ALBERT

Beschreibung: Single-cell sequencing technologies have revealed an unexpectedly broad repertoire of cells required to mediate complex functions in multicellular organisms. Despite the multiple roles of adipose tissue in maintaining systemic metabolic homeostasis, adipocytes are thought to be largely homogenous with only 2 major subtypes recognized in humans so far. Here we report the existence and characteristics of 4 distinct human adipocyte subtypes, and of their respective mesenchymal progenitors. The phenotypes of these distinct adipocyte subtypes are differentially associated with key adipose tissue functions, including thermogenesis, lipid storage, and adipokine secretion. The transcriptomic signature of “brite/beige” thermogenic adipocytes reveals mechanisms for iron accumulation and protection from oxidative stress, necessary for mitochondrial biogenesis and respiration upon activation. Importantly, this signature is enriched in human supraclavicular adipose tissue, confirming that these cells comprise thermogenic depots in vivo, and explain previous findings of a rate-limiting role of iron in adipose tissue browning. The mesenchymal progenitors that give rise to beige/brite adipocytes express a unique set of cytokines and transcriptional regulators involved in immune cell modulation of adipose tissue browning. Unexpectedly, we also find adipocyte subtypes specialized for high-level expression of the adipokines adiponectin or leptin, associated with distinct transcription factors previously implicated in adipocyte differentiation. The finding of a broad adipocyte repertoire derived from a distinct set of mesenchymal progenitors, and of the transcriptional regulators that can control their development, provides a framework for understanding human adipose tissue function and role in metabolic disease.

Beschreibung: Gene expression and metabolism are coupled at numerous levels. Cells must sense and respond to nutrients in their environment, and specialized cells must synthesize metabolic products required for their function. Pluripotent stem cells have the ability to differentiate into a wide variety of specialized cells. How metabolic state contributes to stem cell differentiation is not understood. In this study, we show that RNA-binding by the stem cell translation regulator Musashi-1 (MSI1) is allosterically inhibited by 18–22 carbon ω-9 monounsaturated fatty acids. The fatty acid binds to the N-terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi proteins are critical for development of the brain, blood, and epithelium. We identify stearoyl-CoA desaturase-1 as a MSI1 target, revealing a feedback loop between ω-9 fatty acid biosynthesis and MSI1 activity. We propose that other RRM proteins could act as metabolite sensors to couple gene expression changes to physiological state.

Beschreibung: Abstract 2222 Activated Protein C (APC) is well known for its anticoagulant activity in the coagulation cascade. Inactivation of factors VIIIa (FVIIIa) and Va (FVa) by APC down regulates thrombin generation. The importance of FVa inactivation is seen in individuals with a common genetic mutation in Factor V, known as APC resistant Factor V Leiden (FVL). This missense mutation leads to the elimination of one of three APC cleavage sites on FVa, and FVa inactivation occurs at a slower rate. APC resistance leads to a thrombophilic state and individuals with FVL have a higher risk of thrombosis. Some reports suggest that hemophiliacs with the FVL mutation have reduced clinical severity compared to hemophiliacs without the FVL mutation. They have fewer bleeding episodes and also a delay in age when bleeding episodes begin (Kurnik et al Haematologica 2007;92:982-985). Consistent with this observation, transgenic mice engineered to carry the FVL mutation in combination with a deficiency in FVIII or factor IX (FIX) display normal thrombus formation in a laser injury model compare to no thrombus in the absence of the FVL mutation (Schlachterman et al J Thrombos and Haemostas 3:2730, 2005). Therefore, targeting a therapeutic agent to stabilize FVa by inhibiting APC may help normalize clotting in hemophiliacs. An anti-APC aptamer was discovered by Systematic Evolution of Ligands by EXponential enrichment (SELEX) and optimized for therapeutic use. In vitro assays were used to determine if the anti-APC aptamer blocks the anticoagulant activity of APC. The aptamer inhibited APC cleavage of a chromogenic peptide substrate. Furthermore, the aptamer decreased the clot time in a plasma-based assay and corrected thrombin generation as measured by calibrated automated thrombogram (CAT) following thrombomodulin-mediated protein C activation. These results show that the anti-APC aptamer efficiently blocks the anticoagulant activity of APC. APC also has an important cytoprotective role which can occur when APC binds and interacts with endothelial protein cell receptor (EPCR) and protease activated receptor-1 (PAR-1). In its cytoprotective role, APC can protect cells from apoptosis and inflammation. It is essential for the safety of a procoagulant, anti-APC therapeutic, that it not block this activity. Flow cytometric experiments were used to determine if the anti-APC aptamer affected the cytoprotective activity of APC. Aptamer was either pre-mixed with APC or added to APC-treated HUVEC cells. There was no interference of APC binding to EPCR on HUVEC cells in either type of experiment. In addition, the aptamer did not interfere with APC binding to EPCR on THP-1 cells. These experiments suggest that the anti-APC aptamer should not interfere with the ability of APC to bind to and protect cells. Taken together with the procoagulant activity described above, these data suggest that the anti-APC aptamer should be a promising new agent for the treatment of hemostatic defects. Disclosures: Wagner: Archemix Corportation: Employment. Schwartz: Archemix Corporation: Employment. McGinness: Archemix Corportation: Employment. Genga: Archemix Corporation: Employment. Kurz: Archemix Corporation: Employment. Waters: Archemix Corporation: Employment. Schaub: Archemix Corporation: Employment.

Beschreibung: Hemophilia A and B are caused by deficiencies in coagulation factor VIII (FVIII) and factor IX, respectively, resulting in deficient blood coagulation via the intrinsic pathway. The extrinsic coagulation pathway, mediated by factor VIIa and tissue factor (TF), remains intact but is negatively regulated by tissue factor pathway inhibitor (TFPI), which inhibits both factor VIIa and its product, factor Xa. This inhibition limits clot initiation via the extrinsic pathway, whereas factor deficiency in hemophilia limits clot propagation via the intrinsic pathway. ARC19499 is an aptamer that inhibits TFPI, thereby enabling clot initiation and propagation via the extrinsic pathway. The core aptamer binds tightly and specifically to TFPI. ARC19499 blocks TFPI inhibition of both factor Xa and the TF/factor VIIa complex. ARC19499 corrects thrombin generation in hemophilia A and B plasma and restores clotting in FVIII-neutralized whole blood. In the present study, using a monkey model of hemophilia, FVIII neutralization resulted in prolonged clotting times as measured by thromboelastography and prolonged saphenous-vein bleeding times, which are consistent with FVIII deficiency. ARC19499 restored thromboelastography clotting times to baseline levels and corrected bleeding times. These results demonstrate that ARC19499 inhibition of TFPI may be an effective alternative to current treatments of bleeding associated with hemophilia.

Beschreibung: Abstract 1134 Hemophilia is a bleeding disorder that results from deficiencies in coagulation factor VIII (FVIII; hemophilia A) or factor IX (FIX; hemophilia B), primarily impacting the intrinsic coagulation pathway. The extrinsic coagulation pathway remains intact in hemophiliacs and is negatively regulated by tissue factor pathway inhibitor (TFPI). The primary role of TFPI is regulation of the FVIIa:tissue factor complex through inhibition of FVIIa and FXa. Inhibition of TFPI may provide an effective treatment for hemophilia by allowing sufficient thrombin generation via the extrinsic coagulation pathway to bypass the defect in clot propagation caused by deficiency of FVIII or FIX. ARC19499 is an aptamer that is a potent, specific inhibitor of TFPI. We have employed a variety of in vitro and in vivo methods to demonstrate that ARC19499 mediates a procoagulant effect in hemophilic plasma and in a model of hemophilia A in non-human primates (NHP). Plasma-based experiments that measure thrombin generation over time demonstrate that ARC19499 mediates a procoagulant response in both hemophilia A and hemophilia B plasma, restoring thrombin generation to near normal levels at 10 – 100 nM aptamer. Experiments in TFPI-depleted plasma and in vitro binding experiments demonstrate that this procoagulant effect is dependent on the specific interaction between ARC19499 and TFPI. ARC19499 restores thrombin generation to a level that is equivalent to or better than 14% FVIII replacement at a concentration of 30 nM in hemophilia A plasma and has an additive effect on thrombin generation when used in combination with FVIII. In a NHP model of hemophilia, cynomolgus monkeys acquire a hemophilia A-like state following administration of an anti-FVIII antibody as measured by ex vivo thromboelastography (TEG). TEG coagulation parameters are corrected following administration of ARC19499. In addition, antibody-mediated FVIII depletion moderately prolongs saphenous-vein bleeding time. ARC19499 corrects bleeding times to normal. These experiments demonstrate that the anti-TFPI aptamer ARC19499 mediates a procoagulant hemostatic effect both in vitro and in vivo in hemophilia model systems that is dependent on specific interaction between the aptamer and TFPI. ARC19499 may provide an effective alternative to replacement factors and bypassing agents for the treatment of hemophilia. Additionally, aptamer therapeutics have the advantage of subcutaneous bioavailability which provides an opportunity for improved treatment regimens in hemophilia. Disclosures: McGinness: Archemix Corporation: Employment. Waters:Archemix Corporation: Employment. Genga:Archemix Corporation: Employment. Olson:Archemix Corporation: Employment. Nelson:Archemix Corporation: Employment. Kurz:Archemix Corporation: Employment. Schaub:Archemix Corporation: Employment.

Beschreibung: Abstract 1214 Hemophilia is a bleeding disorder characterized by a deficiency in coagulation factor VIII or IX, rendering the body incapable of maintaining hemostasis. BAX 499 is a therapeutic aptamer that inhibits tissue factor pathway inhibitor (TFPI) and is under investigation for treatment of hemophilia patients. BAX 499 interferes with TFPI inhibition of both factor Xa (FXa) and the tissue factor/factor VIIa complex in vitro and improves thrombin generation and clotting parameters in hemophilia plasma. BAX 499 also improves coagulation in a hemophilia A-like cynomolgus monkey model, as determined by ex vivo thromboelastography and bleeding time measurements. In order to understand the mechanism by which BAX 499 inhibits TFPI, we carried out studies to determine the BAX 499 binding site on TFPI and compared BAX 499 activity to domain-specific anti-TFPI antibodies. The binding site of the aptamer on TFPI was determined by performing binding experiments with truncated TFPI proteins, aptamer-antibody competition-binding experiments, and hydrogen-deuterium exchange mapping experiments. The aptamer binds tightly to full-length TFPI (KD ∼ 3 nM), less well to a protein missing some of the C-terminal region of TFPI, very weakly to a protein that contains only the Kunitz 3 (K3) and C-terminal domains, and not at all to a protein that contains only the Kunitz 1 (K1) and Kunitz 2 (K2) domains. Domain-specific monoclonal antibodies were used as competitors in a nitrocellulose dot blot binding assay using radiolabeled aptamer and in an ELISA assay that monitors aptamer binding to plate-bound TFPI. In both assays, K1 and K3 domain antibodies competed with aptamer for binding, a C-terminal domain antibody partially competed, and a K2 domain antibody did not compete. When hydrogen-deuterium exchange was carried out in the presence of aptamer, residues within the K1, K3, and C-terminal domains of TFPI were protected (residues 15–69 and 191–272). Taken together, these data suggest that the binding epitope of BAX 499 on TFPI spans the entire protein molecule, explicitly requiring the K1 and K3 domains, and is partially dependent on the C-terminal domain. Activity assays demonstrate that the inhibition profile of BAX 499 is similar to that observed with anti-TFPI antibodies that bind to the K1, K3, and C-terminal domains of the protein. In an assay measuring inhibition of FXa activity by TFPI, antibodies against the K2 domain fully inhibited TFPI and restored FXa activity. In contrast, BAX 499 and antibodies against the K1, K3, or C-terminal domains of the protein did not fully inhibit TFPI, resulting in partial restoration of FXa activity. Examination of aptamer binding to TFPI in the presence of FXa suggests that the aptamer and protein can bind to TFPI simultaneously, highlighting a potentially unique mechanism for TFPI inhibition. Binding of BAX 499 to TFPI appears to involve regions across the entirety of the protein, with the exception of the K2 domain. As the K2 domain is primarily responsible for binding to and inhibiting FXa, these experiments suggest that the aptamer has a unique mechanism of interfering with TFPI inhibition of FXa activity. This mechanism of action is able to mediate a procoagulant response in models of hemophilia and may prove effective for the treatment of hemophilia patients. Disclosures: Waters: Baxter Healthcare Corporation: Employment. Genga:Baxter Healthcare Corporation: Employment. Thomson:Archemix Corporation: Employment. Schaub:Archemix Corporation: Employment. Kurz:Baxter Healthcare Corporation: Employment. McGinness:Baxter Healthcare Corporation: Employment.