32 families it was likely that VWD segregated with the VWF gene. In eight families (25%) VWD clearly did not segregate with the VWF gene. We suggest that mutation screening of the VWF gene has limited general utility in genetic diagnostic and family studies in type 1 VWD. If genetic studies are performed, the incomplete penetrance and variable expressivity of type 1 VWD must be taken into account. Unless linkage of VWD phenotype with the VWF gene can be clearly demonstrated, the results of any genetic family studies should be interpreted with caution.
The effect of glycaemic control on fibrin network structure of type 2 diabetic subjects
Authors: Covic Namukolo , Loots Du Toit , van der Westhuizen Francois , van Zyl Danie , Rheeder Paul , Jerling Johann , Weisel John , Pieters Marlien
Published: December 2006

Diabetic subjects have been shown to have altered fibrin network structures. One possible cause may be fibrinogen glycation resulting in altered structure/function properties. We investigated the effect of glucose control on fibrinogen glycation and fibrin network structure in type 2 diabetes. Blood samples were taken from twenty uncontrolled diabetic subjects at baseline to determine the levels of fibrinogen glycation and fibrin network structures. The subjects were then treated with insulin until blood glucose control was achieved before end blood samples were taken. Twenty age- and BMI-matched non-diabetic subjects were included as a reference group. The diabetic subjects had significantly higher mean fibrinogen glycation at baseline than the non-diabetic subjects (7.84 vs. 3.89 mol glucose / mol fibrinogen;p < 0.001).This was significantly reduced during the intervention (7.84 to 5.24 mol glucose / mol fibrinogen; p< 0.0002) in the diabetic group. Both groups had high mean fibrinogen concentrations (4.25 and 4.02 g/l, diabetic and non-diabetic subjects respectively). There was no difference in fibrinogen concentration, porosity, compaction and kinetics of clot formation between the diabetic subjects and non-diabetic subjects at baseline, nor were there any changes during the intervention despite the reduced fibrinogen glycation. Fibrin network characteristics correlated well with fibrinogen but not with any markers of glycaemic control. Improved glycaemic control resulted in decreased fibrinogen glycation but not fibrinogen concentration. It seems as though porosity, compaction and kinetics of clot formation are more related to fibrinogen concentration than fibrinogen glycation in this model.
Ethyl pyruvate exerts combined anti-inflammatory and anticoagulant effects on human monocytic cells
Authors: Bakhtiari Kamran , Dessing Mark , van ’t Veer Cornelis , Spek C. , Tanck Michael , Meijers Joost , van der Poll Tom , van Zoelen Marieke
Published: December 2006

Sepsis is characterized by a concurrent activation of inflammation and coagulation. Recently, recombinant human activated protein C was shown to decrease mortality in patients with severe sepsis presumably due to a combined anti-inflammatory and anticoagulant effect.These promising findings led to a search for other products that influence both the inflammatory and the procoagulant response to severe infection. Ethyl pyruvate (EP) was recently identified as an experimental anti-inflammatory agent during endotoxemia and sepsis. The aim of the present study was to investigate whether EP influences coagulation besides its anti-inflammatory effects. For this we investigated the effects of EP on the expression and function of tissue factor (TF), the principal initiator of coagulation activation in sepsis, in human monocytic (THP-1) cell cultures. EP dose-dependently inhibited the production of tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1α and MIP-1β by lipopolysaccharide (LPS)-stimulated THP-1 cells at mRNA and protein level, thereby confirming its anti-inflammatory properties in this in-vitro system.In addition, EP dose-dependently attenuated the increases in TF mRNA levels,TF-protein-surface expression and cell-surface-associated TF activity in LPS-stimulated THP-1 cells. These results demonstrate for the first time that EP is a compound with combined anti-inflammatory and anticoagulant effects.
Integrin binding characteristics of the disintegrin-like domain of ADAM-15
Authors: Lu Dong , Chung Kian , Xia Min , Scully Mike , Kakkar Vijay , Lu Xinjie
Published: December 2006

Although discovered as potent inhibitors of αIIbβ3-mediated platelet aggregation, snake venom disintegrins are now known to bind to other integrins according to different degrees of potency and specificity. More recently, homologues of the disintegrinlike loop have been found as a discrete domain in the ADAM family, yet the potency and specificity of each of these domains in terms of integrin binding is relatively unknown. In this present study, we have selected the disintegrin-like domain (dd) of ADAM-15 (designated as ddADAM-15), the only RGD containing domain in the ADAM family, for a structure/function study. Experimentally, the ddADAM-15 and a number of mutants in which the RGD-containing loop was substituted by cognate regions from ADAM-2, -12 and -19 were tested in terms of integrin-binding activity. For comparison with ADAM-15, an additional mutant (dddenADAM-15) was designed based upon the RGD-containing loop of snake venom dendroaspin, a disintegrin-like integrin antagonist. The results showed that ddADAM-15 is an inhibitor of platelet aggregation, though with less potency than dddenADAM-15. None of the other mutants exhibited significant inhibition of platelet aggregation. ddADAM-15 was found to have higher binding ability for α2β1 and α9β1 than the ADAM-2 derived mutant which appeared to be more selective for αVβ3 and α4β1 than either ddADAM-15 or its ADAM-19 based mutant. The integrin-binding properties of ddADAM-15 were completely abolished by point mutation within the RGD motif (R64GD→A64GD). These results suggest a more subtle contribution of this loop sequence to defining the functionality of the ADAMs compared to dendroaspin.
Performance goals for the laboratory testing of antithrombin, protein C and protein S
Authors: Haverkate Frits , Kluft Cornelis , Meijer Piet
Published: December 2006

To achieve a reliable analytical quality for both monitoring and diagnostic testing, laboratories need to fulfil the widely accepted analytical performance goals based on the biological variation of the analytes of testing. Not only is the short-term analytical performance, which regularly is assessed by internal quality control procedures, of importance, but also the long-term analytical performance. To assess the long-term analytical performance, data obtained from an external quality assessment programme can be used. In this study we have used the evaluation model designed by the ECAT Foundation for the assessment of the longterm analytical performance, including imprecision, bias and total analytical error. The model was applied to the data from 136 different laboratories for the assay of antithrombin (activity), protein C (activity and antigen) and protein S (activity, total and free antigen). The imprecision (median; range), reflected by the long-term analytical coefficient of variation (LCVA), was the lowest for antithrombin (7.6%; 2.6 – 43.8%) and the highest for protein S activity (17.2%; 4.3 – 88.6%). For bias and total error the same pattern was observed (antithrombin: 3.8%; 0.3 – 17.1% and 9.1%; 3.4 – 34.3%, respectively; protein S activity: 12.8%; 3.1 – 34.8% and 24.5%; 9.9 – 87.0%, respectively). For the majority of the laboratories (70 – 85%) the imprecision contributes considerably more to the total error than the bias. However the effect of the bias on the analytical quality is not negligible. Assays for antithrombin, protein C and protein S are mainly used for diagnostic testing. About 70 – 100% of the laboratories can fulfil the desirable performance goal for imprecision. The desirable performance goal for bias was reached by 50 – 95% of the laboratories. In all cases the highest numbers of laboratories fulfilling performance goals was obtained for the protein C variables. To improve the analytical quality in assays of antithrombin, protein C and protein S it is highly recommended that primarily imprecision (non-systematic failures) be suppressed. However the effect of the bias (systematic failures) on the analytical quality should not be neglected. A useful tool for determining the imprecision (LCVA) and bias is the long-term analytical performance evaluation model as used by the ECAT Foundation.
Thrombin generation, a function test of the haemostaticthrombotic system
Authors: Al Dieri Raed , De Smedt Erik , Béguin Suzette , Hemker H.
Published: December 2006

By the use of a fluorogenic thrombin substrate and continuous calibration of each individual sample, it is now possible to obtain a thrombin generation (TG) curve (or thrombogram) in plasma, with or without platelets, in an easy routine procedure at high throughput and with an acceptable experimental error (< 5%). Evidence is growing that the parameters of the thrombogram, and notably the area under the curve (endogenous thrombin potential, ETP), are useful in assessing bleeding-or thrombotic risk and its modification by antithrombotic-or haemostatic treatment. Available data strongly suggest that conditions (congenital, acquired, drug-induced) that increase TG all cause a thrombotic tendency and that conditions that decrease TG prevent thrombosis but, beyond a limit, cause bleeding. Diminution of TG is a common denominator of all antithrombotic treatment, including anti-platelet drugs. The thrombogram can also be used as a tool in the search for new antithrombotics and reflects the haemorrhagic or thrombotic side effects of other drugs (e.g. oral contraceptives).The thrombogram thus is a promising new approach to clinical management of bleeding and thrombotic disease as well as a tool in drug research and epidemiology. Our experience at this moment is insufficient, however, to already clearly define its limits.
Combined partial exon skipping and cryptic splice site activation as a new molecular mechanism for recessive type 1 von Willebrand disease
Authors: Gallinaro Lisa , Sartorello Francesca , Pontara Elena , Cattini Maria , Bertomoro Antonella , Bartoloni Lucia , Pagnan Antonio , Casonato Alessandra
Published: December 2006

We describe the complex picture associated with a mutated splice junction in intron 13 of von Willebrand factor (VWF) gene. The proband, characterized by a marked decrease in plasma and platelet VWF and near normal multimer organization, was classified as recessive type 1 von Willebrand disease (VWD). Genetic analysis demonstrated that he was homozygous for the 1534–3C>A mutation in the consensus sequence of the acceptor splicing site of intron 13 of the VWF gene. Platelet mRNA analysis documented three VWF transcripts: a wild type generated by the correct recognition of the mutated splice site, a smaller transcript not containing exon 14, and a longer one that, in addition to exons 13 and 14, included a 62bp fragment corresponding to the end of intron 13. The small transcript derives from the skipping of exon 14, the long one from the activation of a cryptic splice site in intron 13; both show a premature stop codon inVWF propeptide, so the probandVWF derives entirely from the correct splice site recognition. Combined incomplete exon skipping and cryptic splice site activation are first recognized in VWD. Since the 1534–3C>A mutation does not abolish the normal processing of mRNA, it is unlikely to be found in type 3VWD. This mutation therefore appears to be peculiar to type 1 VWD.
A non-anticoagulant synthetic pentasaccharide reduces inflammation in a murine model of kidney ischemia-reperfusion injury
Authors: Frank Rolf , Holscher Todd , Schabbauer Gernot , Tencati Michael , Pawlinski Rafal , Weitz Jeffrey , Mackman Nigel
Published: December 2006

Fondaparinux is a synthetic pentasaccharide that selectively inhibits factor Xa (FXa) in an antithrombin-dependent fashion.This newly developed anticoagulant is used in the prevention and treatment of venous thromboembolism. Recently, we showed that fondaparinux reduces inflammation and protects the kidney from ischemia-reperfusion (I/R) injury. However, the relative contributions of the anticoagulant and anti-inflammatory activities of fondaparinux to the observed protection is unknown.To address this, we chemically modified fondaparinux to abolish its affinity for antithrombin and analyzed the effect of this non-anti- coagulant (NAC)-pentasaccharide on binding of U937 cells to P-selectin in vitro and on inflammation in a murine model of kidney I/R injury. NAC-pentasaccharide was as effective as fondaparinux at inhibiting the binding of U937 cells to P-selectin.In addition, NAC-pentasaccharide significantly reduced IL-6 and MIP-2 expression and injury in the kidney I/R model.These findings indicate that the anti-inflammatory activity of fondaparinux can be dissociated from its anticoagulant activity and that NACpentasaccharide is protective in kidney I/R injury.
Focal arterial inflammation is augmented in mice with a deficiency of the protein C gene
Authors: Ganopolsky Jorge , Noria Francisco , Sandoval-Cooper Mayra , Ploplis Victoria , Castellino Francis
Published: December 2006

Increased risk of thrombosis, with propitious conditions for fibrin deposition, along with upregulation of inflammation, are important factors that enhance plaque formation in atherosclerosis. Evidence supporting the role of anticoagulant protein C (PC) as an inflammatory agent has emerged, supplementing its wellknown function as an anticoagulant. Thus, we sought to examine whethera PC deficiency would lead to an enhanced response to an acute arterial hyperplasic challenge. The presentation of early arterial inflammation was studied using a copper/silicone arterial cuff model of accelerated focal neointimal remodeling in mice with a heterozygous total deficiency of PC (PC+/−). Increased inflammation, cell proliferation, cell migration, fibrin elevation, and tissue necrosis were observed in the treated arteries of PC+/−mice, as compared to arteries of equally challenged age- and gender-matched WT mice. These results indicate that PC+/−mice subjected to this challenge displayed enhanced focal arterial inflammation and thrombosis, leading to larger neointimas and subsequent localized occlusion, as compared to their WT counterparts.
Effect of antiplatelet agents clopidogrel, aspirin, and cilostazol on circulating tissue factor procoagulant activity in patients with peripheral arterial disease
Authors: Vaidyula Vijender , Bagga Shagun , Jalagadugula Gauthami , Gaughan John , Wilhite Douglas , Comerota Anthony , Rao A.
Published: December 2006

Tissue factor (TF) is the physiological initiating mechanism for blood coagulation. Platelets play an important role in monocyte TF expression, thrombosis and inflammation. Aspirin, clopidogrel and cilostazol, which inhibit platelet responses by different mechanisms, are widely used in patients with arterial diseases. We tested the hypothesis that platelet-inhibiting agents inhibit the levels of circulatingTF procoagulant activity (TF-PCA) in patients with peripheral arterial disease (PAD).Twenty-six patients with lower extremity PAD, average age 65.9 ± 8.4 years (mean± SEM), were studied at baseline and following sequential twoweek treatment regimens with aspirin (325 mg daily), clopidogrel (75 mg daily) or a phosphodiesterase inhibitor cilostazol (100 mg twice daily) singly, and with each possible combination of these agents. Circulating TF-PCA in whole blood, and plasma factor VIIa, prothrombin fragment F1.2, thrombin-antithrombin complexes (TAT), and P-selectin were measured. Baseline TFPCA levels in the patients were elevated (131 ± 19 U/ml) compared to control subjects (23 ± 2, p<0.0001).TF-PCA levels declined following treatment with clopidogrel alone, and with combinations of clopidogrel with aspirin or cilostazol, with the lowest levels being with the triple-drug combination. Plasma P-selectin declined in all treatment groups. No changes were noted in plasma factorVIIa, F1.2 or TAT.In conclusion, treatment of PAD patients with antiplatelet agents decreases circulatingTF, a molecule with prothrombotic and proinflammatory effects. These findings suggest an unrecognized mechanism, beyond inhibiting aggregation responses, for the efficacy of antiplatelet drugs in patients with arterial diseases.
Palmitoylation at Cys595 is essential for PECAM-1 localisation into membrane microdomains and for efficient PECAM-1-mediated cytoprotection
Authors: Sardjono Caroline , Harbour Stacey , Yip Jana , Paddock Cathy , Tridandapani Susheela , Newman Peter , Jackson Denise
Published: December 2006

The Ig-ITIM superfamily member, PECAM-1 acts as a negative regulator of ITAM-signalling pathways in platelets involving GPVI/FcR gamma chain and FcγRIIa. This negative feedback loop involves regulation of collagen and GPVI-dependent aggregation events, platelet-thrombus-growth on immobilised collagen under flow and FcγRIIa-mediated platelet responses. In this study, we show that PECAM-1 is selectively palmitoylated involving a thioester linkage with an unpaired cysteine residue at amino acid position 595 in its cytoplasmic domain. As palmitoylation is known to target proteins to membrane microdomains, we investigated the microdomain localisation for PECAM-1 in platelets and nucleated cells. In unstimulated platelets, ∼20% of PECAM-1 is localised toTriton-insoluble microdomain fractions and it does not increase with platelet activation by collagen, collagen-related peptide, thrombin-or human-aggregated IgG. PECAM-1 is in close physical proximity with GPVI in platelet microdomains. Removal of platelet cytoskeleton prior to sucrose-density-gradient separation showed that PECAM-1 was associated with both theTriton-soluble and membrane skeleton in microdomain-associated fractions. Disruption of microdomains by membrane-cholesterol depletion resulted in loss of PECAM-1 localisation to membrane microdomains. Mutational analysis of juxtamembrane cysteine residue to alanine (C595A) of human PECAM-1 resulted in loss of palmitoylation and a sixfold decrease in association with membrane microdomains. Functionally, the palmitoylated cysteine 595 residue is required, in part, for efficient PECAM-1-mediated cytoprotection. These results show that cysteine 595 is required for constitutive association of PECAM-1 with membrane microdomains and PECAM-1-mediated cytoprotection, where it may act as a crucial regulator of signaling and apoptosis events.
Comparison of VASP-phosphorylation assay to light-transmission aggregometry in assessing inhibition of the platelet ADP P2Y12 receptor
Authors: Pampuch Agnieszka , de Gaetano Giovanni , Cerletti Chiara
Published: December 2006

There is need to improve platelet function testing to monitor the response to antiplatelet drugs. We compared flow-cytometric analysis of intraplatelet vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) to light-transmission aggregometry for the detection of drug-induced in-vitro inhibition of the platelet P2Y12 ADP receptor on 22 healthy subjects (10 males, 12 females, 28.5 ± 6.6 years). The platelet reactivity index (PRI) of VASP was calculated both from mean fluorescence intensity (MFI) and percent of fluorescence-positive platelets in the presence of PGE1 with or without ADP (10 µM). Platelet aggregation was induced by ADP (1.25, 2.5 and 5 µM). Cangrelor, a competitive inhibitor of the P2Y12 receptor, preincubated 5 minutes, induced a concentration-dependent inhibition of platelet ADP-receptor function in both tests. Indeed PRI (%) based on either MFI or percent platelets gated were highly correlated with each other (r = 0.97, p<0.0001) and with aggregation in- duced by ADP. The IC50 of cangrelor against each of the three ADP concentrations used in aggregometry increased from 5.8 ± 3.4 nM to 23.1 ± 4.0 nM and to 98 ± 25 nM, respectively. The IC50 of cangrelor based on VASP-P was within the same range (25.5 ± 7.7 nM). No correlation was observed between IC50 values of cangrelor and ADP concentrations giving 50% effect (EC50) in the absence of the drug. However, at 10 nM cangrelor seven subjects could be identified by the VASP-P assay as “low responders” to the drug (PRI> 50%), and six of them also had an aggregation response to 5 µM ADP > 50%. These six subjects showed the lowest ADP EC50 values in the absence of the drug, possibly reflecting high sensitivity of their platelet P2Y12 receptors to ADP. In conclusion, both the VASP-P assay and light-transmission aggregometry detect in a comparable way in-vitro pharmacological inhibition of the platelet P2Y12 ADP receptor and its individual variability.
Population-Based Sample Reveals Gene–Gender Interactions in Blood Pressure in White Americans
Authors: Rana Brinda K. , Insel Paul A. , Payne Samuel H. , Abel Kenneth , Beutler Ernest , Ziegler Michael G. , Schork Nicholas J. , O’Connor Daniel T.
Published: December 2006

The influence of genetic contributors, such as common single nucleotide polymorphisms, on blood pressure and essential hypertension may vary with the gender. We used the power of a large, community-based sample to probe whether gender interacts with genes in contributing to extremes of blood pressure in 611 male and 656 female age-matched white Americans within the top and bottom 5th percentiles of blood pressure among >53 000 people in a health maintenance program. This approach has >90% statistical power to detect genes contributing as little as 3% to trait (blood pressure) variation. We scored ≈60 000 genotypes in the subjects: 48 single nucleotide polymorphisms at 33 autosomal and 2 X-linked genes in adrenergic and renal pathways that regulate blood pressure. Six individual variants significantly affected blood pressure and demonstrated gene-by-gender interaction, yielding different effects of the single nucleotide polymorphism on blood pressure in males and females. In females, polymorphisms at β
1
-adrenergic receptor and α
2A
-adrenergic receptor contributed to blood pressure, whereas in men, polymorphisms at β
2
-adrenergic receptor and angiotensinogen were associated. An α
2A
-adrenergic receptor haplotype influenced blood pressure in women, whereas 2 angiotensinogen haplotypes were associated in men. We also detected gene-by-gene, gender-specific interactions (epistasis) in pathophysiological pathways. This study reveals gender-specific effects of single nucleotide polymorphisms, haplotypes, and gene-by-gene interactions that determine blood pressure in white Americans. Such genetic variants may define genetically and etiologically distinct subgroups of men and women with essential hypertension and may have implications for rational treatment selection.
Comparison of Interleukin-6 and C-Reactive Protein for the Risk of Developing Hypertension in Women
Authors: Sesso Howard D. , Wang Lu , Buring Julie E. , Ridker Paul M , Gaziano J. Michael
Published: December 2006

Although markers of systemic inflammation may have a role in the development of hypertension, supportive clinical data remain limited. We, therefore, examined interleukin (IL)-6 and C-reactive protein (CRP) in a nested case-control study of 400 women developing hypertension and an equal number of age-matched normotensive control subjects during 10 years of follow-up as part of the Women’s Health Study. All of the women initially had nonhypertensive blood pressure values and no history of diagnosis or treatment. Subjects provided self-reported risk factors, and IL-6 and CRP were measured from baseline bloods. Case subjects reported elevated systolic (≥140 mm Hg) or diastolic (≥90 mm Hg) blood pressure, newly diagnosed hypertension, or initiating antihypertensive treatment during follow-up. In crude-matched models, IL-6 and CRP quartiles were each strongly associated with hypertension risk (both
P
s for trend <0.0001). In multivariate models, the linear trends became nonsignificant, and the relative risks (95% CIs) of hypertension for IL-6 reduced to 1.00 (ref), 1.29 (0.76 to 2.19), 2.14 (1.23 to 3.73), and 1.70 (0.92 to 3.13) and for CRP were 1.00 (ref), 2.09 (1.16 to 3.76), 2.51 (1.42 to 4.44), and 2.44 (1.29 to 4.64), primarily because of confounding by body mass index. Simultaneous adjustment for IL-6 and CRP modestly attenuated both sets of relative risks, although more for IL-6. Finally, there was no effect modification by baseline blood pressure or other risk factors (all
P
s for interaction >0.05). Therefore, after multivariate adjustment and strong confounding by body mass index, IL-6 was weakly associated and CRP strongly associated with hypertension risk. In models simultaneously examining IL-6 and CRP, only CRP remained strongly associated with an increased risk of hypertension.
Association of Adrenal Steroids With Hypertension and the Metabolic Syndrome in Blacks
Authors: Kidambi Srividya , Kotchen Jane M. , Grim Clarence E. , Raff Hershel , Mao Jingnan , Singh Ravinder J. , Kotchen Theodore A.
Published: December 2006

Blacks have a high prevalence of hypertension and adrenal cortical adenomas/hyperplasia. We evaluated the hypothesis that adrenal steroids are associated with hypertension and the metabolic syndrome in blacks. Ambulatory blood pressures, anthropometric measurements, and measurements of plasma renin activity (PRA), aldosterone, fasting lipids, glucose, and insulin were obtained in 397 subjects (46% hypertensive and 50% female) after discontinuing antihypertensive and lipid-lowering medications. Hypertension was defined as average ambulatory blood pressure >130/85 mm Hg. Late-night and early morning salivary cortisol, 24-hour urine-free cortisol, and cortisone excretion were measured in a consecutive subsample of 97 subjects (40% hypertensive and 52% female). Compared with normotensive subjects, hypertensive subjects had greater waist circumference and unfavorable lipid profiles, were more insulin resistant, and had lower PRA and higher plasma aldosterone and both late-night and early morning salivary cortisol concentrations. Twenty-four–hour urine-free cortisol and cortisone did not differ. Overall, ambulatory blood pressure was positively correlated with plasma aldosterone (
r
=0.22;
P
<0.0001) and late-night salivary cortisol (
r
=0.23;
P
=0.03) and inversely correlated with PRA (
r
=−0.21;
P
<0.001). Plasma aldosterone correlated significantly with waist circumference, total cholesterol, triglycerides, insulin, and the insulin-resistance index. Based on Adult Treatment Panel III criteria, 17% of all of the subjects were classified as having the metabolic syndrome. Plasma aldosterone levels, but not PRA, were elevated in subjects with the metabolic syndrome (
P
=0.0002). The association of aldosterone with blood pressure, waist circumference, and insulin resistance suggests that aldosterone may contribute to obesity-related hypertension in blacks. In addition, we speculate that relatively high aldosterone and low PRA in these hypertensive individuals may reflect a mild variant of primary aldosteronism.
Regulation of Vascular Tone During Pregnancy
Authors: Hagedorn Kathryn A. , Cooke Christy-Lynn , Falck John R. , Mitchell Bryan F. , Davidge Sandra T.
Published: December 2006

During pregnancy, maternal vascular function is altered through mechanisms that remain unclear. Progesterone synthesis and metabolism are also increased. Progesterone metabolites are potent endogenous ligands for the pregnane X receptor (PXR), a nuclear receptor that induces the expression of hepatic cytochrome P450 enzymes. Cytochrome P450 enzymes located in the vasculature can metabolize arachidonic acid to produce epoxyeicosatrienoic acids, known vasodilators. We hypothesized that PXR is present in vascular tissue and contributes to vascular adaptations to pregnancy. PXR mRNA was detected in mouse mesenteric arteries by quantitative RT-PCR. Constrictor and relaxation responses in wildtype (PXR+/+) and PXR-deficient (PXR−/−) mice were compared by wire myography. Relative to nonpregnant controls, arteries from pregnant PXR+/+mice had reduced sensitivity to phenylephrine-induced constriction (EC50: 2.77±0.32 μmol/L versus 5.13±0.36 μmol/L;P=0.009) and enhanced maximal vasorelaxation to bradykinin (26±3% versus 44±16%;P=0.013). However, these pregnancy adaptations were absent in PXR−/−mice. We also hypothesized that PXR is activated by progesterone metabolites. Treatment of PXR+/+and PXR−/−nonpregnant mice with 5β-dihydroprogesterone for 7 days enhanced endothelium-dependent relaxation in only the PXR+/+mice, similarly to that seen in pregnancy. In treated mice, inhibition of cytochrome P450 epoxygenase activity withN-methylsulphonyl-6-(2-propargyloxyphenyl)hexanamide attenuated vasorelaxation in arteries from PXR+/+but not PXR−/−mice. We conclude that PXR contributes to the development of vascular adaptations to pregnancy, likely in response to activation by progesterone metabolites, and that PXR-dependent increases in vasorelaxation may be because of activation of cytochrome P450 epoxygenases.
Predictors of Sudden Cardiac Death and Appropriate Shock in the Comparison of Medical Therapy, Pacing, and Defibrillation in Heart Failure (COMPANION) Trial
Authors: Saxon Leslie A. , Bristow Michael R. , Boehmer John , Krueger Steven , Kass David A. , De Marco Teresa , Carson Peter , DiCarlo Lorenzo , Feldman Arthur M. , Galle Elizabeth , Ecklund Fred
Published: December 2006

Background—

The factors that determine the risk for sudden death or implantable cardioverter defibrillator therapy in patients receiving cardiac resynchronization therapy (CRT) therapies are largely unknown.

Methods and Results—

We hypothesized that clinical measures of heart failure severity and the presence of comorbid conditions would predict the risk of malignant arrhythmias in the 1520 patients enrolled in the Comparison of Medical Therapy, Pacing, and Defibrillation in Heart Failure (COMPANION) Trial. Outcomes in the CRT group after implantable cardioverter defibrillator therapy were also evaluated. The CRT-defibrillator device reduced the risk of sudden death by 56% compared with drug therapy (17 of 595 [2.9%] versus 18 of 308 [5.8%],
P
<0.02). CRT therapy was not associated with sudden death risk reduction (48 of 617 [7.8%]). Other factors associated with reduced sudden death risk were left ventricular ejection fraction >20% (HR, 0.55 [95% CI, 0.35 to 0.87];
P
=0.01), QRS duration >160 ms (HR, 0.63 [95% CI, 0.40 to 0.997];
P
=0.05), and female gender (HR, 0.56 [95% CI, 0.34 to 0.94];
P
=0.003). The risk for sudden death was increased by advanced New York Heart Association class IV heart failure (HR, 2.62 [95% CI, 1.61 to 4.26];
P
<0.011) and renal dysfunction (HR, 1.69 [95% CI, 1.06 to 2.69];
P
=0.03). An appropriate shock was experienced in 88 (15%) of the 595 CTR-D patients. In the CRT-defibrillator patients, female gender (HR, 0.54 [95 % CI, 0.31 to 0.94];
P
=0.03) and use of neurohormonal antagonists were associated with reduced risk. Class IV heart failure status increased risk. Appropriate implantable cardioverter defibrillator therapy was positively associated with risk of death or all-cause hospitalization (HR, 1.57;
P
<0.002), pump failure death or hospitalization (HR, 2.35;
P
<0.001), and sudden death (HR, 2.99;
P
=0.03), but not total mortality (HR, 1.3;
P
=0.28).

Conclusions—

In CRT candidates, sudden cardiac death risk is associated with higher New York Heart Association class and renal dysfunction. In CRT-defibrillator recipients, reduction in the risk of an appropriate shock is associated with medical therapy with neurohormonal antagonists, female gender, and New York Heart Association functional class III versus IV clinical status. Shock therapy was associated with worse outcome.
Osho International Meditation Resort (Pune, 2000s): An Anthropological Analysis of Sannyasin Therapies and The Rajneesh Legacy
Authors: D'Andrea Anthony
Published: December 2006

This article examines a multinational community of meditators and therapists, now called the Osho International Meditation Resort. Based on 6 months of ethnographic fieldwork at this resort located in Pune, India, the article focuses on cathartic therapies and active meditations, analyzed as practices of self-formation affected by relations of power and ideology. The article provides new data about the institutional and ideological contexts that currently inform sannyasin practices, revealing how the organization paradoxically promotes and controls expressive behavior. The study introduces the notion of “psychic deterritorialization” as a way to conceptualize processes of catharsis and self-derailment verified among Western sannyasins and travelers. Moreover, it examines orientalist claims about the Indian self and culture that tend to obstruct Indian nationals from participating in therapy workshops. The efficacy of expressive therapies, as exemplified by the Osho resort, depends on historically shaped tenets of “repression/expression” that forge the Western subject.
Humanistic Psychology and Social Transformation: Building the Path Toward a Livable Today and a Just Tomorrow
Authors: Diaz-Laplante Jeannette
Published: December 2006

This article discusses the relevance of humanistic psychology to the goals of social transformation within the context of globalization and poverty eradication. It discusses the applicability of basic humanistic psychology principles to the training of development workers, the strengths and limitations of Maslow's theory of human needs, and the political and economic implications of humanistic psychology. Suggestions for future directions in research, practice, and interdisciplinary collaboration are presented.
The Courage To Create Rollo May: The Courage To Be Paul Tillich
Authors: Cunningham Roger Jeff
Published: December 2006

This article considers the concepts of courage and creativity in the words of three late-20th-century theologians: Paul Tillich and Rollo May, who focused on psychology, and Reverend Al Carmines, who blended his creative self into his ministry, originating the avant-garde performing arts world of the 1960s and 1970s.