Autophagy and its consequences for platelet biology.

Autophagy, the continuous recycling of intracellular building blocks, molecules, and organelles is necessary to preserve cellular function and homeostasis. In this context, it was demonstrated that autophagy plays an important role in megakaryopoiesis, the development and differentiation of hematopoietic progenitor cells into megakaryocytes. Furthermore, in recent years, autophagic proteins were detected in platelets, anucleate cells generated by megakaryocytes, responsible for hemostasis, thrombosis, and a key cell in inflammation and host immune responses. In the last decade studies have indicated the occurrence of autophagy in platelets. Moreover, autophagy in platelets was subsequently demonstrated to be involved in platelet aggregation, adhesion, and thrombus formation. Here, we review the current knowledge about autophagy in platelets, its function, and clinical implications. However, at the advent of platelet autophagy research, additional discoveries derived from evolving work will be required to precisely define the contributions of autophagy in platelets, and to expand the ever increasing physiologic and pathologic roles these remarkable and versatile blood cells play.

A New Role of NAP1L1 in Megakaryocytes and Human Platelets.

Platelets (PLTs) are anucleate and considered incapable of nuclear functions. Contrastingly, nuclear proteins were detected in human PLTs. For most of these proteins, it is unclear if nuclear or alternatively assigned functions are performed, a question we wanted to address for nuclear assembly protein 1like 1 (NAP1L1). Using a wide array of molecular methods, including RNAseq, co-IP, overexpression and functional assays, we explored expression pattern and functionality of NAP1L1 in PLTs, and CD34+-derived megakaryocytes (MKs). NAP1L1 is expressed in PLTs and MKs. Co-IP experiments revealed that dihydrolipolylysine-residue acetyltransferase (DLAT encoded protein PDC-E2, ODP2) dynamically interacts with NAP1L1. PDC-E2 is part of the mitochondrial pyruvate-dehydrogenase (PDH) multi-enzyme complex, playing a crucial role in maintaining cellular respiration, and promoting ATP-synthesis via the respiratory chain. Since altered mitochondrial function is a hallmark of infectious syndromes, we analyzed PDH activity in PLTs from septic patients demonstrating increased activity, paralleling NAP1L1 expression levels. MKs PDH activity decreased following an LPS-challenge. Furthermore, overexpression of NAP1L1 significantly altered the ability of MKs to form proplatelet extensions, diminishing thrombopoiesis. These results indicate that NAP1L1 performs in other than nucleosome-assembly functions in PTLs and MKs, binding a key mitochondrial protein as a potential chaperone, and gatekeeper, influencing PDH activity and thrombopoiesis.

IFITM3 regulates fibrinogen endocytosis and platelet reactivity in nonviral sepsis.

Platelets and megakaryocytes are critical players in immune responses. Recent reports suggest infection and inflammation alter the megakaryocyte and platelet transcriptome to induce altered platelet reactivity. We determined whether nonviral sepsis induces differential platelet gene expression and reactivity. Nonviral sepsis upregulated IFN-induced transmembrane protein 3 (IFITM3), an IFN-responsive gene that restricts viral replication. As IFITM3 has been linked to clathrin-mediated endocytosis, we determined whether IFITM3 promoted endocytosis of α-granule proteins. IFN stimulation enhanced fibrinogen endocytosis in megakaryocytes and platelets from Ifitm+/+ mice, but not Ifitm-/- mice. IFITM3 overexpression or deletion in megakaryocytes demonstrated IFITM3 was necessary and sufficient to regulate fibrinogen endocytosis. Mechanistically, IFITM3 interacted with clathrin and αIIb and altered their plasma membrane localization into lipid rafts. In vivo IFN administration increased fibrinogen endocytosis, platelet reactivity, and thrombosis in an IFITM-dependent manner. In contrast, Ifitm-/- mice were completely rescued from IFN-induced platelet hyperreactivity and thrombosis. During murine sepsis, platelets from Ifitm+/+ mice demonstrated increased fibrinogen content and platelet reactivity, which was dependent on IFN-α and IFITMs. Platelets from patients with nonviral sepsis had increases in platelet IFITM3 expression, fibrinogen content, and hyperreactivity. These data identify IFITM3 as a regulator of platelet endocytosis, hyperreactivity, and thrombosis during inflammatory stress.

Human platelets display dysregulated sepsis-associated autophagy, induced by altered LC3 protein-protein interaction of the Vici-protein EPG5.

Platelets mediate central aspects of host responses during sepsis, an acute profoundly systemic inflammatory response due to infection. Macroautophagy/autophagy, which mediates critical aspects of cellular responses during inflammatory conditions, is known to be a functional cellular process in anucleate platelets, and is essential for normal platelet functions. Nevertheless, how sepsis may alter autophagy in platelets has never been established. Using platelets isolated from septic patients and matched healthy controls, we show that during clinical sepsis, the number of autophagosomes is increased in platelets, most likely due to an accumulation of autophagosomes, some containing mitochondria and indicative of mitophagy. Therefore, autophagy induction or early-stage autophagosome formation (as compared to decreased later-stage autophagosome maturation or autophagosome-late endosome/lysosome fusion) is normal or increased. This was consistent with decreased fusion of autophagosomes with lysosomes in platelets. EPG5 (ectopic P-granules autophagy protein 5 homolog), a protein essential for normal autophagy, expression did increase, while protein-protein interactions between EPG5 and MAP1LC3/LC3 (which orchestrate the fusion of autophagosomes and lysosomes) were significantly reduced in platelets during sepsis. Furthermore, data from a megakaryocyte model demonstrate the importance of TLR4 (toll like receptor 4), LPS-dependent signaling for regulating this mechanism. Similar phenotypes were also observed in platelets isolated from a patient with Vici syndrome: an inherited condition caused by a naturally occurring, loss-of-function mutation in EPG5. Together, we provide evidence that autophagic functions are aberrant in platelets during sepsis, due in part to reduced EPG5-LC3 interactions, regulated by TLR4 engagement, and the resultant accumulation of autophagosomes.Abbreviations: ACTB: beta actin; CLP: cecal ligation and puncture; Co-IP: co-immunoprecipitation; DAP: death associated protein; DMSO: dimethyl sulfoxide; EPG5: ectopic P-granules autophagy protein 5 homolog; ECL: enhanced chemiluminescence; HBSS: Hanks' balanced salt solution; HRP: horseradish peroxidase; ICU: intensive care unit; LPS: lipopolysaccharide; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; MKs: megakaryocytes; PFA: paraformaldehyde; PBS: phosphate-buffered saline; PLA: proximity ligation assay; pRT-PCR: quantitative real-time polymerase chain reaction; RT: room temperature; SQSTM1/p62: sequestosome 1; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TLR4: toll like receptor 4; TEM: transmission electron microscopy; WGA: wheat germ agglutinin.

Maintaining Myocardial Glucose Utilization in Diabetic Cardiomyopathy Accelerates Mitochondrial Dysfunction.

Cardiac glucose uptake and oxidation are reduced in diabetes despite hyperglycemia. Mitochondrial dysfunction contributes to heart failure in diabetes. It is unclear whether these changes are adaptive or maladaptive. To directly evaluate the relationship between glucose delivery and mitochondrial dysfunction in diabetic cardiomyopathy, we generated transgenic mice with inducible cardiomyocyte-specific expression of the GLUT4. We examined mice rendered hyperglycemic following low-dose streptozotocin prior to increasing cardiomyocyte glucose uptake by transgene induction. Enhanced myocardial glucose in nondiabetic mice decreased mitochondrial ATP generation and was associated with echocardiographic evidence of diastolic dysfunction. Increasing myocardial glucose delivery after short-term diabetes onset exacerbated mitochondrial oxidative dysfunction. Transcriptomic analysis revealed that the largest changes, driven by glucose and diabetes, were in genes involved in mitochondrial function. This glucose-dependent transcriptional repression was in part mediated by O-GlcNAcylation of the transcription factor Sp1. Increased glucose uptake induced direct O-GlcNAcylation of many electron transport chain subunits and other mitochondrial proteins. These findings identify mitochondria as a major target of glucotoxicity. They also suggest that reduced glucose utilization in diabetic cardiomyopathy might defend against glucotoxicity and caution that restoring glucose delivery to the heart in the context of diabetes could accelerate mitochondrial dysfunction by disrupting protective metabolic adaptations.

Longitudinal RNA-Seq Analysis of the Repeatability of Gene Expression and Splicing in Human Platelets Identifies a Platelet SELP Splice QTL.

RATIONALE: Longitudinal studies are required to distinguish within versus between-individual variation and repeatability of gene expression. They are uniquely positioned to decipher genetic signal from environmental noise, with potential application to gene variant and expression studies. However, longitudinal analyses of gene expression in healthy individuals-especially with regards to alternative splicing-are lacking for most primary cell types, including platelets. OBJECTIVE: To assess repeatability of gene expression and splicing in platelets and use repeatability to identify novel platelet expression quantitative trait loci (QTLs) and splice QTLs. METHODS AND RESULTS: We sequenced the transcriptome of platelets isolated repeatedly up to 4 years from healthy individuals. We examined within and between individual variation and repeatability of platelet RNA expression and exon skipping, a readily measured alternative splicing event. We find that platelet gene expression is generally stable between and within-individuals over time-with the exception of a subset of genes enriched for the inflammation gene ontology. We show an enrichment among repeatable genes for associations with heritable traits, including known and novel platelet expression QTLs. Several exon skipping events were also highly repeatable, suggesting heritable patterns of splicing in platelets. One of the most repeatable was exon 14 skipping of SELP. Accordingly, we identify rs6128 as a platelet splice QTL and define an rs6128-dependent association between SELP exon 14 skipping and race. In vitro experiments demonstrate that this single nucleotide variant directly affects exon 14 skipping and changes the ratio of transmembrane versus soluble P-selectin protein production. CONCLUSIONS: We conclude that the platelet transcriptome is generally stable over 4 years. We demonstrate the use of repeatability of gene expression and splicing to identify novel platelet expression QTLs and splice QTLs. rs6128 is a platelet splice QTL that alters SELP exon 14 skipping and soluble versus transmembrane P-selectin protein production.

Sepsis alters the transcriptional and translational landscape of human and murine platelets.

There is increasing recognition that platelets have a functional role in the pathophysiology of sepsis, though this role has not been precisely defined. Whether sepsis alters the human platelet transcriptome and translational landscape has never been established. We used parallel techniques of RNA sequencing and ribosome footprint profiling to interrogate the platelet transcriptome and translatome in septic patients and healthy donors. We identified 1806 significantly differentially expressed (false discovery rate <0.05) transcripts in platelets from septic patients. Platelet translational events during sepsis were also upregulated. To explore the relevance of a murine model of sepsis, cecal ligation and puncture (CLP), we compared sepsis-induced changes in platelet gene expression between septic patients and mice subjected to CLP. Platelet transcriptional (ρ = 0.42, P = 3.2 × 10-285) and translational (ρ = 0.65, P = 1.09 × 10-56) changes were significantly correlated between septic patients and mice. We focused on ITGA2B, tracking and validating the expression, regulation, and functional impact of changes in ITGA2B during sepsis. Increased ITGA2B was identified in bone marrow megakaryocytes within 24 hours of sepsis onset. Subsequent increases in ITGA2B were seen in circulating platelets, suggesting dynamic trafficking of the messenger RNA. Transcriptional changes in ITGA2B were accompanied by de novo protein synthesis of αIIb and integrin αIIbß3 activation. Increased αIIb was associated with mortality in humans and mice. These findings provide previously unrecognized evidence that human and murine sepsis similarly alters the platelet transcriptional and translational landscape. Moreover, ITGA2B is upregulated and functional in sepsis due to trafficking from megakaryocytes and de novo synthesis in platelets and is associated with increased mortality.

Human megakaryocytes possess intrinsic antiviral immunity through regulated induction of IFITM3.

Evolving evidence indicates that platelets and megakaryocytes (MKs) have unexpected activities in inflammation and infection; whether viral infections upregulate biologically active, antiviral immune genes in platelets and MKs is unknown, however. We examined antiviral immune genes in these cells in dengue and influenza infections, viruses that are global public health threats. Using complementary biochemical, pharmacological, and genetic approaches, we examined the regulation and function of interferon-induced transmembrane protein 3 (IFITM3), an antiviral immune effector gene not previously studied in human platelets and MKs. IFITM3 was markedly upregulated in platelets isolated from patients during clinical influenza and dengue virus (DENV) infections. Lower IFITM3 expression in platelets correlated with increased illness severity and mortality in patients. Administering a live, attenuated DENV vaccine to healthy subjects significantly increased platelet IFITM3 expression. Infecting human MKs with DENV selectively increased type I interferons and IFITM3. Overexpression of IFITM3 in MKs was sufficient to prevent DENV infection. In naturally occurring, genetic loss-of-function studies, MKs from healthy subjects harboring a homozygous mutation in IFITM3 (rs12252-C, a common single-nucleotide polymorphism in areas of the world where DENV is endemic) were significantly more susceptible to DENV infection. DENV-induced MK secretion of interferons prevented infection of bystander MKs and hematopoietic stem cells. Thus, viral infections upregulate IFITM3 in human platelets and MKs, and IFITM3 expression is associated with adverse clinical outcomes. These observations establish, for the first time, that human MKs possess antiviral functions, preventing DENV infection of MKs and hematopoietic stem cells after local immune signaling.

Amicus or Adversary Revisited: Platelets in Acute Lung Injury and Acute Respiratory Distress Syndrome.

Platelets are essential cellular effectors of hemostasis and contribute to disease as circulating effectors of pathologic thrombosis. These are their most widely known biologic activities. Nevertheless, recent observations demonstrate that platelets have a much more intricate repertoire beyond these traditional functions and that they are specialized for contributions to vascular barrier integrity, organ repair, antimicrobial host defense, inflammation, and activities across the immune continuum. Paradoxically, on the basis of clinical investigations and animal models of disease, some of these newly discovered activities of platelets appear to contribute to tissue injury. Studies in the last decade indicate unique interactions of platelets and their precursor, the megakaryocyte, in the lung and implicate platelets as essential effectors in experimental acute lung injury and clinical acute respiratory distress syndrome. Additional discoveries derived from evolving work will be required to precisely define the contributions of platelets to complex subphenotypes of acute lung injury and to determine if these remarkable and versatile blood cells are therapeutic targets in acute respiratory distress syndrome.

Endogenous LINE-1 (Long Interspersed Nuclear Element-1) Reverse Transcriptase Activity in Platelets Controls Translational Events Through RNA-DNA Hybrids.

OBJECTIVE: One source of endogenous reverse transcriptase (eRT) activity in nucleated cells is the LINE-1/L1 (long interspersed nuclear element-1), a non-LTR retrotransposon that is implicated in the regulation of gene expression. Nevertheless, the presence and function of eRT activity and LINE-1 in human platelets, an anucleate cell, has not previously been determined. APPROACH AND RESULTS: We demonstrate that human and murine platelets possess robust eRT activity and identify the source as being LINE-1 ribonucleoprotein particles. Inhibition of eRT in vitro in isolated platelets from healthy individuals or in people with HIV treated with RT inhibitors enhanced global protein synthesis and platelet activation. If HIV patients were treated with reverse transcriptase inhibitor, we found that platelets from these patients had increased basal activation. We next discovered that eRT activity in platelets controlled the generation of RNA-DNA hybrids, which serve as translational repressors. Inhibition of platelet eRT lifted this RNA-DNA hybrid-induced translational block and was sufficient to increase protein expression of target RNAs identified by RNA-DNA hybrid immunoprecipitation. CONCLUSIONS: Thus, we provide the first evidence that platelets possess L1-encoded eRT activity. We also demonstrate that platelet eRT activity regulates platelet hyperreactivity and thrombosis and controls RNA-DNA hybrid formation and identify that RNA-DNA hybrids function as a novel translational control mechanism in human platelets.

Granzyme A in Human Platelets Regulates the Synthesis of Proinflammatory Cytokines by Monocytes in Aging.

Dysregulated inflammation is implicated in the pathobiology of aging, yet platelet-leukocyte interactions and downstream cytokine synthesis in aging remains poorly understood. Platelets and monocytes were isolated from healthy younger (age <45, n = 37) and older (age ≥65, n = 30) adults and incubated together under autologous and nonautologous conditions. Synthesis of inflammatory cytokines by monocytes, alone or in the presence of platelets, was examined. Next-generation RNA-sequencing allowed for unbiased profiling of the platelet transcriptome in aging. Basal IL-8 and MCP-1 synthesis by monocytes alone did not differ between older and younger adults. However, in the presence of autologous platelets, monocytes from older adults synthesized greater IL-8 (41 ± 5 versus 9 ± 2 ng/ml, p < 0.0001) and MCP-1 (867 ± 150 versus 216 ± 36 ng/ml, p < 0.0001) than younger adults. Platelets from older adults were sufficient for upregulating the synthesis of inflammatory cytokines by monocytes. Using RNA-sequencing of platelets followed by validation via RT-PCR and immunoblot, we discovered that granzyme A (GrmA), a serine protease not previously identified in human platelets, increases with aging (∼9-fold versus younger adults, p < 0.05) and governs increased IL-8 and MCP-1 synthesis through TLR4 and caspase-1. Inhibiting GrmA reduced excessive IL-8 and MCP-1 synthesis in aging to levels similar to younger adults. In summary, human aging is associated with changes in the platelet transcriptome and proteome. GrmA is present and bioactive in human platelets, is higher in older adults, and controls the synthesis of inflammatory cytokines by monocytes. Alterations in the platelet molecular signature and signaling to monocytes may contribute to dysregulated inflammatory syndromes in older adults.

Serotype 3 pneumococci sequester platelet-derived human thrombospondin-1 via the adhesin and immune evasion protein Hic.

Streptococcus pneumoniae serotype 3 strains emerge frequently within clinical isolates of invasive diseases. Bacterial invasion into deeper tissues is associated with colonization and immune evasion mechanisms. Thus, pneumococci express a versatile repertoire of surface proteins sequestering and interacting specifically with components of the human extracellular matrix and serum. Hic, a PspC-like pneumococcal surface protein, possesses vitronectin and factor H binding activity. Here, we show that heterologously expressed Hic domains interact, similar to the classical PspC molecule, with human matricellular thrombospondin-1 (hTSP-1). Binding studies with isolated human thrombospondin-1 and various Hic domains suggest that the interaction between hTSP-1 and Hic differs from binding to vitronectin and factor H. Binding of Hic to hTSP-1 is inhibited by heparin and chondroitin sulfate A, indicating binding to the N-terminal globular domain or type I repeats of hTSP-1. Competitive inhibition experiments with other pneumococcal hTSP-1 adhesins demonstrated that PspC and PspC-like Hic recognize similar domains, whereas PavB and Hic can bind simultaneously to hTSP-1. In conclusion, Hic binds specifically hTSP-1; however, truncation in the N-terminal part of Hic decreases the binding activity, suggesting that the full length of the α-helical regions of Hic is required for an optimal interaction.

Neonatal NET-inhibitory factor and related peptides inhibit neutrophil extracellular trap formation.

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMNs), extrude molecular lattices of decondensed chromatin studded with histones, granule enzymes, and antimicrobial peptides that are referred to as neutrophil extracellular traps (NETs). NETs capture and contain bacteria, viruses, and other pathogens. Nevertheless, experimental evidence indicates that NETs also cause inflammatory vascular and tissue damage, suggesting that identifying pathways that inhibit NET formation may have therapeutic implications. Here, we determined that neonatal NET-inhibitory factor (nNIF) is an inhibitor of NET formation in umbilical cord blood. In human neonatal and adult neutrophils, nNIF inhibits key terminal events in NET formation, including peptidyl arginine deiminase 4 (PAD4) activity, neutrophil nuclear histone citrullination, and nuclear decondensation. We also identified additional nNIF-related peptides (NRPs) that inhibit NET formation. nNIFs and NRPs blocked NET formation induced by pathogens, microbial toxins, and pharmacologic agonists in vitro and in mouse models of infection and systemic inflammation, and they improved mortality in murine models of systemic inflammation, which are associated with NET-induced collateral tissue injury. The identification of NRPs as neutrophil modulators that selectively interrupt NET generation at critical steps suggests their potential as therapeutic agents. Furthermore, our results indicate that nNIF may be an important regulator of NET formation in fetal and neonatal inflammation.

Further insights into the anti-PF4/heparin IgM immune response.

Anti-platelet factor 4 (PF4)/heparin antibodies are not only the cause of heparin-induced thrombocytopenia but might also play a role in the antibacterial host defence. Recently, marginal zone (MZ) B cells were identified to be crucial for anti-PF4/heparin IgG antibody production in mice. Combining human studies and a murine model of polymicrobial sepsis we further characterised the far less investigated anti-PF4/heparin IgM immune response. We detected anti-PF4/heparin IgM antibodies in the sera of paediatric patients < 6 months of age after cardiac surgery and in sera of splenectomised mice subjected to polymicrobial sepsis. In addition, PF4/heparin-specific IgM B cells were not only found in murine spleen, but also in peritoneum and bone marrow upon in vitro stimulation. Together, this indicates involvement of additional B cell populations, as MZ B cells are not fully developed in humans until the second year of life and are restricted to the spleen in mice. Moreover, PF4/heparin-specific B cells were detected in human cord blood upon in vitro stimulation and PF4-/- mice produced anti-PF4/heparin IgM antibodies after polymicrobial sepsis. In conclusion, the anti-PF4/heparin IgM response is a potential innate immune reaction driven by a B cell population distinct from MZ B cells.

Partially desulfated heparin modulates the interaction between anti-protamine/heparin antibodies and platelets.

Protamine (PRT) is the standard drug to neutralise heparin. PRT/heparin complexes induce an immune response similar to that observed in heparin-induced thrombocytopenia (HIT). Partially desulfated heparin (ODSH) was shown to interfere with anti-platelet factor 4/heparin antibodies (Abs), which are responsible for HIT. In this study, we analyse the impact of ODSH on the interaction between anti-PRT/heparin Abs and platelets. The ability of ODSH to prevent anti-PRT/heparin Ab-induced platelet destruction in vivo was investigated using the NOD/SCID mouse model. ODSH improved platelet survival in the presence of PRT, heparin and anti-PRT/heparin Abs (median platelet survival after 300 minutes (min) with 20 µg/ml ODSH: 75%, range 70-81% vs without ODSH: 49%, range 44-59%, p=0.006). Furthermore, when ODSH was applied 60 min after Ab injection platelet survival was improved (median platelet survival after 300 min with ODSH: 83%, range 77-93% vs without ODSH: 59%, range 29-61%, p=0.02). In in vitro experiments ODSH inhibited platelet activation at concentrations >16 µg/mL (p<0.001), as well as PRT/heparin complex binding to platelets (mean fluorescence intensity [MFI] without ODSH: 85 ± 14 vs with ODSH: 15 ± 0.6, p=0.013). ODSH also displaced pre-bound complexes from the platelet surface (MFI without ODSH: 324 ± 43 vs with 32 µg/ml ODSH: 53 ± 9, p<0.001). While interfering with platelet activation by anti-PRT/heparin Abs, up to a concentration of 16 µg/ml, ODSH had only minimal impact on neutralisation of heparin by PRT. In conclusion, our study shows that ODSH is able to inhibit platelet activation and destruction suggesting a potential clinical use to reduce anti-PRT/heparin Ab-mediated adverse effects.

Human neutrophil antigen-3a antibodies induce neutrophil stiffening and conformational activation of CD11b without shedding of L-selectin.

BACKGROUND: HNA-3a antibodies induce severe transfusion-related acute lung injury (TRALI) in which neutrophils play a major role. As neutrophil passage through the pulmonary microvasculature is a critical step in the pathogenesis of TRALI, we investigated the impact of HNA-3a antibodies on two important factors that could impair granulocyte passage through lung capillaries: the elasticity of neutrophils and the expression and activation of adhesion molecules. STUDY DESIGN AND METHODS: The impact of HNA-3a antibodies on the elasticity of neutrophils was investigated using atomic force microscopy (AFM). Neutrophils were settled on poly-2-hydroxyethyl-methacrylate-coated glass slides before treatment with anti-HNA-3a plasma samples, control plasma, or control plasma containing formyl-methionyl-leucyl-phenylalanine (fMLP). Elasticity measurements were carried out in a temperature-controlled perfusion chamber using an atomic force microscopy (AFM) device. The impact of HNA-3a antibodies on the surface expression of total CD11b, activation of CD11b, and L-selectin (CD62L) shedding was investigated by flow cytometry. The functional impact of HNA-3a antibodies on neutrophil adhesion was assessed using fibrinogen-coated plates. RESULTS: HNA-3a antibodies induced stiffening of neutrophils (+24%-40%; p < 0.05) to a similar extent as fMLP. This effect was blocked by treatment of neutrophils with cytochalasin D. While total surface expression of CD11b and L-selectin on neutrophils was largely unaffected, HNA-3a antibodies induced alloantigen-specific activation of CD11b (+72%-107%; p < 0.05) and increased adhesion of neutrophils to fibrinogen. CONCLUSION: Accumulation of neutrophils in the pulmonary microvasculature during severe TRALI is likely mediated by increased rigidity and CD11b-mediated adhesion of neutrophils leading to retention of neutrophils.

Pneumococcal Adhesins PavB and PspC Are Important for the Interplay with Human Thrombospondin-1.

The human matricellular glycoprotein thrombospondin-1 (hTSP-1) is released by activated platelets and mediates adhesion of Gram-positive bacteria to various host cells. In staphylococci, the adhesins extracellular adherence protein (Eap) and autolysin (Atl), both surface-exposed proteins containing repeating structures, were shown to be involved in the acquisition of hTSP-1 to the bacterial surface. The interaction partner(s) on the pneumococcal surface was hitherto unknown. Here, we demonstrate for the first time that pneumococcal adherence and virulence factor B (PavB) and pneumococcal surface protein C (PspC) are key players for the interaction of Streptococcus pneumoniae with matricellular hTSP-1. PavB and PspC are pneumococcal surface-exposed adhesins and virulence factors exhibiting repetitive sequences in their core structure. Heterologously expressed fragments of PavB and PspC containing repetitive structures exhibit hTSP-1 binding activity as shown by ELISA and surface plasmon resonance studies. Binding of hTSP-1 is charge-dependent and inhibited by heparin. Importantly, the deficiency in PavB and PspC reduces the recruitment of soluble hTSP-1 by pneumococci and decreases hTSP-1-mediated pneumococcal adherence to human epithelial cells. Platelet activation assays suggested that PavB and PspC are not involved in the activation of purified human platelets by pneumococci. In conclusion, this study indicates a pivotal role of PavB and PspC for pneumococcal recruitment of soluble hTSP-1 to the bacterial surface and binding of pneumococci to host cell-bound hTSP-1 during adhesion.