CD40 signalling in platelet function

Le CD40 est un membre de la famille des récepteurs du facteur de nécrose tumorale ("Tumour necrosis factor", TNF), initialement identifié sur des cellules de carcinome de la vessie. L'interaction du CD40 avec son ligand (CD40L) est d'une importance cruciale pour le développement des cellules B et de la commutation d'isotype au cours de la réponse immunitaire acquise. L'expression du complexe CD40/CD40L était initialement cru d'être limiter aux cellules du système immunitaire, mais aujourd'hui il est bien connu que ce complexe est également exprimé sur les cellules du système circulatoire et vasculaire, et est impliqué dans diverses réactions inflammatoires; de sorte que le CD40L est maintenant considéré comme une molécule thrombo-inflammatoire prédictive des événements cardiovasculaires. Les plaquettes expriment constitutivement le CD40, alors que le CD40L n'est exprimé que suite à leur l'activation. Il est ensuite clivé en sa forme soluble (sCD40L) qui représente la majorité du sCD40L en circulation. Il fut démontré que le sCD40L influence l'activation plaquettaire mais son effet exact sur la fonction plaquettaire, ainsi que les mécanismes cellulaires et moléculaires sous-jacents à son action demeurent inconnus. Ainsi, ce projet a été entrepris dans le but d’adresser les objectifs spécifiques suivants: 1) évaluer les effets in vitro du sCD40L sur l'activation et l'agrégation plaquettaire; 2) identifier les récepteurs plaquettaires impliqués dans l’action du sCD40L; 3) élucider les voies signalétiques intracellulaires induits par le sCD40L; 4) évaluer les effets du sCD40L sur la formation de thrombus in vivo. Nous avons trouvé que le sCD40L augmente fortement l'activation et l'agrégation des plaquettes en réponse à de faibles concentrations d'agonistes. Les plaquettes humaines traitées avec une forme mutante du sCD40L qui n'interagit pas avec le CD40, et les plaquettes de souris déficientes en CD40 ne furent pas en mesure d'induire de telles réponses, indiquant que le récepteur principal du sCD40L au niveau des plaquettes est le CD40. En plus, nous avons identifié la présence de plusieurs membres de la famille du facteur associé du récepteur du TNF ("TNF receptor-associated factor", TRAF) dans les plaquettes et nous avons montré que seulement le TRAF2 s'associe avec le CD40 suite à la stimulation par le sCD40L. Nos résultats indiquent aussi que le sCD40L agisse sur les plaquettes au repos par l'entremise de deux voies signalétiques distinctes. La première voie implique l'activation de la petite GTPase Rac1 et de sa cible en aval, soit la protéine kinase p38 activée par le mitogène ("p38 mitogen-activated protein kinase", p38 MAPK ), menant au changement de forme plaquettaire et à la polymérisation de l'actine; alors que la deuxième voie implique l'activation de la cascade signalétique du NF-kB. Par ailleurs, à la suite d'une lésion artérielle induite par le chlorure de fer, le sCD40L exacerbe la formation de thrombus et l'infiltration leucocytaire au sein du thrombus dans les souris du type sauvage, mais pas chez les souris déficientes en CD40. En conclusion, ce projet a permis d'identifier pour la première fois deux voies signalétiques distinctes en aval du CD40 plaquettaire et a permis d'établir leur implication dans l'activation et l'agrégation plaquettaire en réponse au sCD40L. De manière plus importante, ce projet nous a permis d'établir un lien direct entre les niveaux élevés du sCD40L circulant et la formation de thrombus in vivo, tout en soulignant l'importance du CD40 dans ce processus. Par conséquent, l'axe CD40/CD40L joue un rôle important dans l'activation des plaquettes, les prédisposant à une thrombose accrue en réponse à une lésion vasculaire. Ces résultats peuvent expliquer en partie la corrélation entre les taux circulants élevés du sCD40L et l'incidence des maladies cardiovasculaires.

Behavioral Measures and their Correlation with IPM Iteration Counts on Semi-Definite Programming Problems

We study four measures of problem instance behavior that might account for the observed differences in interior-point method (IPM) iterations when these methods are used to solve semidefinite programming (SDP) problem instances: (i) an aggregate geometry measure related to the primal and dual feasible regions (aspect ratios) and norms of the optimal solutions, (ii) the (Renegar-) condition measure C(d) of the data instance, (iii) a measure of the near-absence of strict complementarity of the optimal solution, and (iv) the level of degeneracy of the optimal solution. We compute these measures for the SDPLIB suite problem instances and measure the correlation between these measures and IPM iteration counts (solved using the software SDPT3) when the measures have finite values. Our conclusions are roughly as follows: the aggregate geometry measure is highly correlated with IPM iterations (CORR = 0.896), and is a very good predictor of IPM iterations, particularly for problem instances with solutions of small norm and aspect ratio. The condition measure C(d) is also correlated with IPM iterations, but less so than the aggregate geometry measure (CORR = 0.630). The near-absence of strict complementarity is weakly correlated with IPM iterations (CORR = 0.423). The level of degeneracy of the optimal solution is essentially uncorrelated with IPM iterations.

Bayesian tools for zero counts in compositional data

The log-ratio methodology makes available powerful tools for analyzing compositional data. Nevertheless, the use of this methodology is only possible for those data sets without null values. Consequently, in those data sets where the zeros are present, a previous treatment becomes necessary. Last advances in the treatment of compositional zeros have been centered especially in the zeros of structural nature and in the rounded zeros. These tools do not contemplate the particular case of count compositional data sets with null values. In this work we deal with \count zeros" and we introduce a treatment based on a mixed Bayesian-multiplicative estimation. We use the Dirichlet probability distribution as a prior and we estimate the posterior probabilities. Then we apply a multiplicative modi¯cation for the non-zero values. We present a case study where this new methodology is applied. Key words: count data, multiplicative replacement, composition, log-ratio analysis

What counts as response and intervention in RTI? : A sociocultural an??lisis

Resumen tomado de la publicaci??n

A Field Evaluation of the Time-of-Detection Method to Estimate Population Size and Density for Aural Avian Point Counts

The time-of-detection method for aural avian point counts is a new method of estimating abundance, allowing for uncertain probability of detection. The method has been specifically designed to allow for variation in singing rates of birds. It involves dividing the time interval of the point count into several subintervals and recording the detection history of the subintervals when each bird sings. The method can be viewed as generating data equivalent to closed capture–recapture information. The method is different from the distance and multiple-observer methods in that it is not required that all the birds sing during the point count. As this method is new and there is some concern as to how well individual birds can be followed, we carried out a field test of the method using simulated known populations of singing birds, using a laptop computer to send signals to audio stations distributed around a point. The system mimics actual aural avian point counts, but also allows us to know the size and spatial distribution of the populations we are sampling. Fifty 8-min point counts (broken into four 2-min intervals) using eight species of birds were simulated. Singing rate of an individual bird of a species was simulated following a Markovian process (singing bouts followed by periods of silence), which we felt was more realistic than a truly random process. The main emphasis of our paper is to compare results from species singing at (high and low) homogenous rates per interval with those singing at (high and low) heterogeneous rates. Population size was estimated accurately for the species simulated, with a high homogeneous probability of singing. Populations of simulated species with lower but homogeneous singing probabilities were somewhat underestimated. Populations of species simulated with heterogeneous singing probabilities were substantially underestimated. Underestimation was caused by both the very low detection probabilities of all distant individuals and by individuals with low singing rates also having very low detection probabilities.

Data assimilation: From photon counts to Earth System forecasts

Data assimilation – the set of techniques whereby information from observing systems and models is combined optimally – is rapidly becoming prominent in endeavours to exploit Earth Observation for Earth sciences, including climate prediction. This paper explains the broad principles of data assimilation, outlining different approaches (optimal interpolation, three-dimensional and four-dimensional variational methods, the Kalman Filter), together with the approximations that are often necessary to make them practicable. After pointing out a variety of benefits of data assimilation, the paper then outlines some practical applications of the exploitation of Earth Observation by data assimilation in the areas of operational oceanography, chemical weather forecasting and carbon cycle modelling. Finally, some challenges for the future are noted.

Platelet-mediated metabolism of the common dietary flavonoid, quercetin.

BACKGROUND: Flavonoid metabolites remain in blood for periods of time potentially long enough to allow interactions with cellular components of this tissue. It is well-established that flavonoids are metabolised within the intestine and liver into methylated, sulphated and glucuronidated counterparts, which inhibit platelet function. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate evidence suggesting platelets which contain metabolic enzymes, as an alternative location for flavonoid metabolism. Quercetin and a plasma metabolite of this compound, 4'-O-methyl quercetin (tamarixetin) were shown to gain access to the cytosolic compartment of platelets, using confocal microscopy. High performance liquid chromatography (HPLC) and mass spectrometry (MS) showed that quercetin was transformed into a compound with a mass identical to tamarixetin, suggesting that the flavonoid was methylated by catechol-O-methyl transferase (COMT) within platelets. CONCLUSIONS/SIGNIFICANCE: Platelets potentially mediate a third phase of flavonoid metabolism, which may impact on the regulation of the function of these cells by metabolites of these dietary compounds.

Tweaking the gain on platelet regulation: The tachykinin connection

Soluble factors such as ADP and thromboxane (TX) A(2) that are secreted or released by platelets at sites of tissue injury, mediate autocrine and paracrine regulation of platelet function, resulting in rapid localised thrombus formation. The suppression of platelet function, particularly through targeting such secondary regulatory mechanisms, that serve to 'fine-tune' the platelet response, has proven effective in the prevention of inappropriate platelet activation that results in thrombosis. The most commonly used anti-platelet approaches (ADP receptor antagonism or inhibition of TXA(2) synthesis), however, lack efficacy in many patients, suggesting the existence of additional uncharacterised mechanisms for the regulation of platelet function. Recent data, which form a focus of this review, have identified peripheral tachykinin peptide family members, such as substance P and the newly identified endokinins, as physiologically important positive feedback regulators of platelet function. The actions of tachykinins that are released from platelets during activation are mediated by the neurokinin-1 receptor. Initial analysis of the role of this receptor in platelet thrombus formation, and thrombosis in the mouse, indicate this to be a promising new target for the development of anti-thrombotic drugs. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

A functional genomics approach reveals novel quantitative trait loci associated with platelet signalling pathways

Platelet response to activation varies widely between individuals but shows interindividual consistency and strong heritability. The genetic basis of this variation has not been properly explored. We therefore systematically measured the effect on function of sequence variation in 97 candidate genes in the collagen and adenosine-diphosphate (ADP) signaling pathways. Resequencing of the genes in 48 European DNA samples nearly doubled the number of known single nucleotide polymorphisms (SNPs) and informed the selection of 1327 SNPs for genotyping in 500 healthy Northern European subjects with known platelet responses to collagen-related peptide (CRP-XL) and ADP. This identified 17 novel associations with platelet function (P < .005) accounting for approximately 46% of the variation in response. Further investigations with platelets of known genotype explored the mechanisms behind some of the associations. SNPs in PEAR1 associated with increased platelet response to CRP-XL and increased PEAR1 protein expression after platelet degranulation. The minor allele of a 3' untranslated region (UTR) SNP (rs2769668) in VAV3 was associated with higher protein expression (P = .03) and increased P-selectin exposure after ADP activation (P = .004). Furthermore the minor allele of the intronic SNP rs17786144 in ITPR1 modified Ca2+ levels after activation with ADP (P < .004). These data provide novel insights into key hubs within platelet signaling networks.

PECAM-1 expression and activity negatively regulate multiple platelet signaling pathways

Platelet endothelial cell adhesion molecule-1 (PECAM-1) inhibits platelet response to collagen and may also inhibit two other major platelet agonists ADP and thrombin although this has been less well explored. We hypothesized that the combined effect of inhibiting these three platelet activating pathways may act to significantly inhibit thrombus formation. We demonstrate a negative relationship between PECAM-1 surface expression and platelet response to cross-linked collagen related peptide (CRP-XL) and ADP, and an inhibitory effect of PECAM-1 clustering on platelet response to CRP-XL, ADP and thrombin. This combined inhibition of multiple signaling pathways results in a marked reduction in thrombus formation. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Future innovations in anti-platelet therapies

Platelets have long been recognized to be of central importance in haemostasis, but their participation in pathological conditions such as thrombosis, atherosclerosis and inflammation is now also well established. The platelet has therefore become a key target in therapies to combat cardiovascular disease. Anti-platelet therapies are used widely, but current approaches lack efficacy in a proportion of patients, and are associated with side effects including problem bleeding. In the last decade, substantial progress has been made in understanding the regulation of platelet function, including the characterization of new ligands, platelet-specific receptors and cell signalling pathways. It is anticipated this progress will impact positively on the future innovations towards more effective and safer anti-platelet agents. In this review, the mechanisms of platelet regulation and current anti-platelet therapies are introduced, and strong, and some more speculative, potential candidate target molecules for future anti-platelet drug development are discussed.

The neurokinin 1 receptor: a potential new target for anti-platelet therapy?

The important role of platelets in the development of arterial thrombosis and cardiovascular disease is well established. Current treatments for arterial thrombosis include anti-platelet agents such as aspirin, thienopyridines and glycoprotein IIb-IIIa inhibitors. Despite these drugs being effective there remains a substantial unmet clinical demand for more effective therapeutic approaches, which may reflect the existence of alternative underlying regulatory mechanisms to those already targeted. Recent publications have demonstrated a key role for tachykinins in the positive feedback regulation of platelet aggregation and thrombus formation. The pro-thrombotic effects of tachykinins on platelets are mediated through the neurokinin 1 receptor, which may therefore offer a novel therapeutic drug target in the prevention and the treatment of arterial thrombosis.

Peripheral tachykinins and the neurokinin receptor NK1 are required for platelet thrombus formation

Platelets play an important role in hemostasis, with inappropriate platelet activation being a major contributor to debilitating and often fatal thrombosis by causing myocardial infarction and stroke. Although current antithrombotic treatment is generally well tolerated and effective, many patients still experience cardiovascular problems, which may reflect the existence of alternative underlying regulatory mechanisms in platelets to those targeted by existing drugs. In this study, we define a role for peripherally distributed members of the tachykinin family of peptides, namely substance P and the newly discovered endokinins A and B that are present in platelets, in the activation of platelet function and thrombus formation. We have reported previously that the preferred pharmacologically characterized receptor for these peptides, the NK1 receptor, is present on platelets. Inhibition or deficiency of the NK1 receptor, or SP agonist activity, resulted in substantially reduced thrombus formation in vitro under arterial flow conditions, increased bleeding time in mice, and a decrease in experimentally induced thromboembolism. Inhibition of the NK1 receptor may therefore provide benefit in patients vulnerable to thrombosis and may offer an alternative therapeutic target.