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Store-operated [Ca.sup.2+] entry (SOCE) is the major route of [Ca.sup.2+] influx in platelets. The [Ca.sup.2+] sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the [Ca.sup.2+] channel Orai1 and the inositol trisphosphate receptor ([IP.sub.3]R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and [IP.sub.3]R in platelets. Deletion of platelet BIN2 ([Bin2.sup.fl/fl,Pf4,Cre] mice) resulted in reduced [Ca.sup.2+] store release and [Ca.sup.2+] influx in response to all tested platelet agonists. These defects were a consequence of impaired [IP.sub.3]R function in combination with defective STIM1-mediated SOC channel activation, while [Ca.sup.2+] store content and agonist-induced [IP.sub.3] production were unaltered. This severely defective [Ca.sup.2+] signaling translated into impaired thrombus formation under flow and a protection of [Bin2.sup.fl/fl,Pf4,Cre] mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo- inflammatory disease settings., Introduction Platelet activation and aggregation at the injured vessel wall is essential for primary hemostasis, but may also trigger occlusion of diseased vessels, resulting in acute ischemia and infarction of [...]

Hirschsprung disease (HSCR, OMIM 142623) involves congenital intestinal obstruction caused by dysfunction of neural crest cells and their progeny during enteric nervous system (ENS) development. HSCR is a multifactorial disorder; pathogenetic variants accounting for disease phenotype are identified only in a minority of cases, and the identification of novel disease-relevant genes remains challenging. In order to identify and to validate a potential disease-causing relevance of novel HSCR candidate genes, we established a complementary study approach, combining whole exome sequencing (WES) with transcriptome analysis of murine embryonic ENS-related tissues, literature and database searches, in silico network analyses, and functional readouts using candidate gene-specific genome-edited cell clones. WES datasets of two patients with HSCR and their non-affected parents were analysed, and four novel HSCR candidate genes could be identified: ATP7A, SREBF1, ABCD1 and PIAS2. Further rare variants in these genes were identified in additional HSCR patients, suggesting disease relevance. Transcriptomics revealed that these genes are expressed in embryonic and fetal gastrointestinal tissues. Knockout of these genes in neuronal cells demonstrated impaired cell differentiation, proliferation and/or survival. Our approach identified and validated candidate HSCR genes and provided further insight into the underlying pathomechanisms of HSCR. Author summary: Hirschsprung disease (HSCR) is a rare developmental disorder. It leads to the absence of enteric nerve cells (aganglionosis) in the large intestine and is caused by functional defects of neuronal precursor cells during embryonic development of the gut nervous system. The aganglionosis manifests as a variety of symptoms including impaired peristalsis and the formation of a pathogenic dilatation of the intestine (megacolon). The etiology of HSCR is considered to be multifactorial. Variants in more than 20 genes have been reported to be overrepresented in HSCR and replicated in independent cohorts. However, variants in those risk genes account for only 30% of all cases, suggesting that many more genes have to be implicated in the development of HSCR. As the identification and the subsequent validation of novel gene variants to be disease-causing or not, still remains a major challenge, we established and applied a complementary study pipeline. This enabled us to identify four novel candidate genes in two HSCR patients and to validate their potential disease relevance. Our approach represents a suitable way to dissect the complex genetic architecture underlying HSCR. [ABSTRACT FROM AUTHOR]

Platelets are essential for normal hemostasis; however, pathological conditions can also trigger unwanted platelet activation precipitating thrombosis and ischemic damage of vital organs such as the heart or brain. Glycoprotein (GP)VI- and C-type lectin-like receptor 2 (CLEC-2)-mediated (hem)immunoreceptor tyrosine-based activation motif (ITAM) signaling represents a major pathway for platelet activation. The two members of the Growth-factor receptor-bound protein 2 (Grb2) family of adapter proteins expressed in platelets – Grb2 and Grb2-related adapter protein downstream of Shc (Gads) – are part of the hem(ITAM) signaling cascade by forming an adapter protein complex with linker for activation of T cells (LAT). To date, a possible functional redundancy between these two adapters in platelet activation has not been investigated. We here generated megakaryocyte- and platelet-specific Grb2/Gads double knockout (DKO) mice and analyzed their platelet function in vitro and in vivo. The DKO platelets exhibited virtually abolished (hem)ITAM signaling whereas only partial defects were seen in Grb2 or Gads single-deficient platelets. This was based on impaired phosphorylation of key molecules in the (hem)ITAM signaling cascade and translated into impaired hemostasis and partially defective arterial thrombosis, thereby exceeding the defects in either Grb2 KO or Gads KO mice. Despite this severe (hem)ITAM signaling defect, CLEC-2 dependent regulation of blood-lymphatic vessel separation was not affected in the DKO animals. These results provide direct evidence for critically redundant roles of Grb2 and Gads for platelet function in hemostasis and thrombosis, but not development. [ABSTRACT FROM AUTHOR]

Maintenance of tumor vasculature integrity is indispensable for tumor growth and thus affects tumor progression. Previous studies have identified platelets as major regulators of tumor vascular integrity, as their depletion selectively rendered tumor vessels highly permeable and caused massive intratumoral hemorrhage. While these results established platelets as potential targets for antitumor therapy, their depletion is not a treatment option due to their essential role in hemostasis. Thus, a detailed understanding of how platelets safeguard vascular integrity in tumors is urgently demanded. Here, we show for the first time that functional inhibition of glycoprotein VI (GPVI) on the platelet surface with an antibody (JAQ1) F(ab) 2 fragment rapidly induces tumor hemorrhage and diminishes tumor growth similar to complete platelet depletion while not inducing systemic bleeding complications. The intratumor bleeding and tumor growth arrest could be reverted by depletion of Ly6G + cells, confirming them to be responsible for the induction of bleeding and necrosis within the tumor. In addition, JAQ1 F(ab) 2 -mediated GPVI inhibition increased intratumoral accumulation of coadministered chemotherapeutic agents, such as Doxil and paclitaxel, thereby resulting in a profound antitumor effect. In summary, our findings identify platelet GPVI as a key regulator of vascular integrity specifically in growing tumors and could serve as a basis for the development of antitumor strategies based on the interference with platelet function., (© 2019 by The American Society of Hematology.)

The proinflammatory microenvironment in the respiratory airway induces maturation of both resident and infiltrating dendritic cells (DCs) upon influenza A virus (IAV) infection. This results in upregulation of antiviral pathways as well as modulation of endocytic processes, which affect the susceptibility of DCs to IAV infection. Therefore, it is highly relevant to understand how IAV interacts with and infects mature DCs. To investigate how different subsets of human myeloid DCs (MDCs) involved in tissue inflammation are affected by inflammatory stimulation during IAV infection, we stimulated primary blood MDCs and inflammatory monocyte-derived DCs (MDDCs) with TLR ligands, resulting in maturation. Interestingly, MDDCs but not MDCs were protected against IAV infection after LPS (TLR4) stimulation. In contrast, stimulation with TLR7/8 ligand protected MDCs but not MDDCs from IAV infection. The reduced susceptibility to IAV infection correlated with induction of type I IFNs. We found that differential expression of TLR4, TRIF, and MyD88 in the two MDC subsets regulated the ability of the cells to enter an antiviral state upon maturation. This difference was functionally confirmed using small interfering RNA and inhibitors. Our data show that different human MDC subsets may play distinct roles during IAV infection, as their capacity to induce type I IFNs is dependent on TLR-specific maturation, resulting in differential susceptibility to IAV infection., (Copyright © 2015 by The American Association of Immunologists, Inc.)