TNF Receptor-Associated Factor 4 (TRAF4) is a novel binding partner of glycoprotein Ib and glycoprotein VI in human platelets.

Background: Reactive oxygen species generation is one consequence of ligand engagement of platelet glycoprotein (GP) receptors GPIb-IX-V and GPVI, which bind VWF/collagen and initiate thrombosis at arterial shear, however the precise molecular mechanism coupling redox pathway activation to engagement of these receptors is unknown. Objective: The objective of this study was to identify novel binding partners for GPIb-IX-V and GPVI that could provide a potential link between redox pathways and early platelet signalling events. Methods and Results: Using protein array analysis and affinity-binding assays, we demonstrated that the orphan TNF receptor-associated factor (TRAF) family member, TRAF4, selectively binds cytoplasmic sequences of GPIbβ and GPVI. TRAF4, p47(phox) (of the NADPH oxidase (Nox2) enzyme complex), and other redox relevant signalling proteins such as Hic-5, co-immunoprecipitate with GPIb/GPVI from human platelet lysates whilst MBP-TRAF4 or MBP-p47(phox) fusion proteins specifically pull-down GPIb/GPVI. GPIb- or GPVI-selective agonists induce phosphorylation of the TRAF4-associated proteins, Hic-5 and Pyk2, with phosphorylation attenuated by Nox2 inhibition. Conclusion: These results describe the first direct association of TRAF4 with a receptor, and identify a novel binding partner for GPIb-IX-V and GPVI, providing a potential link between these platelet receptors and downstream TRAF4/Nox2-dependent redox pathways

Stimulation of platelet glycoprotein IIb-IIIa (αIIbβ3-integrin) functional activity by a monoclonal antibody to the N-terminal region of glycoprotein IIIa

AbstractPlatelet glycoprotein (GP) IIb-IIIa complex (αIIbβ3-integrin) changes its conformation upon platelet activation that results in binding of RGD-containing ligands and expression of ligand-induced binding site (LIBS) neoepitopes. Anti-GIIb-IIIa monoclonal antibody (monAB) CRC54 bound to ≤10% of GPIIb-IIIa on resting platelets but binding was enhanced by the occupation of GPIIb-IIIa with RGDS peptide and by platelet activation indicating that CRC54 is directed against LIBS epitope. The epitope was located within the first 100 N-terminal residues of GPIIIa and differed from other LIBS epitopes. CRC54 as well as its Fab fragments were able to induce platelet aggregation. CRC54 also stimulated interaction of GPIIb-IIIa with its ligands (fibrinogen and fibronectin) and conformation-dependent antibodies. The results indicated that changes of GPIIb-IIIa conformation, binding of ligands and platelet aggregation could be stimulated via interaction of anti-LIBS antibody with the N-terminal part of GPIIIa

Platelet adhesion and degranulation induce pro-survival and pro-angiogenic signalling in ovarian cancer cells

Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells

Chemistry of hot springs along the Eastern Lau Spreading Center

Author Posting. © The Author(s), 2010. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Geochimica et Cosmochimica Acta 75 (2011): 1013-1038, doi:10.1016/j.gca.2010.12.008.The Eastern Lau Spreading Center (ELSC) is the southernmost part of the back-arc spreading axis in the Lau Basin, west of the Tonga trench and the active Tofua volcanic arc. Over its 397-km length it exhibits large and systematic changes in spreading rate, magmatic/tectonic processes, and proximity to the volcanic arc. In 2005 we collected 81 samples of vent water from six hydrothermal fields along the ELSC. The chemistry of these waters varies both within and between vent fields, in response to changes in substrate composition, temperature and pressure, pH, water/rock ratio, and input from magmatic gases and subducted sediment. Hot-spring temperatures range from 229º to 363ºC at the five northernmost fields, with a general decrease to the south that is reversed at the Mariner field. The southernmost field, Vai Lili, emitted water at up to 334°C in 1989 but had a maximum venting temperature of only 121ºC in 2005, due to waning activity and admixture of bottom seawater into the subseafloor plumbing system. Chloride varies both within fields and from one field to another, from a low of 528 mmol/kg to a high of 656 mmol/kg, and may be enriched by phase separation and/or leaching of Cl from the rock. Concentrations of the soluble elements K, Rb, Cs, and B likewise increase southward as the volcanic substrate becomes more silica-rich, especially on the Valu Fa Ridge. Iodine and δ7Li increase southward, and δ11B decreases as B increases, apparently in response to increased input from subducted sediment as the arc is approached. Species that decrease southward as temperature falls are Si, H2S, Li, Na/Cl, Fe, Mn, and 87Sr/86Sr, whereas pH, alkalinity, Ca, and Sr increase. Oxygen isotopes indicate a higher water/rock ratio in the three systems on Valu Fa Ridge, consistent with higher porosity in more felsic volcanic rocks. Vent waters at the Mariner vent field on the Valu Fa Ridge are significantly hotter, more acid and metal-rich, less saline, and richer in dissolved gases and other volatiles, including H2S, CO2, and F, than the other vent fields, consistent with input of magmatic gases. The large variations in geologic and geophysical parameters produced by back-arc spreading along the ELSC, which exceed those along mid-ocean ridge spreading axes, produce similar large variations in the composition of vent waters, and thus provide new insights into the processes that control the chemistry of submarine hot springs.We thank the U.S. National Science Foundation and its RIDGE 2000 Program for funding this study via grants OCE0241826 (to MJM), OCE0242902 (to PJM), OCE0241796 (to JSS, MKT), and OCE0242088 (to CGW), as well as the Deep Ocean Exploration Institute at WHOI (to GP, ER)

Constitutive dimerization of glycoprotein VI (GPVI) in resting platelets is essential for binding to collagen and activation in flowing blood

The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (K(d)) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triple-helical (GPO)(10)-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ~29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ~39 and ~44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation

Improved Imputation of Common and Uncommon Single Nucleotide Polymorphisms (SNPs) with a New Reference Set

Statistical imputation of genotype data is an important technique for analysis of genome-wide association studies (GWAS). We have built a reference dataset to improve imputation accuracy for studies of individuals of primarily European descent using genotype data from the Hap1, Omni1, and Omni2.5 human SNP arrays (Illumina). Our dataset contains 2.5-3.1 million variants for 930 European, 157 Asian, and 162 African/African-American individuals. Imputation accuracy of European data from Hap660 or OmniExpress array content, measured by the proportion of variants imputed with R^2^>0.8, improved by 34%, 23% and 12% for variants with MAF of 3%, 5% and 10%, respectively, compared to imputation using publicly available data from 1,000 Genomes and International HapMap projects. The improved accuracy with the use of the new dataset could increase the power for GWAS by as much as 8% relative to genotyping all variants. This reference dataset is available to the scientific community through the NCBI dbGaP portal. Future versions will include additional genotype data as well as non-European populations

The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin

SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline–rich domain. To identify proteins that bind to the SHIP-2 proline–rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton