Single-cell analyses demonstrate that a heme-GATA1 feedback loop regulates red cell differentiation.

Erythropoiesis is the complex, dynamic, and tightly regulated process that generates all mature red blood cells. To understand this process, we mapped the developmental trajectories of progenitors from wild-type, erythropoietin-treated, and Flvcr1-deleted mice at single-cell resolution. Importantly, we linked the quantity of each cell's surface proteins to its total transcriptome, which is a novel method. Deletion of Flvcr1 results in high levels of intracellular heme, allowing us to identify heme-regulated circuitry. Our studies demonstrate that in early erythroid cells (CD71+Ter119neg-lo), heme increases ribosomal protein transcripts, suggesting that heme, in addition to upregulating globin transcription and translation, guarantees ample ribosomes for globin synthesis. In later erythroid cells (CD71+Ter119lo-hi), heme decreases GATA1, GATA1-target gene, and mitotic spindle gene expression. These changes occur quickly. For example, in confirmatory studies using human marrow erythroid cells, ribosomal protein transcripts and proteins increase, and GATA1 transcript and protein decrease, within 15 to 30 minutes of amplifying endogenous heme synthesis with aminolevulinic acid. Because GATA1 initiates heme synthesis, GATA1 and heme together direct red cell maturation, and heme stops GATA1 synthesis, our observations reveal a GATA1-heme autoregulatory loop and implicate GATA1 and heme as the comaster regulators of the normal erythroid differentiation program. In addition, as excessive heme could amplify ribosomal protein imbalance, prematurely lower GATA1, and impede mitosis, these data may help explain the ineffective (early termination of) erythropoiesis in Diamond Blackfan anemia and del(5q) myelodysplasia, disorders with excessive heme in colony-forming unit-erythroid/proerythroblasts, explain why these anemias are macrocytic, and show why children with GATA1 mutations have DBA-like clinical phenotypes.

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.

Glycoprotein Ib-IX-V Complex Transmits Cytoskeletal Forces That Enhance Platelet Adhesion.

Platelets bind to exposed vascular matrix at a wound site through a highly specialized surface receptor, glycoprotein (GP) Ib-IX-V complex, which recognizes von Willebrand factor (VWF) in the matrix. GPIb-IX-V is a catch bond for it becomes more stable as force is applied to it. After attaching to the wound site, platelets generate cytoskeletal forces to compact and reinforce the hemostatic plug. Here, we evaluated the role of the GPIb-IX-V complex in the transmission of cytoskeletal forces. We used arrays of flexible, silicone nanoposts to measure the contractility of individual platelets on VWF. We found that a significant proportion of cytoskeletal forces were transmitted to VWF through GPIb-IX-V, an unexpected finding given the widely held notion that platelet forces are transmitted exclusively through its integrins. In particular, we found that the interaction between GPIbα and the A1 domain of VWF mediates this force transmission. We also demonstrate that the binding interaction between GPIbα and filamin A is involved in force transmission. Furthermore, our studies suggest that cytoskeletal forces acting through GPIbα are involved in maintaining platelet adhesion when external forces are absent. Thus, the GPIb-IX-V/VWF bond is able to transmit force, and uses this force to strengthen the bond through a catch-bond mechanism. This finding expands our understanding of how platelets attach to sites of vascular injury, describing a new, to the best of our knowledge, mechanism in which the catch bonds of GPIb-IX-V/VWF can be supported by internal forces produced by cytoskeletal tension.

Transfusion-induced alloimmunization and platelet refractoriness in a mouse model: mechanisms and interventions.

BACKGROUND: Platelet (PLT) transfusions can be an essential therapy for patients with thrombocytopenia to maintain hemostasis. However, some patients become alloimmunized to antigens on PLTs (typically HLA), which can prevent efficacy of PLT transfusion due to antibody-mediated clearance. In extreme cases, patients with alloimmunization to multiple HLAs can become "refractory" to PLT transfusion, such that insufficient compatible PLT units can be found to meet transfusion needs. MATERIALS AND METHODS: An in vivo murine model of PLT-induced alloimmunization was refined so as to include both transfusion with allogeneic leukoreduced PLTs and studies of posttransfusion PLT recoveries, allowing assessment of alloimmunization and refractoriness. Basic mechanisms of antibody-mediated PLT clearance were investigated using recipients missing either the C3 complement gene or the common gamma chain for Fc receptors. In addition, the efficacy of using costimulatory blockade as a therapeutic intervention was assessed by testing CTLA4-Ig administration before PLT transfusion. RESULTS: Fcγ receptors (but not complement C3) are required for alloantibody-mediated PLT refractoriness. In addition, levels of anti-MHC predict the extent of refractoriness in a given animal. Finally, costimulatory blockade as a therapeutic modality prevents transfusion-induced PLT refractoriness. CONCLUSIONS: Together these findings introduce new experimental methods, basic mechanistic understanding, and a potential therapeutic intervention for alloimmunization to MHC-based antigens on transfused PLTs.

GAGE-seq concurrently profiles multiscale 3D genome organization and gene expression in single cells.

The organization of mammalian genomes features a complex, multiscale three-dimensional (3D) architecture, whose functional significance remains elusive because of limited single-cell technologies that can concurrently profile genome organization and transcriptional activities. Here, we introduce genome architecture and gene expression by sequencing (GAGE-seq), a scalable, robust single-cell co-assay measuring 3D genome structure and transcriptome simultaneously within the same cell. Applied to mouse brain cortex and human bone marrow CD34+ cells, GAGE-seq characterized the intricate relationships between 3D genome and gene expression, showing that multiscale 3D genome features inform cell-type-specific gene expression and link regulatory elements to target genes. Integration with spatial transcriptomic data revealed in situ 3D genome variations in mouse cortex. Observations in human hematopoiesis unveiled discordant changes between 3D genome organization and gene expression, underscoring a complex, temporal interplay at the single-cell level. GAGE-seq provides a powerful, cost-effective approach for exploring genome structure and gene expression relationships at the single-cell level across diverse biological contexts.

Concurrent profiling of multiscale 3D genome organization and gene expression in single mammalian cells.

The organization of mammalian genomes within the nucleus features a complex, multiscale three-dimensional (3D) architecture. The functional significance of these 3D genome features, however, remains largely elusive due to limited single-cell technologies that can concurrently profile genome organization and transcriptional activities. Here, we report GAGE-seq, a highly scalable, robust single-cell co-assay that simultaneously measures 3D genome structure and transcriptome within the same cell. Employing GAGE-seq on mouse brain cortex and human bone marrow CD34+ cells, we comprehensively characterized the intricate relationships between 3D genome and gene expression. We found that these multiscale 3D genome features collectively inform cell type-specific gene expressions, hence contributing to defining cell identity at the single-cell level. Integration of GAGE-seq data with spatial transcriptomic data revealed in situ variations of the 3D genome in mouse cortex. Moreover, our observations of lineage commitment in normal human hematopoiesis unveiled notable discordant changes between 3D genome organization and gene expression, underscoring a complex, temporal interplay at the single-cell level that is more nuanced than previously appreciated. Together, GAGE-seq provides a powerful, cost-effective approach for interrogating genome structure and gene expression relationships at the single-cell level across diverse biological contexts.

The transcriptomic landscape of normal and ineffective erythropoiesis at single-cell resolution.

The anemias of myelodysplastic syndrome (MDS) and Diamond Blackfan anemia (DBA) are generally macrocytic and always reflect ineffective erythropoiesis yet result from diverse genetic mutations. To delineate shared mechanisms that lead to cell death, we studied the fate of single erythroid marrow cells from individuals with DBA or MDS-5q. We defined an unhealthy (vs healthy) differentiation trajectory using transcriptional pseudotime and cell surface proteins. The pseudotime trajectories diverge immediately after cells upregulate transferrin receptor (CD71), import iron, and initiate heme synthesis, although cell death occurs much later. Cells destined to die express high levels of heme-responsive genes, including ribosomal protein and globin genes, whereas surviving cells downregulate heme synthesis and upregulate DNA damage response, hypoxia, and HIF1 pathways. Surprisingly, 24% ± 12% of cells from control subjects follow the unhealthy trajectory, implying that heme might serve as a rheostat directing cells to live or die. When heme synthesis was inhibited with succinylacetone, more DBA cells followed the healthy trajectory and survived. We also noted high numbers of messages with retained introns that increased as erythroid cells matured, confirmed the rapid cycling of colony forming unit-erythroid, and demonstrated that cell cycle timing is an invariant property of differentiation stage. Including unspliced RNA in pseudotime determinations allowed us to reliably align independent data sets and accurately query stage-specific transcriptomic changes. MDS-5q (unlike DBA) results from somatic mutation, so many normal (unmutated) erythroid cells persist. By independently tracking erythroid differentiation of cells with and without chromosome 5q deletions, we gained insight into why 5q+ cells cannot expand to prevent anemia.

Changes in cardiorespiratory function and fatigue following 12 weeks of exercise training in women with systemic lupus erythematosus: a pilot study.

OBJECTIVE: In patients with systemic lupus erythematosus (SLE), fatigue is a debilitating symptom with poorly understood pathophysiology. Cardiorespiratory dysfunction has been hypothesised as a contributor to SLE-fatigue. The purpose of this exploratory study was to examine changes in cardiorespiratory function, following an exercise training programme in women with SLE, together with patient reported outcomes and other pathophysiological measures that may underlie SLE-fatigue. METHODS: Sixteen women with SLE and fatigue (Fatigue Severity Scale (FSS) ≥3) were enrolled in a supervised aerobic exercise training programme of vigorous intensity. The primary outcome was time to reach anaerobic threshold (AT-Time) during a cardiopulmonary exercise test (CPET). Secondary outcomes included changes in the 10-minute walk test (10MWT), FSS scores and the Patient Reported Outcomes Measurement Information System (PROMIS-57) survey. Mitochondrial function was assessed by the oxygen consumption rate (OCR)/extracellular acidification rate (ECAR) metabolic potential ratio. RESULTS: Following 12 weeks of exercise training, AT-Time increased by 93±82 (mean±SD) s (p<0.001), 10MWT increased by 84±66 m (p<0.001) and peak oxygen uptake (VO2) increased by 1.4±2.0 mL/kg/min (p=0.013). There were improvements in FSS score (-1.4±1.0, p<0.0001) and in most of the PROMIS-57 domains. The decrease in FSS scores correlated with an increase in the OCR/ECAR ratio (Pearson's correlation r=-0.59, p=0.03). A subset of subjects (9/15) had significant reduction in their Interferon Stimulated Genes (ISG) (p=0.007) accompanied by a significant increase in the OCR/ECAR ratio (p=0.013). CONCLUSIONS: Cardiorespiratory function was improved in concomitance with reductions in fatigue following a 12-week aerobic exercise programme. The reduction in fatigue scores correlated with improvements in mitochondrial function.

Tissue factor, lipid rafts, and microparticles.

Evidence is emerging that tissue-factor-bearing microparticles and other microparticles arise from regions of the parent cell's plasma membrane that are rich in lipid rafts. In this brief review, we summarize the evidence for the raft origins of microparticles and the implications of these origins for the biological and medical consequences of microparticle production and for therapeutic strategies to diminish their production and potential to do harm.

A functional 14-3-3zeta-independent association of PI3-kinase with glycoprotein Ib alpha, the major ligand-binding subunit of the platelet glycoprotein Ib-IX-V complex.

Engagement of the adhesion receptor glycoprotein (GP) Ib-IX-V by von Willebrand factor (VWF) mediates platelet adhesion to damaged vessels and triggers platelet activation and thrombus formation in heart attack and stroke. GPIb-IX-V contains distinct 14-3-3zeta-binding sites at the GPIb alpha C-terminus involving phosphorylation of Ser609, an upstream site involving phosphorylated Ser587/Ser590, and a protein kinase A (PKA)-dependent site on GPIb beta involving Ser166. 14-3-3zeta regulates the VWF-binding affinity of GPIb-IX-V and inhibiting 14-3-3zeta association blocks receptor signaling, suggesting a key functional role for 14-3-3zeta. We used deletion mutants of GPIb alpha expressed in Chinese hamster ovary (CHO) cells to define the relationship of 14-3-3zeta binding to another GPIb-IX-V-associated signaling protein, phosphoinositide 3-kinase (PI3-kinase). Pull-down experiments involving glutathione S-transferase (GST)-PI3-kinase/p85-subunit and GST-14-3-3zeta indicated that both proteins interacted with contiguous GPIb alpha sequences 580 to 590/591 to 610. Deleting these, but not upstream sequences of GPIb alpha expressed in CHO cells, inhibited VWF/ristocetin-dependent Akt phosphorylation, relative to wild-type receptor, confirming this region encompassed a functional PI3-kinase-binding site. Pull-down experiments with GST-p85 truncates indicated the GPIb alpha-binding region involved the p85 breakpoint cluster region (BCR) domain, containing RSXSXP. However, pull-down of GPIb-IX was unaltered by mutation/deletion/phosphorylation of this potential 14-3-3zeta-binding sequence in mutant constructs of GST-p85, suggesting PI3-kinase bound GPIb alpha independently of 14-3-3zeta; 14-3-3zeta inhibitor peptide R18 also blocked pull-down of receptor by GST-14-3-3zeta but not GST-p85, and GST-p85 pull-downs were unaffected by excess 14-3-3zeta. Together, these data suggest the GPIb alpha C-terminus regulates signaling through independent association of 14-3-3zeta and PI3-kinase.

Structural basis of the leukocyte integrin Mac-1 I-domain interactions with the platelet glycoprotein Ib.

Cell-surface receptor interactions between leukocyte integrin macrophage-1 antigen (Mac-1, also known as CR3, αMß2, CD11b/CD18) and platelet glycoprotein Ibα (GPIbα) are critical to vascular inflammation. To define the key residues at the binding interface, we used nuclear magnetic resonance (NMR) to assign the spectra of the mouse Mac-1 I-domain and mapped the residues contacting the mouse GPIbα N-terminal domain (GPIbαN) to the locality of the integrin metal ion-dependant adhesion site (MIDAS) surface. We next determined the crystal structures of the mouse GPIbαN and Mac-1 I-domain to 2 Å and 2.5 Å resolution, respectively. The mouse Mac-1 I-domain crystal structure reveals an active conformation that is stabilized by a crystal contact from the α7-helix with a glutamate side chain completing the octahedral coordination sphere of the MIDAS Mg2+ ion. The amino acid sequence of the α7-helix and disposition of the glutamic acid matches the C-terminal capping region α-helix of GPIbα effectively acting as a ligand mimetic. Using these crystal structures in combination with NMR measurements and docking analysis, we developed a model whereby an acidic residue from the GPIbα leucine-rich repeat (LRR) capping α-helix coordinates directly to the Mac-1 MIDAS Mg2+ ion. The Mac-1:GPIbαN complex involves additional interactions consolidated by an elongated pocket flanking the GPIbαN LRR capping α-helix. The GPIbαN α-helix has an HxxxE motif, which is equivalent by homology to RxxxD from the human GPIbαN. Subsequent mutagenesis of residues at this interface, coupled with surface plasmon resonance studies, confirmed the importance of GPIbαN residues H218, E222, and the Mac-1 MIDAS residue T209 to formation of the complex.

The type Ialpha inositol polyphosphate 4-phosphatase generates and terminates phosphoinositide 3-kinase signals on endosomes and the plasma membrane.

Endosomal trafficking is regulated by the recruitment of effector proteins to phosphatidylinositol 3-phosphate [PtdIns(3)P] on early endosomes. At the plasma membrane, phosphatidylinositol-(3,4)-bisphosphate [PtdIns(3,4)P2] binds the pleckstrin homology (PH) domain-containing proteins Akt and TAPP1. Type Ialpha inositol polyphosphate 4-phosphatase (4-phosphatase) dephosphorylates PtdIns(3,4)P2, forming PtdIns(3)P, but its subcellular localization is unknown. We report here in quiescent cells, the 4-phosphatase colocalized with early and recycling endosomes. On growth factor stimulation, 4-phosphatase endosomal localization persisted, but in addition the 4-phosphatase localized at the plasma membrane. Overexpression of the 4-phosphatase in serum-stimulated cells increased cellular PtdIns(3)P levels and prevented wortmannin-induced endosomal dilatation. Furthermore, mouse embryonic fibroblasts from homozygous Weeble mice, which have a mutation in the type I 4-phosphatase, exhibited dilated early endosomes. 4-Phosphatase translocation to the plasma membrane upon growth factor stimulation inhibited the recruitment of the TAPP1 PH domain. The 4-phosphatase contains C2 domains, which bound PtdIns(3,4)P2, and C2-domain-deletion mutants lost PtdIns(3,4)P2 4-phosphatase activity, did not localize to endosomes or inhibit TAPP1 PH domain membrane recruitment. The 4-phosphatase therefore both generates and terminates phosphoinositide 3-kinase signals at distinct subcellular locations.

Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIbα.

Inflammation and thrombosis occur together in many diseases. The leukocyte integrin Mac-1 (also known as integrin αMß2, or CD11b/CD18) is crucial for leukocyte recruitment to the endothelium, and Mac-1 engagement of platelet GPIbα is required for injury responses in diverse disease models. However, the role of Mac-1 in thrombosis is undefined. Here we report that mice with Mac-1 deficiency (Mac-1-/-) or mutation of the Mac-1-binding site for GPIbα have delayed thrombosis after carotid artery and cremaster microvascular injury without affecting parameters of haemostasis. Adoptive wild-type leukocyte transfer rescues the thrombosis defect in Mac-1-/- mice, and Mac-1-dependent regulation of the transcription factor Foxp1 contributes to thrombosis as evidenced by delayed thrombosis in mice with monocyte-/macrophage-specific overexpression of Foxp1. Antibody and small-molecule targeting of Mac-1:GPIbα inhibits thrombosis. Our data identify a new pathway of thrombosis involving leukocyte Mac-1 and platelet GPIbα, and suggest that targeting this interaction has anti-thrombotic therapeutic potential with reduced bleeding risk.

Corrigendum: Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIbα.

This corrects the article DOI: 10.1038/ncomms15559.

Molecular Imaging of Platelet-Endothelial Interactions and Endothelial von Willebrand Factor in Early and Mid-Stage Atherosclerosis.

BACKGROUND: Nonthrombotic platelet-endothelial interactions may contribute to atherosclerotic plaque development, although in vivo studies examining mechanism without platelet preactivation are lacking. Using in vivo molecular imaging at various stages of atherosclerosis, we quantified platelet-endothelial interactions and evaluated the contribution of major adhesion pathways. METHODS AND RESULTS: Mice deficient for the low-density lipoprotein receptor and Apobec-1 were studied as an age-dependent model of atherosclerosis at 10, 20, 30, and 40 weeks of age, which provided progressive increase in stage from early fatty streak (10 weeks) to large complex plaques without rupture (40 weeks). Platelet-targeted contrast ultrasound molecular imaging of the thoracic aorta performed with microbubbles targeted to GPIbα demonstrated selective signal enhancement as early as 10 weeks of age. This signal increased progressively with age (almost 8-fold increase from 10 to 40 weeks, analysis of variance P<0.001). Specificity for platelet targeting was confirmed by the reduction in platelet-targeted signal commensurate with the decrease in platelet count after immunodepletion with anti-GPIb or anti-CD41 antibody. Inhibition of P-selectin in 20 and 40 weeks atherosclerotic mice resulted in a small (15% to 30%) reduction in platelet signal. Molecular imaging with microbubbles targeted to the A1 domain of von Willebrand factor demonstrated selective signal enhancement at all time points, which did not significantly increase with age. Treatment of 20 and 40 week mice with recombinant ADAMTS13 eliminated platelet and von Willebrand factor molecular imaging signal. CONCLUSIONS: Platelet-endothelial interactions occur in early atherosclerosis. These interactions are in part caused by endothelial von Willebrand factor large multimers, which can be reversed with exogenous ADAMTS13.

The structure of the GPIb-filamin A complex.

Filamin A (FLNa), a dimeric actin cross-linking and scaffold protein with numerous intracellular binding partners, anchors the platelet adhesion glycoprotein (GP) Ib-IX-V receptor to actin cytoskeleton. We mapped the GPIbalpha binding site to a single domain of FLNa and resolved the structure of this domain and its interaction complex with the corresponding GPIbalpha cytoplasmic domain. This is the first atomic structure of this class of membrane glycoprotein-cytoskeleton connection. GPIbalpha binds in a groove formed between the C and D beta strands of FLNa domain 17. The interaction is strikingly similar to that between the beta7 integrin tail and a different FLNa domain, potentially defining a conserved motif for FLNa binding. Nevertheless, the structures also reveal specificity of the interfaces, which explains different regulatory mechanisms. To verify the topology of GPIb-FLNa interaction we also purified the native complex from platelets and showed that GPIb interacts with the C-terminus of FLNa, which is in accordance with our biochemical and structural data.