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and the effects on the downstream signaling pathways of FcgRIIa favor an intracellular mechanism for 12-LOX either directly through PLCg2 or via an upstream ITAM signaling complex (see figure). What is important is that this is the first demonstration that 12-LOX is involved in platelet activation through the immune Fc receptor FcgRIIa and as such may provide a viable therapeutic target for patients with HIT. It is also worth noting our own observation that following infusion of heparin, there is a significant release of 12-HETE in vivo, despite aspirin therapy,14 suggesting 12-LOX may be even more intrinsically involved in the pathology of HIT. However, because 12-HETE has roles in other cells, the effect of inhibiting 12-LOX may have wider ranging effects that will be revealed through in vivo studies with inhibitors such as ML355. Conflict-of-interest disclosure: The author declares no competing financial interests. n REFERENCES 1. Yeung J, Tourdot BE, Fernandez-Perez P, et al. Platelet 12-LOX is essential for FcgRIIa-mediated platelet activation. Blood 2014;124(14):2271-2279. 2. Greinacher A, Althaus K, Krauel K, Selleng S. Heparin-induced thrombocytopenia. Hamostaseologie. 2010;30(1):17-18, 20-28.
12. Nie D, Krishnamoorthy S, Jin R, et al. Mechanisms regulating tumor angiogenesis by 12-lipoxygenase in prostate cancer cells. J Biol Chem. 2006;281(27):18601-18609. 13. Baba A, Sakuma S, Okamoto H, Inoue T, Iwata H. Calcium induces membrane translocation of 12-lipoxygenase in rat platelets. J Biol Chem. 1989;264(27): 15790-15795.
14. McMahon GS, Jones CI, Hayes PD, Naylor AR, Goodall AH. Transient heparin-induced platelet activation linked to generation of platelet 12-lipoxygenase. Findings from a randomised controlled trial. Thromb Haemost. 2013; 109(6):1099-1107. © 2014 by The American Society of Hematology
l l l PLATELETS & THROMBOPOIESIS
Comment on Meyer et al, page 2280
Less Jak2 makes more platelets ----------------------------------------------------------------------------------------------------Radek C. Skoda
UNIVERSITY HOSPITAL BASEL
In this issue of Blood, Meyer et al report that deleting Jak2 selectively in megakaryocytes and platelets results in an unexpected thrombocytosis phenotype. Their results demonstrate that Jak2 is dispensable for megakaryocyte differentiation and platelet formation but is required for suppressing circulating thrombopoietin (Tpo).1
J
ak2 is an intracellular tyrosine kinase that associates with hematopoietic cytokine receptors and is essential for mediating signaling by Tpo, erythropoietin, and in part also granulocyte colony-stimulating factor. Jak2 inhibitors can be used to suppress excess hematopoiesis in patients with
myeloproliferative neoplasms, although at higher doses anemia and thrombocytopenia are frequently observed. Jak2 knockout mice die during embryogenesis due to absence of definitive erythropoiesis.2 Similarly, induced conditional knockout of Jak2 in adult hematopoiesis was lethal due to severe anemia
3. Boulaftali Y, Hess PR, Kahn ML, Bergmeier W. Platelet immunoreceptor tyrosine-based activation motif (ITAM) signaling and vascular integrity. Circ Res. 2014;114(7):1174-1184. 4. Porro B, Songia P, Squellerio I, Tremoli E, Cavalca V. Analysis, physiological and clinical significance of 12-HETE: a neglected platelet-derived 12-lipoxygenase product. J Chromatogr B Analyt Technol Biomed Life Sci. 2014;964:26-40. 5. Luci DK, Jameson JB II, Yasgar A, et al. Synthesis and structure-activity relationship studies of 4-((2-hydroxy-3methoxybenzyl)amino)benzenesulfonamide derivatives as potent and selective inhibitors of 12-lipoxygenase. J Med Chem. 2014;57(2):495-506. 6. Yeung J, Apopa PL, Vesci J, et al. 12-lipoxygenase activity plays an important role in PAR4 and GPVI-mediated platelet reactivity. Thromb Haemost. 2013;110(3):569-581. 7. Yeung J, Apopa PL, Vesci J, et al. Protein kinase C regulation of 12-lipoxygenase-mediated human platelet activation. Mol Pharmacol. 2012;81(3):420-430. 8. Guo Y, Zhang W, Giroux C, et al. Identification of the orphan G protein-coupled receptor GPR31 as a receptor for 12-(S)-hydroxyeicosatetraenoic acid. J Biol Chem. 2011; 286(39):33832-33840. 9. Thomas CP, Morgan LT, Maskrey BH, et al. Phospholipid-esterified eicosanoids are generated in agonist-activated human platelets and enhance tissue factor-dependent thrombin generation. J Biol Chem. 2010; 285(10):6891-6903. 10. Jankun J, Aleem AM, Malgorzewicz S, et al. Synthetic curcuminoids modulate the arachidonic acid metabolism of human platelet 12-lipoxygenase and reduce sprout formation of human endothelial cells. Mol Cancer Ther. 2006;5(5):1371-1382. 11. Kandouz M, Nie D, Pidgeon GP, Krishnamoorthy S, Maddipati KR, Honn KV. Platelet-type 12-lipoxygenase activates NF-kappaB in prostate cancer cells. Prostaglandins Other Lipid Mediat. 2003;71(3-4):189-204.
2168
Model for the regulation of megakaryopoiesis and platelet numbers by Tpo. (A) Normal steady-state situation. Tpo (blue circles) is produced in the liver at a constant rate and reaches the bone marrow via the blood stream. Tpo enters the bone marrow microenvironment and binds to its receptor, Mpl (drawn in red), that is expressed on HSCs and megakaryocytic progenitors. Signaling requires the presence of Jak2 (green circles) and results in expansion of the HSCs and megakaryocytic progenitor pool. The megakaryocytic differentiation and polyploidization begins at the stage of promegakaryoblasts (pro-Meg) and ends with fully differentiated megakaryocytes (Meg), which deliver platelets (PLT) to the lumen of the blood vessels (yellow arrow). The bone marrow cells that express Mpl and platelets bind, internalize, and degrade Tpo, thereby lowering the available Tpo. (B) Megakaryocyte and platelet-specific knockout of Jak2. Expression of Cre-recombinase driven by the Pf4 regulatory elements begins in late megakaryocytic progenitors and deletes Jak2 in megakaryocytes and platelets. Mpl expression remains normal throughout megakaryopoiesis, but Tpo cannot signal in late megakaryopoetic cells or platelets. Mpl without Jak2 cannot efficiently remove and degrade Tpo. As a consequence, more Tpo is available in the bone marrow, leading to an expansion of HSCs, early megakaryocytebiased progenitors, and colony-forming unit Meg. Thrombocytosis is observed in the peripheral blood.
BLOOD, 2 OCTOBER 2014 x VOLUME 124, NUMBER 14
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and thrombocytopenia.3,4 Therefore, at first sight, thrombocytosis in mice lacking Jak2 in megakaryocytes may seem paradoxical. However, mice selectively lacking the Tpo receptor, Mpl, in megakaryocytes and platelets also displayed thrombocytosis.5 The genetic and functional data presented by Meyer et al and the recent report on Mpl-deficient mice by Ng et al5 shed new light into a delicately balanced system that keeps the levels of circulating platelets constant. Together these data suggest that the primary function of Jak2 and Mpl in late megakaryopoiesis and platelets is to reduce the availability of Tpo rather than to promote terminal megakaryocyte differentiation and platelet production. Because Mpl is dependent on Jak2 for signaling, the 2 stories are intricately connected. Although Jak2 expression is ubiquitous, Mpl is expressed in hematopoietic stem cells (HSCs) and early progenitors and at later stages becomes limited to the megakaryocytic lineage (panel A). The circulating Tpo concentration is directly regulated by a negative feedback mechanism exerted by platelets and megakaryocytes.6 Mpl protein expressed on the surface of platelets and megakaryocytes binds Tpo, which is then internalized and degraded.7,8 Consequently, Mpl-deficient mice are thrombocytopenic with very high Tpo serum levels. However, thrombocytosis was observed in 2 mouse models with decreased Mpl expression on differentiated megakaryocytes and platelets as a first hint that the main function of Mpl on megakaryocytes and platelets could be to down-regulate Tpo availability.9,10 The studies by Ng et al and Meyer et al both use Pf4-Cre mice, which express the Cre-recombinase primarily in megakaryocytes and platelets, allowing lineage-specific deletion of conditional alleles during late megakaryocyte maturation but preserving expression in HSCs and progenitors. Both studies now firmly demonstrate that the absence of Mpl and Jak2 in megakaryocytes and platelets does not interfere with terminal differentiation and platelet formation. Thus, with hindsight, it was good to abandon the initially proposed alternative name for Tpo: megakaryocyte growth and development factor. Moreover, both studies found that the HSC and progenitor pool was expanded, suggesting that the stimulatory effects of Tpo mediated by Mpl and Jak2 are due to increased numbers of committed megakaryocytic progenitors (panel B).
Although it is easy to see how loss of Mpl expression on megakaryocytes and platelets reduces Tpo binding and internalization, the loss of Jak2 in late megakaryopoiesis could equally well result in normal platelet numbers. So why do the Pf4-Cre Jak2-deficient mice display such a pronounced thrombocytosis? One hypothetical scenario is that Jak2 deficiency reduces Mpl surface expression, eg, because Jak2 protein may be needed as a chaperone to guide Mpl from the endoplasmatic reticulum through the Golgi to reach the cell surface. However, surface expression of Mpl on megakaryocytes and platelets in Pf4-Cre Jak2-deficient mice was comparable with wild-type controls (see Figure 2J in Meyer et al). The Tpo serum levels in Pf4-Cre Jak2-deficient mice were equal to controls, but given that the platelets numbers were fivefold increased, the Tpo levels in these mice are expected to be lower than in the controls. This suggests that the Jak2-deficient megakaryocytes and platelets are less capable of binding and removing Tpo, as indeed shown by Meyer et al (see Figure 2K in Meyer et al). The mechanism of how Jak2 is involved in this process remains to be determined. Together, the results of the 2 studies by Meyer et al and Ng et al demonstrate that Mpl and Jak2 are dispensable for terminal megakaryocyte differentiation and platelet formation and suggest that the key regulatory step controlled by Tpo and Mpl is to stimulate a megakaryocyte-biased stem and progenitor cells and thereby to determine the number of committed megakaryocytic progenitors and undifferentiated megakaryocytic precursors.
Conflict-of-interest disclosure: The author declares no competing financial interests. n REFERENCES 1. Meyer SC, Keller MD, Woods BA, et al. Genetic studies reveal an unexpected negative regulatory role for Jak2 in thrombopoiesis. Blood. 2014;124(14):2280-2284. 2. Neubauer H, Cumano A, Mu¨ ller M, Wu H, Huffstadt U, Pfeffer K. Jak2 deficiency defines an essential developmental checkpoint in definitive hematopoiesis. Cell. 1998;93(3):397-409. 3. Park SO, Wamsley HL, Bae K, et al. Conditional deletion of Jak2 reveals an essential role in hematopoiesis throughout mouse ontogeny: implications for Jak2 inhibition in humans. PLoS ONE. 2013;8(3):e59675. 4. Grisouard J, Hao-Shen H, Dirnhofer S, Wagner KU, Skoda RC. Selective deletion of Jak2 in adult mouse hematopoietic cells leads to lethal anemia and thrombocytopenia. Haematologica. 2014;99(4):e52-e54. 5. Ng AP, Kauppi M, Metcalf D, et al. Mpl expression on megakaryocytes and platelets is dispensable for thrombopoiesis but essential to prevent myeloproliferation. Proc Natl Acad Sci USA. 2014;111(16):5884-5889. 6. Kuter DJ, Rosenberg RD. The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit. Blood. 1995;85(10):2720-2730. 7. Stoffel R, Wiestner A, Skoda RC. Thrombopoietin in thrombocytopenic mice: evidence against regulation at the mRNA level and for a direct regulatory role of platelets. Blood. 1996;87(2):567-573. 8. Fielder PJ, Gurney AL, Stefanich E, et al. Regulation of thrombopoietin levels by c-mpl-mediated binding to platelets. Blood. 1996;87(6):2154-2161. 9. Lannutti BJ, Epp A, Roy J, Chen J, Josephson NC. Incomplete restoration of Mpl expression in the mpl-/- mouse produces partial correction of the stem cell-repopulating defect and paradoxical thrombocytosis. Blood. 2009; 113(8):1778-1785. 10. Tiedt R, Coers J, Ziegler S, et al. Pronounced thrombocytosis in transgenic mice expressing reduced levels of Mpl in platelets and terminally differentiated megakaryocytes. Blood. 2009;113(8):1768-1777. © 2014 by The American Society of Hematology
l l l RED CELLS, IRON, & ERYTHROPOIESIS
Comment on McIver et al, page 2285
Exosome complex and erythropoiesis ----------------------------------------------------------------------------------------------------Xiuli An1
1
NEW YORK BLOOD CENTER
In this issue of Blood, McIver et al demonstrate that components of the exosome complex are endogenous suppressors of erythroid maturation, revealing a new and novel regulatory mechanism for erythroid development.1
E
rythropoiesis is a process during which pluripotent hematopoietic stem cells proliferate, differentiate, and eventually form mature erythrocytes. Eight distinct developmental stages including burst-forming unit-erythroid, colony-forming unit-
BLOOD, 2 OCTOBER 2014 x VOLUME 124, NUMBER 14
erythroid, proerythroblast, basophilic erythroblast, polychromatic erythroblast, orthochromatic erythroblast, reticulocyte, and mature erythrocyte can be distinguished during this process, which is accompanied by many characteristic changes including
2169
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2014 124: 2168-2169 doi:10.1182/blood-2014-08-596361
Less Jak2 makes more platelets Radek C. Skoda
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