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Direct imaging of the lung microcirculation in mice indicates that it is a major site of mature platelet production from megakaryocytes. A breath of fresh blood Platelets, which are essential for generating clots to stop bleeding, are produced to a large extent in the bone marrow, but indirect evidence points to a pulmonary contribution to their genesis. Emma Lefrançais et al . imaged microcirculation in the lungs of mice and report that the lung serves as a reservoir for haematopoietic progenitors and platelet-releasing megakaryocytes that can be recruited to compensate for bone marrow deficiencies. They estimate that the lung is responsible for around 50% of total platelet production in mice, a finding that positions it as an organ of notable haematopoietic potential. Platelets are critical for haemostasis, thrombosis, and inflammatory responses 1 , 2 , but the events that lead to mature platelet production remain incompletely understood 3 . 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 4 , 5 , 6 , 7 . Here, by directly imaging the lung microcirculation in mice 8 , 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 9 , 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.

Protease-activated receptor 1 (PAR1) is the prototypical member of a family of G-protein-coupled receptors that mediate cellular responses to thrombin and related proteases. Thrombin irreversibly activates PAR1 by cleaving the amino-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the heptahelical bundle of the receptor to affect G-protein activation. Here we report the 2.2-Å-resolution crystal structure of human PAR1 bound to vorapaxar, a PAR1 antagonist. The structure reveals an unusual mode of drug binding that explains how a small molecule binds virtually irreversibly to inhibit receptor activation by the tethered ligand of PAR1. In contrast to deep, solvent-exposed binding pockets observed in other peptide-activated G-protein-coupled receptors, the vorapaxar-binding pocket is superficial but has little surface exposed to the aqueous solvent. Protease-activated receptors are important targets for drug development. The structure reported here will aid the development of improved PAR1 antagonists and the discovery of antagonists to other members of this receptor family. The X-ray crystal structure of the human G-protein-coupled receptor protease-activated receptor 1 (PAR1) bound to the antagonist vorapaxar is solved, revealing an unusual method of drug binding that should facilitate the development of improved PAR1-selective antagonists. Thrombin receptor structure The X-ray crystal structure of the human protease-activated receptor 1 (PAR1) bound to vorapaxar, a PAR1 antagonist, has been determined at 2.2 Å resolution. PAR1, also known as the thrombin receptor, is a G protein-coupled receptor that mediates cellular responses to the coagulation protease thrombin and related proteases. Vorapaxar was recently shown to prevent myocardial infarction in at-risk patients, and knowledge of the PAR structure will be relevant to the design of PAR1 antagonists with better drug properties.

Activation of protease-activated receptor-2 (PAR2) has been implicated in inflammation, pruritus, and skin barrier regulation, all characteristics of atopic dermatitis (AD), as well as Netherton syndrome which has similar characteristics. However, understanding the precise role of PAR2 on neuro-immune communication in AD has been hampered by the lack of appropriate animal models. We used a recently established mouse model with epidermal overexpression of PAR2 (PAR2OE) and littermate WT mice to study the impact of increased PAR2 expression in epidermal cells on spontaneous and house dust mite (HDM)-induced skin inflammation, itch, and barrier dysfunction in AD, and . PAR2OE newborns displayed no overt abnormalities, but spontaneously developed dry skin, severe pruritus, and eczema. Dermatological, neurophysiological, and immunological analyses revealed the hallmarks of AD-like skin disease. Skin barrier defects were observed before onset of skin lesions. Application of HDM onto PAR2OE mice triggered pruritus and the skin phenotype. PAR2OE mice displayed an increased density of nerve fibers, increased nerve growth factor and endothelin-1 expression levels, alloknesis, enhanced scratching (hyperknesis), and responses of dorsal root ganglion cells to non-histaminergic pruritogens. PAR2 in keratinocytes, activated by exogenous and endogenous proteases, is sufficient to drive barrier dysfunction, inflammation, and pruritus and sensitize skin to the effects of HDM in a mouse model that mimics human AD. PAR2 signaling in keratinocytes appears to be sufficient to drive several levels of neuro-epidermal communication, another feature of human AD.

Atrial fibrillation (AF), the most common arrhythmia, is a growing epidemic with substantial morbidity and economic burden. Mechanisms underlying vulnerability to AF remain poorly understood, which contributes to the current lack of highly effective therapies. Recognizing mechanistic subtypes of AF may guide an individualized approach to patient management. Here, we describe a family with a previously unreported syndrome characterized by early-onset AF (age <35 years), conduction disease and signs of a primary atrial myopathy. Phenotypic penetrance was complete in all mutation carriers, although complete disease expressivity appears to be age-dependent. We show that this syndrome is caused by a novel, heterozygous p.Glu11Lys mutation in the atrial-specific myosin light chain gene MYL4 . In zebrafish, mutant MYL4 leads to disruption of sarcomeric structure, atrial enlargement and electrical abnormalities associated with human AF. These findings describe the cause of a rare subtype of AF due to a primary, atrial-specific sarcomeric defect. Here, Michael Gollob and colleagues perform a whole exome sequencing study to identify a mutation in the atrial-specific myosin light chain gene MYL4 in a small family with autosomal dominant familial atrial fibrillation. They also test the functionality of this MYL4 mutation in zebrafish cardiac function and recapitulate disease-related phenotypes.

Maintenance of vascular integrity is critical for homeostasis, and temporally and spatially regulated vascular leak is a central feature of inflammation. Sphingosine-1-phosphate (S1P) can regulate endothelial barrier function, but the sources of the S1P that provide this activity in vivo and its importance in modulating different inflammatory responses are unknown. We report here that mutant mice engineered to selectively lack S1P in plasma displayed increased vascular leak and impaired survival after anaphylaxis, administration of platelet-activating factor (PAF) or histamine, and exposure to related inflammatory challenges. Increased leak was associated with increased interendothelial cell gaps in venules and was reversed by transfusion with wild-type erythrocytes (which restored plasma S1P levels) and by acute treatment with an agonist for the S1P receptor 1 (S1pr1). S1pr1 agonist did not protect wild-type mice from PAF-induced leak, consistent with plasma S1P levels being sufficient for S1pr1 activation in wild-type mice. However, an agonist for another endothelial cell Gi-coupled receptor, Par2, did protect wild-type mice from PAF-induced vascular leak, and systemic treatment with pertussis toxin prevented rescue by Par2 agonist and sensitized wild-type mice to leak-inducing stimuli in a manner that resembled the loss of plasma S1P. Our results suggest that the blood communicates with blood vessels via plasma S1P to maintain vascular integrity and regulate vascular leak. This pathway prevents lethal responses to leak-inducing mediators in mouse models.

The environmental cues involved in regulating germinal center size are not fully understood. Cyster and colleagues show that the sphingosine 1-phosphate receptor S1P 2 controls the survival and localization of B cells in germinal centers by antagonizing signaling by the kinase Akt and follicular chemoattractants. Mice deficient in sphingosine 1-phosphate receptor type 2 (S1P 2 ) develop diffuse large B cell lymphoma. However, the role of S1P 2 in normal germinal center (GC) physiology is unknown. Here we show that S1P 2 -deficient GC B cells outgrew their wild-type counterparts in chronically established GCs. We found that antagonism of the kinase Akt mediated by S1P 2 and its downstream mediators Gα 12 , Gα 13 and p115RhoGEF regulated cell viability and was required for growth control in chronically proliferating GCs. Moreover, S1P 2 inhibited GC B cell responses to follicular chemoattractants and helped confine cells to the GC. In addition, S1P 2 overexpression promoted the centering of activated B cells in the follicle. We suggest that by inhibiting Akt activation and migration, S1P 2 helps restrict GC B cell survival and localization to an S1P-low niche at the follicle center.

A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris- N -nitrilotriacetic acid (tris-NTA) group binding to a His 10 -tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution. Functional analysis of membrane proteins would benefit from the combination of imaging with ligand characterisation. Here, Pfreundschuh et al . use specialised atomic force microscopy tips to image and quantify the binding of two ligands at the same time.

How does a protease act like a hormone to regulate cellular functions? The coagulation protease thrombin (EC 3.4.21.5) activates platelets and regulates the behavior of other cells by means of G protein-coupled protease-activated receptors (PARs). PAR1 is activated when thrombin binds to and cleaves its amino-terminal exodomain to unmask a new receptor amino terminus. This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body of the receptor to effect transmembrane signaling. The irreversibility of PAR1's proteolytic activation mechanism stands in contrast to the reversible ligand binding that activates classical G protein-coupled receptors and compels special mechanisms for desensitization and resensitization. In endothelial cells and fibroblasts, activated PAR1 rapidly internalizes and then sorts to lysosomes rather than recycling to the plasma membrane as do classical G protein-coupled receptors. This trafficking behavior is critical for termination of thrombin signaling. An intracellular pool of thrombin receptors refreshes the cell surface with naive receptors, thereby maintaining thrombin responsiveness. Thus cells have evolved a trafficking solution to the signaling problem presented by PARs. Four PARs have now been identified. PAR1, PAR3, and PAR4 can all be activated by thrombin. PAR2 is activated by trypsin and by trypsin-like proteases but not by thrombin. Recent studies with knockout mice, receptor-activating peptides, and blocking antibodies are beginning to define the role of these receptors in vivo.

The in vivo roles of the hundreds of mammalian G protein-coupled receptors (GPCRs) are incompletely understood. To explore these roles, we generated mice expressing the S1 subunit of pertussis toxin, a known inhibitor of G(i/o) signaling, under the control of the ROSA26 locus in a Cre recombinase-dependent manner (ROSA26(PTX)). Crossing ROSA26(PTX) mice to mice expressing Cre in pancreatic beta cells produced offspring with constitutive hyperinsulinemia, increased insulin secretion in response to glucose, and resistance to diet-induced hyperglycemia. This phenotype underscored the known importance of G(i/o) and hence of GPCRs for regulating insulin secretion. Accordingly, we quantified mRNA for each of the approximately 373 nonodorant GPCRs in mouse to identify receptors highly expressed in islets and examined the role of several. We report that 3-iodothyronamine, a thyroid hormone metabolite, could negatively and positively regulate insulin secretion via the G(i)-coupled alpha(2A)-adrenergic receptor and the G(s)-coupled receptor Taar1, respectively, and protease-activated receptor-2 could negatively regulate insulin secretion and may contribute to physiological regulation of glucose metabolism. The ROSA26(PTX) system used in this study represents a new genetic tool to achieve tissue-specific signaling pathway modulation in vivo that can be applied to investigate the role of G(i/o)-coupled GPCRs in multiple cell types and processes.

Significance This paper shows that a class of receptors known to modulate insulin release by pancreatic β cells also regulates the proliferation of these cells and restrains the perinatal β-cell expansion that establishes adult β-cell mass, suggesting that alterations in signaling by these receptors could contribute to the decreased β-cell numbers seen in patients with type 2 diabetes. Further, inhibition of signaling through these receptors potentially could be used to generate more β cells for people with diabetes. Gi-GPCRs, G protein-coupled receptors that signal via Gα proteins of the i/o class (Gα ᵢ/ₒ), acutely regulate cellular behaviors widely in mammalian tissues, but their impact on the development and growth of these tissues is less clear. For example, Gi-GPCRs acutely regulate insulin release from pancreatic β cells, and variants in genes encoding several Gi-GPCRs—including the α-2a adrenergic receptor, ADRA2A—increase the risk of type 2 diabetes mellitus. However, type 2 diabetes also is associated with reduced total β-cell mass, and the role of Gi-GPCRs in establishing β-cell mass is unknown. Therefore, we asked whether Gi-GPCR signaling regulates β-cell mass. Here we show that Gi-GPCRs limit the proliferation of the insulin-producing pancreatic β cells and especially their expansion during the critical perinatal period. Increased Gi-GPCR activity in perinatal β cells decreased β-cell proliferation, reduced adult β-cell mass, and impaired glucose homeostasis. In contrast, Gi-GPCR inhibition enhanced perinatal β-cell proliferation, increased adult β-cell mass, and improved glucose homeostasis. Transcriptome analysis detected the expression of multiple Gi-GPCRs in developing and adult β cells, and gene-deletion experiments identified ADRA2A as a key Gi-GPCR regulator of β-cell replication. These studies link Gi-GPCR signaling to β-cell mass and diabetes risk and identify it as a potential target for therapies to protect and increase β-cell mass in patients with diabetes.