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“BackgroundProtein kinase C (PKC) is a major regulator of platelet function and secretion. The underlying molecular pathway from PKC to secretion, however, is poorly understood. By a proteomics screen we identified the guanine nucleotide exchange factor cytohesin-2 as a candidate PKC substrate. ObjectivesWe aimed to validate cytohesin-2 as a PKC

substrate in platelets and to determine its role in granule secretion and other platelet responses. Methods and resultsImmunoprecipitation was performed with a phosphoserine PKC substrate antibody followed by mass spectrometry, leading to the identification of cytohesin-2. By western blotting we showed that different agonists induced cytohesin-2 phosphorylation by PKC. Protein function MK-1775 in vivo was investigated using a pharmacological approach. The cytohesin inhibitor SecinH3 significantly enhanced platelet dense granule secretion SB203580 chemical structure and aggregation, as measured by lumi-aggregometry. Flow cytometry data indicate that -granule release and integrin (IIb3) activation were not affected by cytohesin-2 inhibition. Lysosome secretion was assessed by a colorimetric assay and was also unchanged. As shown by western blotting, ARF6 interacted with cytohesin-2 and was present in an active GTP-bound form under basal conditions. Upon platelet stimulation, this interaction was largely lost and ARF6 activation

decreased, both of which could be rescued by PKC inhibition. ConclusionsCytohesin-2 constitutively suppresses platelet dense granule secretion and aggregation by keeping ARF6 in a GTP-bound state. PKC-mediated phosphorylation of cytohesin-2 PFTα relieves this inhibitory effect, thereby promoting platelet secretion and aggregation.”
“AimColorectal cancer

(CRC) screening programmes detect early cancers but unfortunately have limited sensitivity and specificity. Mass spectrometry-based determination of serum peptide and protein profiles provides a new approach for improved screening. MethodSerum samples were obtained from 126 CRC patients before treatment and 277 control individuals. An additional group of samples from 50 CRC patients and 82 controls was used for validation. Peptide and protein enrichments were carried out using reverse-phase C18 and weak-cation exchange magnetic beads in an automated solid-phase extraction and spotting procedure. Profiles were acquired on a matrix-assisted laser desorption/ionization time-of-flight system. Discriminant rules using logistic regression were calibrated for the peptide and protein signatures separately, followed by combining the classifications to obtain double cross-validated predicted class probabilities. Results were validated on an identical patient set. ResultsA discriminative power was found for patients with CRC representative for all histopathological stages compared with controls with an area under the curve of 0.95 in the test set (0.