Amyloid precursor protein is required for in vitro platelet adhesion to amyloid peptides and potentiation of thrombus formation.

Amyloid precursor protein (APP) is the precursor of amyloid ß (Aß) peptides, whose accumulation in the brain is associated with Alzheimer's disease. APP is also expressed on the platelet surface and Aß peptides are platelet agonists. The physiological role of APP is largely unknown. In neurons, APP acts as an adhesive receptor, facilitating integrin-mediated cell adhesion, while in platelets it regulates coagulation and venous thrombosis. In this work, we analyzed platelets from APP KO mice to investigate whether membrane APP supports platelet adhesion to physiological and pathological substrates. We found that APP-null platelets adhered and spread normally on collagen, von Willebrand Factor or fibrinogen. However, adhesion on immobilized Aß peptides Aß1-40, Aß1-42 and Aß25-35 was completely abolished in platelets lacking APP. By contrast, platelet activation and aggregation induced by Aß peptides occurred normally in the absence of APP. Adhesion of APP-transfected HEK293 to Aß peptides was significantly higher than that of control cells expressing low levels of APP. Co-coating of Aß1-42 and Aß25-35 with collagen strongly potentiated platelet adhesion when whole blood from wild type mice was perfused at arterial shear rate, but had no effects with blood from APP KO mice. These results demonstrate that APP selectively mediates platelet adhesion to Aß under static condition but not platelet aggregation, and is responsible for Aß-promoted potentiation of thrombus formation under flow. Therefore, APP may facilitate an early step in thrombus formation when Aß peptides accumulate in cerebral vessel walls or atherosclerotic plaques.

Release of Prometastatic Platelet-Derived Microparticles Induced by Breast Cancer Cells: A Novel Positive Feedback Mechanism for Metastasis.

Circulating platelets and platelet-derived microparticles are regulators of cancer metastasis. In this study, we show that breast cancer cells induce platelet aggregation and lead to the release of platelet-derived microparticles. Although able to cause comparable aggregation, the highly aggressive MDA-MB-231 cells were more potent than the poorly aggressive MCF7 cells in inducing platelet-derived microparticles release, which was comparable to that promoted by thrombin. MDA-MB-231 cells were able to bind and internalize both MCF7- and MDA-MB-231-induced platelet-derived microparticles with comparable efficiency. By contrast, MCF7 cells did not interact with either type of platelet-derived microparticles. Upon internalization, only platelet-derived microparticles released by platelet stimulation with MDA-MB-231 cells, but not those released upon stimulation with MCF7 cells, caused activation of MDA-MB-231 cells and promoted the phosphorylation of selected signaling proteins, including p38MAPK and myosin light chain. Accordingly, MDA-MB-231-induced, but not MCF7-induced, platelet-derived microparticles dose-dependently stimulated migration and invasion of targeted MDA-MB-231 cells. These results identify a novel paracrine positive feedback mechanism initiated by aggressive breast cancer cell types to potentiate their invasive phenotype through the release of platelet-derived microparticles.

γ-PGA Hydrolases of Phage Origin in Bacillus subtilis and Other Microbial Genomes.

Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection.

Cellular properties of human erythrocytes preserved in saline-adenine-glucose-mannitol in the presence of L-carnitine.

L-Carnitine (LC) in the preservation medium during storage of red blood cells (RBC) can improve the mean 24-hr percent recovery in vivo and increase RBC life-span after reinfusion. The purpose of the study was to investigate the differences in the biochemical properties of RBCs stored in the presence or absence of LC, and the cell-age related responses to storage conditions and to LC. RBC concentrates in saline-adenine-glucose-mannitol (SAG-M) were stored in the presence or absence of 5 mM LC at 4 degrees C for up to 8 weeks. RBC subpopulations of different densities were prepared by centrifugation on Stractan density gradient. Cells were sampled at 0, 3, 6, and 8 weeks, and hematological and cellular properties analyzed (MCV, MCHC, 4.1a/4.1b ratio as a cell age parameter, intracellular Na(+) and K(+)). After 6 weeks, MCV of RBC stored in the presence of LC was lower than that of controls (6 weeks MCV: controls 95.4 +/- 1.8 fl; LC 91.5 +/- 2.0 fl; n = 6; P < 0.005). This was due to swelling of control cells, and affected mainly older RBCs. LC appeared to reduce or retard cell swelling. Among the osmotically active substances whose changes during storage could contribute to cell swelling, only intracellular Na(+) and K(+) differed between stored control RBCs and LC-treated cells. LC reduces the swelling of older cells during storage at 4 degrees C in SAG-M, possibly by acting on the permeability of cell membrane to monovalent cations.

Proteomic analysis of erythrocyte membranes by soft Immobiline gels combined with differential protein extraction.

Improvements in proteome analysis of erythrocyte membrane proteins by two-dimensional electrophoresis are here reported. In particular, a differential extraction procedure was set up allowing separation of integral membrane proteins from peripheral species. Moreover, the use of dilute Immobiline gels (down to as low as 3% T matrix) permitted a better penetration and transfer inside the gel of proteins with large M(r). These protocol modifications, combined with sample delipidation and alkylation prior to electrophoresis, which prevented generation of homo- and hetero-oligomers following disulfide scrambling phenomena, allowed the display of more than 500 spots in the pI/M(r) plane. Among those, noteworthy was the presence of high levels of filamentous proteins, such as alpha-spectrin and ankyrins, or integral membrane proteins, such as band 3, band 4.1 and 4.2, not displayed or barely present in other maps exploiting immobilized pH gradients in the first dimension. Accordingly, our results show that this 2D mapping technique is a valuable tool in exploring pathologies related to genetic defects associated to membrane proteins.