The P2Y12 receptor induces platelet aggregation through weak activation of the αIIbβ3 integrin – a phosphoinositide 3-kinase-dependent mechanism

AbstractHigh concentrations of adenosine-5′-diphosphate ADP are able to induce partial aggregation without shape change of P2Y1 receptor-deficient mouse platelets through activation of the P2Y12 receptor. In the present work we studied the transduction pathways selectively involved in this phenomenon. Flow cytometric analyses using R-phycoerythrin-conjugated JON/A antibody (JON/A-PE), an antibody which recognizes activated mouse αIIbβ3 integrin, revealed a low level activation of αIIbβ3 in P2Y1 receptor-deficient platelets in response to 100 μM ADP or 1 μM 2MeS-ADP. Adrenaline induced no such activation but strongly potentiated the effect of ADP in a dose-dependent manner. Global phosphorylation of 32P-labeled platelets showed that P2Y12-mediated aggregation was not accompanied by an increase in the phosphorylation of myosin light chain (P20) or pleckstrin (P47) and was not affected by the protein kinase C (PKC) inhibitor staurosporine. On the other hand, two unrelated phosphoinositide 3-kinase inhibitors, wortmannin and LY294002, inhibited this aggregation. Our results indicate that (i) the P2Y12 receptor is able to trigger a P2Y1 receptor-independent inside-out signal leading to αIIbβ3 integrin activation and platelet aggregation, (ii) ADP and adrenaline use different signaling pathways which synergize to activate the αIIbβ3 integrin, and (iii) the transduction pathway triggered by the P2Y12 receptor is independent of PKC but dependent on phosphoinositide 3-kinase

The Effects of Micro-vessel Curvature Induced Elongational Flows on Platelet Adhesion

The emerging profile of blood flow and the cross-sectional distribution of blood cells have far reaching biological consequences in various diseases and vital internal processes, such as platelet adhesion. The effects of several essential blood flow parameters, such as red blood cell free layer width, wall shear rate, and hematocrit on platelet adhesion were previously explored to great lengths in straight geometries. In the current work, the effects of channel curvature on cellular blood flow are investigated by simulating the accurate cellular movement and interaction of red blood cells and platelets in a half-arc channel for multiple wall shear rate and hematocrit values. The results show significant differences in the emerging shear rate values and distributions between the inner and outer arc of the channel curve, while the cell distributions remain predominantly uninfluenced. The simulation predictions are also compared to experimental platelet adhesion in a similar curved geometry. The inner side of the arc shows elevated platelet adhesion intensity at high wall shear rate, which correlates with increased shear rate and shear rate gradient sites in the simulation. Furthermore, since the platelet availability for binding seems uninfluenced by the curvature, these effects might influence the binding mechanics rather than the probability. The presence of elongational flows is detected in the simulations and the link to increased platelet adhesion is discussed in the experimental results

MAARS: a novel high-content acquisition software for the analysis of mitotic defects in fission yeast

Faithful segregation of chromosomes during cell division relies on multiple processes such as chromosome attachment and correct spindle positioning. Yet mitotic progression is defined by multiple parameters, which need to be quantitatively evaluated. To study the spatiotemporal control of mitotic progression, we developed a high-content analysis (HCA) approach that combines automated fluorescence microscopy with real-time quantitative image analysis and allows the unbiased acquisition of multiparametric data at the single-cell level for hundreds of cells simultaneously. The Mitotic Analysis and Recording System (MAARS) provides automatic and quantitative single-cell analysis of mitotic progression on an open-source platform. It can be used to analyze specific characteristics such as cell shape, cell size, metaphase/anaphase delays, and mitotic abnormalities including spindle mispositioning, spindle elongation defects, and chromosome segregation defects. Using this HCA approach, we were able to visualize rare and unexpected events of error correction during anaphase in wild-type or mutant cells. Our study illustrates that such an expert system of mitotic progression is able to highlight the complexity of the mechanisms required to prevent chromosome loss during cell division

Production and characterization of Orpinomyces mutant xylanases with improved temperature and pH stabilities

The error-prone PCR technique has been widely used in order to obtain thermostable enzymes more suitable for industrial conditions. The Orpinomyces xynA mutant library allowed the selection of four thermostable mutants (M1-M4). Molecular dynamics (MD) predicted an N-terminal tail as being a destabilizing structural region and allowed further enhancing of the mutant xylanases thermostability. Thus, removal of the 27 amino acid residues enabled an increase in the enzyme half-life values (t1/2). However, besides the improved thermostability, the large enzyme production and high catalytic performance are also relevant for the biotechnological application of enzymes. During the mutant enzymes production in E. coli, the IPTG induction protocol allowed high expression levels of soluble and active xylanases. The mutant xylanases without the 27 amino acid residues showed improved thermostability and the shorter versions of M2 and M4 (named as SM2 and SM4) also presented a good performance in more extreme pH conditions

Treatment of Low-flow Vascular Malformations by Ultrasound-guided Sclerotherapy with Polidocanol Foam: 24 Cases and Literature Review

AbstractObjectivesTreatment by sclerotherapy has been suggested as a first-line treatment of low-flow vascular malformations. This study reports our experience in treating low-flow vascular malformations by ultrasound-guided sclerosis with polidocanol foam at the Vascular Medicine Department in Grenoble, France.DesignRetrospective single-centre consecutive series.Materials and methodsBetween January 2006 and December 2009, we analysed the complete records of patients with symptomatic low-flow vascular malformations of venous, lymphatic or complex type (Klippel–Trenaunay syndrome, KTS) treated by ultrasound-guided sclerosis. The therapeutic indication was always validated by the Consultative Committee for vascular malformations of the University Hospital of Grenoble. All vascular malformations were classified according to the Hamburg Classification. The sclerosing agent was polidocanol used as foam.ResultsA total of 24 patients between 7 and 78 years were treated (19 venous malformations, three KTSs and two venous-lymphatic malformations). The concentrations of polidocanol used ranged from 0.25% to 3%. The average number of sessions was 2.3 (1–16). After a median follow-up at 5 months after the last session, 23 out of 24 patients reported a decrease in pain; in nine cases (37.5%), over 50% reduction in size was observed, and in 14 cases (58.3%), a reduction of less than 50% of the original size was obtained. Two minor side effects were reported.ConclusionsTreatment by ultrasound-guided sclerosis using polidocanol foam seems to be well tolerated and can improve the symptoms of low-flow malformations without the risks of more aggressive sclerosing agents, such as ethanol

The use of tire rubber in the production of high-performance concrete

The advances in concrete technology lead to the search for alternative materials that provide improvements in concrete properties while at the same time collaborating with sustainable practices in construction. In this study, the influence of the incorporation of waste tire rubber on the mechanical properties of high-performance concrete was discussed. The waste rubber from the tire retreading process was used in partial substitution of the fine aggregate (sand) in the percentages of 7.5%, 15% and 30% with respect to the mass of the sand. For the characterization of the concrete, the following tests were carried out: water absorption, void index, specific density, compressive strength, flexural tensile strength, modulus of elasticity and microscopy analysis. The incorporation of rubber as aggregate in high-performance concrete proved to be promising for the production of a structural concrete with special characteristics, besides collaborating with the proper disposal of waste tires651110114sem informaçã

Traumatic vessel injuries initiating hemostasis generate high shear conditions

Blood flow is a major regulator of hemostasis and arterial thrombosis. The current view is that low and intermediate flows occur in intact healthy vessels, whereas high shear levels (>2000 s−1) are reached in stenosed arteries, notably during thrombosis. To date, the shear rates occurring at the edge of a lesion in an otherwise healthy vessel are nevertheless unknown. The aim of this work was to measure the shear rates prevailing in wounds in a context relevant to hemostasis. Three models of vessel puncture and transection were developed and characterized for a study that was implemented in mice and humans. Doppler probe measurements supplemented by a computational model revealed that shear rates at the edge of a wound reached high values, with medians of 22 000 s−1, 25 000 s−1, and 7000 s−1 after puncture of the murine carotid artery, aorta, or saphenous vein, respectively. Similar shear levels were observed after transection of the mouse spermatic artery. These results were confirmed in a human venous puncture model, where shear rates in a catheter implanted in the cubital vein reached 2000 to 27 000 s−1. In all models, the high shear conditions were accompanied by elevated levels of elongational flow exceeding 1000 s−1. In the puncture model, the shear rates decreased steeply with increasing injury size. This phenomenon could be explained by the low hydrodynamic resistance of the injuries as compared with that of the downstream vessel network. These findings show that high shear rates (>3000 s−1) are relevant to hemostasis and not exclusive to arterial thrombosis

Platelet FcγRIIA-induced serotonin release exacerbates the severity of Transfusion-Related Acute Lung Injury in mice

Transfusion-related acute lung injury (TRALI) remains a major cause of transfusion-related fatalities. The mechanism of human antibody-mediated TRALI, especially the involvement of the Fcγ receptors, is not clearly established. Contrary to mice, human platelets are unique in their expression of the FcγRIIA/CD32A receptor, suggesting that our understanding of the pathogenesis of antibody-mediated TRALI is partial, as the current murine models incompletely recapitulate the human immunology. We evaluated the role of FcγRIIA/CD32A in TRALI using a humanized mouse model expressing the FcγRIIA/CD32A receptor. When challenged with a recombinant chimeric human immunoglobulin G1/mouse anti–major histocompatibility complex class I monoclonal antibody, these mice exhibited exacerbated alveolar edema and higher mortality compared with wild-type (WT) mice. Unlike in WT mice, monocytes/macrophages in CD32A(+) mice were accessory for TRALI initiation, indicating the decisive contribution of another cell type. Platelet activation was dramatically increased in CD32A(+) animals, resulting in their increased consumption and massive release of their granule contents. Platelet depletion prevented the exacerbation of TRALI in CD32A(+) mice but did not affect TRALI in WT animals. By blocking platelet serotonin uptake with fluoxetine, we showed that the severity of TRALI in CD32A(+) mice resulted from the serotonin released by the activated platelets. Furthermore, inhibition of 5-hydroxytryptamine 2A serotonin receptor with sarpogrelate, before or after the induction of TRALI, abolished the aggravation of lung edema in CD32A(+) mice. Our findings show that platelet FcγRIIA/CD32A activation exacerbates antibody-mediated TRALI and provide a rationale for designing prophylactic and therapeutic strategies targeting the serotonin pathway to attenuate TRALI in patients

Severe bleeding and absent ADP-induced platelet aggregation associated with inherited combined CalDAG-GEFI and P2Y12 deficiencies

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