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CD47 on erythrocytes inhibits phagocytosis through interaction with the inhibitory immunoreceptor SIRP alpha expressed by macrophages. Thus, the CD47-SIRP alpha interaction constitutes a negative signal for erythrocyte phagocytosis. However, we report here that CD47 does not only function as a "do not eat me" signal for uptake but can also act as an "eat me" signal. In particular, a subset of old erythrocytes present in whole blood was shown to bind and to be phagocytosed via CD47-SIRP alpha interactions. Furthermore, we provide evidence that experimental aging of erythrocytes induces a conformational change in CD47 that switches the molecule from an inhibitory signal into an activating one. Preincubation of experimentally aged erythrocytes with human serum before the binding assay was required for this activation. We also demonstrate that aged erythrocytes have the capacity to bind the CD47-binding partner thrombospondin-1 (TSP-1) and that treatment of aged erythrocytes with a TSP-1 derived peptide enabled their phagocytosis by human red pulp macrophages. Finally, CD47 on erythrocytes that had been stored for prolonged time was shown to undergo a conformational change and bind TSP-1. These findings reveal a more complex role for CD47-SIRP alpha interactions in erythrocyte phagocytosis, with CD47 acting as a molecular switch for controlling erythrocyte phagocytosis. (Blood. 2012;119(23):5512-5521)

UV-C irradiation has been shown to be effective for pathogen reduction in platelet concentrates, but preliminary work indicated that UV-C irradiation of platelets can induce platelet aggregation. In this study, the mechanism underlying this phenomenon was investigated. Irradiation of platelets with UV-C light (1500 J/m(2)) caused platelet aggregation, which was dependent on integrin alphaIIbbeta3 activation (GPIIb/IIIa). This activation occurred despite treatment with several signal transduction inhibitors known to block platelet activation. UV-C also induced activation of recombinant alphaIIbbeta3 in Chinese hamster ovary (CHO) cells, an environment in which physiologic agonists fail to activate. Activation of alphaIIbbeta3 requires talin binding to the beta3 tail, yet alphaIIbbeta3-Delta724 (lacking the talin binding site) was activated by UV-C irradiation, excluding a requirement for talin binding. The UV-C effect appears to be general in that beta(1) and beta(2) integrins are also activated by UV-C. To explain these findings, we investigated the possibility of UV-C-induced photolysis of disulfide bonds, in analogy with the activating effect of reducing agents on integrins. Indeed, UV-C induced a marked increase in free thiol groups in platelet surface proteins including alphaIIbbeta3. Thus, UV-C appears to activate alphaIIbbeta3 not by affecting intracellular signal transduction, but by reduction of disulfide bonds regulating integrin conformation.

Background Platelet concentrates (PCs) are stored at room temperature to preserve their biological activity. To minimize the risk of bacterial outgrowth, storage time is limited to 7 days. It is well-established that prolonged storage of PCs results in modifications that result in a decreased hemostatic efficacy. This loss of platelet functionality during storage is commonly referred to as the platelet storage lesion (PSL). Typical events linked to development of the PSL are shape changes, platelet activation and loss of receptors crucial for platelet functionality. Two-dimensional (2D) differential gel electrophoresis (DIGE), isotope tagging and isotope-coded affinity tagging (ICAT) have been previously used to monitor changes in protein composition during storage. These studies have provided valuable insights into the changes associated with the PSL, however, these studies generally focused on a limited set of proteins. Aim We aimed to generate an overview of changes in the platelet proteome during storage using label free quantitative mass spectrometry. Furthermore, we employed Gene ontology (GO) enrichment analysis to identify pathways and biological processes that were linked to development of the PSL. Methods Three independently pooled PCs were stored in plasma under standard blood bank conditions for 16 days. Tryptic peptides were separated by nanoscale C18 reverse phase chromatography coupled on line to an Orbitrap Fusion Tribrid mass spectrometer. The RAW mass spectrometry files were processed with the MaxQuant computational platform. The global changes in protein level during platelet storage were assessed employing the analysis-of-variance functions of PERSEUS. Gene ontology enrichment analysis of biological processes, molecular functions and cellular compartments of the significantly different proteins was performed using the Cytoscape plug-in BiNGO. Results A total number of 2501 proteins was detected in all 3 biological replicates in at least one of the time points analyzed. The analysis showed that 18 proteins were down-regulated over time, whereas the level of 3 proteins was found to increase. CytoScape BinGo analysis of these significantly downregulated proteins revealed that the majority of this set was linked to GO-terms platelet degranulation, secretion and regulated exocytosis. This set of proteins included von Willebrand factor (VWF), serglycin (SRGN), SPARC, amyloid beta A4 protein (APP), multimerin-1 (MMRN1) and platelet factor 4 (PF4). A significant decline in these protein levels was observed at day 5 of storage, suggesting that release of α-granules is a relatively early event during platelet storage. At day 5 also a marked decline in S100A9 was observed. S100A9 has been implicated in degranulation in neutrophils, and may therefore also be linked to platelet granule release. Levels of membrane surface platelet glycoproteins such as glycoprotein Ibα did not significantly change at day 5. Only one single protein, histone H2A, was found to be consistently decreased already at two days of storage, but the significance of this finding is not clear. Upon prolonged storage (13 and 16 days) an increase in the level of α-2-macroglobulin (A2M), immunoglobulin M (IGM) and glycogenin-1 (GYG1) was observed suggesting that platelets acquire an (increased) potential to bind and/or internalize proteins from their environment. Consistent with this notion we also detected significant levels of several serine protease inhibitors, although levels of these proteins did not change upon storage. Conclusions Overall, our findings highlight dynamic changes in protein composition of platelets during storage. Our data provide evidence for sustained release of α-granules over time which becomes significant at day 5. Our data also suggest that during storage, platelets can bind or ingest proteins from their environment which may have impact on the hemostatic properties of stored platelets. Disclosures Leebeek: CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxalta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Dutch Hemphilia Foundation: Research Funding.

Sepsis is a life threatening organ dysfunction caused by a dysregulated host response to infection. It is a global health care problem and the leading cause of death in ICU patients. A rather unknown but effective mechanism to protect ourselves from such pathogen invasion is immune-adherence clearance (IAC). During IAC pathogens (including bacteria, fungi and viruses) can bind to red blood cells (RBCs) through complement opsonization and subsequent binding to complement receptor 1 (CR1, CD35) expressed on their cell surface. After binding of the pathogen to CR1, the RBCs deliver the pathogens to macrophages of the spleen and liver, where they are phagocytosed and degraded. To study the underlying mechanisms of IAC in more detail, we have developed an assay to monitor the transfer of opsonized pathogens bound to RBCs to human monocytes and/or macrophages under flow conditions, using confocal microscopy. The transfer of a variety of different pathogens, including S. aureus, E. coli and C. albicans, was studied in this assay. When looking at the transfer process in detail, it was noticed that RBC attach to the phagocyte prior to the transfer and remain attached shortly after the transfer, suggesting that RBC might be interacting with phagocytes through adhesion molecules such as integrins to establish firm binding between these two cell types. To test this hypothesis several adhesion molecules, on the phagocytes as well as on the RBC, were blocked by monoclonal antibodies and IAC was quantified. First, complement receptor 3 (CR3, amb2 CD11b/CD18 integrin) an important receptor for adhesion, migration and phagocytosis was blocked by monoclonal antibodies on the phagocytes, after which bacterial transfer was impaired. This indicated that CR3 is crucial for efficient IAC. To confirm the involvement of CR3 in IAC, we analyzed the monocytes of a known LAD-1 patient. This rare immunodeficiency is characterized by an inherited molecular defect of the β2integrin subunit (CD18) which results in impaired adhesion and migration of the patient's leukocytes and clinically manifests in recurrent infections. Our results showed a highly reduced interaction between the phagocytes and the RBCs, resulting in a decrease in bacterial transfer of >70%. Next, we tested the effects of blocking antibodies directed against several RBC adhesion molecules on the transfer process. When blocking CD147 (Basigin, Ok blood group) and ICAM-4 (CD242, Landsteiner-Wiener), but not Glycophorin A (GPA, CD235a) on the RBC the transfer process was largely inhibited. These findings demonstrate the involvement of direct cell-cell interactions between RBC and macrophages in IAC and provide evidence that RBC adopt a "sticky" phenotype after binding a pathogen through CR1, which enables phagocytes to bind them under flow. We anticipated that blood bank filters (currently used for leukocyte depletion) might be used to bind and filter RBC-pathogen complexes from blood due to their "sticky" phenotype. This was found to be possible in a series of experiments, not only using in vitrogenerated RBC-pathogen complexes, but also using blood from septic patients. Filtration using a standard leukocyte reduction filter showed reduction of RBC-pathogen complexes close to 100%, independent of the type of pathogen. We foresee that this knowledge can be used to develop a generic approach to deplete RBCs carrying pathogens specifically from the blood stream, and thereby filter RBC-pathogen complexes from the blood of patients suffering from sepsis. Disclosures No relevant conflicts of interest to declare.

Introduction: One of the properties of platelets is the ability, upon activation, to expose phosphatidylserine (PS), providing a negative surface necessary for the docking of the blood coagulation complexes. During storage, PS exposure should be kept to a minimum to achieve optimal transfusion recoveries. Many events underlying PS exposure are still unknown despite intensive research over the last 20 years. The involvement of a phosphorylation event has been suggested and we investigated the involvement of AMP-dependent kinase (AMPK) as a candidate involved in the cascade leading to PS exposure. Methods: Platelets were washed free of plasma by three consecutive wash steps in substrate-free medium, preventing aggregation during the first two steps by acidification of the platelets with acid citrate dextrose (ACD). Platelets were subsequently incubated with different metabolic substrates for 18 h. The nucleotide levels in the cytosolic fractions of the platelets were analyzed by HPLC, PS exposure via flow cytometry using AnnexinV-FITC as label and phosphorylated AMPK (reflecting activation of AMPK) was determined by immunoblotting using antibodies against phosphorylated AMPK (P-AMPK). Results: When glucose was present, hardly any phosphorylation of AMPK was observed. Under substrate-deprived conditions, AMPK becomes highly phosphorylated. In the presence of mitochondrial substrates (pyruvate, acetate) AMPK phosphorylation was clearly higher than with glucose. The analysis of the nucleotide profiles showed a good correlation between phosphorylated AMPK and AMP/ATP ratios. Interestingly, PS exposure showed a strong correlation with AMP/ATP ratios and the phosphorylation of AMPK. Inhibition of LKB1, the upstream kinase of AMPK, with 5-iodotubericidin partially suppressed AMPK phosphorylation and PS exposure in response to substrate deprivation. Conclusion: In human platelets, activation of AMPK induced by metabolic variation strongly correlates with PS exposure, suggesting a role for AMPK in the induction of PS exposure.