Tovo David

Alternatively activated macrophages produce catecholamines to sustain adaptive thermogenesis

All homeotherms use thermogenesis to maintain their core body temperature, ensuring that cellular functions and physiological processes can continue in cold environments. In the prevailing model of thermogenesis, when the hypothalamus senses cold temperatures it triggers sympathetic discharge, resulting in the release of noradrenaline in brown adipose tissue and white adipose tissue. Acting via the β3-adrenergic receptors, noradrenaline induces lipolysis in white adipocytes, whereas it stimulates the expression of thermogenic genes, such as PPAR-γ coactivator 1a (Ppargc1a), uncoupling protein 1 (Ucp1) and acyl-CoA synthetase long-chain family member 1 (Acsl1), in brown adipocytes. However, the precise nature of all the cell types involved in this efferent loop is not well established. Here we report in mice an unexpected requirement for the interleukin-4 (IL-4)-stimulated program of alternative macrophage activation in adaptive thermogenesis. Exposure to cold temperature rapidly promoted alternative activation of adipose tissue macrophages, which secrete catecholamines to induce thermogenic gene expression in brown adipose tissue and lipolysis in white adipose tissue. Absence of alternatively activated macrophages impaired metabolic adaptations to cold, whereas administration of IL-4 increased thermogenic gene expression, fatty acid mobilization and energy expenditure, all in a macrophage-dependent manner. Thus, we have discovered a role for alternatively activated macrophages in the orchestration of an important mammalian stress response, the response to cold.

TMEM16F Forms a Ca2+-Activated Cation Channel Required for Lipid Scrambling in Platelets during Blood Coagulation

Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca(2+)-activated Cl(-) channels (CaCCs) is linked to Scott syndrome with deficient Ca(2+)-dependent lipid scrambling. We generated TMEM16F knockout mice that exhibit bleeding defects and protection in an arterial thrombosis model associated with platelet deficiency in Ca(2+)-dependent phosphatidylserine exposure and procoagulant activity and lack a Ca(2+)-activated cation current in the platelet precursor megakaryocytes. Heterologous expression of TMEM16F generates a small-conductance Ca(2+)-activated nonselective cation (SCAN) current with subpicosiemens single-channel conductance rather than a CaCC. TMEM16F-SCAN channels permeate both monovalent and divalent cations, including Ca(2+), and exhibit synergistic gating by Ca(2+) and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a Ca(2+)-activated channel permeable to Ca(2+) and critical for Ca(2+)-dependent scramblase activity during blood coagulation. PAPERFLICK:

The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors

Platelets are critical for haemostasis, thrombosis, and inflammatory responses, but the events that lead to mature platelet production remain incompletely understood. The bone marrow has been proposed to be a major site of platelet production, although there is indirect evidence that the lungs might also contribute to platelet biogenesis. Here, by directly imaging the lung microcirculation in mice, we show that a large number of megakaryocytes circulate through the lungs, where they dynamically release platelets. Megakaryocytes that release platelets in the lungs originate from extrapulmonary sites such as the bone marrow; we observed large megakaryocytes migrating out of the bone marrow space. The contribution of the lungs to platelet biogenesis is substantial, accounting for approximately 50% of total platelet production or 10 million platelets per hour. Furthermore, we identified populations of mature and immature megakaryocytes along with haematopoietic progenitors in the extravascular spaces of the lungs. Under conditions of thrombocytopenia and relative stem cell deficiency in the bone marrow, these progenitors can migrate out of the lungs, repopulate the bone marrow, completely reconstitute blood platelet counts, and contribute to multiple haematopoietic lineages. These results identify the lungs as a primary site of terminal platelet production and an organ with considerable haematopoietic potential.

Carcinoma mucins trigger reciprocal activation of platelets and neutrophils in a murine model of Trousseau syndrome

Trousseau syndrome is classically defined as migratory, heparin-sensitive but warfarin-resistant microthrombi in patients with occult, mucinous adenocarcinomas. Injecting carcinoma mucins into mice generates platelet-rich microthrombi dependent on P- and L-selectin but not thrombin. Heparin prevents mucin binding to P- and L-selectin and mucin-induced microthrombi. This model of Trousseau syndrome explains resistance to warfarin, which inhibits fluid-phase coagulation but not selectins. Here we found that carcinoma mucins do not generate microthrombi in mice lacking P-selectin glycoprotein ligand-1 (PSGL-1), the leukocyte ligand for P- and L-selectin. Furthermore, mucins did not activate platelets in blood from PSGL-1-deficient mice. Mucins induced microthrombi in radiation chimeras lacking endothelial P-selectin but not in chimeras lacking platelet P-selectin. Mucins caused leukocytes to release cathepsin G, but only if platelets were present. Mucins failed to generate microthrombi in cathepsin G-deficient mice. Mucins did not activate platelets in blood from mice lacking cathepsin G or protease-activated receptor-4 (PAR4), indicating that cathepsin G activates platelets through PAR4. Using knockout mice and blocking antibodies, we found that mucin-triggered cathepsin G release requires L-selectin and PSGL-1 on neutrophils, P-selectin on platelets, and Src family kinases in both cell types. Thus, carcinoma mucins promote thrombosis through adhesion-dependent, bidirectional signaling in neutrophils and platelets.

Decreased Thrombotic Tendency in Mouse Models of the Bernard-Soulier Syndrome

Objective—The platelet glycoprotein (GP)Ib-V-IX complex is a receptor required for normal hemostasis deficient in the Bernard-Soulier bleeding disorder. To evaluate the consequences of GPIb-V-IX deficiency in thrombosis we generated mouse models of the disease by targeting the GPIb&bgr; subunit. Methods and Results—Complete deletion (GPIb&bgr;−/−) or an intracellular truncation (GPIb&bgr;&Dgr;IC−/−) reproduced typical and variant forms of Bernard-Soulier, with absent and partial (20%) expression of the complex on the platelet surface. Both strains exhibited thrombocytopenia and enlarged platelets with abnormal microtubular structures but normal granule composition. They exhibited prolonged tail bleeding times, which were less pronounced in GPIb&bgr;&Dgr;IC−/−. Decreased thrombus formation was observed after blood perfusion over a collagen coated surface at high shear. Resistance to vascular occlusion and an abnormal thrombus composition were observed in a model of FeCl3-induced lesion of carotid arteries. In a model of laser-induced lesion of mesenteric arterioles, thrombosis was strongly reduced in GPIb&bgr;−/− mice, while a more modest effect was observed in GPIb&bgr;&Dgr;IC−/− animals. Finally, the two strains were protected against death in a model of systemic thromboembolism. Conclusions—This study provides in vivo evidence of a decreased thrombotic tendency linked to defective platelet GPIb-V-IX in mouse models of Bernard-Soulier syndrome.

Roles and interactions among protease-activated receptors and P2ry12 in hemostasis and thrombosis

Toward understanding their redundancies and interactions in hemostasis and thrombosis, we examined the roles of thrombin receptors (protease-activated receptors, PARs) and the ADP receptor P2RY12 (purinergic receptor P2Y G protein-coupled 12) in human and mouse platelets ex vivo and in mouse models. Par3−/− and Par4+/− mouse platelets showed partially decreased responses to thrombin, resembling those in PAR1 antagonist-treated human platelets. P2ry12+/− mouse platelets showed partially decreased responses to ADP, resembling those in clopidogrel-treated human platelets. Par3−/− mice showed nearly complete protection against carotid artery thrombosis caused by low FeCl3 injury. Par4+/− and P2ry12+/− mice showed partial protection. Increasing FeCl3 injury abolished such protection; combining partial attenuation of thrombin and ADP signaling, as in Par3−/−:P2ry12+/− mice, restored it. Par4−/− mice, which lack platelet thrombin responses, showed still better protection. Our data suggest that (i) the level of thrombin driving platelet activation and carotid thrombosis was low at low levels of arterial injury and increased along with the contribution of thrombin-independent pathways of platelet activation with increasing levels of injury; (ii) although P2ry12 acts downstream of PARs to amplify platelet responses to thrombin ex vivo, P2ry12 functioned in thrombin/PAR-independent pathways in our in vivo models; and (iii) P2ry12 signaling was more important than PAR signaling in hemostasis models; the converse was noted for arterial thrombosis models. These results make predictions being tested by ongoing human trials and suggest hypotheses for new antithrombotic strategies.

Mice expressing a mutant form of fibrinogen that cannot support fibrin formation exhibit compromised antimicrobial host defense

Fibrin(ogen) is central to hemostasis and thrombosis and also contributes to multiple physiologic and pathologic processes beyond coagulation. However, the precise contribution of soluble fibrinogen vs insoluble fibrin matrices to vascular integrity, tissue repair, inflammation, and disease has been undefined and unapproachable. To establish the means to distinguish fibrinogen- and fibrin-dependent processes in vivo, Fib(AEK) mice were generated that carry normal levels of circulating fibrinogen but lack the capacity for fibrin polymer formation due to a germ-line mutation in the Aα chain thrombin cleavage site. Homozygous Fib(AEK) mice developed to term and exhibited postnatal survival superior to that of fibrinogen-deficient mice. Unlike fibrinogen-deficient mice, platelet-rich plasma from Fib(AEK) mice supported normal platelet aggregation in vitro, highlighting that fibrinogen(AEK) retains the functional capacity to support interactions with platelets. Thrombin failed to release fibrinopeptide-A from fibrinogen(AEK) and failed to induce polymer formation with Fib(AEK) plasma or purified fibrinogen(AEK) in 37°C mixtures regardless of incubation time. Fib(AEK) mice displayed both an absence of fibrin polymer formation following liver injury, as assessed by electron microscopy, and a failure to generate stable occlusive thrombi following FeCl3 injury of carotid arteries. Fib(AEK) mice exhibited a profound impediment in Staphylococcus aureus clearance following intraperitoneal infection similar to fibrinogen-deficient mice, yet Fib(AEK) mice displayed a significant infection dose-dependent survival advantage over fibrinogen-deficient mice following peritonitis challenge. Collectively, these findings establish for the first time that fibrin polymer is the molecular form critical for antimicrobial mechanisms while simultaneously highlighting biologically meaningful contributions and functions of the soluble molecule.

Synthesis of GPIbβ with novel transmembrane and cytoplasmic sequences in a Bernard–Soulier patient resulting in GPIb-defective signaling in CHO cells

Summary.  The molecular defect of a new Bernard–Soulier patient, originating from Morocco and presenting thrombocytopenia with large platelets and an absence of ristocetin‐induced platelet agglutination, has been identified and reproduced in transfected heterologous cells. Gene sequencing revealed insertion of a guanine in the domain coding for the transmembrane region of the glycoprotein (GP) Ibβ subunit. This mutation causes a translational frame shift, which creates putative novel transmembrane and cytoplasmic 37 and 125 amino acids domains, respectively. A 34 kDa immunoreactive GPIbβ band, instead of the normal 26 kDa subunit, was detected by Western blotting in lysates from the patients platelets and from transfected cells and in immunoprecipitates of metabolically labeled cells. The abnormal subunit did not associate with GPIbα and was mainly intracellular, although a significant fraction could reach the cell surface. Cells expressing the mutant GPIb–IX complex adhered to a von Willebrand factor matrix but were unable to change shape, unlike cells expressing the wild‐type receptor. These results strongly suggest a novel role of the GPIbβ subunit and its transmembrane‐intracellular region in GPIb‐VWF‐dependent signaling, in addition to a role in correct assembly and cell surface targeting of the GPIb–V–IX complex.

Identification of five novel 14-3-3 isoforms interacting with the GPIb-IX complex in platelets.

Summary.  Background: Binding of von Willebrand factor to the platelet glycoprotein (GP)Ib–IX complex initiates a signaling cascade leading to integrin αIIbβ3 activation, a key process in hemostasis and thrombosis. Interaction of 14‐3‐3ζ with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway. Objective: The aim of our study was to determine whether other members of the 14‐3‐3 family bind to GPIb–IX. Results: In this study, western blot analyses showed that platelets also contain the 14‐3‐3β, 14‐3‐3γ, 14‐3‐3ε, 14‐3‐3η and 14‐3‐3θ isoforms, but lack 14‐3‐3σ. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14‐3‐3 isoforms expressed in platelets, including, as previously reported, 14‐3‐3ζ, bind to GPIb–IX. In addition, their interaction was found to critically require the same GPIbα domains (580–590 and 605–610) already identified as essential for 14‐3‐3ζ binding, in agreement with the conservation of the sequence of the I‐helix among these different isoforms. Pull‐down experiments indicated that all six 14‐3‐3 isoforms present in platelets bind to GPIbβ. In contrast, deletion or mutation of the GPIbβ intracytoplasmic tail did not affect the interaction of GPIb–IX with the 14‐3‐3 isoforms, questioning the importance of this domain. Conclusions: Our study suggests that, to inhibit GPIb‐induced integrin αIIbβ3 activation, a more appropriate strategy than inhibiting individual 14‐3‐3 isoforms would be to target the 14‐3‐3‐binding motif on GPIb or, alternatively, the conserved 14‐3‐3 I‐helix.

Inhibition of adhesive and signaling functions of the platelet GPIb‐V‐IX complex by a cell penetrating GPIbα peptide

Summary.  Background: Interaction between the platelet glycoprotein (GP)Ib‐V‐IX complex and von Willebrand factor (VWF) is critical for initiating platelet‐vessel wall contacts, particularly under high shear conditions. This interaction also plays an important role in initiating platelet activation through the generation of intracellular signals resulting in platelet shape change and integrin αIIbβ3 activation. Objective: A cell‐penetrating peptide strategy was used to study the role of the intracellular domain of the GPIbα subunit in VWF/GPIb‐V‐IX‐dependent adhesion and activation. Methods: Peptides of 11–13 amino acids, covering the 557–610 region, were coupled to a nine‐arginine permeating tag (R9) and the effects of their cell entry on VWF‐dependent responses were analyzed. Results: The R9α557 peptide corresponding to the 557–569 segment reduced platelet agglutination in response to VWF, while the other peptides had no effect. The decreased platelet agglutination appeared to be an indirect consequence of adenosine diphosphate release as a normal response was restored by apyrase or a P2Y1 receptor antagonist. A more direct effect of R9α557 on GPIb VWF‐dependent functions was observed in adhesion studies on a VWF matrix, where it decreased platelet adhesion and profoundly inhibited filopodia formation. In addition, cell adhesion was reduced and shape change absent when Chinese hamster ovary cells expressing the GPIb‐IX complex were incubated with R9α557. Conclusion: This study performed in intact platelets suggests a functional role of the 557–569 domain of GPIbα in controlling VWF‐dependent adhesion and signaling.