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Thromb Haemost 2017; 117(12): 2322-2333
DOI: 10.1160/TH17-06-0433
Cellular Haemostasis and Platelets
Schattauer GmbH Stuttgart

Developmental Stage–Specific Manifestations of Absent TPO/c-MPL Signalling in Newborn Mice

Viola Lorenz   *
,
Haley Ramsey   *
,
Zhi-Jian Liu
,
Joseph Italiano Jr.
,
Karin Hoffmeister
,
Sihem Bihorel
,
Donald Mager
,
Zhongbo Hu
,
William B. Slayton
,
Benjamin T. Kile
,
Martha Sola-Visner   *
,
Francisca Ferrer-Marin   *
Further Information

Publication History

23 June 2017

08 September 2017

Publication Date:
06 December 2017 (online)

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Abstract

Congenital amegakaryocytic thrombocytopaenia (CAMT) is a disorder caused by c-MPL mutations that impair thrombopoietin (TPO) signalling, resulting in a near absence of megakaryocytes (MKs). While this phenotype is consistent in adults, neonates with CAMT can present with severe thrombocytopaenia despite normal MK numbers. To investigate this, we characterized MKs and platelets in newborn c-MPL –/– mice. Liver MKs in c-MPL –/– neonates were reduced in number and size compared with wild-type (WT) age-matched MKs, and exhibited ultrastructural abnormalities not found in adult c-MPL –/– MKs. Platelet counts were lower in c-MPL –/– compared with WT mice at birth and did not increase over the first 2 weeks of life. In vivo biotinylation revealed a significant reduction in the platelet half-life of c-MPL –/– newborn mice (P2) compared with age-matched WT pups, which was not associated with ultrastructural abnormalities. Genetic deletion of the pro-apoptotic Bak did not rescue the severely reduced platelet half-life of c-MPL –/– newborn mice, suggesting that it was due to factors other than platelets entering apoptosis early. Indeed, adult GFP+ (green fluorescent protein transgenic) platelets transfused into thrombocytopenic c-MPL –/– P2 pups also had a shortened lifespan, indicating the importance of cell-extrinsic factors. In addition, neonatal platelets from WT and c-MPL –/– mice exhibited reduced P-selectin surface expression following stimulation compared with adult platelets of either genotype, and platelets from c-MPL –/– neonates exhibited reduced glycoprotein IIb/IIIa (GPIIb/IIIa) activation in response to thrombin compared with age-matched WT platelets. Taken together, our findings indicate that c-MPL deficiency is associated with abnormal maturation of neonatal MKs and developmental stage-specific defects in platelet function.

Keywords

animal models - bone marrow - megakaryocytes - platelet physiology - thrombocytopaenia

Authors' Contributions

V.L., H.R, Z-J.L., S. B., D.M., Z.H. and F.F-M. designed and performed experiments, collected and analysed data, and wrote the manuscript; J.I. Jr. performed and interpreted the electron microscopy studies; K.H., W.B.S. and B.T.K. assisted with experimental design, data interpretation and preparation of the manuscript; M.S-V. and F.F-M. supervised and designed experiments, interpreted data and wrote the manuscript.


* Viola Lorenz, Haley Ramsey, Martha Sola-Visner and Francisca Ferrer-Marin contributed equally to this work.


Supplementary Material

 

  • References

  • 1 Sola MC, Du Y, Hutson AD, Christensen RD. Dose-response relationship of megakaryocyte progenitors from the bone marrow of thrombocytopenic and non-thrombocytopenic neonates to recombinant thrombopoietin. Br J Haematol 2000; 110 (02) 449-453
    CrossrefPubMedGoogle Scholar
  • 2 Pastos KM, Slayton WB, Rimsza LM, Young L, Sola-Visner MC. Differential effects of recombinant thrombopoietin and bone marrow stromal-conditioned media on neonatal versus adult megakaryocytes. Blood 2006; 108 (10) 3360-3362
    CrossrefPubMedGoogle Scholar
  • 3 Liu ZJ, Sola-Visner M. Neonatal and adult megakaryopoiesis. Curr Opin Hematol 2011; 18 (05) 330-337
    CrossrefPubMedGoogle Scholar
  • 4 Liu ZJ, Italiano Jr J, Ferrer-Marin F. , et al. Developmental differences in megakaryocytopoiesis are associated with up-regulated TPO signaling through mTOR and elevated GATA-1 levels in neonatal megakaryocytes. Blood 2011; 117 (15) 4106-4117
    CrossrefPubMedGoogle Scholar
  • 5 Klusmann JH, Godinho FJ, Heitmann K. , et al. Developmental stage-specific interplay of GATA1 and IGF signaling in fetal megakaryopoiesis and leukemogenesis. Genes Dev 2010; 24 (15) 1659-1672
    CrossrefPubMedGoogle Scholar
  • 6 Woo AJ, Wieland K, Huang H. , et al. Developmental differences in IFN signaling affect GATA1s-induced megakaryocyte hyperproliferation. J Clin Invest 2013; 40609
    PubMedGoogle Scholar
  • 7 Bluteau O, Langlois T, Rivera-Munoz P. , et al. Developmental changes in human megakaryopoiesis. J Thromb Haemost 2013; 11 (09) 1730-1741
    CrossrefPubMedGoogle Scholar
  • 8 Majewski IJ, Metcalf D, Mielke LA. , et al. A mutation in the translation initiation codon of Gata-1 disrupts megakaryocyte maturation and causes thrombocytopenia. Proc Natl Acad Sci U S A 2006; 103 (38) 14146-14151
    CrossrefPubMedGoogle Scholar
  • 9 Hollanda LM, Lima CS, Cunha AF. , et al. An inherited mutation leading to production of only the short isoform of GATA-1 is associated with impaired erythropoiesis. Nat Genet 2006; 38 (07) 807-812
    CrossrefPubMedGoogle Scholar
  • 10 Letestu R, Vitrat N, Massé A. , et al. Existence of a differentiation blockage at the stage of a megakaryocyte precursor in the thrombocytopenia and absent radii (TAR) syndrome. Blood 2000; 95 (05) 1633-1641
    PubMedGoogle Scholar
  • 11 al-Jefri AH, Dror Y, Bussel JB, Freedman MH. Thrombocytopenia with absent radii: frequency of marrow megakaryocyte progenitors, proliferative characteristics, and megakaryocyte growth and development factor responsiveness. Pediatr Hematol Oncol 2000; 17 (04) 299-306
    CrossrefPubMedGoogle Scholar
  • 12 Dreyfus M, Tchernia G. Thrombocytopenia with absent radii (TAR syndrome): new advances in the mechanism of thrombocytopenia. Pediatr Hematol Oncol 2000; 17 (07) 521-522
    CrossrefPubMedGoogle Scholar
  • 13 Ballmaier M, Germeshausen M, Schulze H. , et al. c-mpl mutations are the cause of congenital amegakaryocytic thrombocytopenia. Blood 2001; 97 (01) 139-146
    CrossrefPubMedGoogle Scholar
  • 14 Geddis AE. Congenital amegakaryocytic thrombocytopenia and thrombocytopenia with absent radii. Hematol Oncol Clin North Am 2009; 23 (02) 321-331
    CrossrefPubMedGoogle Scholar
  • 15 Stoddart MT, Connor P, Germeshausen M, Ballmaier M, Steward CG. Congenital amegakaryocytic thrombocytopenia (CAMT) presenting as severe pancytopenia in the first month of life. Pediatr Blood Cancer 2013; 60 (09) E94-E96
    CrossrefPubMedGoogle Scholar
  • 16 Ihara K, Ishii E, Eguchi M. , et al. Identification of mutations in the c-mpl gene in congenital amegakaryocytic thrombocytopenia. Proc Natl Acad Sci U S A 1999; 96 (06) 3132-3136
    CrossrefPubMedGoogle Scholar
  • 17 Germeshausen M, Ballmaier M, Welte K. MPL mutations in 23 patients suffering from congenital amegakaryocytic thrombocytopenia: the type of mutation predicts the course of the disease. Hum Mutat 2006; 27 (03) 296
    PubMedGoogle Scholar
  • 18 King S, Germeshausen M, Strauss G, Welte K, Ballmaier M. Congenital amegakaryocytic thrombocytopenia: a retrospective clinical analysis of 20 patients. Br J Haematol 2005; 131 (05) 636-644
    CrossrefPubMedGoogle Scholar
  • 19 Fox NE, Chen R, Hitchcock I, Keates-Baleeiro J, Frangoul H, Geddis AE. Compound heterozygous c-Mpl mutations in a child with congenital amegakaryocytic thrombocytopenia: functional characterization and a review of the literature. Exp Hematol 2009; 37 (04) 495-503
    CrossrefPubMedGoogle Scholar
  • 20 Rose MJ, Nicol KK, Skeens MA, Gross TG, Kerlin BA. Congenital amegakaryocytic thrombocytopenia: the diagnostic importance of combining pathology with molecular genetics. Pediatr Blood Cancer 2008; 50 (06) 1263-1265
    CrossrefPubMedGoogle Scholar
  • 21 Henter JI, Winiarski J, Ljungman P, Ringdén O, Ost A. Bone marrow transplantation in two children with congenital amegakaryocytic thrombocytopenia. Bone Marrow Transplant 1995; 15 (05) 799-801
    PubMedGoogle Scholar
  • 22 Gurney AL, Carver-Moore K, de Sauvage FJ, Moore MW. Thrombocytopenia in c-mpl-deficient mice. Science 1994; 265 (5177): 1445-1447
    CrossrefPubMedGoogle Scholar
  • 23 Alexander WS, Roberts AW, Nicola NA, Li R, Metcalf D. Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietic receptor c-Mpl. Blood 1996; 87 (06) 2162-2170
    PubMedGoogle Scholar
  • 24 Bunting S, Widmer R, Lipari T. , et al. Normal platelets and megakaryocytes are produced in vivo in the absence of thrombopoietin. Blood 1997; 90 (09) 3423-3429
    PubMedGoogle Scholar
  • 25 Liu ZJ, Hoffmeister KM, Hu Z. , et al. Expansion of the neonatal platelet mass is achieved via an extension of platelet lifespan. Blood 2014; 123 (22) 3381-3389
    CrossrefPubMedGoogle Scholar
  • 26 Bentfeld-Barker ME, Bainton DF. Ultrastructure of rat megakaryocytes after prolonged thrombocytopenia. J Ultrastruct Res 1977; 61 (02) 201-214
    CrossrefPubMedGoogle Scholar
  • 27 Poujol C, Ware J, Nieswandt B, Nurden AT, Nurden P. Absence of GPIbalpha is responsible for aberrant membrane development during megakaryocyte maturation: ultrastructural study using a transgenic model. Exp Hematol 2002; 30 (04) 352-360
    CrossrefPubMedGoogle Scholar
  • 28 Hu Z, Slayton WB, Rimsza LM, Bailey M, Sallmon H, Sola-Visner MC. Differences between newborn and adult mice in their response to immune thrombocytopenia. Neonatology 2010; 98 (01) 100-108
    CrossrefPubMedGoogle Scholar
  • 29 Sparger KA, Ramsey H, Lorenz V. , et al. Developmental differences between newborn and adult mice in response to romiplostim. Platelets 2017; 1-8
    PubMedGoogle Scholar
  • 30 Josefsson EC, James C, Henley KJ. , et al. Megakaryocytes possess a functional intrinsic apoptosis pathway that must be restrained to survive and produce platelets. J Exp Med 2011; 208 (10) 2017-2031
    CrossrefPubMedGoogle Scholar
  • 31 Potts KS, Sargeant TJ, Dawson CA. , et al. Mouse prenatal platelet-forming lineages share a core transcriptional program but divergent dependence on MPL. Blood 2015; 126 (06) 807-816
    CrossrefPubMedGoogle Scholar
  • 32 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 U S A 2014; 111 (16) 5884-5889
    CrossrefPubMedGoogle Scholar
  • 33 Lebois M, Dowling MR, Gangatirkar P. , et al. Regulation of platelet lifespan in the presence and absence of thrombopoietin signaling. J Thromb Haemost 2016; 14 (09) 1882-1887
    CrossrefPubMedGoogle Scholar
  • 34 Coupland LA, Cromer D, Davenport MP, Parish CR. A novel fluorescent-based assay reveals that thrombopoietin signaling and Bcl-X(L) influence, respectively, platelet and erythrocyte lifespans. Exp Hematol 2010; 38 (06) 453-461.e1
    CrossrefPubMedGoogle Scholar
  • 35 Zhang H, Nimmer PM, Tahir SK. , et al. Bcl-2 family proteins are essential for platelet survival. Cell Death Differ 2007; 14 (05) 943-951
    PubMedGoogle Scholar
  • 36 Debrincat MA, Josefsson EC, James C. , et al. Mcl-1 and Bcl-x(L) coordinately regulate megakaryocyte survival. Blood 2012; 119 (24) 5850-5858
    CrossrefPubMedGoogle Scholar
  • 37 Kodama T, Hikita H, Kawaguchi T. , et al. Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages. Cell Death Differ 2012; 19 (11) 1856-1869
    CrossrefPubMedGoogle Scholar
  • 38 Kirito K, Watanabe T, Sawada K, Endo H, Ozawa K, Komatsu N. Thrombopoietin regulates Bcl-xL gene expression through Stat5 and phosphatidylinositol 3-kinase activation pathways. J Biol Chem 2002; 277 (10) 8329-8337
    CrossrefPubMedGoogle Scholar
  • 39 Mason KD, Carpinelli MR, Fletcher JI. , et al. Programmed anuclear cell death delimits platelet life span. Cell 2007; 128 (06) 1173-1186
    CrossrefPubMedGoogle Scholar
  • 40 Morowski M, Vögtle T, Kraft P, Kleinschnitz C, Stoll G, Nieswandt B. Only severe thrombocytopenia results in bleeding and defective thrombus formation in mice. Blood 2013; 121 (24) 4938-4947
    CrossrefPubMedGoogle Scholar
  • 41 Hanson SR, Slichter SJ. Platelet kinetics in patients with bone marrow hypoplasia: evidence for a fixed platelet requirement. Blood 1985; 66 (05) 1105-1109
    PubMedGoogle Scholar
  • 42 Dowling MR, Josefsson EC, Henley KJ, Kile BT, Hodgkin PD. A model for studying the hemostatic consumption or destruction of platelets. PLoS One 2013; 8 (03) e57783
    CrossrefPubMedGoogle Scholar
  • 43 Ballabh P. Intraventricular hemorrhage in premature infants: mechanism of disease. Pediatr Res 2010; 67 (01) 1-8
    CrossrefPubMedGoogle Scholar
  • 44 Fu Z, Heldt GP, West JB. Increased fragility of pulmonary capillaries in newborn rabbit. Am J Physiol Lung Cell Mol Physiol 2003; 284 (05) L703-L709
    CrossrefPubMedGoogle Scholar
  • 45 Sola-Visner M. Platelets in the neonatal period: developmental differences in platelet production, function, and hemostasis and the potential impact of therapies. Hematology (Am Soc Hematol Educ Program) 2012; 2012: 506-511
    PubMedGoogle Scholar
  • 46 Stolla MC, Leyens K, Catherman SC. , et al. P-selectin expression and platelet function are developmentally regulated. Blood 2014; 124 (21) 1439
    PubMedGoogle Scholar
  • 47 Margraf A, Nussbaum C, Rohwedder I. , et al. Maturation of platelet function during murine fetal development in vivo. Arterioscler Thromb Vasc Biol 2017; 37 (06) 1076-1086
    CrossrefPubMedGoogle Scholar
  • 48 Berger G, Hartwell DW, Wagner DD. P-selectin and platelet clearance. Blood 1998; 92 (11) 4446-4452
    CrossrefPubMedGoogle Scholar
  • 49 Subramaniam M, Frenette PS, Saffaripour S, Johnson RC, Hynes RO, Wagner DD. Defects in hemostasis in P-selectin-deficient mice. Blood 1996; 87 (04) 1238-1242
    PubMedGoogle Scholar
  • 50 Sparger KA, Assmann SF, Granger S. , et al. Platelet transfusion practices among very-low-birth-weight infants. JAMA Pediatr 2016; 170 (07) 687-694
    CrossrefPubMedGoogle Scholar