The snake venom rhodocytin has been reported to bind to integrin alpha2beta1 and glycoprotein (GP) Ibalpha on platelets, but it is also able to induce activation independent of the 2 receptors and of GPVI. Using rhodocytin affinity chromatography, we have identified a novel C-type lectin receptor, CLEC-2, in platelets that confers signaling responses to rhodocytin when expressed in a cell line. CLEC-2 has a single tyrosine residue in a YXXL motif in its cytosolic tail, which undergoes tyrosine phosphorylation upon platelet activation by rhodocytin or an antibody to CLEC-2, but not to collagen, thrombin receptor agonist peptide (TRAP), or convulxin. Tyrosine phosphorylation of CLEC-2 and other signaling proteins by rhodocytin is inhibited by the Src family kinase inhibitor PP2. Further, activation of murine platelets by rhodocytin is abolished in the absence of Syk and PLCgamma2, and partially reduced in the absence of LAT, SLP-76, and Vav1/Vav3. These findings define a novel signaling pathway in platelets whereby activation of CLEC-2 by rhodocytin leads to tyrosine phosphorylation of its cytosolic tail, binding of Syk and initiation of downstream tyrosine phosphorylation events, and activation of PLCgamma2. CLEC-2 is the first C-type lectin receptor to be found on platelets which signals through this novel pathway.

We have applied a proteomics approach to analyze signaling cascades in human platelets stimulated by thrombin receptor activating peptide (TRAP). By analyzing basal and TRAP-activated platelets using 2-dimensional gel electrophoresis (2-DE), we detected 62 differentially regulated protein features. From these, 41 could be identified by liquid chromatographycoupled tandem mass spectrometry (LC-MS/MS) and were found to derive from 31 different genes, 8 of which had not previously been reported in platelets, including the adapter downstream of tyrosine kinase 2 (Dok-2). Further studies revealed that the change in mobility of Dok-2 was brought about by tyrosine phosphorylation. Dok-2 tyrosine phosphorylation was also found to be involved in collagen receptor, glycoprotein VI (GPVI), signaling as well as in outside-in signaling through the major platelet integrin, ␣ IIb ␤ 3 . These studies also provided the first demonstration of posttranslational modification of 2 regulator of G protein signaling (RGS) proteins, RGS10 and 18. Phosphorylation of RGS18 was mapped to Ser49 by MS/MS analysis. This study provides a new approach for the identification of novel signaling molecules in activated platelets, providing new insights into the mechanisms of platelet activation and building the basis for the development of therapeutic agents for thrombotic

The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi) screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division.

Hospital at home care allows an important reduction in the costs during the index episode compared with hospital care, whilst maintaining similar outcomes with respect to cardiovascular mortality and morbidity and quality of life at 1 year follow-up.