X-linked macrocytic dyserythropoietic anemia in females with an ALAS2 mutation

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Abstract

Macrocytic anemia with abnormal erythropoiesis is a common feature of megaloblastic anemias, congenital dyserythropoietic anemias, and myelodysplastic syndromes. Here, we characterized a family with multiple female individuals who have macrocytic anemia. The proband was noted to have dyserythropoiesis and iron overload. After an extensive diagnostic evaluation that did not provide insight into the cause of the disease, whole-exome sequencing of multiple family members revealed the presence of a mutation in the X chromosomal gene ALAS2, which encodes 5′-aminolevulinate synthase 2, in the affected females. We determined that this mutation (Y365C) impairs binding of the essential cofactor pyridoxal 5′-phosphate to ALAS2, resulting in destabilization of the enzyme and consequent loss of function. X inactivation was not highly skewed in wbc from the affected individuals. In contrast, and consistent with the severity of the ALAS2 mutation, there was a complete skewing toward expression of the WT allele in mRNA from reticulocytes that could be recapitulated in primary erythroid cultures. Together, the results of the X inactivation and mRNA studies illustrate how this X-linked dominant mutation in ALAS2 can perturb normal erythropoiesis through cell-nonautonomous effects. Moreover, our findings highlight the value of whole-exome sequencing in diagnostically challenging cases for the identification of disease etiology and extension of the known phenotypic spectrum of disease.

Authors

Vijay G. Sankaran, Jacob C. Ulirsch, Vassili Tchaikovskii, Leif S. Ludwig, Aoi Wakabayashi, Senkottuvelan Kadirvel, R. Coleman Lindsley, Rafael Bejar, Jiahai Shi, Scott B. Lovitch, David F. Bishop, David P. Steensma

Caspase-1–mediated pathway promotes generation of thromboinflammatory microparticles

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Abstract

Extracellular ATP is a signal of tissue damage and induces macrophage responses that amplify inflammation and coagulation. Here we demonstrate that ATP signaling through macrophage P2X7 receptors uncouples the thioredoxin (TRX)/TRX reductase (TRXR) system and activates the inflammasome through endosome-generated ROS. TRXR and inflammasome activity promoted filopodia formation, cellular release of reduced TRX, and generation of extracellular thiol pathway–dependent, procoagulant microparticles (MPs). Additionally, inflammasome-induced activation of an intracellular caspase-1/calpain cysteine protease cascade degraded filamin, thereby severing bonds between the cytoskeleton and tissue factor (TF), the cell surface receptor responsible for coagulation activation. This cascade enabled TF trafficking from rafts to filopodia and ultimately onto phosphatidylserine-positive, highly procoagulant MPs. Furthermore, caspase-1 specifically facilitated cell surface actin exposure, which was required for the final release of highly procoagulant MPs from filopodia. Together, the results of this study delineate a thromboinflammatory pathway and suggest that components of this pathway have potential as pharmacological targets to simultaneously attenuate inflammation and innate immune cell–induced thrombosis.

Authors

Andrea S. Rothmeier, Patrizia Marchese, Brian G. Petrich, Christian Furlan-Freguia, Mark H. Ginsberg, Zaverio M. Ruggeri, Wolfram Ruf

RASA3 is a critical inhibitor of RAP1-dependent platelet activation

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Abstract

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.

Authors

Lucia Stefanini, David S. Paul, Raymond F. Robledo, E. Ricky Chan, Todd M. Getz, Robert A. Campbell, Daniel O. Kechele, Caterina Casari, Raymond Piatt, Kathleen M. Caron, Nigel Mackman, Andrew S. Weyrich, Matthew C. Parrott, Yacine Boulaftali, Mark D. Adams, Luanne L. Peters, Wolfgang Bergmeier

Protein tyrosine phosphatase–σ regulates hematopoietic stem cell–repopulating capacity

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Abstract

Hematopoietic stem cell (HSC) function is regulated by activation of receptor tyrosine kinases (RTKs). Receptor protein tyrosine phosphatases (PTPs) counterbalance RTK signaling; however, the functions of receptor PTPs in HSCs remain incompletely understood. We found that a receptor PTP, PTPσ, was substantially overexpressed in mouse and human HSCs compared with more mature hematopoietic cells. Competitive transplantation of bone marrow cells from PTPσ-deficient mice revealed that the loss of PTPσ substantially increased long-term HSC-repopulating capacity compared with BM cells from control mice. While HSCs from PTPσ-deficient mice had no apparent alterations in cell-cycle status, apoptosis, or homing capacity, these HSCs exhibited increased levels of activated RAC1, a RhoGTPase that regulates HSC engraftment capacity. shRNA-mediated silencing of PTPσ also increased activated RAC1 levels in wild-type HSCs. Functionally, PTPσ-deficient BM cells displayed increased cobblestone area–forming cell (CAFC) capacity and augmented transendothelial migration capacity, which was abrogated by RAC inhibition. Specific selection of human cord blood CD34⁺CD38^–CD45RA^–lin^– PTPσ^– cells substantially increased the repopulating capacity of human HSCs compared with CD34⁺CD38^–CD45RA^–lin^– cells and CD34⁺CD38^–CD45RA^–lin^–PTPσ⁺ cells. Our results demonstrate that PTPσ regulates HSC functional capacity via RAC1 inhibition and suggest that selecting for PTPσ-negative human HSCs may be an effective strategy for enriching human HSCs for transplantation.

Authors

Mamle Quarmyne, Phuong L. Doan, Heather A. Himburg, Xiao Yan, Mai Nakamura, Liman Zhao, Nelson J. Chao, John P. Chute

Pleiotrophin mediates hematopoietic regeneration via activation of RAS

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Abstract

Hematopoietic stem cells (HSCs) are highly susceptible to ionizing radiation–mediated death via induction of ROS, DNA double-strand breaks, and apoptotic pathways. The development of therapeutics capable of mitigating ionizing radiation–induced hematopoietic toxicity could benefit both victims of acute radiation sickness and patients undergoing hematopoietic cell transplantation. Unfortunately, therapies capable of accelerating hematopoietic reconstitution following lethal radiation exposure have remained elusive. Here, we found that systemic administration of pleiotrophin (PTN), a protein that is secreted by BM-derived endothelial cells, substantially increased the survival of mice following radiation exposure and after myeloablative BM transplantation. In both models, PTN increased survival by accelerating the recovery of BM hematopoietic stem and progenitor cells in vivo. PTN treatment promoted HSC regeneration via activation of the RAS pathway in mice that expressed protein tyrosine phosphatase receptor-zeta (PTPRZ), whereas PTN treatment did not induce RAS signaling in PTPRZ-deficient mice, suggesting that PTN-mediated activation of RAS was dependent upon signaling through PTPRZ. PTN strongly inhibited HSC cycling following irradiation, whereas RAS inhibition abrogated PTN-mediated induction of HSC quiescence, blocked PTN-mediated recovery of hematopoietic stem and progenitor cells, and abolished PTN-mediated survival of irradiated mice. These studies demonstrate the therapeutic potential of PTN to improve survival after myeloablation and suggest that PTN-mediated hematopoietic regeneration occurs in a RAS-dependent manner.

Authors

Heather A. Himburg, Xiao Yan, Phuong L. Doan, Mamle Quarmyne, Eva Micewicz, William McBride, Nelson J. Chao, Dennis J. Slamon, John P. Chute

Plasma fibronectin supports hemostasis and regulates thrombosis

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Abstract

Plasma fibronectin (pFn) has long been suspected to be involved in hemostasis; however, direct evidence has been lacking. Here, we demonstrated that pFn is vital to control bleeding in fibrinogen-deficient mice and in WT mice given anticoagulants. At the site of vessel injury, pFn was rapidly deposited and initiated hemostasis, even before platelet accumulation, which is considered the first wave of hemostasis. This pFn deposition was independent of fibrinogen, von Willebrand factor, β3 integrin, and platelets. Confocal and scanning electron microscopy revealed pFn integration into fibrin, which increased fibrin fiber diameter and enhanced the mechanical strength of clots, as determined by thromboelastography. Interestingly, pFn promoted platelet aggregation when linked with fibrin but inhibited this process when fibrin was absent. Therefore, pFn may gradually switch from supporting hemostasis to inhibiting thrombosis and vessel occlusion following the fibrin gradient that decreases farther from the injured endothelium. Our data indicate that pFn is a supportive factor in hemostasis, which is vital under both genetic and therapeutic conditions of coagulation deficiency. By interacting with fibrin and platelet β3 integrin, pFn plays a self-limiting regulatory role in thrombosis, suggesting pFn transfusion may be a potential therapy for bleeding disorders, particularly in association with anticoagulant therapy.

Authors

Yiming Wang, Adili Reheman, Christopher M. Spring, Jalil Kalantari, Alexandra H. Marshall, Alisa S. Wolberg, Peter L. Gross, Jeffrey I. Weitz, Margaret L. Rand, Deane F. Mosher, John Freedman, Heyu Ni

Disposable platform provides visual and color-based point-of-care anemia self-testing

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Abstract

BACKGROUND. Anemia, or low blood hemoglobin (Hgb) levels, afflicts 2 billion people worldwide. Currently, Hgb levels are typically measured from blood samples using hematology analyzers, which are housed in hospitals, clinics, or commercial laboratories and require skilled technicians to operate. A reliable, inexpensive point-of-care (POC) Hgb test would enable cost-effective anemia screening and chronically anemic patients to self-monitor their disease. We present a rapid, stand-alone, and disposable POC anemia test that, via a single drop of blood, outputs color-based visual results that correlate with Hgb levels.

METHODS. We tested blood from 238 pediatric and adult patients with anemia of varying degrees and etiologies and compared hematology analyzer Hgb levels with POC Hgb levels, which were estimated via visual interpretation using a color scale and an optional smartphone app for automated analysis.

RESULTS. POC Hgb levels correlated with hematology analyzer Hgb levels (r = 0.864 and r = 0.856 for visual interpretation and smartphone app, respectively), and both POC test methods yielded comparable sensitivity and specificity for detecting any anemia (n = 178) (<11 g/dl) (sensitivity: 90.2% and 91.1%, specificity: 83.7% and 79.2%, respectively) and severe anemia (n = 10) (<7 g/dl) (sensitivity: 90.0% and 100%, specificity: 94.6% and 93.9%, respectively).

CONCLUSIONS. These results demonstrate the feasibility of this POC color-based diagnostic test for self-screening/self-monitoring of anemia.

TRIAL REGISTRATION. Not applicable.

FUNDING. This work was funded by the FDA-funded Atlantic Pediatric Device Consortium, the Georgia Research Alliance, Children’s Healthcare of Atlanta, the Georgia Center of Innovation for Manufacturing, and the InVenture Prize and Ideas to Serve competitions at the Georgia Institute of Technology.

Authors

Erika A. Tyburski, Scott E. Gillespie, William A. Stoy, Robert G. Mannino, Alexander J. Weiss, Alexa F. Siu, Rayford H. Bulloch, Karthik Thota, Anyela Cardenas, Wilena Session, Hanna J. Khoury, Siobhán O’Connor, Silvia T. Bunting, Jeanne Boudreaux, Craig R. Forest, Manila Gaddh, Traci Leong, L. Andrew Lyon, Wilbur A. Lam

TMEM14C is required for erythroid mitochondrial heme metabolism

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Abstract

The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.

Authors

Yvette Y. Yien, Raymond F. Robledo, Iman J. Schultz, Naoko Takahashi-Makise, Babette Gwynn, Daniel E. Bauer, Abhishek Dass, Gloria Yi, Liangtao Li, Gordon J. Hildick-Smith, Jeffrey D. Cooney, Eric L. Pierce, Kyla Mohler, Tamara A. Dailey, Non Miyata, Paul D. Kingsley, Caterina Garone, Shilpa M. Hattangadi, Hui Huang, Wen Chen, Ellen M. Keenan, Dhvanit I. Shah, Thorsten M. Schlaeger, Salvatore DiMauro, Stuart H. Orkin, Alan B. Cantor, James Palis, Carla M. Koehler, Harvey F. Lodish, Jerry Kaplan, Diane M. Ward, Harry A. Dailey, John D. Phillips, Luanne L. Peters, Barry H. Paw

Proteasome function is required for platelet production

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Abstract

The proteasome inhibiter bortezomib has been successfully used to treat patients with relapsed multiple myeloma; however, many of these patients become thrombocytopenic, and it is not clear how the proteasome influences platelet production. Here we determined that pharmacologic inhibition of proteasome activity blocks proplatelet formation in human and mouse megakaryocytes. We also found that megakaryocytes isolated from mice deficient for PSMC1, an essential subunit of the 26S proteasome, fail to produce proplatelets. Consistent with decreased proplatelet formation, mice lacking PSMC1 in platelets (Psmc1^fl/fl Pf4-Cre mice) exhibited severe thrombocytopenia and died shortly after birth. The failure to produce proplatelets in proteasome-inhibited megakaryocytes was due to upregulation and hyperactivation of the small GTPase, RhoA, rather than NF-κB, as has been previously suggested. Inhibition of RhoA or its downstream target, Rho-associated protein kinase (ROCK), restored megakaryocyte proplatelet formation in the setting of proteasome inhibition in vitro. Similarly, fasudil, a ROCK inhibitor used clinically to treat cerebral vasospasm, restored platelet counts in adult mice that were made thrombocytopenic by tamoxifen-induced suppression of proteasome activity in megakaryocytes and platelets (Psmc1^fl/fl Pdgf-Cre-ER mice). These results indicate that proteasome function is critical for thrombopoiesis, and suggest inhibition of RhoA signaling as a potential strategy to treat thrombocytopenia in bortezomib-treated multiple myeloma patients.

Authors

Dallas S. Shi, Matthew C.P. Smith, Robert A. Campbell, Patrick W. Zimmerman, Zechariah B. Franks, Bjorn F. Kraemer, Kellie R. Machlus, Jing Ling, Patrick Kamba, Hansjörg Schwertz, Jesse W. Rowley, Rodney R. Miles, Zhi-Jian Liu, Martha Sola-Visner, Joseph E. Italiano Jr., Hilary Christensen, Walter H.A. Kahr, Dean Y. Li, Andrew S. Weyrich

Factor XIII activity mediates red blood cell retention in venous thrombi

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Abstract

Venous thrombi, fibrin- and rbc-rich clots triggered by inflammation and blood stasis, underlie devastating, and sometimes fatal, occlusive events. During intravascular fibrin deposition, rbc are thought to become passively trapped in thrombi and therefore have not been considered a modifiable thrombus component. In the present study, we determined that activity of the transglutaminase factor XIII (FXIII) is critical for rbc retention within clots and directly affects thrombus size. Compared with WT mice, mice carrying a homozygous mutation in the fibrinogen γ chain (Fibγ^390–396A) had a striking 50% reduction in thrombus weight due to reduced rbc content. Fibrinogen from mice harboring the Fibγ^390–396A mutation exhibited reduced binding to FXIII, and plasma from these mice exhibited delayed FXIII activation and fibrin crosslinking, indicating these residues mediate FXIII binding and activation. FXIII-deficient mice phenocopied mice carrying Fibγ^390–396A and produced smaller thrombi with fewer rbc than WT mice. Importantly, FXIII-deficient human clots also exhibited reduced rbc retention. The addition of FXIII to FXIII-deficient clots increased rbc retention, while inhibition of FXIII activity in normal blood reduced rbc retention and produced smaller clots. These findings establish the FXIII-fibrinogen axis as a central determinant in venous thrombogenesis and identify FXIII as a potential therapeutic target for limiting venous thrombosis.

Authors

Maria M. Aleman, James R. Byrnes, Jian-Guo Wang, Reginald Tran, Wilbur A. Lam, Jorge Di Paola, Nigel Mackman, Jay L. Degen, Matthew J. Flick, Alisa S. Wolberg