Analysis of protein networks in resting and collagen receptor (GPVI)-stimulated platelet sub-proteomes.

Proteomics approaches have made important contributions to the characterisation of platelet regulatory mechanisms. A common problem encountered with this method, however, is the masking of low-abundance (e.g. signalling) proteins in complex mixtures by highly abundant proteins. In this study, subcellular fractionation of washed human platelets either inactivated or stimulated with the glycoprotein (GP) VI collagen receptor agonist, collagen-related peptide, reduced the complexity of the platelet proteome. The majority of proteins identified by tandem mass spectrometry are involved in signalling. The effect of GPVI stimulation on levels of specific proteins in subcellular compartments was compared and analysed using in silico quantification, and protein associations were predicted using STRING (the search tool for recurring instances of neighbouring genes/proteins). Interestingly, we observed that some proteins that were previously unidentified in platelets including teneurin-1 and Van Gogh-like protein 1, translocated to the membrane upon GPVI stimulation. Newly identified proteins may be involved in GPVI signalling nodes of importance for haemostasis and thrombosis.

Regulation of platelet biology by platelet endothelial cell adhesion molecule-1.

Platelet endothelial cell adhesion molecule-1 (PECAM-1), an immunoreceptor tyrosine-based inhibitory motif containing receptor, plays diverse and apparently contradictory roles in regulating the response of platelets to stimuli; inhibiting platelet response to immunoreceptor tyrosine-based activation motif and G protein-coupled receptor signalling following stimulation with collagen, adenosine diphosphate, and thrombin, as well as enhancing integrin outside-in signalling. These dual, and opposing, roles suggest an important and complex role for PECAM-1 in orchestrating platelet response to vascular damage. Indeed, during thrombus formation, the influence of PECAM-1 on the multiple signalling pathways combines leading to a relatively large inhibitory effect on thrombus formation.

Benchmarking and setting intervention targets for key cell count parameters

Herd Companion uses routine milk‐recording records to generate twelve‐month rolling averages that indicate performance trends. This article looks at Herd Somatic Cell Count (SCC) and four other SCC‐related parameters from 252 National Milk Records (NMR) recorded herds to assess how each parameter correlates with the Herd SCC. The analysis provides evidence for the importance of targeting individual cows with high SCC recordings (>200,000 cells/ml and >500,000 cells/ml) and/or individual cows with repeatedly high SCC recordings (chronic high SCC) and/or cows that begin lactation with a high SCC recording (dry period infection) in order to achieve bulk milk Herd SCC below 200,000 cells/ml.

Wide-angle X-ray scattering study of structural parameters in non-crystalline polymers

The paper presents an analysis of WAXS (wide-angle X-ray scattering) data which aids an understanding of the structure of non-crystalline polymers. Experimental results are compared with calculations of scattering from possible models. Evidence is presented which supports the view that the chains in molten PE do not lie parallel but have a conformation in accord with the predictions of energy calculations. However, the evidence indicates that in “molten” PTFE the chains lie parallel over distances well in excess of their diameters. WAXS-based proposals are made for the conformations of a-PMMA and a-PS.

Extracting quantitative structural parameters for disordered polymers from neutron scattering data

The organization of non-crystalline polymeric materials at a local level, namely on a spatial scale between a few and 100 a, is still unclear in many respects. The determination of the local structure in terms of the configuration and conformation of the polymer chain and of the packing characteristics of the chain in the bulk material represents a challenging problem. Data from wide-angle diffraction experiments are very difficult to interpret due to the very large amount of information that they carry, that is the large number of correlations present in the diffraction patterns.We describe new approaches that permit a detailed analysis of the complex neutron diffraction patterns characterizing polymer melts and glasses. The coupling of different computer modelling strategies with neutron scattering data over a wide Q range allows the extraction of detailed quantitative information on the structural arrangements of the materials of interest. Proceeding from modelling routes as diverse as force field calculations, single-chain modelling and reverse Monte Carlo, we show the successes and pitfalls of each approach in describing model systems, which illustrate the need to attack the data analysis problem simultaneously from several fronts.

Direct Computation of Stochastic Flow in Reservoirs with Uncertain Parameters

A direct method is presented for determining the uncertainty in reservoir pressure, flow, and net present value (NPV) using the time-dependent, one phase, two- or three-dimensional equations of flow through a porous medium. The uncertainty in the solution is modelled as a probability distribution function and is computed from given statistical data for input parameters such as permeability. The method generates an expansion for the mean of the pressure about a deterministic solution to the system equations using a perturbation to the mean of the input parameters. Hierarchical equations that define approximations to the mean solution at each point and to the field covariance of the pressure are developed and solved numerically. The procedure is then used to find the statistics of the flow and the risked value of the field, defined by the NPV, for a given development scenario. This method involves only one (albeit complicated) solution of the equations and contrasts with the more usual Monte-Carlo approach where many such solutions are required. The procedure is applied easily to other physical systems modelled by linear or nonlinear partial differential equations with uncertain data.

Profiling dehydrin gene sequence and physiological parameters in drought tolerant and susceptible spring wheat cultivars.

Physiological and yield traits such as stomatal conductance (mmol m-2s-1), Leaf relative water content (RWC %) and grain yield per plant were studied in a separate experiment. Results revealed that five out of sixteen cultivars viz. Anmol, Moomal, Sarsabz, Bhitai and Pavan, appeared to be relatively more drought tolerant. Based on morphophysiological results, studies were continued to look at these cultivars for drought tolerance at molecular level. Initially, four well recognized primers for dehydrin genes (DHNs) responsible for drought induction in T. durum L., T. aestivum L. and O. sativa L. were used for profiling gene sequence of sixteen wheat cultivars. The primers amplified the DHN genes variably like Primer WDHN13 (T. aestivum L.) amplified the DHN gene in only seven cultivars whereas primer TdDHN15 (T. durum L.) amplified all the sixteen cultivars with even different DNA banding patterns some showing second weaker DNA bands. Third primer TdDHN16 (T. durum L.) has shown entirely different PCR amplification prototype, specially showing two strong DNA bands while fourth primer RAB16C (O. sativa L.) failed to amplify DHN gene in any of the cultivars. Examination of DNA sequences revealed several interesting features. First, it identified the two exon/one intron structure of this gene (complete sequences were not shown), a feature not previously described in the two database cDNA sequences available from T. aestivum L. (gi|21850). Secondly, the analysis identified several single nucleotide polymorphisms (SNPs), positions in gene sequence. Although complete gene sequence was not obtained for all the cultivars, yet there were a total of 38 variable positions in exonic (coding region) sequence, from a total gene length of 453 nucleotides. Matrix of SNP shows these 37 positions with individual sequence at positions given for each of the 14 cultivars (sequence of two cultivars was not obtained) included in this analysis. It demonstrated a considerable diversity for this gene with only three cultivars i.e. TJ-83, Marvi and TD-1 being similar to the consensus sequence. All other cultivars showed a unique combination of SNPs. In order to prove a functional link between these polymorphisms and drought tolerance in wheat, it would be necessary to conduct a more detailed study involving directed mutation of this gene and DHN gene expression.

Staphylococcal extracellular adherence protein induces platelet activation by stimulation of thiol isomerases

OBJECTIVE: Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation. The staphylococcal extracellular adherence protein (Eap) functions as an adhesin for host plasma proteins. Therefore we tested the effect of Eap on platelets. METHODS AND RESULTS: We found a strong stimulation of the platelet-surface thiol isomerases protein disulfide isomerase, endoplasmic reticulum stress proteins 57 and 72 by Eap. Eap induced thiol isomerase-dependent glycoprotein IIb/IIIa activation, granule secretion, and platelet aggregation. Treatment of platelets with thiol blockers, bacitracin, and anti-protein disulfide isomerase antibody inhibited Eap-induced platelet activation. The effect of Eap on platelets and protein disulfide isomerase activity was completely blocked by glycosaminoglycans. Inhibition by the hydrophobic probe bis(1-anilinonaphthalene 8-sulfonate) suggested the involvement of hydrophobic sites in protein disulfide isomerase and platelet activation by Eap. CONCLUSIONS: In the present study, we found an additional and yet unknown mechanism of platelet activation by a bacterial adhesin, involving stimulation of thiol isomerases. The thiol isomerase stimulatory and prothrombotic features of a microbial secreted protein are probably not restricted to S aureus and Eap. Because many microorganisms are coated with amyloidogenic proteins, it is likely that the observed mechanism is a more general one.

Temporal constraints on linear BRDF model parameters

Linear models of bidirectional reflectance distribution are useful tools for understanding the angular variability of surface reflectance as observed by medium-resolution sensors such as the Moderate Resolution Imaging Spectrometer. These models are operationally used to normalize data to common view and illumination geometries and to calculate integral quantities such as albedo. Currently, to compensate for noise in observed reflectance, these models are inverted against data collected during some temporal window for which the model parameters are assumed to be constant. Despite this, the retrieved parameters are often noisy for regions where sufficient observations are not available. This paper demonstrates the use of Lagrangian multipliers to allow arbitrarily large windows and, at the same time, produce individual parameter sets for each day even for regions where only sparse observations are available.

Rhinocetin, a venom-derived integrin-specific antagonist inhibits collagen-induced platelet and endothelial cell functions

Snaclecs are small non-enzymatic proteins present in viper venoms reported to modulate haemostasis of victims through effects on platelets, vascular endothelial and smooth muscle cells. In this study, we have isolated and functionally characterised a snaclec which we named rhinocetin from the venom of West African gaboon viper, Bitis gabonica rhinoceros. Rhinocetin was shown to comprise α and β chains with the molecular masses of 13.5 and 13kDa respectively. Sequence and immunoblot analysis of rhinocetin confirmed this to be a novel snaclec. Rhinocetin inhibited collagen-stimulated activation of human platelets in dose dependent manner, but displayed no inhibitory effects on glycoprotein VI (collagen receptor) selective agonist, CRP-XL-, ADP- or thrombin-induced platelet activation. Rhinocetin antagonised the binding of monoclonal antibodies against the α2 subunit of integrin α2β1 to platelets and coimmunoprecipitation analysis confirmed integrin α2β1 as a target for this venom protein. Rhinocetin inhibited a range of collagen induced platelet functions such as fibrinogen binding, calcium mobilisation, granule secretion, aggregation and thrombus formation. It also inhibited integrin α2β1 dependent functions of human endothelial cells. Together, our data suggest rhinocetin to be a modulator of integrin α2β1 function and thus may provide valuable insights into the role of this integrin in physiological and pathophysiological scenarios including haemostasis, thrombosis and envenomation.

Platelet endothelial cell adhesion molecule-1 signaling inhibits the activation of human platelets

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a 130-kd transmembrane glycoprotein and a member of the growing family of receptors with immunoreceptor tyrosine-based inhibitory motifs (ITIMs). PECAM-1 is expressed on platelets, certain T cells, monocytes, neutrophils, and vascular endothelial cells and is involved in a range of cellular processes, though the role of PECAM-1 in platelets is unclear. Cross-linking of PECAM-1 results in phosphorylation of the ITIM allowing the recruitment of signaling proteins that bind by way of Src-homology domain 2 interactions. Proteins that have been implicated in the negative regulation of cellular activation by ITIM-bearing receptors include the tyrosine phosphatases SHP-1 and SHP-2. Tyrosine phosphorylation of immunoreceptor tyrosine-based activatory motif (ITAM)-bearing receptors such as the collagen receptor GPVI-Fc receptor gamma-chain complex on platelets leads to activation. Increasing evidence suggests that ITIM- and ITAM-containing receptors may act antagonistically when expressed on the same cell. In this study it is demonstrated that cross-linking PECAM-1 inhibits the aggregation and secretion of platelets in response to collagen and the GPVI-selective agonist convulxin. In these experiments thrombin-mediated platelet aggregation and secretion were also reduced, albeit to a lesser degree than for collagen, suggesting that PECAM-1 function may not be restricted to the inhibition of ITAM-containing receptor pathways. PECAM-1 activation also inhibited platelet protein tyrosine phosphorylation stimulated by convulxin and thrombin; this was accompanied by inhibition of the mobilization of calcium from intracellular stores. These data suggest that PECAM-1 may play a role in the regulation of platelet function in vivo.

The negative regulation of platelet function: extending the role of the ITIM

The central role of immune-receptorlike signaling mechanisms in the activation of platelets at sites of vascular injury is well established. Of equal importance to the regulatory systems that control the activation of platelets are those systems that negatively regulate platelets and thereby prevent inappropriate platelet activation and thrombosis. Recent reports have identified a new mechanism through which this may be achieved, which involves signaling via a receptor that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM). The role of ITIMs in the control of platelet function is the subject of this review.

The integration of proteomics and systems approaches to map regulatory mechanisms underpinning platelet function.

Platelets in the circulation are triggered by vascular damage to activate, aggregate and form a thrombus that prevents excessive blood loss. Platelet activation is stringently regulated by intracellular signalling cascades, which when activated inappropriately lead to myocardial infarction and stroke. Strategies to address platelet dysfunction have included proteomics approaches which have lead to the discovery of a number of novel regulatory proteins of potential therapeutic value. Global analysis of platelet proteomes may enhance the outcome of these studies by arranging this information in a contextual manner that recapitulates established signalling complexes and predicts novel regulatory processes. Platelet signalling networks have already begun to be exploited with interrogation of protein datasets using in silico methodologies that locate functionally feasible protein clusters for subsequent biochemical validation. Characterization of these biological systems through analysis of spatial and temporal organization of component proteins is developing alongside advances in the proteomics field. This focused review highlights advances in platelet proteomics data mining approaches that complement the emerging systems biology field. We have also highlighted nucleated cell types as key examples that can inform platelet research. Therapeutic translation of these modern approaches to understanding platelet regulatory mechanisms will enable the development of novel anti-thrombotic strategies.