Interferons prime the endothelium for toll-like receptor-mediated thrombin generation.

BACKGROUND: Respiratory infection is associated with microvascular thrombus formation and marked elevation in cytokine levels. The role of cytokines elaborated by the pulmonary epithelium in thrombotic responses is poorly understood. OBJECTIVES: Our goal was to identify cytokines of pulmonary epithelial cell origin that enhance thrombin generation in the endothelium at concentrations equal to or less than those found in the circulation during infection. METHODS: We screened multiple cytokines produced by the pulmonary epithelium for the ability to enhance toll-like receptor (TLR)-mediated endothelial thrombin generation. Effects of cytokines on tissue factor and thrombomodulin expression, cytokine selectivity for different TLRs, and prothrombotic activity of endogenous cytokines in conditioned medium from pulmonary human epithelial cells were evaluated. RESULTS: MIP-1ß, MCP-1, IL-10, IL-6, IL-1ß, TNFα, IFNα, IFNß, and IFNγ were tested for their ability to enhance TLR3-mediated thrombin generation on endothelial cells. Only interferons (IFNs) and TNFα promoted TLR3-mediated thrombin generation at levels that circulate during infection. IFNs robustly enhanced tissue factor expression when used in conjunction with TLR agonists and reduced thrombomodulin expression in the endothelium independently of TLRs. IFNα, which is typically elevated with viral infection, only synergized with TLR3 agonists mimicking viral pathogen-associated molecular patterns. In contrast, IFNγ, which is typically observed in bacterial infection, synergized more effectively with TLR4 agonists released by bacteria. Conditioned media from inflamed pulmonary epithelial cells primed the endothelium for TLR-mediated thrombin generation. Anti-IFN type I antibodies blocked this effect, indicating that endogenous IFNs prime the endothelium for TLR-mediated thrombin generation. CONCLUSION: IFNs elaborated by the pulmonary epithelium are necessary and sufficient to enhance TLR-mediated thrombin generation.

PACSIN2 regulates platelet integrin ß1 hemostatic function.

BACKGROUND: Upon vessel injury, platelets adhere to exposed matrix constituents via specific membrane receptors, including the von Willebrand factor receptor glycoprotein (GP)Ib-IX-V complex and integrins ß1 and ß3. In platelets, the Fes/CIP4-homology Bin-Amphiphysin-Rvs protein PACSIN2 associates with the cytoskeletal and scaffolding protein filamin A (FlnA), linking GPIbα and integrins to the cytoskeleton. OBJECTIVES: Here we investigated the role of PACSIN2 in platelet function. METHODS: Platelet parameters were evaluated in mice lacking PACSIN2 and platelet integrin ß1. RESULTS: Pacsin2-/- mice displayed mild thrombocytopenia, prolonged bleeding time, and delayed thrombus formation in a ferric chloride-mediated carotid artery injury model, which was normalized by injection of control platelets. Pacsin2-/- platelets formed unstable thrombi that embolized abruptly in a laser-induced cremaster muscle injury model. Pacsin2-/- platelets had hyperactive integrin ß1, as evidenced by increased spreading onto surfaces coated with the collagen receptor α2ß1-specific peptide GFOGER and increased binding of the antibody 9EG7 directed against active integrin ß1. By contrast, Pacsin2-/- platelets had normal integrin αIIbß3 function and expressed P-selectin normally following stimulation through the collagen receptor GPVI or with thrombin. Deletion of platelet integrin ß1 in Pacsin2-/- mice normalized platelet count, hemostasis, and thrombus formation. A PACSIN2 peptide mimicking the FlnA-binding site mediated the pull-down of a FlnA rod 2 construct by integrin ß7, a model for integrin ß-subunits. CONCLUSIONS: Pacsin2-/- mice displayed severe thrombus formation defects due to hyperactive platelet integrin ß1. The data suggest that PACSIN2 binding to FlnA negatively regulates platelet integrin ß1 hemostatic function.

Endocytosis of the thrombopoietin receptor Mpl regulates megakaryocyte and erythroid maturation in mice.

Dnm2fl/fl Pf4-Cre (Dnm2Plt-/- ) mice lacking the endocytic GTPase dynamin 2 (DNM2) in platelets and megakaryocytes (MKs) develop hallmarks of myelofibrosis. At the cellular level, the tyrosine kinase JAK2 is constitutively active but decreased in expression in Dnm2Plt-/- platelets. Additionally, Dnm2Plt-/- platelets cannot endocytose the thrombopoietin (TPO) receptor Mpl, leading to elevated circulating TPO levels. Here, we assessed whether the hyperproliferative phenotype of Dnm2Plt-/- mice was due to JAK2 constitutive activation or to elevated circulating TPO levels. In unstimulated Dnm2Plt-/- platelets, STAT3 and, to a lower extent, STAT5 were phosphorylated, but their phosphorylation was slowed and diminished upon TPO stimulation. We further crossed Dnm2Plt-/- mice in the Mpl-/- background to generate Mpl-/-Dnm2Plt-/- mice lacking Mpl ubiquitously and DNM2 in platelets and MKs. Mpl-/- Dnm2Plt-/- platelets had severely reduced JAK2 and STAT3 but normal STAT5 expression. Mpl-/- Dnm2Plt-/- mice had severely reduced bone marrow MK and hematopoietic stem and progenitor cell numbers. Additionally, Mpl-/- Dnm2Plt-/- mice had severe erythroblast (EB) maturation defects, decreased expression of hemoglobin and heme homeostasis genes and increased expression of ribosome biogenesis and protein translation genes in spleen EBs, and developed anemia with grossly elevated plasma erythropoietin (EPO) levels, leading to early fatality by postnatal day 25. Mpl-/- Dnm2Plt+/+ mice had impaired EB development at three weeks of age, which normalized with adulthood. Together, the data shows that DNM2-dependent Mpl-mediated endocytosis in platelets and MKs is required for steady-state hematopoiesis and provides novel insights into a developmentally controlled role for Mpl in normal erythropoiesis, regulating hemoglobin and heme production.

Exposure to fine particulate matter (PM2.5) during landscape fire events and the risk of cardiorespiratory emergency department attendances: a time-series study in Perth, Western Australia.

BACKGROUND: Landscape fires (LFs) are the main source of elevated particulate matter (PM2.5) in Australian cities and towns. This study examined the associations between daily exposure to fine PM2.5 during LF events and daily emergency department attendances (EDA) for all causes, respiratory and cardiovascular outcomes. METHODS: Daily PM2.5 was estimated using a model that included PM2.5 measurements on the previous day, remotely sensed aerosols and fires, hand-drawn tracing of smoke plumes from satellite images, fire danger ratings and the atmosphere venting index. Daily PM2.5 was then categorised as high (≥99th percentile), medium (96th-98th percentile) and low (≤95th percentile). Daily EDA for all-cause and cardiorespiratory conditions were obtained from the Western Australian Emergency Department Data Collection. We used population-based cohort time-series multivariate regressions with 95% CIs to assess modelled daily PM2.5 and EDA associations from 2015 to 2017. We estimated the lag-specific associations and cumulative risk ratios (RR) at lags of 0-3 days, adjusted for sociodemographic factors, weather and time. RESULTS: All-cause EDA and overall cardiovascular presentations increased on all lagged days and up to 5% (RR 1.05, 95% CI 1.03 to 1.06) and 7% (RR 1.07, 95% CI 1.01 to 1.12), respectively, at the high level. High-level exposure was also associated with increased acute lower respiratory tract infections at 1 (RR 1.19, 95% CI 1.10 to 1.29) and 3 (RR 1.17, 95% CI 1.10 to 1.23) days lags and transient ischaemic attacks at 1 day (RR 1.25, 95% CI 1.02 to 1.53) and 2 (RR 1.20, 95% CI 1.01 to 1.42) days lag. CONCLUSIONS: Exposure to PM2.5 concentrations during LFs was associated with an increased risk of all-cause EDA, overall EDA cardiovascular diseases, acute respiratory tract infections and transient ischaemic attacks.

Bleeding diathesis in mice lacking JAK2 in platelets.

The tyrosine kinase JAK2 is a critical component of intracellular JAK/STAT cytokine signaling cascades that is prevalent in hematopoietic cells, such as hematopoietic stem cells and megakaryocytes (MKs). Individuals expressing the somatic JAK2 V617F mutation commonly develop myeloproliferative neoplasms (MPNs) associated with venous and arterial thrombosis, a leading cause of mortality. The role of JAK2 in hemostasis remains unclear. We investigated the role of JAK2 in platelet hemostatic function using Jak2fl/fl Pf4-Cre (Jak2Plt-/-) mice lacking JAK2 in platelets and MKs. Jak2Plt-/- mice developed MK hyperplasia and splenomegaly associated with severe thrombocytosis and bleeding. This notion was supported by failure to occlude in a ferric chloride carotid artery injury model and by a cremaster muscle laser-induced injury assay, in which Jak2Plt-/- platelets failed to form stable thrombi. Jak2Plt-/- platelets formed thrombi poorly after adhesion to type 1 collagen under arterial shear rates. Jak2Plt-/- platelets spread poorly on collagen under static conditions or on fibrinogen in response to the collagen receptor GPVI-specific agonist, collagen-related peptide (CRP). After activation with collagen, CRP, or the CLEC-2 agonist rhodocytin, Jak2Plt-/- platelets displayed decreased α-granule secretion and integrin αIIbß3 activation or aggregation, but showed normal responses to thrombin. Jak2Plt-/- platelets had impaired intracellular signaling when activated via GPVI, as assessed by tyrosine phosphorylation. Together, the results show that JAK2 deletion impairs platelet immunoreceptor tyrosine-based activation motif signaling and hemostatic function in mice and suggest that aberrant JAK2 signaling in patients with MPNs affects GPVI signaling, leading to hemostatic platelet function.

Antiplatelet properties of Pim kinase inhibition are mediated through disruption of thromboxane A2 receptor signaling.

Pim kinases are upregulated in several forms of cancer, contributing to cell survival and tumour development, but their role in platelet function and thrombotic disease has not been explored. We report for the first time that Pim-1 is expressed in human and mouse platelets. Genetic deletion or pharmacological inhibition of Pim kinase results in reduced thrombus formation but is not associated with impaired haemostasis. Attenuation of thrombus formation was found to be due to inhibition of the thromboxane A2 receptor as effects on platelet function was non-additive to inhibition caused by the cyclooxygenase inhibitor indomethacin or thromboxane A2 receptor antagonist GR32191. Treatment with Pim kinase inhibitors caused reduced surface expression of the thromboxane A2 receptor and resulted in reduced responses to thromboxane A2 receptor agonists, indicating a role for Pim kinase in the regulation of thromboxane A2 receptor function. Our research identifies a novel, Pim kinase dependent regulatory mechanism for the thromboxane A2 receptor and represents a new targeting strategy that is independent of COX-1 inhibition or direct antagonism of the thromboxane A2 receptor that whilst attenuating thrombosis does not increase bleeding.

Dynamin 2 is required for GPVI signaling and platelet hemostatic function in mice.

Receptor-mediated endocytosis, which contributes to a wide range of cellular functions, including receptor signaling, cell adhesion, and migration, requires endocytic vesicle release by the large GTPase dynamin 2. Here, the role of dynamin 2 was investigated in platelet hemostatic function using both pharmacological and genetic approaches. Dnm2fl/fl Pf4-Cre (Dnm2Plt - / -) mice specifically lacking dynamin 2 within the platelet lineage developed severe thrombocytopenia and bleeding diathesis and Dnm2Plt - / - platelets adhered poorly to collagen under arterial shear rates. Signaling via the collagen receptor GPVI was impaired in platelets treated with the dynamin GTPase inhibitor dynasore, as evidenced by poor protein tyrosine phosphorylation, including that of the proximal tyrosine kinase Lyn on its activating tyrosine 396 residue. Platelet stimulation via GPVI resulted in a slight decrease in GPVI, which was maintained by dynasore treatment. Dynasore-treated platelets had attenuated function when stimulated via GPVI, as evidenced by reduced GPIbα downregulation, α-granule release, integrin αIIbß3 activation, and spreading onto immobilized fibrinogen. By contrast, responses to the G-protein coupled receptor agonist thrombin were minimally affected by dynasore treatment. GPVI expression was severely reduced in Dnm2Plt-/- platelets, which were dysfunctional in response to stimulation via GPVI, and to a lesser extent to thrombin. Dnm2Plt-/- platelets lacked fibrinogen in their α-granules, but retained von Willebrand factor. Taken together, the data show that dynamin 2 plays a proximal role in signaling via the collagen receptor GPVI and is required for fibrinogen uptake and normal platelet hemostatic function.

Carbonic Anhydrases Function in Anther Cell Differentiation Downstream of the Receptor-Like Kinase EMS1.

Plants extensively employ leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest family of RLKs, to control a wide range of growth and developmental processes as well as defense responses. To date, only a few direct downstream effectors for LRR-RLKs have been identified. We previously showed that the LRR-RLK EMS1 (EXCESS MICROSPOROCYTES1) and its ligand TPD1 (TAPETUM DETERMINANT1) are required for the differentiation of somatic tapetal cells and reproductive microsporocytes during early anther development in Arabidopsis thaliana Here, we report the identification of ß-carbonic anhydrases (ßCAs) as the direct downstream targets of EMS1. EMS1 biochemically interacts with ßCA proteins. Loss of function of ßCA genes caused defective tapetal cell differentiation, while overexpression of ßCA1 led to the formation of extra tapetal cells. EMS1 phosphorylates ßCA1 at four sites, resulting in increased ßCA1 activity. Furthermore, phosphorylation-blocking mutations impaired the function of ßCA1 in tapetal cell differentiation; however, a phosphorylation mimic mutation promoted the formation of tapetal cells. ßCAs are also involved in pH regulation in tapetal cells. Our findings highlight the role of ßCA in controlling cell differentiation and provide insights into the posttranslational modification of carbonic anhydrases via receptor-like kinase-mediated phosphorylation.