Clustering of glycoprotein VI (GPVI) dimers upon adhesion to collagen as a mechanism to regulate GPVI signaling in platelets.

Essentials Dimeric high-affinity collagen receptor glycoprotein VI (GPVI) is present on resting platelets. Spatio-temporal organization of platelet GPVI-dimers was evaluated using advanced microscopy. Upon platelet adhesion to collagenous substrates, GPVI-dimers coalesce to form clusters. Clustering of GPVI-dimers may increase avidity and facilitate platelet activation SUMMARY: Background Platelet glycoprotein VI (GPVI) binding to subendothelial collagen exposed upon blood vessel injury initiates thrombus formation. Dimeric GPVI has high affinity for collagen, and occurs constitutively on resting platelets. Objective To identify higher-order oligomerization (clustering) of pre-existing GPVI dimers upon interaction with collagen as a mechanism to initiate GPVI-mediated signaling. Methods GPVI was located by use of fluorophore-conjugated GPVI dimer-specific Fab (antigen-binding fragment). The tested substrates include Horm collagen I fibers, soluble collagen III, GPVI-specific collagen peptides, and fibrinogen. GPVI dimer clusters on the platelet surface interacting with these substrates were visualized with complementary imaging techniques: total internal reflection fluorescence microscopy to monitor real-time interactions, and direct stochastic optical reconstruction microscopy (dSTORM), providing relative quantification of GPVI cluster size and density. Confocal microscopy was used to locate GPVI dimer clusters, glycoprotein Ib, integrin α2 ß1 , and phosphotyrosine. Results Upon platelet adhesion to all collagenous substrates, GPVI dimers coalesced to form clusters; notably clusters formed along the fibers of Horm collagen. dSTORM revealed that GPVI density within clusters depended on the substrate, collagen III being the most effective. Clusters on fibrinogen-adhered platelets were much smaller and more numerous; whether these are pre-existing oligomers of GPVI dimers or fibrinogen-induced is not clear. Some GPVI dimer clusters colocalized with areas of phosphotyrosine, indicative of signaling activity. Integrin α2 ß1 was localized to collagen fibers close to GPVI dimer clusters. GPVI clustering depends on a dynamic actin cytoskeleton. Conclusions Platelet adhesion to collagen induces GPVI dimer clustering. GPVI clustering increases both avidity for collagen and the proximity of GPVI-associated signaling molecules, which may be crucial for the initiation and persistence of signaling.

CLEC-2-dependent activation of mouse platelets is weakly inhibited by cAMP but not by cGMP.

BACKGROUND: The activation of platelet CLEC-2 by podoplanin on lymphatic endothelial cells (LECs) has a critical role in prevention of mixing of lymphatic and blood vasculatures during embryonic development. Paradoxically, LECs release cAMP and cGMP-elevating agents, prostacyclin (PGI2 ) and nitric oxide (NO), respectively, which are powerful inhibitors of platelet activation. This raises the question of how podoplanin is able to activate CLEC-2 in the presence of the inhibitory cyclic nucleotides. OBJECTIVES: We investigated the influence of cyclic nucleotides on CLEC-2 signaling in platelets. METHODS: We used rhodocytin, CLEC-2 monoclonal antibody, LECs and recombinant podoplanin as CLEC-2 agonists on mouse platelets. The effects of the cyclic nucleotide-elevating agents PGI2 , forskolin and the NO-donor GSNO were assessed with light transmission aggregometry, flow cytometry, protein phosphorylation and fluorescent imaging of platelets on LECs. RESULTS: We show that platelet aggregation induced by CLEC-2 agonists is resistant to GSNO but inhibited by PGI2 . The effect of PGI2 is mediated through decreased phosphorylation of CLEC-2, Syk and PLCγ2. In contrast, adhesion and spreading of platelets on recombinant podoplanin, CLEC-2 antibody and LECs is not affected by PGI2 and GSNO. Consistent with this, CLEC-2 activation of Rac, which is required for platelet spreading, is not altered in the presence of PGI2 . CONCLUSIONS: The present results demonstrate that platelet adhesion and activation on CLEC-2 ligands or LECs is maintained in the presence of PGI2 and NO.

GPVI and CLEC-2 in hemostasis and vascular integrity.

SUMMARY: The glycoprotein VI (GPVI)-FcR gamma-chain complex initiates powerful activation of platelets by the subendothelial matrix proteins collagen and laminin through an immunoreceptor tyrosine-based activation motif (ITAM)-regulated signaling pathway. ITAMs are characterized by two YxxL sequences separated by 6-12 amino acids and are found associated with several classes of immunoglobulin (Ig) and C-type lectin receptors in hematopoietic cells, including Fc receptors. Cross-linking of the Ig GPVI leads to phosphorylation of two conserved tyrosines in the FcR gamma-chain ITAM by Src family tyrosine kinases, followed by binding and activation of the tandem SH2 domain-containing Syk tyrosine kinase and stimulation of a downstream signaling cascade that culminates in activation of phospholipase Cgamma2 (PLCgamma2). In contrast, the C-type lectin receptor CLEC-2 mediates powerful platelet activation through Src and Syk kinases, but regulates Syk through a novel dimerization mechanism via a single YxxL motif known as a hemITAM. CLEC-2 is a receptor for podoplanin, which is expressed at high levels in several tissues, including type 1 lung alveolar cells, lymphatic endothelial cells, kidney podocytes and some tumors, but is absent from vascular endothelial cells and platelets. In this article, we compare the mechanism of platelet activation by GPVI and CLEC-2 and consider their functional roles in hemostasis and other vascular processes, including maintenance of vascular integrity, angiogenesis and lymphogenesis.

Impact of a change in antibiotic prophylaxis on total antibiotic use in a surgical intensive care unit.

OBJECTIVE: The aim of this study was to evaluate the impact of reducing the length of antibiotic prophylaxis for cerebro spinal shunts on total antibiotic use and key resistant pathogens. METHODS: In January 2004, the use of antibiotic prophylaxis was reduced to a single shot dose with cefuroxime in an intensive care unit (ICU). Prior to this intervention, prophylaxis with second-generation cephalosporins was administered during the entire period of external cerebro spinal fluid (CSF) drainage. The effect on the antibiotic use density (AD: DDD [defined daily doses] per 1,000 patient-days[pd]) was calculated prior to (January 2002-December 2003) and following implementation of the intervention(January 2004-December 2006) by segmented regression analysis of an interrupted time series. Resistance proportions(RP) and resistance densities (RD), defined as resistant pathogen/1,000 pd of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis or E. faecium, third-generation-resistant (3GC) Escherichia coli and Klebsiella pneumoniae, and imipenem-resistant Pseudomonus aeruginosa, were compared by the Fisher's exact test before and after the intervention. RESULTS: Total antibiotic use by 147 DDD/1,000 pd decreased after the intervention when pre-operative prophylaxis was changed into single shot prophylaxis, from an estimated mean of 1,036 DDD/1,000 pd before the intervention to 887DDD/1,000 pd post-intervention. This decrease was primarily due to a significant reduction in the amount of cefuroxime used for prophylaxis. The reduction in total antibiotic consumption was sustainable, and it did not increase again during the next 36 months. The RR and RD of third-generation cephalosporin-resistant E. coli increased after January 2004, whereas the percentage of MRSA significantly decreased. CONCLUSION: Change to single shot prophylaxis along with an ongoing antibiotic stewardship program resulted in a cut-back in total antibiotic use amounting to as much as 15%. It would therefore appear that targeting interventions aimed at reducing antibiotic prophylaxis in surgical ICUs may be very worthwhile.

The novel Syk inhibitor R406 reveals mechanistic differences in the initiation of GPVI and CLEC-2 signaling in platelets.

BACKGROUND: Syk is a key mediator of signaling pathways downstream of several platelet surface receptors including GPVI/FcRgamma collagen receptor, the C-type lectin receptor CLEC-2, and integrin alphaIIbbeta3. A recent study identified the novel small molecule R406 as a selective inhibitor of Syk. OBJECTIVES: The present study evaluates the role of Syk in human platelets using the novel inhibitor R406. METHODS: Agonist-induced GPVI and CLEC-2 signaling were assessed using aggregometry, immunoprecipitation and western blotting to determine the effects of R406 on platelet activation. RESULTS: We demonstrate R406 to be a powerful inhibitor of Syk in human platelets. R406 abrogated shape change and aggregation induced by activation of GPVI and CLEC-2, and reduced platelet spreading on fibrinogen. The inhibitory effect of R406 was associated with inhibition of tyrosine phosphorylation of signaling proteins that lay downstream of Syk for all three receptors, including PLCgamma2. Strikingly, R406 markedly inhibited tyrosine phosphorylation of CLEC-2 and Syk downstream of CLEC-2 activation, whereas phosphorylation of Syk downstream of GPVI and integrin alphaIIbbeta3 was unaffected. CONCLUSIONS: The inhibitory effect of R406 provides direct evidence of a role for Syk in GPVI, CLEC-2 and integrin alphaIIbbeta3 signaling in human platelets. Further, the results demonstrate a critical role for Syk in mediating tyrosine phosphorylation of CLEC-2, suggesting a novel model in which both Src and Syk kinases regulate tyrosine phosphorylation of the C-type lectin receptor leading to platelet activation.

Regulation of actin assembly by SCAR/WAVE proteins.

Actin reorganization is a tightly regulated process that co-ordinates complex cellular events, such as cell migration, chemotaxis, phagocytosis and adhesion, but the molecular mechanisms that underlie these processes are not well understood. SCAR (suppressor of cAMP receptor)/WAVE [WASP (Wiskott-Aldrich syndrome protein)-family verprolin homology protein] proteins are members of the conserved WASP family of cytoskeletal regulators, which play a critical role in actin dynamics by triggering Arp2/3 (actin-related protein 2/3)-dependent actin nucleation. SCAR/WAVEs are thought to be regulated by a pentameric complex which also contains Abi (Abl-interactor), Nap (Nck-associated protein), PIR121 (p53-inducible mRNA 121) and HSPC300 (haematopoietic stem progenitor cell 300), but the structural organization of the complex and the contribution of its individual components to the regulation of SCAR/WAVE function remain unclear. Additional features of SCAR/WAVE regulation are highlighted by the discovery of other interactors and distinct complexes. It is likely that the combinatorial assembly of different components of SCAR/WAVE complexes will prove to be vital for their roles at the centre of dynamic actin reorganization.

Diode array digital radiography: initial clinical experience.

Initial clinical results are described for a new method of digital radiography based on high-detail self-scanning linear diode arrays which overcome many of the limitations of present film or other digital methods. The technique uses a fan-shaped x-ray beam to produce a nearly scatter-free image on a phosphor strip that is fiber-optically coupled to six self-scanning arrays of light-sensitive diodes spaced 0.025 mm, thus providing 6,144 discrete sensors across the field of view. Because these diodes have a greatly expanded dynamic range and operate at very low noise, it becomes possible to visualize small density differences or contrast below 1% both in the light and dark areas of the image. Because of the efficiency of detection and display, radiation doses can be reduced for a given information content. Our preliminary clinical studies have shown to broad application of our method in examining the chest and abdomen and in performing intravenous digital arteriography.